Brian Kobilka

All Publications

• Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody. Nature communications Toyoda, Y., Zhu, A., Kong, F., Shan, S., Zhao, J., Wang, N., Sun, X., Zhang, L., Yan, C., Kobilka, B. K., Liu, X. 2023; 14 (1): 3655

  Abstract

  The α1A-adrenergic receptor (α1AAR) belongs to the family of G protein-coupled receptors that respond to adrenaline and noradrenaline. α1AAR is involved in smooth muscle contraction and cognitive function. Here, we present three cryo-electron microscopy structures of human α1AAR bound to the endogenous agonist noradrenaline, its selective agonist oxymetazoline, and the antagonist tamsulosin, with resolutions range from 2.9 Å to 3.5 Å. Our active and inactive α1AAR structures reveal the activation mechanism and distinct ligand binding modes for noradrenaline compared with other adrenergic receptor subtypes. In addition, we identified a nanobody that preferentially binds to the extracellular vestibule of α1AAR when bound to the selective agonist oxymetazoline. These results should facilitate the design of more selective therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

  View details for DOI 10.1038/s41467-023-39310-x

  View details for PubMedID 37339967

  View details for PubMedCentralID 8185284

• Structure based approaches on fentanyl template to design novel mu opioid modulators Ople, R., Wang, H., Li, Q., Polacco, B., Bernhard, S., Appourchaux, K., Sribhashyam, S., Eans, S., Huttenhain, R., McLaughlin, J., Kobilka, B., Majumdar, S. AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS. 2023

  View details for DOI 10.1124/jpet.122.527690

  View details for Web of Science ID 001043657700524

• Structural basis for activation of CB1 by an endocannabinoid analog. Nature communications Krishna Kumar, K., Robertson, M. J., Thadhani, E., Wang, H., Suomivuori, C. M., Powers, A. S., Ji, L., Nikas, S. P., Dror, R. O., Inoue, A., Makriyannis, A., Skiniotis, G., Kobilka, B. 2023; 14 (1): 2672

  Abstract

  Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the "toggle switch" residues F2003.36 and W3566.48. Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator.

  View details for DOI 10.1038/s41467-023-37864-4

  View details for PubMedID 37160876

  View details for PubMedCentralID PMC10169858

• Constrained catecholamines gain β2AR selectivity through allosteric effects on pocket dynamics. Nature communications Xu, X., Shonberg, J., Kaindl, J., Clark, M. J., Stößel, A., Maul, L., Mayer, D., Hübner, H., Hirata, K., Venkatakrishnan, A. J., Dror, R. O., Kobilka, B. K., Sunahara, R. K., Liu, X., Gmeiner, P. 2023; 14 (1): 2138

  Abstract

  G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β1 and β2 adrenergic receptors (β1AR and β2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β2AR over the β1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β2AR, as well as a less stable binding pocket for constrained epinephrine in the β1AR. The differences in the amino acid sequence of the extracellular vestibule of the β1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

  View details for DOI 10.1038/s41467-023-37808-y

  View details for PubMedID 37059717

  View details for PubMedCentralID PMC10104803

• Function and dynamics of the intrinsically disordered carboxyl terminus of beta2 adrenergic receptor. Nature communications Heng, J., Hu, Y., Perez-Hernandez, G., Inoue, A., Zhao, J., Ma, X., Sun, X., Kawakami, K., Ikuta, T., Ding, J., Yang, Y., Zhang, L., Peng, S., Niu, X., Li, H., Guixa-Gonzalez, R., Jin, C., Hildebrand, P. W., Chen, C., Kobilka, B. K. 2023; 14 (1): 2005

  Abstract

  Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The beta2 adrenergic receptor's (beta2AR) 71 amino acid CT is a substrate for GPCR kinases and binds beta-arrestins to regulate signaling. Here we show that the beta2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking beta-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged beta2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

  View details for DOI 10.1038/s41467-023-37233-1

  View details for PubMedID 37037825

• Negative allosteric modulation of the glucagon receptor by RAMP2. Cell Krishna Kumar, K., O'Brien, E. S., Habrian, C. H., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. 2023; 186 (7): 1465-1477.e18

  Abstract

  Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

  View details for DOI 10.1016/j.cell.2023.02.028

  View details for PubMedID 37001505

• Negative allosteric modulation of the glucagon receptor by RAMP2 O'Brien, E. S., Kumar, K., Habrian, C., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. CELL PRESS. 2023: 161A

  View details for Web of Science ID 000989629700786

• Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature communications Xu, J., Wang, Q., Hübner, H., Hu, Y., Niu, X., Wang, H., Maeda, S., Inoue, A., Tao, Y., Gmeiner, P., Du, Y., Jin, C., Kobilka, B. K. 2023; 14 (1): 376

  Abstract

  The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

  View details for DOI 10.1038/s41467-022-35726-z

  View details for PubMedID 36690613

  View details for PubMedCentralID PMC9870890

• Structure-based design of bitopic ligands for the µ-opioid receptor. Nature Faouzi, A., Wang, H., Zaidi, S. A., DiBerto, J. F., Che, T., Qu, Q., Robertson, M. J., Madasu, M. K., El Daibani, A., Varga, B. R., Zhang, T., Ruiz, C., Liu, S., Xu, J., Appourchaux, K., Slocum, S. T., Eans, S. O., Cameron, M. D., Al-Hasani, R., Pan, Y. X., Roth, B. L., McLaughlin, J. P., Skiniotis, G., Katritch, V., Kobilka, B. K., Majumdar, S. 2022

  Abstract

  Mu opioid receptor (µOR) agonists like fentanyl have long been used for pain management, but are considered a major public health concern due to their adverse side effects, including lethal overdose.1 To design safer therapeutics, we report a conceptually novel approach targeting conserved sodium (Na+) binding site2, observed in µOR3 and many other class A GPCRs, by bitopic fentanyl derivatives functionalized via a linker with a positively charged guanidino group. Cryo-EM structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the ligand's guanidine and the key Asp2.50 residue in the Na+ site. While the lead bitopics maintain nanomolar potency and high efficacy at Gi subtypes, they show strongly reduced arrestin recruitment, one also shows the lowest Gz-efficacy among the panel of µOR agonists, including partial and biased, morphinan and fentanyl analogs. In mice, the best bitopic ligand displayed µOR dependent antinociception with attenuated adverse effects supporting the µOR Na+ site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands engaging the Na+ pocket in class A GPCRs can be designed to control their efficacy and functional selectivity profiles for Gi/o/z subtypes and arrestins, thus modulating their in vivo pharmacology.

  View details for DOI 10.1038/s41586-022-05588-y

  View details for PubMedID 36450356

• Insights into distinct signaling profiles of the OR activated by diverse agonists. Nature chemical biology Qu, Q., Huang, W., Aydin, D., Paggi, J. M., Seven, A. B., Wang, H., Chakraborty, S., Che, T., DiBerto, J. F., Robertson, M. J., Inoue, A., Suomivuori, C., Roth, B. L., Majumdar, S., Dror, R. O., Kobilka, B. K., Skiniotis, G. 2022

  Abstract

  Drugs targeting the mu-opioid receptor (muOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two muOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and beta-arrestin recruitment. Cryo-EM structures of muOR-Gi1 complex with MP (2.5A) and LFT (3.2A) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and beta-arrestins bind. These observations highlight how drugs engaging different parts of the muOR orthosteric pocket can lead to distinct signaling outcomes.

  View details for DOI 10.1038/s41589-022-01208-y

  View details for PubMedID 36411392

• Membrane phosphoinositides regulate GPCR-beta-arrestin complex assembly and dynamics. Cell Janetzko, J., Kise, R., Barsi-Rhyne, B., Siepe, D. H., Heydenreich, F. M., Kawakami, K., Masureel, M., Maeda, S., Garcia, K. C., von Zastrow, M., Inoue, A., Kobilka, B. K. 2022

  Abstract

  Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with beta-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and invitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in beta-arrestin recruitment and GPCR-beta-arrestin complexdynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for beta-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of beta-arrestin and stabilize GPCR-beta-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-beta-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for beta-arrestin recruitment, this provides a mechanism for beta-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

  View details for DOI 10.1016/j.cell.2022.10.018

  View details for PubMedID 36368322

• A cholesterol analog stabilizes the human beta2-adrenergic receptor nonlinearly with temperature. Science signaling Serdiuk, T., Manna, M., Zhang, C., Mari, S. A., Kulig, W., Pluhackova, K., Kobilka, B. K., Vattulainen, I., Muller, D. J. 2022; 15 (737): eabi7031

  Abstract

  In cell membranes, G protein-coupled receptors (GPCRs) interact with cholesterol, which modulates their assembly, stability, and conformation. Previous studies have shown how cholesterol modulates the structural properties of GPCRs at ambient temperature. Here, we characterized the mechanical, kinetic, and energetic properties of the human beta2-adrenergic receptor (beta2AR) in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) at room temperature (25°C), at physiological temperature (37°C), and at high temperature (42°C). We found that CHS stabilized various structural regions of beta2AR differentially, which changed nonlinearly with temperature. Thereby, the strongest effects were observed for structural regions that are important for receptor signaling. Moreover, at 37°C, but not at 25° or 42°C, CHS caused beta2AR to increase and stabilize conformational substates to adopt to basal activity. These findings indicate that the nonlinear, temperature-dependent action of CHS in modulating the structural and functional properties of this GPCR is optimized for 37°C.

  View details for DOI 10.1126/scisignal.abi7031

  View details for PubMedID 35671340

• Structure-based Evolution of G protein-biased mu-opioid Receptor Agonists. Angewandte Chemie (International ed. in English) Gmeiner, P., Wang, H., Hetzer, F., Huang, W., Qu, Q., Meyerowitz, J., Kaindl, J., Hubner, H., Skiniotis, G., Kobilka, B. K. 2022

  Abstract

  The mu-opioid receptor (muOR) is the major target for opioid analgesics. Activation of muOR initiates signaling through G protein pathways as well as through beta-arrestin recruitment. muOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased muOR agonist. Here we report the cryoEM structure of PZM21 bound muOR in complex with G i protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G i protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to muOR in complex with G i protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce beta-arrestin recruitment by the muOR, and hold promise for developing next-generation analgesics with fewer adverse effects.

  View details for DOI 10.1002/anie.202200269

  View details for PubMedID 35385593

• Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins. Science advances Tsutsumi, N., Maeda, S., Qu, Q., Vogele, M., Jude, K. M., Suomivuori, C., Panova, O., Waghray, D., Kato, H. E., Velasco, A., Dror, R. O., Skiniotis, G., Kobilka, B. K., Garcia, K. C. 1800; 8 (3): eabl5442

  Abstract

  Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

  View details for DOI 10.1126/sciadv.abl5442

  View details for PubMedID 35061538

• Translating science to medicine: The case for physician-scientists. Science translational medicine Utz, P. J., Jain, M. K., Cheung, V. G., Kobilka, B. K., Lefkowitz, R., Yamada, T., Dzau, V. J. 2022; 14 (632): eabg7852

  Abstract

  As the number of physician-scientists continues to decline, action must be taken to support them as they embark on their careers.

  View details for DOI 10.1126/scitranslmed.abg7852

  View details for PubMedID 35171650

• Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-alpha-MSH and setmelanotide. Cell research Heyder, N. A., Kleinau, G., Speck, D., Schmidt, A., Paisdzior, S., Szczepek, M., Bauer, B., Koch, A., Gallandi, M., Kwiatkowski, D., Burger, J., Mielke, T., Beck-Sickinger, A. G., Hildebrand, P. W., Spahn, C. M., Hilger, D., Schacherl, M., Biebermann, H., Hilal, T., Kuhnen, P., Kobilka, B. K., Scheerer, P. 2021

  Abstract

  The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by theFDA, the peptide agonists NDP-alpha-MSH and setmelanotide, with 2.9A and 2.6A resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-alpha-MSH, a high-affinity linear variant of the endogenous agonist alpha-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

  View details for DOI 10.1038/s41422-021-00569-8

  View details for PubMedID 34561620

• Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1. Immunity Tsutsumi, N., Qu, Q., Mavri, M., Baggesen, M. S., Maeda, S., Waghray, D., Berg, C., Kobilka, B. K., Rosenkilde, M. M., Skiniotis, G., Garcia, K. C. 2021

  Abstract

  Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

  View details for DOI 10.1016/j.immuni.2021.06.001

  View details for PubMedID 34216564

• Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist. Nature communications Sun, B., Feng, D., Chu, M. L., Fish, I., Lovera, S., Sands, Z. A., Kelm, S., Valade, A., Wood, M., Ceska, T., Kobilka, T. S., Lebon, F., Kobilka, B. K. 2021; 12 (1): 3305

  Abstract

  Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8A resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

  View details for DOI 10.1038/s41467-021-23519-9

  View details for PubMedID 34083522

• G-protein activation by a metabotropic glutamate receptor. Nature Seven, A. B., Barros-Álvarez, X., de Lapeyrière, M., Papasergi-Scott, M. M., Robertson, M. J., Zhang, C., Nwokonko, R. M., Gao, Y., Meyerowitz, J. G., Rocher, J. P., Schelshorn, D., Kobilka, B. K., Mathiesen, J. M., Skiniotis, G. 2021

  Abstract

  Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

  View details for DOI 10.1038/s41586-021-03680-3

  View details for PubMedID 34194039

• How GPCR Phosphorylation Patterns Orchestrate Arrestin-Mediated Signaling. Cell Latorraca, N. R., Masureel, M., Hollingsworth, S. A., Heydenreich, F. M., Suomivuori, C., Brinton, C., Townshend, R. J., Bouvier, M., Kobilka, B. K., Dror, R. O. 2020

  Abstract

  Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.

  View details for DOI 10.1016/j.cell.2020.11.014

  View details for PubMedID 33296703

• Viewing rare conformations of the beta2 adrenergic receptor with pressure-resolved DEER spectroscopy. Proceedings of the National Academy of Sciences of the United States of America Lerch, M. T., Matt, R. A., Masureel, M., Elgeti, M., Kumar, K. K., Hilger, D., Foys, B., Kobilka, B. K., Hubbell, W. L. 2020

  Abstract

  The beta2 adrenergic receptor (beta2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 A) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The beta2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free beta2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in beta2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

  View details for DOI 10.1073/pnas.2013904117

  View details for PubMedID 33257561

• Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist. Proceedings of the National Academy of Sciences of the United States of America Kawai, T., Sun, B., Yoshino, H., Feng, D., Suzuki, Y., Fukazawa, M., Nagao, S., Wainscott, D. B., Showalter, A. D., Droz, B. A., Kobilka, T. S., Coghlan, M. P., Willard, F. S., Kawabe, Y., Kobilka, B. K., Sloop, K. W. 2020

  Abstract

  Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over beta-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

  View details for DOI 10.1073/pnas.2014879117

  View details for PubMedID 33177239

• Analysis of beta2AR-Gs and beta2AR-Gi complex formation by NMR spectroscopy. Proceedings of the National Academy of Sciences of the United States of America Ma, X., Hu, Y., Batebi, H., Heng, J., Xu, J., Liu, X., Niu, X., Li, H., Hildebrand, P. W., Jin, C., Kobilka, B. K. 2020

  Abstract

  The beta2-adrenergic receptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the beta2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the beta2AR-Gs complex revealed the interaction interface of beta2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the beta2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the beta2AR in response to the full agonist BI-167107 and Gs and Gi1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the beta2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the beta2AR-Gs and the beta2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs These differences between the beta2AR-Gs and beta2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

  View details for DOI 10.1073/pnas.2009786117

  View details for PubMedID 32868434

• Author Correction: Structural insights into mu-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2020

  Abstract

  An amendment to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41586-020-2542-z

  View details for PubMedID 32724208

• Structural insights into probe-dependent positive allosterism of the GLP-1 receptor. Nature chemical biology Bueno, A. B., Sun, B., Willard, F. S., Feng, D., Ho, J. D., Wainscott, D. B., Showalter, A. D., Vieth, M., Chen, Q., Stutsman, C., Chau, B., Ficorilli, J., Agejas, F. J., Cumming, G. R., Jimenez, A., Rojo, I., Kobilka, T. S., Kobilka, B. K., Sloop, K. W. 2020

  Abstract

  Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a 'molecular glue'. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein-protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

  View details for DOI 10.1038/s41589-020-0589-7

  View details for PubMedID 32690941

• An allosteric modulator binds to a conformational hub in the beta2 adrenergic receptor. Nature chemical biology Liu, X., Kaindl, J., Korczynska, M., StoSSel, A., Dengler, D., Stanek, M., Hubner, H., Clark, M. J., Mahoney, J., Matt, R. A., Xu, X., Hirata, K., Shoichet, B. K., Sunahara, R. K., Kobilka, B. K., Gmeiner, P. 2020

  Abstract

  Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the beta2-adrenergic receptor (beta2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for beta2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

  View details for DOI 10.1038/s41589-020-0549-2

  View details for PubMedID 32483378

• Activation of the alpha2B adrenoceptor by the sedative sympatholytic dexmedetomidine. Nature chemical biology Yuan, D., Liu, Z., Kaindl, J., Maeda, S., Zhao, J., Sun, X., Xu, J., Gmeiner, P., Wang, H., Kobilka, B. K. 2020

  Abstract

  The alpha2 adrenergic receptors (alpha2ARs) are G protein-coupled receptors (GPCRs) that respond to adrenaline and noradrenaline and couple to the Gi/o family of G proteins. alpha2ARs play important roles in regulating the sympathetic nervous system. Dexmedetomidine is a highly selective alpha2AR agonist used in post-operative patients as an anxiety-reducing, sedative medicine that decreases the requirement for opioids. As is typical for selective alphaAR agonists, dexmedetomidine consists of an imidazole ring and a substituted benzene moiety lacking polar groups, which is in contrast to betaAR-selective agonists, which share an ethanolamine group and an aromatic system with polar, hydrogen-bonding substituents. To better understand the structural basis for the selectivity and efficacy of adrenergic agonists, we determined the structure of the alpha2BAR in complex with dexmedetomidine and Go at a resolution of 2.9A by single-particle cryo-EM. The structure reveals the mechanism of alpha2AR-selective activation and provides insights into Gi/o coupling specificity.

  View details for DOI 10.1038/s41589-020-0492-2

  View details for PubMedID 32152538

• Structure and selectivity engineering of the M1 muscarinic receptor toxin complex. Science (New York, N.Y.) Maeda, S. n., Xu, J. n., N Kadji, F. M., Clark, M. J., Zhao, J. n., Tsutsumi, N. n., Aoki, J. n., Sunahara, R. K., Inoue, A. n., Garcia, K. C., Kobilka, B. K. 2020; 369 (6500): 161–67

  Abstract

  Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.

  View details for DOI 10.1126/science.aax2517

  View details for PubMedID 32646996

• Structure of the neurotensin receptor 1 in complex with β-arrestin 1. Nature Huang, W. n., Masureel, M. n., Qianhui, Q. n., Janetzko, J. n., Inoue, A. n., Kato, H. E., Robertson, M. J., Nguyen, K. C., Glenn, J. S., Skiniotis, G. n., Kobilka, B. K. 2020

  Abstract

  Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization, and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information delineating the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-EM structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1ΔCT). We found that phosphorylation of NTSR1 was critical for obtaining a stable complex with βarr1ΔCT, and identified phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observed a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared to a structure of rhodopsin-arrestin-1, our structure displays an approximately 85° rotation of arrestin relative to the receptor. These findings highlight both conserved aspects but also the plasticity of arrestin-receptor interactions.

  View details for DOI 10.1038/s41586-020-1953-1

  View details for PubMedID 31945771

• Structural insights into differences in G protein activation by family A and family B GPCRs. Science (New York, N.Y.) Hilger, D. n., Kumar, K. K., Hu, H. n., Pedersen, M. F., O'Brien, E. S., Giehm, L. n., Jennings, C. n., Eskici, G. n., Inoue, A. n., Lerch, M. n., Mathiesen, J. M., Skiniotis, G. n., Kobilka, B. K. 2020; 369 (6503)

  Abstract

  Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

  View details for DOI 10.1126/science.aba3373

  View details for PubMedID 32732395

• Binding pathway determines norepinephrine selectivity for the human β1AR over β2AR. Cell research Xu, X. n., Kaindl, J. n., Clark, M. J., Hübner, H. n., Hirata, K. n., Sunahara, R. K., Gmeiner, P. n., Kobilka, B. K., Liu, X. n. 2020

  Abstract

  Beta adrenergic receptors (βARs) mediate physiologic responses to the catecholamines epinephrine and norepinephrine released by the sympathetic nervous system. While the hormone epinephrine binds β1AR and β2AR with similar affinity, the smaller neurotransmitter norepinephrine is approximately tenfold selective for the β1AR. To understand the structural basis for this physiologically important selectivity, we solved the crystal structures of the human β1AR bound to an antagonist carazolol and different agonists including norepinephrine, epinephrine and BI-167107. Structural comparison revealed that the catecholamine-binding pockets are identical between β1AR and β2AR, but the extracellular vestibules have different shapes and electrostatic properties. Metadynamics simulations and mutagenesis studies revealed that these differences influence the path norepinephrine takes to the orthosteric pocket and contribute to the different association rates and thus different affinities.

  View details for DOI 10.1038/s41422-020-00424-2

  View details for PubMedID 33093660

• Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms. Cell reports Seven, A. B., Hilger, D. n., Papasergi-Scott, M. M., Zhang, L. n., Qu, Q. n., Kobilka, B. K., Tall, G. G., Skiniotis, G. n. 2020

  Abstract

  Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A.

  View details for DOI 10.1016/j.celrep.2020.02.086

  View details for PubMedID 32126208

• Conformational transitions of a neurotensin receptor1-Gi1complex. Nature Kato, H. E., Zhang, Y., Hu, H., Suomivuori, C., Kadji, F. M., Aoki, J., Krishna Kumar, K., Fonseca, R., Hilger, D., Huang, W., Latorraca, N. R., Inoue, A., Dror, R. O., Kobilka, B. K., Skiniotis, G. 2019

  Abstract

  Neurotensin receptor1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3A. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts moreflexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

  View details for DOI 10.1038/s41586-019-1337-6

  View details for PubMedID 31243364

• Assembly of a GPCR-G Protein Complex CELL Du, Y., Duc, N., Rasmussen, S. F., Hilger, D., Kubiak, X., Wang, L., Bohon, J., Kim, H., Wegrecki, M., Asuru, A., Jeong, K., Lee, J., Chance, M. R., Lodowski, D. T., Kobilka, B. K., Chung, K. 2019; 177 (5): 1232-+

  View details for DOI 10.1016/j.cell.2019.04.022

  View details for Web of Science ID 000468103800015

• Structural Insights into the Process of GPCR-G Protein Complex Formation CELL Liu, X., Xu, X., Hilger, D., Aschauer, P., Tiemann, J. S., Du, Y., Liu, H., Hirata, K., Sun, X., Guixa-Gonzalez, R., Mathiesen, J. M., Hildebrand, P. W., Kobilka, B. K. 2019; 177 (5): 1243-+

  View details for DOI 10.1016/j.cell.2019.04.021

  View details for Web of Science ID 000468103800016

• Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes SCIENCE Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G., Kobilka, B. K. 2019; 364 (6440): 552-+

  View details for DOI 10.1126/science.aaw5188

  View details for Web of Science ID 000467631800036

• Selective modulation of the cannabinoid type 1 (CB1) receptor as an emerging platform for the treatment of neuropathic pain MEDCHEMCOMM Banister, S. D., Kumar, K., Kumar, V., Kobilka, B. K., Malhotra, S. V. 2019; 10 (5): 647–59

  View details for DOI 10.1039/c8md00595h

  View details for Web of Science ID 000468790800002

• Structural insights into the activation of metabotropic glutamate receptors (vol 566, pg 79, 2019) NATURE Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T., Laeremans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019; 567 (7747): E10

  View details for DOI 10.1038/s41586-019-0983-z

  View details for Web of Science ID 000461126600009

• Diverse GPCRs exhibit conserved water networks for stabilization and activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Venkatakrishnan, A. J., Ma, A. K., Fonseca, R., Latorraca, N. R., Kelly, B., Betz, R. M., Asawa, C., Kobilka, B. K., Dror, R. O. 2019; 116 (8): 3288–93

  View details for DOI 10.1073/pnas.1809251116

  View details for Web of Science ID 000459074400073

• Structural Insights into the Subtype-Selective Antagonist Binding to the M2 Muscarinic Receptor Lee, S., Ryoji, S., Shoji, M., Kobayashi, T., Kobilka, B. K., Vaidehi, N. CELL PRESS. 2019: 206A

  View details for DOI 10.1016/j.bpj.2018.11.1135

  View details for Web of Science ID 000460779801035

• Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex CELL Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H., Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E., Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O., Malhotra, S. V., Kobilka, B. K., Skiniotis, G. 2019; 176 (3): 448-+

  View details for DOI 10.1016/j.cell.2018.11.040

  View details for Web of Science ID 000456526100007

• An improved yeast surface display platform for the screening of nanobody immune libraries. Scientific reports Uchanski, T., Zogg, T., Yin, J., Yuan, D., Wohlkonig, A., Fischer, B., Rosenbaum, D. M., Kobilka, B. K., Pardon, E., Steyaert, J. 2019; 9 (1): 382

  Abstract

  Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.

  View details for PubMedID 30674983

• Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations. Cell Wingler, L. M., Elgeti, M., Hilger, D., Latorraca, N. R., Lerch, M. T., Staus, D. P., Dror, R. O., Kobilka, B. K., Hubbell, W. L., Lefkowitz, R. J. 2019

  Abstract

  "Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or beta-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. beta-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

  View details for PubMedID 30639099

• Saving the Endangered Physician-Scientist - A Plan for Accelerating Medical Breakthroughs. The New England journal of medicine Jain, M. K., Cheung, V. G., Utz, P. J., Kobilka, B. K., Yamada, T. n., Lefkowitz, R. n. 2019; 381 (5): 399–402

  View details for DOI 10.1056/NEJMp1904482

  View details for PubMedID 31365796

• Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding. Structure (London, England : 1993) Spoerri, P. M., Sapra, K. T., Zhang, C. n., Mari, S. A., Kato, H. E., Kobilka, B. K., Müller, D. J. 2019

  Abstract

  G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states.

  View details for DOI 10.1016/j.str.2019.07.014

  View details for PubMedID 31422910

• Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy. Molecular cell Xu, J. n., Hu, Y. n., Kaindl, J. n., Risel, P. n., Hübner, H. n., Maeda, S. n., Niu, X. n., Li, H. n., Gmeiner, P. n., Jin, C. n., Kobilka, B. K. 2019

  Abstract

  The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

  View details for DOI 10.1016/j.molcel.2019.04.028

  View details for PubMedID 31103421

• Assembly of a GPCR-G Protein Complex. Cell Du, Y. n., Duc, N. M., Rasmussen, S. G., Hilger, D. n., Kubiak, X. n., Wang, L. n., Bohon, J. n., Kim, H. R., Wegrecki, M. n., Asuru, A. n., Jeong, K. M., Lee, J. n., Chance, M. R., Lodowski, D. T., Kobilka, B. K., Chung, K. Y. 2019

  Abstract

  The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

  View details for PubMedID 31080064

• Mechanism of β2AR regulation by an intracellular positive allosteric modulator. Science (New York, N.Y.) Liu, X. n., Masoudi, A. n., Kahsai, A. W., Huang, L. Y., Pani, B. n., Staus, D. P., Shim, P. J., Hirata, K. n., Simhal, R. K., Schwalb, A. M., Rambarat, P. K., Ahn, S. n., Lefkowitz, R. J., Kobilka, B. n. 2019; 364 (6447): 1283–87

  Abstract

  Drugs targeting the orthosteric, primary binding site of G protein-coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor's inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2- over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

  View details for DOI 10.1126/science.aaw8981

  View details for PubMedID 31249059

• Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex. Cell Krishna Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H., Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E., Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O., Malhotra, S. V., Kobilka, B. K., Skiniotis, G. 2018

  Abstract

  Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), aG protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

  View details for PubMedID 30639101

• Structural insights into the subtype-selective antagonist binding to the M-2 muscarinic receptor NATURE CHEMICAL BIOLOGY Suno, R., Lee, S., Maeda, S., Yasuda, S., Yamashita, K., Hirata, K., Horita, S., Tawaramoto, M. S., Tsujimoto, H., Murata, T., Kinoshita, M., Yamamoto, M., Kobilka, B. K., Vaidehi, N., Iwata, S., Kobayashi, T. 2018; 14 (12): 1150-+

  View details for DOI 10.1038/s41589-018-0152-y

  View details for Web of Science ID 000450230300017

• Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, H., Hofmann, J., Fish, I., Schaake, B., Eitel, K., Bartuschat, A., Kaindl, J., Rampp, H., Banerjee, A., Huebner, H., Clark, M. J., Vincent, S. G., Fisher, J. T., Heinrich, M. R., Hirata, K., Liu, X., Sunahara, R. K., Shoichet, B. K., Kobilka, B. K., Gmeiner, P. 2018; 115 (47): 12046–50

  View details for DOI 10.1073/pnas.1813988115

  View details for Web of Science ID 000450642800061

• Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor. Nature chemical biology Suno, R., Lee, S., Maeda, S., Yasuda, S., Yamashita, K., Hirata, K., Horita, S., Tawaramoto, M. S., Tsujimoto, H., Murata, T., Kinoshita, M., Yamamoto, M., Kobilka, B. K., Vaidehi, N., Iwata, S., Kobayashi, T. 2018

  Abstract

  Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

  View details for PubMedID 30420692

• Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists. Proceedings of the National Academy of Sciences of the United States of America Liu, H., Hofmann, J., Fish, I., Schaake, B., Eitel, K., Bartuschat, A., Kaindl, J., Rampp, H., Banerjee, A., Hubner, H., Clark, M. J., Vincent, S. G., Fisher, J. T., Heinrich, M. R., Hirata, K., Liu, X., Sunahara, R. K., Shoichet, B. K., Kobilka, B. K., Gmeiner, P. 2018

  Abstract

  Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

  View details for PubMedID 30404914

• Structural insights into binding specificity, efficacy and bias of a beta2AR partial agonist. Nature chemical biology Masureel, M., Zou, Y., Picard, L., van der Westhuizen, E., Mahoney, J. P., Rodrigues, J. P., Mildorf, T. J., Dror, R. O., Shaw, D. E., Bouvier, M., Pardon, E., Steyaert, J., Sunahara, R. K., Weis, W. I., Zhang, C., Kobilka, B. K. 2018; 14 (11): 1059–66

  Abstract

  Salmeterol is a partial agonist for the beta2 adrenergic receptor (beta2AR) and the first long-acting beta2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound beta2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between beta1AR and beta2AR explain the high receptor-subtype selectivity. A structural comparison with the beta2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited beta-arrestin recruitment for salmeterol.

  View details for PubMedID 30327561

• Rationally Engineered Tandem Facial Amphiphiles for Improved Membrane Protein Stabilization Efficacy CHEMBIOCHEM Das, M., Du, Y., Mortensen, J. S., Hariharan, P., Lee, H., Byrne, B., Loland, C. J., Guan, L., Kobilka, B. K., Chae, P. 2018; 19 (20): 2225–32

  Abstract

  A new family of tandem facial glucosides/maltosides (TFGs/TFMs) for membrane protein manipulation was prepared. The best detergent varied depending on the hydrophobic thickness of the target protein, but ether-based TFMs (TFM-C0E, TFM-C3E, and TFM-C5E) were notable for their ability to confer higher membrane protein stability than the previously developed amide-based TFA-1 (P. S. Chae, K. Gotfryd, J. Pacyna, L. J. W. Miercke, S. G. F. Rasmussen, R. A. Robbins, R. R. Rana, C. J. Loland, B. Kobilka, R. Stroud, B. Byrne, U. Gether, S. H. Gellman, J. Am. Chem. Soc. 2010, 132, 16750-16752). Thus, this study not only introduces novel agents with the potential to be used in membrane protein research but also highlights the importance of both the hydrophobic length and linker functionality of the detergent in stabilizing membrane proteins.

  View details for PubMedID 30070754

• A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability. The Analyst Hussain, H., Helton, T., Du, Y., Mortensen, J. S., Hariharan, P., Ehsan, M., Byrne, B., Loland, C. J., Kobilka, B. K., Guan, L., Chae, P. S. 2018

  Abstract

  The study of membrane proteins is extremely challenging, mainly because of the incompatibility of the hydrophobic surfaces of membrane proteins with an aqueous medium. Detergents are essential agents used to maintain membrane protein stability in non-native environments. However, conventional detergents fail to stabilize the native structures of many membrane proteins. Development of new amphipathic agents with enhanced efficacy for membrane protein stabilization is necessary to address this important problem. We have designed and synthesized linear and branched mannitol-based amphiphiles (MNAs), and comparative studies showed that most of the branched MNAs had advantages over the linear agents in terms of membrane protein stability. In addition, a couple of the new MNAs displayed favorable behaviors compared to n-dodecyl-beta-d-maltoside and the previously developed MNAs in maintaining the native protein structures, indicating potential utility of these new agents in membrane protein study.

  View details for DOI 10.1039/c8an01408f

  View details for PubMedID 30334564

• Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nature communications Maeda, S., Koehl, A., Matile, H., Hu, H., Hilger, D., Schertler, G. F., Manglik, A., Skiniotis, G., Dawson, R. J., Kobilka, B. K. 2018; 9 (1): 3712

  Abstract

  Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Galpha and Gbetagamma subunits in the heterotrimer, and confers resistance to GTPgammaS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.

  View details for PubMedID 30213947

• Structural mechanisms of selectivity and gating in anion channelrhodopsins. Nature Kato, H. E., Kim, Y. S., Paggi, J. M., Evans, K. E., Allen, W. E., Richardson, C., Inoue, K., Ito, S., Ramakrishnan, C., Fenno, L. E., Yamashita, K., Hilger, D., Lee, S. Y., Berndt, A., Shen, K., Kandori, H., Dror, R. O., Kobilka, B. K., Deisseroth, K. 2018

  Abstract

  Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anionchannels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

  View details for PubMedID 30158697

• Crystal structure of the natural anion-conducting channelrhodopsin GtACR1. Nature Kim, Y. S., Kato, H. E., Yamashita, K., Ito, S., Inoue, K., Ramakrishnan, C., Fenno, L. E., Evans, K. E., Paggi, J. M., Dror, R. O., Kandori, H., Kobilka, B. K., Deisseroth, K. 2018

  Abstract

  The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anionconductance and high anionselectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9A resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.

  View details for PubMedID 30158696

• Indole-based positive allosteric modulators for targeting CB1 receptor to overcome neuropathic pain Resendez, A., Kumar, K., Kumar, V., Kobilka, B., Malhotra, S. AMER CHEMICAL SOC. 2018

  View details for Web of Science ID 000447609102071

• Single Proteoliposome High-Content Analysis Reveals Differences in the Homo-Oligomerization of GPCRs Walsh, S. M., Mathiasen, S., Christensen, S. M., Fay, J. F., King, C., Provasi, D., Borrero, E., Rasmussen, S. F., Fung, J., Filizola, M., Hristova, K., Kobilka, B., Farrens, D. L., Stamou, D. CELL PRESS. 2018: 300–312

  Abstract

  G-protein-coupled receptors (GPCRs) control vital cellular signaling pathways. GPCR oligomerization is proposed to increase signaling diversity. However, many reports have arrived at disparate conclusions regarding the existence, stability, and stoichiometry of GPCR oligomers, partly because of cellular complexity and ensemble averaging of intrareconstitution heterogeneities that complicate the interpretation of oligomerization data. To overcome these limitations, we exploited fluorescence-microscopy-based high-content analysis of single proteoliposomes. This allowed multidimensional quantification of intrinsic monomer-monomer interactions of three class A GPCRs (β2-adrenergic receptor, cannabinoid receptor type 1, and opsin). Using a billion-fold less protein than conventional assays, we quantified oligomer stoichiometries, association constants, and the influence of two ligands and membrane curvature on oligomerization, revealing key similarities and differences for three GPCRs with decidedly different physiological functions. The assays introduced here will assist with the quantitative experimental observation of oligomerization for transmembrane proteins in general.

  View details for PubMedID 30021106

  View details for PubMedCentralID PMC6050755

• An Engineered Lithocholate-Based Facial Amphiphile Stabilizes Membrane Proteins: Assessing the Impact of Detergent Customizability on Protein Stability CHEMISTRY-A EUROPEAN JOURNAL Das, M., Du, Y., Mortensen, J. S., Bae, H., Byrne, B., Loland, C. J., Kobilka, B. K., Chae, P. 2018; 24 (39): 9860–68

  Abstract

  Amphiphiles are critical tools for the structural and functional study of membrane proteins. Membrane proteins encapsulated by conventional head-to-tail detergents tend to undergo structural degradation, necessitating the development of structurally novel agents with improved efficacy. In recent years, facial amphiphiles have yielded encouraging results in terms of membrane protein stability. Herein, we report a new facial detergent (i.e., LFA-C4) that confers greater stability to tested membrane proteins than the bola form analogue. Owing to the increased facial property and the adaptability of the detergent micelles in complex with different membrane proteins, LFA-C4 yields increased stability compared to n-dodecyl-β-d-maltoside (DDM). Thus, this study not only describes a novel maltoside detergent with enhanced protein-stabilizing properties, but also shows that the customizable nature of a detergent plays an important role in the stabilization of membrane proteins. Owing to both synthetic convenience and enhanced stabilization efficacy for a range of membrane proteins, the new agent has major potential in membrane protein research.

  View details for DOI 10.1002/chem.201801141

  View details for Web of Science ID 000438198000023

  View details for PubMedID 29741269

• The Molecular Basis of G Protein-Coupled Receptor Activation. Annual review of biochemistry Weis, W. I., Kobilka, B. K. 2018; 87: 897–919

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into alpha and betagamma subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

  View details for PubMedID 29925258

• Structure of the µ-opioid receptor-Gi protein complex. Nature Koehl, A., Hu, H., Maeda, S., Zhang, Y., Qu, Q., Paggi, J. M., Latorraca, N. R., Hilger, D., Dawson, R., Matile, H., Schertler, G. F., Granier, S., Weis, W. I., Dror, R. O., Manglik, A., Skiniotis, G., Kobilka, B. K. 2018

  Abstract

  The mu-opioid receptor (muOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by muOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5A resolution cryo-electron microscopy structure of the muOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its Nterminus interacting with conserved receptor residues and its Cterminus engaging regions important for opioid-ligand selectivity. Comparison of the muOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein alpha-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the OR.

  View details for PubMedID 29899455

• Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist STRUCTURE Spoerri, P. M., Kato, H. E., Pfreundschuh, M., Mari, S. A., Serdiuk, T., Thoma, J., Sapra, K., Zhang, C., Kobilka, B. K., Muller, D. J. 2018; 26 (6): 829-+

  Abstract

  The protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) involved in hemostasis, thrombosis, and inflammation, is activated by thrombin or other coagulation proteases. This activation is inhibited by the irreversible antagonist vorapaxar used for anti-platelet therapy. Despite detailed structural and functional information, how vorapaxar binding alters the structural properties of PAR1 to prevent activation is hardly known. Here we apply dynamic single-molecule force spectroscopy to characterize how vorapaxar binding changes the mechanical, kinetic, and energetic properties of human PAR1 under physiologically relevant conditions. We detect structural segments stabilizing PAR1 and quantify their properties in the unliganded and the vorapaxar-bound state. In the presence of vorapaxar, most structural segments increase conformational variability, lifetime, and free energy, and reduce mechanical rigidity. These changes highlight a general trend in how GPCRs are affected by strong antagonists.

  View details for PubMedID 29731231

• Structure-based discovery of selective positive allosteric modulators of antagonists for the M-2 muscarinic acetylcholine receptor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Korczynska, M., Clark, M. J., Valant, C., Xu, J., Von Moo, E., Albold, S., Weiss, D. R., Torosyan, H., Huang, W., Kruse, A. C., Lyda, B. R., May, L. T., Baltos, J., Sexton, P. M., Kobilka, B. K., Christopoulos, A., Shoichet, B. K., Sunahara, R. K. 2018; 115 (10): E2419–E2428

  Abstract

  Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

  View details for PubMedID 29453275

• Structure and dynamics of GPCR signaling complexes NATURE STRUCTURAL & MOLECULAR BIOLOGY Hilger, D., Masureel, M., Kobilka, B. K. 2018; 25 (1): 4–12

  Abstract

  G-protein-coupled receptors (GPCRs) relay numerous extracellular signals by triggering intracellular signaling through coupling with G proteins and arrestins. Recent breakthroughs in the structural determination of GPCRs and GPCR-transducer complexes represent important steps toward deciphering GPCR signal transduction at a molecular level. A full understanding of the molecular basis of GPCR-mediated signaling requires elucidation of the dynamics of receptors and their transducer complexes as well as their energy landscapes and conformational transition rates. Here, we summarize current insights into the structural plasticity of GPCR-G-protein and GPCR-arrestin complexes that underlies the regulation of the receptor's intracellular signaling profile.

  View details for PubMedID 29323277

• The cubicon method for concentrating membrane proteins in the cubic mesophase NATURE PROTOCOLS Ma, P., Weichert, D., Aleksandrov, L. A., Jensen, T. J., Riordan, J. R., Liu, X., Kobilka, B. K., Caffrey, M. 2017; 12 (9): 1745–62

  Abstract

  The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

  View details for PubMedID 28771236

• Single-molecule analysis of ligand efficacy in beta(2)AR-G-protein activation NATURE Gregorio, G., Masureel, M., Hilger, D., Terry, D. S., Juette, M., Zhao, H., Zhou, Z., Perez-Aguilar, J., Hauge, M., Mathiasen, S., Javitch, J. A., Weinstein, H., Kobilka, B. K., Blanchard, S. C. 2017; 547 (7661): 68-+

  Abstract

  G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR-Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

  View details for PubMedID 28607487

  View details for PubMedCentralID PMC5502743

• Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature Zhang, Y., Sun, B., Feng, D., Hu, H., Chu, M., Qu, Q., Tarrasch, J. T., Li, S., Sun Kobilka, T., Kobilka, B. K., Skiniotis, G. 2017; 546 (7657): 248-253

  Abstract

  Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near atomic resolution. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding.

  View details for DOI 10.1038/nature22394

  View details for PubMedID 28538729

• Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature Liang, Y., Khoshouei, M., Radjainia, M., Zhang, Y., Glukhova, A., Tarrasch, J., Thal, D. M., Furness, S. G., Christopoulos, G., Coudrat, T., Danev, R., Baumeister, W., Miller, L. J., Christopoulos, A., Kobilka, B. K., Wootten, D., Skiniotis, G., Sexton, P. M. 2017; 546 (7656): 118-123

  Abstract

  Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsβγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

  View details for DOI 10.1038/nature22327

  View details for PubMedID 28437792

• Structural and Functional Analysis of a beta(2)-Adrenergic Receptor Complex with GRK5 Cell Komolov, K. E., Du, Y., Duc, N. M., Betz, R. M., Rodrigues, J. P., Leib, R. D., Patra, D., Skiniotis, G., Adams, C. M., Dror, R. O., Chung, K. Y., Kobilka, B. K., Benovic, J. L. 2017; 169 (3): 407-421 e16

  Abstract

  The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

  View details for DOI 10.1016/j.cell.2017.03.047

  View details for PubMedID 28431242

• Crystal structure of the adenosine A(2A) receptor bound to an antagonist reveals a potential allosteric pocket PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, B., Bachhawat, P., Chu, M. L., Wood, M., Ceska, T., Sands, Z. A., Mercier, J., Lebon, F., Kobilka, T. S., Kobilka, B. K. 2017; 114 (8): 2066-2071

  Abstract

  The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

  View details for DOI 10.1073/pnas.1621423114

  View details for Web of Science ID 000395099500093

  View details for PubMedCentralID PMC5338372

• Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket. Proceedings of the National Academy of Sciences of the United States of America Sun, B., Bachhawat, P., Chu, M. L., Wood, M., Ceska, T., Sands, Z. A., Mercier, J., Lebon, F., Kobilka, T. S., Kobilka, B. K. 2017; 114 (8): 2066-2071

  Abstract

  The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

  View details for DOI 10.1073/pnas.1621423114

  View details for PubMedID 28167788

  View details for PubMedCentralID PMC5338372

• Nanobodies to Study G Protein-Coupled Receptor Structure and Function. Annual review of pharmacology and toxicology Manglik, A., Kobilka, B. K., Steyaert, J. 2017; 57: 19-37

  Abstract

  Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.

  View details for DOI 10.1146/annurev-pharmtox-010716-104710

  View details for PubMedID 27959623

• Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature Liu, X. n., Ahn, S. n., Kahsai, A. W., Meng, K. C., Latorraca, N. R., Pani, B. n., Venkatakrishnan, A. J., Masoudi, A. n., Weis, W. I., Dror, R. O., Chen, X. n., Lefkowitz, R. J., Kobilka, B. K. 2017; 548 (7668): 480–84

  Abstract

  G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the β2 adrenergic receptor (β2AR) that was recently isolated from a DNA-encoded small-molecule library. Orthosteric β-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the β2AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the β2AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.

  View details for PubMedID 28813418

• Structure-based discovery of opioid analgesics with reduced side effects NATURE Manglik, A., Lin, H., Aryal, D. K., McCorvy, J. D., Dengler, D., Corder, G., Levit, A., Kling, R. C., Bernat, V., Huebner, H., Huang, X., Sassano, M. F., Giguere, P. M., Loeber, S., Duan, D., Scherrer, G., Kobilka, B. K., Gmeiner, P., Roth, B. L., Shoichet, B. K. 2016; 537 (7619): 185-?

  Abstract

  Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

  View details for DOI 10.1038/nature19112

  View details for Web of Science ID 000382539100038

• Structure-based discovery of opioid analgesics with reduced side effects. Nature Manglik, A., Lin, H., Aryal, D. K., McCorvy, J. D., Dengler, D., Corder, G., Levit, A., Kling, R. C., Bernat, V., Hübner, H., Huang, X., Sassano, M. F., Giguère, P. M., Löber, S., Da Duan, Scherrer, G., Kobilka, B. K., Gmeiner, P., Roth, B. L., Shoichet, B. K. 2016; 537 (7619): 185-190

  Abstract

  Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

  View details for DOI 10.1038/nature19112

  View details for PubMedID 27533032

• Allosteric coupling from G protein to the agonist-binding pocket in GPCRs NATURE DeVree, B. T., Mahoney, J. P., Velez-Ruiz, G. A., Rasmussen, S. G., Kuszak, A. J., Edwald, E., Fung, J., Manglik, A., Masureel, M., Du, Y., Matt, R. A., Pardon, E., Steyaert, J., Kobilka, B. K., Sunahara, R. K. 2016; 535 (7610): 182-?

  Abstract

  G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

  View details for DOI 10.1038/nature18324

  View details for Web of Science ID 000379015600049

  View details for PubMedID 27362234

• Accessible Mannitol-Based Amphiphiles (MNAs) for Membrane Protein Solubilisation and Stabilisation CHEMISTRY-A EUROPEAN JOURNAL Hussain, H., Du, Y., Scull, N. J., Mortensen, J. S., Tarrasch, J., Bae, H. E., Loland, C. J., Byrne, B., Kobilka, B. K., Chae, P. S. 2016; 22 (21): 7068-7073

  Abstract

  Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the β2 adrenergic receptor (β2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the β2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.

  View details for DOI 10.1002/chem.201600533

  View details for Web of Science ID 000377604100011

  View details for PubMedID 27072057

• Structural Basis for Allosteric Coupling Between G Protein and the Agonist-Binding Pocket in GPCRs Mahoney, J., DeVree, B., Velez-Ruiz, G., Rasmussen, S., Kuszak, A., Edwald, E., Manglik, A., Masureel, M., Du, Y., Matt, R., Pardon, E., Steyaert, J., Kobilka, B., Sunahara, R. FEDERATION AMER SOC EXP BIOL. 2016

  View details for Web of Science ID 000406444704368

• Efficacy at the Mu Opioid Receptor: Insights from Orthosteric and Allosteric Ligands Livingston, K. E., Mahoney, J., Manglik, A., Kobilka, B., Sunahara, R., Traynor, J. R. FEDERATION AMER SOC EXP BIOL. 2016

  View details for Web of Science ID 000406444704374

• Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ehsan, M., Du, Y., Scull, N. J., Tikhonova, E., Tarrasch, J., Mortensen, J. S., Loland, C. J., Skiniotis, G., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2016; 138 (11): 3789-3796

  Abstract

  Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

  View details for DOI 10.1021/jacs.5b13233

  View details for Web of Science ID 000372854200027

• Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature Thal, D. M., Sun, B., Feng, D., Nawaratne, V., Leach, K., Felder, C. C., Bures, M. G., Evans, D. A., Weis, W. I., Bachhawat, P., Kobilka, T. S., Sexton, P. M., Kobilka, B. K., Christopoulos, A. 2016; 531 (7594): 335-340

  Abstract

  Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

  View details for DOI 10.1038/nature17188

  View details for PubMedID 26958838

  View details for PubMedCentralID PMC4915387

• Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature Thal, D. M., Sun, B., Feng, D., Nawaratne, V., Leach, K., Felder, C. C., Bures, M. G., Evans, D. A., Weis, W. I., Bachhawat, P., Kobilka, T. S., Sexton, P. M., Kobilka, B. K., Christopoulos, A. 2016; 531 (7594): 335-340

  View details for DOI 10.1038/nature17188

  View details for PubMedID 26958838

• Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry Nguyen Minh Duc, Du, Y., Lee, S., Zhang, C., Kobilka, B. K., Chung, K. CELL PRESS. 2016: 396A

  View details for DOI 10.1016/j.bpj.2015.11.2140

  View details for Web of Science ID 000375142200426

• High-density grids for efficient data collection from multiple crystals. Acta crystallographica. Section D, Structural biology Baxter, E. L., Aguila, L., Alonso-Mori, R., Barnes, C. O., Bonagura, C. A., Brehmer, W., Brunger, A. T., Calero, G., Caradoc-Davies, T. T., Chatterjee, R., DeGrado, W. F., Fraser, J. S., Ibrahim, M., Kern, J., Kobilka, B. K., Kruse, A. C., Larsson, K. M., Lemke, H. T., Lyubimov, A. Y., Manglik, A., McPhillips, S. E., Norgren, E., Pang, S. S., Soltis, S. M., Song, J., Thomaston, J., Tsai, Y., Weis, W. I., Woldeyes, R. A., Yachandra, V., Yano, J., Zouni, A., Cohen, A. E. 2016; 72: 2-11

  Abstract

  Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

  View details for DOI 10.1107/S2059798315020847

  View details for PubMedID 26894529

  View details for PubMedCentralID PMC4756618

• Allosteric regulation of G protein-coupled receptor activity by phospholipids NATURE CHEMICAL BIOLOGY Dawaliby, R., Trubbia, C., Delporte, C., Masureel, M., Van Antwerpen, P., Kobilka, B. K., Govaerts, C. 2016; 12 (1): 35-?

  Abstract

  Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.

  View details for DOI 10.1038/NCHEMBIO.1960

  View details for Web of Science ID 000366674400009

  View details for PubMedID 26571351

  View details for PubMedCentralID PMC4718399

• Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation CHEMICAL COMMUNICATIONS Bae, H. E., Mortensen, J. S., Ribeiro, O., Du, Y., Ehsan, M., Kobilka, B. K., Loland, C. J., Byrne, B., Chae, P. S. 2016; 52 (81): 12104-12107

  Abstract

  A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

  View details for DOI 10.1039/c6cc06147h

  View details for Web of Science ID 000385156700022

  View details for PubMedID 27711401

• In meso in situ In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Huang, C., Olieric, V., Ma, P., Howe, N., Vogeley, L., Liu, X., Warshamanage, R., Weinert, T., Panepucci, E., Kobilka, B., Diederichs, K., Wang, M., Caffrey, M. 2016; 72: 93-112

  Abstract

  Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

  View details for DOI 10.1107/S2059798315021683

  View details for Web of Science ID 000371707300011

  View details for PubMedID 26894538

  View details for PubMedCentralID PMC4756617

• Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape NATURE METHODS Alsteens, D., Pfreundschuh, M., Zhang, C., Spoerri, P. M., Coughlin, S. R., Kobilka, B. K., Mueller, D. J. 2015; 12 (9): 845-?

  Abstract

  Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors.

  View details for DOI 10.1038/NMETH.3479

  View details for Web of Science ID 000360586700030

• Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape. Nature methods Alsteens, D., Pfreundschuh, M., Zhang, C., Spoerri, P. M., Coughlin, S. R., Kobilka, B. K., Müller, D. J. 2015; 12 (9): 845-851

  Abstract

  Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors.

  View details for DOI 10.1038/nmeth.3479

  View details for PubMedID 26167642

• Structural insights into mu-opioid receptor activation NATURE Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2015; 524 (7565): 315-?

  Abstract

  Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

  View details for DOI 10.1038/nature14886

  View details for Web of Science ID 000359714000028

• Propagation of conformational changes during µ-opioid receptor activation. Nature Sounier, R., Mas, C., Steyaert, J., Laeremans, T., Manglik, A., Huang, W., Kobilka, B. K., Déméné, H., Granier, S. 2015; 524 (7565): 375-378

  Abstract

  µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.

  View details for DOI 10.1038/nature14680

  View details for PubMedID 26245377

• Propagation of conformational changes during mu-opioid receptor activation NATURE Sounier, R., Mas, C., Steyaert, J., Laeremans, T., Manglik, A., Huang, W., Kobilka, B. K., Demene, H., Granier, S. 2015; 524 (7565): 375-?

  Abstract

  µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.

  View details for DOI 10.1038/nature14680

  View details for Web of Science ID 000359714000041

• Structural insights into µ-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2015; 524 (7565): 315-321

  Abstract

  Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

  View details for DOI 10.1038/nature14886

  View details for PubMedID 26245379

• Novel Xylene-Linked Maltoside Amphiphiles (XMAs) for Membrane Protein Stabilisation. Chemistry (Weinheim an der Bergstrasse, Germany) Cho, K. H., Du, Y., Scull, N. J., Hariharan, P., Gotfryd, K., Loland, C. J., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2015; 21 (28): 10008-10013

  Abstract

  Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, a novel class of maltoside-bearing amphiphiles, with a xylene linker in the central region, designated xylene-linked maltoside amphiphiles (XMAs) was developed. When these novel agents were evaluated with a number of membrane proteins, it was found that XMA-4 and XMA-5 have particularly favourable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study.

  View details for DOI 10.1002/chem.201501083

  View details for PubMedID 26013293

• SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science Dror, R. O., Mildorf, T. J., Hilger, D., Manglik, A., Borhani, D. W., Arlow, D. H., Philippsen, A., Villanueva, N., Yang, Z., Lerch, M. T., Hubbell, W. L., Kobilka, B. K., Sunahara, R. K., Shaw, D. E. 2015; 348 (6241): 1361-1365

  Abstract

  G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

  View details for DOI 10.1126/science.aaa5264

  View details for PubMedID 26089515

• Structural basis for nucleotide exchange in heterotrimeric G proteins SCIENCE Dror, R. O., Mildorf, T. J., Hilger, D., Manglik, A., Borhani, D. W., Arlow, D. H., Philippsen, A., Villanueva, N., Yang, Z., Lerch, M. T., Hubbell, W. L., Kobilka, B. K., Sunahara, R. K., Shaw, D. E. 2015; 348 (6241): 1361-1365

  Abstract

  G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

  View details for DOI 10.1126/science.aaa5264

  View details for Web of Science ID 000356449500051

• Structural Insights into the Dynamic Process of beta(2)-Adrenergic Receptor Signaling CELL Manglik, A., Kim, T. H., Masureel, M., Altenbach, C., Yang, Z., Hilger, D., Lerch, M. T., Kobilka, T. S., Thian, F. S., Hubbell, W. L., Prosser, R. S., Kobilka, B. K. 2015; 161 (5): 1101-1111

  Abstract

  G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

  View details for DOI 10.1016/j.cell.2015.04.043

  View details for Web of Science ID 000355152600017

  View details for PubMedID 25981665

  View details for PubMedCentralID PMC4441853

• Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Nguyen Minh Duc, N. M., Du, Y., Thorsen, T. S., Lee, S. Y., Zhang, C., Kato, H., Kobilka, B. K., Chung, K. Y. 2015; 26 (5): 808-817

  Abstract

  G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

  View details for DOI 10.1007/s13361-015-1083-4

  View details for Web of Science ID 000352876100014

• Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry. Journal of the American Society for Mass Spectrometry Duc, N. M., Du, Y., Thorsen, T. S., Lee, S. Y., Zhang, C., Kato, H., Kobilka, B. K., Chung, K. Y. 2015; 26 (5): 808-17

  Abstract

  G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

  View details for DOI 10.1007/s13361-015-1083-4

  View details for PubMedID 25740347

• Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM. Nature communications Pfreundschuh, M., Alsteens, D., Wieneke, R., Zhang, C., Coughlin, S. R., Tampé, R., Kobilka, B. K., Müller, D. J. 2015; 6: 8857-?

  Abstract

  A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  View details for DOI 10.1038/ncomms9857

  View details for PubMedID 26561004

  View details for PubMedCentralID PMC4660198

• Development and Characterization of Pepducins as Gs-biased Allosteric Agonists. journal of biological chemistry Carr, R., Du, Y., Quoyer, J., Panettieri, R. A., Janz, J. M., Bouvier, M., Kobilka, B. K., Benovic, J. L. 2014; 289 (52): 35668-35684

  Abstract

  The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β2AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β2AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β2AR and provides valuable tools for the study of β2AR function.

  View details for DOI 10.1074/jbc.M114.618819

  View details for PubMedID 25395624

  View details for PubMedCentralID PMC4276837

• Goniometer-based femtosecond crystallography with X-ray free electron lasers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Soltis, S. M., Gonzalez, A., Aguila, L., Alonso-Mori, R., Barnes, C. O., Baxter, E. L., Brehmer, W., Brewster, A. S., Brunger, A. T., Calero, G., Chang, J. F., Chollet, M., Ehrensberger, P., Eriksson, T. L., Feng, Y., Hattne, J., Hedman, B., Hollenbeck, M., Holton, J. M., Keable, S., Kobilka, B. K., Kovaleva, E. G., Kruse, A. C., Lemke, H. T., Lin, G., Lyubimov, A. Y., Manglik, A., Mathews, I. I., McPhillips, S. E., Nelson, S., Peters, J. W., Sauter, N. K., Smith, C. A., Song, J., Stevenson, H. P., Tsai, Y., Uervirojnangkoorn, M., Vinetsky, V., Wakatsuki, S., Weis, W. I., Zadvornyy, O. A., Zeldin, O. B., Zhu, D., Hodgson, K. O. 2014; 111 (48): 17122-17127

  Abstract

  The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

  View details for DOI 10.1073/pnas.1418733111

  View details for Web of Science ID 000345920800042

  View details for PubMedID 25362050

  View details for PubMedCentralID PMC4260607

• Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis STRUCTURE Thorsen, T. S., Matt, R., Weis, W. I., Kobilka, B. K. 2014; 22 (11): 1657-1664

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution.

  View details for DOI 10.1016/j.str.2014.08.022

  View details for Web of Science ID 000344934300013

  View details for PubMedCentralID PMC4408211

• Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis. Structure (London, England : 1993) Thorsen, T. S., Matt, R., Weis, W. I., Kobilka, B. K. 2014; 22 (11): 1657-64

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution.

  View details for DOI 10.1016/j.str.2014.08.022

  View details for PubMedID 25450769

  View details for PubMedCentralID PMC4408211

• Brian Kobilka Stuck on structure NATURE Azouz, H., Kobilka, B. 2014; 514 (7522): S12–S13

  View details for Web of Science ID 000342988600008

• Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes. Nature methods Mathiasen, S., Christensen, S. M., Fung, J. J., Rasmussen, S. G., Fay, J. F., Jorgensen, S. K., Veshaguri, S., Farrens, D. L., Kiskowski, M., Kobilka, B., Stamou, D. 2014; 11 (9): 931-934

  Abstract

  Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the β2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.

  View details for DOI 10.1038/nmeth.3062

  View details for PubMedID 25086504

• Visualization of arrestin recruitment by a G-protein-coupled receptor NATURE Shukla, A. K., Westfield, G. H., Xiao, K., Reis, R. I., Huang, L., Tripathi-Shukla, P., Qian, J., Li, S., Blanc, A., Oleskie, A. N., Dosey, A. M., Su, M., Liang, C., Gu, L., Shan, J., Chen, X., Hanna, R., Choi, M., Yao, X. J., Klink, B. U., Kahsai, A. W., Sidhu, S. S., Koide, S., Penczek, P. A., Kossiakoff, A. A., Woods, V. L., Kobilka, B. K., Skiniotis, G., Lefkowitz, R. J. 2014; 512 (7513): 218-?

  Abstract

  G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

  View details for DOI 10.1038/nature13430

  View details for Web of Science ID 000340200700037

  View details for PubMedID 25043026

  View details for PubMedCentralID PMC4134437

• Covalent agonists for studying G protein-coupled receptor activation. Proceedings of the National Academy of Sciences of the United States of America Weichert, D., Kruse, A. C., Manglik, A., Hiller, C., Zhang, C., Hübner, H., Kobilka, B. K., Gmeiner, P. 2014; 111 (29): 10744-10748

  Abstract

  Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.

  View details for DOI 10.1073/pnas.1410415111

  View details for PubMedID 25006259

  View details for PubMedCentralID PMC4115510

• Novel Insights into M-3 Muscarinic Acetylcholine Receptor Physiology and Structure 14th International Symposium on Cholinergic Mechanisms (ISCM) Kruse, A. C., Li, J., Hu, J., Kobilka, B. K., Wess, J. HUMANA PRESS INC. 2014: 316–23

  Abstract

  Recent studies with M3 muscarinic acetylcholine receptor (M3R) mutant mice suggest that drugs selectively targeting this receptor subtype may prove useful for the treatment of various pathophysiological conditions. Moreover, the use of M3R-based designer G protein-coupled receptors (GPCRs) has provided novel insights into how Gq-coupled GPCRs can modulate whole-body glucose homeostasis by acting on specific peripheral cell types. More recently, we succeeded in using X-ray crystallography to determine the structure of the M3R bound to the bronchodilating drug tiotropium, a muscarinic antagonist (inverse agonist). This new structural information should facilitate the development of orthosteric or allosteric M3R-selective drugs that are predicted to have considerable therapeutic potential.

  View details for DOI 10.1007/s12031-013-0127-0

  View details for Web of Science ID 000339956700005

  View details for PubMedID 24068573

  View details for PubMedCentralID PMC3966988

• Muscarinic acetylcholine receptors: novel opportunities for drug development NATURE REVIEWS DRUG DISCOVERY Kruse, A. C., Kobilka, B. K., Gautam, D., Sexton, P. M., Christopoulos, A., Wess, J. 2014; 13 (7): 549-560

  Abstract

  The muscarinic acetylcholine receptors are a subfamily of G protein-coupled receptors that regulate numerous fundamental functions of the central and peripheral nervous system. The past few years have witnessed unprecedented new insights into muscarinic receptor physiology, pharmacology and structure. These advances include the first structural views of muscarinic receptors in both inactive and active conformations, as well as a better understanding of the molecular underpinnings of muscarinic receptor regulation by allosteric modulators. These recent findings should facilitate the development of new muscarinic receptor subtype-selective ligands that could prove to be useful for the treatment of many severe pathophysiological conditions.

  View details for DOI 10.1038/nrd4295

  View details for Web of Science ID 000338845000016

  View details for PubMedID 24903776

• Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development CURRENT OPINION IN PHARMACOLOGY Kruse, A. C., Hu, J., Kobilka, B. K., Wess, J. 2014; 16: 24-30

  Abstract

  Muscarinic acetylcholine receptor antagonists are widely used as bronchodilating drugs in pulmonary medicine. The therapeutic efficacy of these agents depends on the blockade of M3 muscarinic receptors expressed on airway smooth muscle cells. All muscarinic antagonists currently used as bronchodilating agents show high affinity for all five muscarinic receptor subtypes, thus increasing the likelihood of unwanted side effects. Recent X-ray crystallographic studies have provided detailed structural information about the nature of the orthosteric muscarinic binding site (the conventional acetylcholine binding site) and an 'outer' receptor cavity that can bind allosteric (non-orthosteric) drugs. These new findings should guide the development of selective M3 receptor blockers that have little or no effect on other muscarinic receptor subtypes.

  View details for DOI 10.1016/j.coph.2014.02.006

  View details for Web of Science ID 000338094300006

  View details for PubMedID 24662799

  View details for PubMedCentralID PMC4065632

• The role of protein dynamics in GPCR function: insights from the beta(2)AR and rhodopsin CURRENT OPINION IN CELL BIOLOGY Manglik, A., Kobilka, B. 2014; 27: 136-143

  Abstract

  G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Recent advances in GPCR crystallography have provided inactive and active state structures for rhodopsin and the β2 adrenergic receptor (β2AR). Although these structures suggest a two-state 'on-off' mechanism of receptor activation, other biophysical studies and observed signaling versatility suggest that GPCRs are highly dynamic and exist in a multitude of functionally distinct conformations. To fully understand how GPCRs work, we must characterize these conformations and determine how ligands affect their energetics and rates of interconversion. This brief review will compare and contrast the dynamic properties of rhodopsin and β2AR that shed light on the role of structural dynamics in their distinct signaling behaviors.

  View details for DOI 10.1016/j.ceb.2014.01.008

  View details for Web of Science ID 000335106100019

  View details for PubMedID 24534489

  View details for PubMedCentralID PMC3986065

• A general protocol for the generation of Nanobodies for structural biology NATURE PROTOCOLS Pardon, E., Laeremans, T., Triest, S., Rasmussen, S. G., Wohlkoenig, A., Ruf, A., Muyldermans, S., Hol, W. G., Kobilka, B. K., Steyaert, J. 2014; 9 (3): 674-693

  Abstract

  There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo-matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6-12 months.

  View details for DOI 10.1038/nprot.2014.039

  View details for Web of Science ID 000332224000014

  View details for PubMedID 24577359

  View details for PubMedCentralID PMC4297639

• Regulation of beta(2)-Adrenergic Receptor Function by Conformationally Selective Single-Domain Intrabodies MOLECULAR PHARMACOLOGY Staus, D. P., Wingler, L. M., Strachan, R. T., Rasmussen, S. G., Pardon, E., Ahn, S., Steyaert, J., Kobilka, B. K., Lefkowitz, R. J. 2014; 85 (3): 472-481

  Abstract

  The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.

  View details for DOI 10.1124/mol.113.089516

  View details for Web of Science ID 000332865500009

  View details for PubMedID 24319111

  View details for PubMedCentralID PMC3935154

• Activation and allosteric modulation of a muscarinic acetylcholine receptor NATURE Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Huebner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K. 2013; 504 (7478): 101-?

  Abstract

  Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

  View details for DOI 10.1038/nature12735

  View details for Web of Science ID 000327851700039

  View details for PubMedID 24256733

  View details for PubMedCentralID PMC4020789

• Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Hübner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K. 2013; 504 (7478): 101-106

  Abstract

  Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

  View details for DOI 10.1038/nature12735

  View details for PubMedID 24256733

• Novel Tripod Amphiphiles for Membrane Protein Analysis CHEMISTRY-A EUROPEAN JOURNAL Chae, P. S., Kruse, A. C., Gotfryd, K., Rana, R. R., Cho, K. H., Rasmussen, S. G., Bae, H. E., Chandra, R., Gether, U., Guan, L., Kobilka, B. K., Loland, C. J., Byrne, B., Gellman, S. H. 2013; 19 (46): 15645-15651

  Abstract

  Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to an enhanced protein solubilisation and stabilisation compared to both conventional detergents and previously described tripod amphiphiles.

  View details for DOI 10.1002/chem.201301423

  View details for Web of Science ID 000326311400030

  View details for PubMedCentralID PMC3947462

• Applications of molecular replacement to G protein-coupled receptors ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Kruse, A. C., Manglik, A., Kobilka, B. K., Weis, W. I. 2013; 69: 2287-2292

  Abstract

  G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

  View details for DOI 10.1107/S090744491301322X

  View details for Web of Science ID 000326648900016

  View details for PubMedID 24189241

  View details for PubMedCentralID PMC3817703

• Adrenaline-activated structure of ß2-adrenoceptor stabilized by an engineered nanobody. Nature Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C., Kobilka, B. K. 2013; 502 (7472): 575-579

  Abstract

  G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

  View details for DOI 10.1038/nature12572

  View details for PubMedID 24056936

  View details for PubMedCentralID PMC3822040

• Adrenaline-activated structure of ß2-adrenoceptor stabilized by an engineered nanobody. Nature Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C., Kobilka, B. K. 2013; 502 (7472): 575-579

  Abstract

  G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

  View details for DOI 10.1038/nature12572

  View details for PubMedID 24056936

• Muscarinic receptors as model targets and antitargets for structure-based ligand discovery. Molecular pharmacology Kruse, A. C., Weiss, D. R., Rossi, M., Hu, J., Hu, K., Eitel, K., Gmeiner, P., Wess, J., Kobilka, B. K., Shoichet, B. K. 2013; 84 (4): 528-540

  Abstract

  G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

  View details for DOI 10.1124/mol.113.087551

  View details for PubMedID 23887926

  View details for PubMedCentralID PMC3781386

• Muscarinic Receptors as Model Targets and Antitargets for Structure-Based Ligand Discovery MOLECULAR PHARMACOLOGY Kruse, A. C., Weiss, D. R., Rossi, M., Hu, J., Hu, K., Eitel, K., Gmeiner, P., Wess, J., Kobilka, B. K., Shoichet, B. K. 2013; 84 (4): 528-540

  Abstract

  G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, 8 bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse β-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

  View details for DOI 10.1124/mol.113.087551

  View details for Web of Science ID 000324322900005

  View details for PubMedCentralID PMC3781386

• Correction to "tandem facial amphiphiles for membrane protein stabilization". Journal of the American Chemical Society Chae, P. S., Gotfryd, K., Pacyna, J., Miercke, L. J., Rasmussen, S. G., Robbins, R. A., Rana, R. R., Loland, C. J., Kobilka, B., Stroud, R., Byrne, B., Gether, U., Gellman, S. H. 2013; 135 (34): 12922-?

  View details for DOI 10.1021/ja407245m

  View details for PubMedID 23941361

• Quantifying and localizing interactions guiding the structural and functional properties of GPCRs 9th European-Biophysical-Societies-Association Congress Zocher, M., Kawamura, S., Cheng, Z., Paul, P. S., Kobilka, B. K., Muller, D. J. SPRINGER. 2013: S108–S108

  View details for Web of Science ID 000330215300278

• The role of ligands on the equilibria between functional States of a g protein-coupled receptor. Journal of the American Chemical Society Kim, T. H., Chung, K. Y., Manglik, A., Hansen, A. L., Dror, R. O., Mildorf, T. J., Shaw, D. E., Kobilka, B. K., Prosser, R. S. 2013; 135 (25): 9465-9474

  Abstract

  G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed two distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Analysis of the spectra as a function of temperature revealed that for all ligands, the activation intermediate is entropically favored and enthalpically disfavored. β2AR enthalpy changes toward activation are notably lower than those observed with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR are weaker. Positive entropy changes toward activation likely reflect greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter analysis. Ligands greatly influence the overall changes in enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.

  View details for DOI 10.1021/ja404305k

  View details for PubMedID 23721409

• THE ROLE OF LIGANDS ON THE THERMAL EQUILIBRIA BETWEEN FUNCTIONAL STATES OF A G PROTEIN-COUPLED RECEPTOR Kim, T., Chung, K., Manglik, A., Dror, R. O., Shaw, D. E., Kobilka, B. K., Prosser, R. INFORMA HEALTHCARE. 2013: 193

  View details for Web of Science ID 000319749200039

• Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature Shukla, A. K., Manglik, A., Kruse, A. C., Xiao, K., Reis, R. I., Tseng, W., Staus, D. P., Hilger, D., Uysal, S., Huang, L., Paduch, M., Tripathi-Shukla, P., Koide, A., Koide, S., Weis, W. I., Kossiakoff, A. A., Kobilka, B. K., Lefkowitz, R. J. 2013; 497 (7447): 137-141

  Abstract

  The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

  View details for DOI 10.1038/nature12120

  View details for PubMedID 23604254

  View details for PubMedCentralID PMC3654799

• G-protein coupled receptors in virtual screening: Functional fidelity and selectivity Weiss, D. R., Ahn, S., Sassano, M. F., Kruse, A. C., Karpiak, J., Roth, B. L., Kobilka, B. K., Shoichet, B. K., Lefkowitz, R. J. AMER CHEMICAL SOC. 2013

  View details for Web of Science ID 000323851304408

• Structural insights into the dynamic process of G-protein- coupled receptor activation Kobilka, B. FEDERATION AMER SOC EXP BIOL. 2013

  View details for Web of Science ID 000319883500113

• The Dynamic Process of beta(2)-Adrenergic Receptor Activation CELL Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Fung, J. J., Bokoch, M. P., Thian, F. S., Kobilka, T. S., Shaw, D. E., Mueller, L., Prosser, R. S., Kobilka, B. K. 2013; 152 (3): 532-542

  Abstract

  G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins.

  View details for DOI 10.1016/j.cell.2013.01.008

  View details for Web of Science ID 000314362800022

  View details for PubMedID 23374348

  View details for PubMedCentralID PMC3586676

• Membrane Curvature Regulates the Oligomerization of Human beta(2)-Adrenergic Receptors 57th Annual Meeting of the Biophysical-Society Mathiasen, S., Tonnesen, A., Christensen, S., Fung, J. J., Rasmussen, S. G., Borrero, E., Provasi, D., Filizola, M., Kobilka, B., Stamou, D. CELL PRESS. 2013: 42A–42A

  View details for Web of Science ID 000316074300213

• Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach PLOS ONE Chung, K. Y., Day, P. W., Velez-Ruiz, G., Sunahara, R. K., Kobilka, B. K. 2013; 8 (1)

  Abstract

  G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β(2)-adrenergic receptor (β(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β(2)AR pull-down, 242 proteins in the inverse agonist-activated β(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β(2)AR-interacting proteins isolated was confirmed by Western blot; three known β(2)AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β(2)AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.

  View details for DOI 10.1371/journal.pone.0054942

  View details for Web of Science ID 000315210400043

  View details for PubMedID 23372797

  View details for PubMedCentralID PMC3556083

• Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study CHEMICAL COMMUNICATIONS Chae, P. S., Rana, R. R., Gotfryd, K., Rasmussen, S. G., Kruse, A. C., Cho, K. H., Capaldi, S., Carlsson, E., Kobilka, B., Loland, C. J., Gether, U., Banerjee, S., Byrne, B., Lee, J. K., Gellman, S. H. 2013; 49 (23): 2287-2289

  Abstract

  The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. (Science, 2012, 337, 473).

  View details for DOI 10.1039/c2cc36844g

  View details for Web of Science ID 000315169400003

  View details for PubMedID 23165475

  View details for PubMedCentralID PMC3578972

• The Structural Basis of G-Protein-Coupled Receptor Signaling (Nobel Lecture) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Kobilka, B. 2013; 52 (25): 6380-6388

  Abstract

  Cells from different parts of our bodies communicate with each other using chemical messengers in the form of hormones and neurotransmitters. They process information encoded in these chemical messages using G-protein-coupled receptors (GPCRs) located in the plasma membrane. The Nobel Prize for Chemistry 2012 was awarded for studies on GPCRs.

  View details for DOI 10.1002/anie.201302116

  View details for Web of Science ID 000320378800002

  View details for PubMedID 23650120

• High-resolution crystal structure of human protease-activated receptor 1 NATURE Zhang, C., Srinivasan, Y., Arlow, D. H., Fung, J. J., Palmer, D., Zheng, Y., Green, H. F., Pandey, A., Dror, R. O., Shaw, D. E., Weis, W. I., Coughlin, S. R., Kobilka, B. K. 2012; 492 (7429): 387-?

  Abstract

  Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.

  View details for DOI 10.1038/nature11701

  View details for Web of Science ID 000312488200047

  View details for PubMedID 23222541

  View details for PubMedCentralID PMC3531875

• Cholesterol increases kinetic, energetic, and mechanical stability of the human beta(2)-adrenergic receptor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zocher, M., Zhang, C., Rasmussen, S. G., Kobilka, B. K., Mueller, D. J. 2012; 109 (50): E3463-E3472

  Abstract

  The steroid cholesterol is an essential component of eukaryotic membranes, and it functionally modulates membrane proteins, including G protein-coupled receptors. To reveal insight into how cholesterol modulates G protein-coupled receptors, we have used dynamic single-molecule force spectroscopy to quantify the mechanical strength and flexibility, conformational variability, and kinetic and energetic stability of structural segments stabilizing the human β(2)-adrenergic receptor (β(2)AR) in the absence and presence of the cholesterol analog cholesteryl hemisuccinate (CHS). CHS considerably increased the kinetic, energetic, and mechanical stability of almost every structural segment at sufficient magnitude to alter the structure and functional relationship of β(2)AR. One exception was the structural core segment of β(2)AR, which establishes multiple ligand binding sites, and its properties were not significantly influenced by CHS.

  View details for DOI 10.1073/pnas.1210373109

  View details for Web of Science ID 000312605600014

  View details for PubMedCentralID PMC3528585

• Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor JOURNAL OF BIOLOGICAL CHEMISTRY Chung, K. Y., Kim, T. H., Manglik, A., Alvares, R., Kobilka, B. K., Prosser, R. S. 2012; 287 (43): 36305-36311

  Abstract

  The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

  View details for DOI 10.1074/jbc.M112.406371

  View details for Web of Science ID 000310364000051

  View details for PubMedID 22893704

  View details for PubMedCentralID PMC3476297

• N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor PLOS ONE Zou, Y., Weis, W. I., Kobilka, B. K. 2012; 7 (10)

  Abstract

  A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

  View details for DOI 10.1371/journal.pone.0046039

  View details for Web of Science ID 000309580800006

  View details for PubMedID 23056231

  View details for PubMedCentralID PMC3464249

• Ligand-Specific Interactions Modulate Kinetic, Energetic, and Mechanical Properties of the Human beta(2) Adrenergic Receptor STRUCTURE Zocher, M., Fung, J. J., Kobilka, B. K., Mueller, D. J. 2012; 20 (8): 1391-1402

  Abstract

  G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β2 adrenergic receptor (β2 AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β2 AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.

  View details for DOI 10.1016/j.str.2012.05.010

  View details for Web of Science ID 000307419200015

  View details for PubMedCentralID PMC4506644

• Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human ß2 adrenergic receptor. Structure Zocher, M., Fung, J. J., Kobilka, B. K., Müller, D. J. 2012; 20 (8): 1391-1402

  Abstract

  G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β2 adrenergic receptor (β2 AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β2 AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.

  View details for DOI 10.1016/j.str.2012.05.010

  View details for PubMedID 22748765

• A new era of GPCR structural and chemical biology NATURE CHEMICAL BIOLOGY Granier, S., Kobilka, B. 2012; 8 (8): 670-673

  Abstract

  G protein-coupled receptors (GPCRs) are versatile molecular machines that regulate the majority of physiological responses to chemically diverse hormones and neurotransmitters. Recent breakthroughs in structural studies have advanced our understanding of GPCR signaling, particularly the selectivity of ligand recognition and receptor activation of G proteins.

  View details for DOI 10.1038/nchembio.1025

  View details for Web of Science ID 000306527600002

  View details for PubMedID 22810761

• A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins CHEMISTRY-A EUROPEAN JOURNAL Chae, P. S., Rasmussen, S. G., Rana, R. R., Gotfryd, K., Kruse, A. C., Manglik, A., Cho, K. H., Nurva, S., Gether, U., Guan, L., Loland, C. J., Byrne, B., Kobilka, B. K., Gellman, S. H. 2012; 18 (31): 9485-9490

  View details for DOI 10.1002/chem.201200069

  View details for Web of Science ID 000306670200005

  View details for PubMedID 22730191

  View details for PubMedCentralID PMC3493560

• Crystal structure of the mu-opioid receptor bound to a morphinan antagonist NATURE Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., Pardo, L., Weis, W. I., Kobilka, B. K., Granier, S. 2012; 485 (7398): 321-U170

  Abstract

  Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  View details for DOI 10.1038/nature10954

  View details for Web of Science ID 000304099100032

  View details for PubMedID 22437502

  View details for PubMedCentralID PMC3523197

• Structure of the delta-opioid receptor bound to naltrindole NATURE Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., Kobilka, B. K. 2012; 485 (7398): 400-U171

  Abstract

  The opioid receptor family comprises three members, the µ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  View details for DOI 10.1038/nature11111

  View details for Web of Science ID 000304099100049

  View details for PubMedID 22596164

  View details for PubMedCentralID PMC3523198

• Structure-based drug screening for G-protein-coupled receptors TRENDS IN PHARMACOLOGICAL SCIENCES Shoichet, B. K., Kobilka, B. K. 2012; 33 (5): 268-272

  Abstract

  G-protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past 4 years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.

  View details for DOI 10.1016/j.tips.2012.03.007

  View details for Web of Science ID 000304232100007

  View details for PubMedID 22503476

  View details for PubMedCentralID PMC3523194

• Structure and dynamics of the M3 muscarinic acetylcholine receptor NATURE Kruse, A. C., Hu, J., Pan, A. C., Arlow, D. H., Rosenbaum, D. M., Rosemond, E., Green, H. F., Liu, T., Chae, P. S., Dror, R. O., Shaw, D. E., Weis, W. I., Wess, J., Kobilka, B. K. 2012; 482 (7386): 552-556

  Abstract

  Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  View details for DOI 10.1038/nature10867

  View details for Web of Science ID 000300770500056

  View details for PubMedID 22358844

  View details for PubMedCentralID PMC3529910

• Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist NATURE Haga, K., Kruse, A. C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W. I., Okada, T., Kobilka, B. K., Haga, T., Kobayashi, T. 2012; 482 (7386): 547-U147

  Abstract

  The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  View details for DOI 10.1038/nature10753

  View details for Web of Science ID 000300770500055

  View details for PubMedID 22278061

  View details for PubMedCentralID PMC3345277

• Highly Accurate Quantification of the Oligomerization of the beta 2 Adrenergic Receptor using FRET Mathiasen, S., Tonnesen, A., Christensen, S. M., Fung, J., Kobilka, B., Stamou, D. CELL PRESS. 2012: 232A–233A

  View details for DOI 10.1016/j.bpj.2011.11.1276

  View details for Web of Science ID 000321561201474

• Anti-Brownian ELectrokinetic (ABEL) Trapping of Single beta(2)-Adrenergic Receptors in the Absence and Presence of Agonist Conference on Single Molecule Spectroscopy and Super-Resolution Imaging V Bockenhauer, S., Fuerstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. SPIE-INT SOC OPTICAL ENGINEERING. 2012

  View details for DOI 10.1117/12.910018

  View details for Web of Science ID 000305624900003

• Structure of the muscarinic acetylcholine receptor M2 subtype bound with an antagonist QNB (3-quinuclidinyl benzilate) Haga, T., Haga, K., Kruse, A. C., Kobilka, B. K., Asada, H., Kobayashi, T. JAPANESE PHARMACOLOGICAL SOC. 2012: 37P

  View details for Web of Science ID 000301883300112

• My early days in GPCR structural biology: from clone to crystal NATURE STRUCTURAL & MOLECULAR BIOLOGY Kobilka, B. 2011; 18 (12): 1312–13

  View details for Web of Science ID 000298011600011

• Conformational dynamics of single G protein-coupled receptors in solution. journal of physical chemistry. B Bockenhauer, S., Fürstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. 2011; 115 (45): 13328-13338

  Abstract

  G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.

  View details for DOI 10.1021/jp204843r

  View details for PubMedID 21928818

  View details for PubMedCentralID PMC3213290

• Conformational Dynamics of Single G Protein-Coupled Receptors in Solution JOURNAL OF PHYSICAL CHEMISTRY B Bockenhauer, S., Fuerstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. 2011; 115 (45): 13328-13338

  Abstract

  G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.

  View details for DOI 10.1021/jp204843r

  View details for Web of Science ID 000296686000025

  View details for PubMedCentralID PMC3213290

• Crystal structure of the beta(2) adrenergic receptor-Gs protein complex NATURE Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., Mathiesen, J. M., Shah, S. T., Lyons, J. A., Caffrey, M., Gellman, S. H., Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., Kobilka, B. K. 2011; 477 (7366): 549-U311

  Abstract

  G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  View details for DOI 10.1038/nature10361

  View details for Web of Science ID 000295320900031

  View details for PubMedID 21772288

  View details for PubMedCentralID PMC3184188

• Conformational changes in the G protein Gs induced by the beta(2) adrenergic receptor NATURE Chung, K. Y., Rasmussen, S. G., Liu, T., Li, S., DeVree, B. T., Chae, P. S., Calinski, D., Kobilka, B. K., Woods, V. L., Sunahara, R. K. 2011; 477 (7366): 611-U143

  Abstract

  G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

  View details for DOI 10.1038/nature10488

  View details for Web of Science ID 000295320900044

  View details for PubMedID 21956331

  View details for PubMedCentralID PMC3448949

• Structural flexibility of the Gas alpha-helical domain in the beta(2)-adrenoceptor Gs complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Westfield, G. H., Rasmussen, S. G., Su, M., Dutta, S., DeVree, B. T., Chung, K. Y., Calinski, D., Velez-Ruiz, G., Oleskie, A. N., Pardon, E., Chae, P. S., Liu, T., Li, S., Woods, V. L., Steyaert, J., Kobilka, B. K., Sunahara, R. K., Skiniotis, G. 2011; 108 (38): 16086-16091

  Abstract

  The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

  View details for DOI 10.1073/pnas.1113645108

  View details for Web of Science ID 000295030000081

  View details for PubMedID 21914848

  View details for PubMedCentralID PMC3179071

• Nanobody stabilization of G protein-coupled receptor conformational states CURRENT OPINION IN STRUCTURAL BIOLOGY Steyaert, J., Kobilka, B. K. 2011; 21 (4): 567-572

  Abstract

  Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.

  View details for DOI 10.1016/j.sbi.2011.06.011

  View details for Web of Science ID 000295073800018

  View details for PubMedID 21782416

  View details for PubMedCentralID PMC3166880

• Structural insights into adrenergic receptor function and pharmacology TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B. K. 2011; 32 (4): 213-218

  Abstract

  It has been over 50years since Sir James Black developed the first beta adrenergic receptor (βAR) blocker to treat heart disease. At that time, the concept of cell surface receptors was relatively new and not widely accepted, and most of the tools currently used to characterize plasma membrane receptors had not been developed. There has been remarkable progress in receptor biology since then, including the development of radioligand binding assays, the biochemical characterization of receptors as discrete membrane proteins, and the cloning of the first G-protein-coupled receptors (GPCRs), which led to the identification of other members of the large family of GPCRs. More recently, progress in GPCR structural biology has led to insights into the three-dimensional structures of βARs in both active and inactive states. Despite all of this progress, the process of developing a drug for a particular GPCR target has become more complex, time-consuming and expensive.

  View details for DOI 10.1016/j.tips.2011.02.005

  View details for Web of Science ID 000290189200007

  View details for PubMedID 21414670

  View details for PubMedCentralID PMC3090711

• Structure and function of an irreversible agonist-beta(2) adrenoceptor complex NATURE Rosenbaum, D. M., Zhang, C., Lyons, J. A., Holl, R., Aragao, D., Arlow, D. H., Rasmussen, S. G., Choi, H., DeVree, B. T., Sunahara, R. K., Chae, P. S., Gellman, S. H., Dror, R. O., Shaw, D. E., Weis, W. I., Caffrey, M., Gmeiner, P., Kobilka, B. K. 2011; 469 (7329): 236-240

  Abstract

  G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

  View details for DOI 10.1038/nature09665

  View details for Web of Science ID 000286143400043

  View details for PubMedID 21228876

  View details for PubMedCentralID PMC3074335

• Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor NATURE Rasmussen, S. G., Choi, H., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., DeVree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I., Kobilka, B. K. 2011; 469 (7329): 175-180

  Abstract

  G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

  View details for DOI 10.1038/nature09648

  View details for Web of Science ID 000286143400030

  View details for PubMedID 21228869

  View details for PubMedCentralID PMC3058308

• Tandem Facial Amphiphiles for Membrane Protein Stabilization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chae, P. S., Gotfryd, K., Pacyna, J., Miercke, L. J., Rasmussen, S. G., Robbins, R. A., Rana, R. R., Loland, C. J., Kobilka, B., Stroud, R., Byrne, B., Gether, U., Gellman, S. H. 2010; 132 (47): 16750-16752

  Abstract

  We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

  View details for DOI 10.1021/ja1072959

  View details for Web of Science ID 000284972400009

  View details for PubMedID 21049926

  View details for PubMedCentralID PMC3050673

• Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins NATURE METHODS Chae, P. S., Rasmussen, S. G., Rana, R. R., Gotfryd, K., Chandra, R., Goren, M. A., Kruse, A. C., Nurva, S., Loland, C. J., Pierre, Y., Drew, D., Popot, J., Picot, D., Fox, B. G., Guan, L., Gether, U., Byrne, B., Kobilka, B., Gellman, S. H. 2010; 7 (12): 1003-U90

  Abstract

  The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

  View details for DOI 10.1038/NMETH.1526

  View details for Web of Science ID 000284686300016

  View details for PubMedID 21037590

  View details for PubMedCentralID PMC3063152

• Energy Landscapes as a Tool to Integrate GPCR Structure, Dynamics, and Function PHYSIOLOGY Deupi, X., Kobilka, B. K. 2010; 25 (5): 293-303

  Abstract

  G protein-coupled receptors (GPCRs) are versatile signaling molecules that mediate the majority of physiological responses to hormones and neurotransmitters. Recent high-resolution structural insights into GPCR structure and dynamics are beginning to shed light on the molecular basis of this versatility. We use energy landscapes to conceptualize the link between structure and function.

  View details for DOI 10.1152/physiol.00002.2010

  View details for Web of Science ID 000282880000005

  View details for PubMedID 20940434

  View details for PubMedCentralID PMC3056154

• International Workshop at the Nobel Forum, Karolinska Institutet on G protein-coupled receptors: finding the words to describe monomers, oligomers, and their molecular mechanisms and defining their meaning. Can a consensus be reached? JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION Kenakin, T., Agnati, L. F., Caron, M., Fredholm, B., Guidoli, D., Kobilka, B., Lefkowitz, R. W., Lohse, M., Woods, A., Fuxe, K. 2010; 30 (5): 284-286

  Abstract

  A meeting was held May 19, 2010 at the Karolinski Institute on Nomenclature in Pharmacology. This meeting occurred in conjunction with the Symposium The Changing World of G Protein Coupled Receptors: From Monomers to Dimers and Receptor Mosaics (Higher-order Oligomers) held the previous day at the Royal Swedish Academy of Science. Two broad topics of nomenclature were discussed; ligand nomenclature and the definition of 'receptor-receptor' interactions. This paper summarizes discussions on these topics along with a consensus definition of the term 'receptor-receptor' interaction.

  View details for DOI 10.3109/10799893.2010.512438

  View details for Web of Science ID 000283476500003

  View details for PubMedID 20858022

• A Device for Separated and Reversible Co-Culture of Cardiomyocytes BIOTECHNOLOGY PROGRESS Chen, M. Q., Whittington, R. H., Day, P. W., Kobilka, B. K., Giovangrandi, L., Kovacs, G. T. 2010; 26 (4): 1164-1171

  Abstract

  A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co-culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co-cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild-type and beta-adrenergic receptor (beta-AR) knockout cardiomyocytes and simultaneously exposed them with the beta-AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of beta-receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co-culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction.

  View details for DOI 10.1002/btpr.431

  View details for PubMedID 20730771

• Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal TRAFFIC Angelotti, T., Daunt, D., Shcherbakova, O. G., Kobilka, B., Hurt, C. M. 2010; 11 (4): 560-578

  Abstract

  Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  View details for DOI 10.1111/j.1600-0854.2010.01033.x

  View details for Web of Science ID 000275530700012

  View details for PubMedID 20059747

  View details for PubMedCentralID PMC2919199

• Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor NATURE Bokoch, M. P., Zou, Y., Rasmussen, S. G., Liu, C. W., Nygaard, R., Rosenbaum, D. M., Fung, J. J., Choi, H., Thian, F. S., Kobilka, T. S., Puglisi, J. D., Weis, W. I., Pardo, L., Prosser, R. S., Mueller, L., Kobilka, B. K. 2010; 463 (7277): 108-U121

  Abstract

  G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  View details for DOI 10.1038/nature08650

  View details for PubMedID 20054398

• Conformational Changes in GPCR Surface and Core Probed by [C-13]-Methyl NMR Spectroscopy Bokoch, M. P., Nygaard, R., Zou, Y., Rasmussen, S. F., Pardo, L., Prosser, R., Mueller, L., Kobilka, B. K. CELL PRESS. 2010: 418A

  View details for DOI 10.1016/j.bpj.2009.12.2257

  View details for Web of Science ID 000208762004083

• Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer EMBO JOURNAL Fung, J. J., Deupi, X., Pardo, L., Yao, X. J., Velez-Ruiz, G. A., DeVree, B. T., Sunahara, R. K., Kobilka, B. K. 2009; 28 (21): 3315-3328

  Abstract

  The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.

  View details for DOI 10.1038/emboj.2009.267

  View details for Web of Science ID 000271891400005

  View details for PubMedID 19763081

  View details for PubMedCentralID PMC2748299

• The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proceedings of the National Academy of Sciences of the United States of America Yao, X. J., Vélez Ruiz, G., Whorton, M. R., Rasmussen, S. G., DeVree, B. T., Deupi, X., Sunahara, R. K., Kobilka, B. 2009; 106 (23): 9501-9506

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

  View details for DOI 10.1073/pnas.0811437106

  View details for PubMedID 19470481

  View details for PubMedCentralID PMC2685739

• The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yao, X. J., Ruiz, G. V., Whorton, M. R., Rasmussen, S. G., DeVree, B. T., Deupi, X., Sunahara, R. K., Kobilka, B. 2009; 106 (23): 9501-9506

  Abstract

  G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

  View details for DOI 10.1073/pnas.0811437106

  View details for Web of Science ID 000266817500071

  View details for PubMedCentralID PMC2685739

• The structure and function of G-protein-coupled receptors NATURE Rosenbaum, D. M., Rasmussen, S. G., Kobilka, B. K. 2009; 459 (7245): 356-363

  Abstract

  G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive activity.

  View details for DOI 10.1038/nature08144

  View details for Web of Science ID 000266243700033

  View details for PubMedID 19458711

• Structure-based discovery of beta(2)-adrenergic receptor ligands PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kolb, P., Rosenbaum, D. M., Irwin, J. J., Fung, J. J., Kobilka, B. K., Shoichet, B. K. 2009; 106 (16): 6843-6848

  Abstract

  Aminergic G protein-coupled receptors (GPCRs) have been a major focus of pharmaceutical research for many years. Due partly to the lack of reliable receptor structures, drug discovery efforts have been largely ligand-based. The recently determined X-ray structure of the beta(2)-adrenergic receptor offers an opportunity to investigate the advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR targets. Approximately 1 million commercially available, "lead-like" molecules were docked against the beta(2)-adrenergic receptor structure. On testing of 25 high-ranking molecules, 6 were active with binding affinities <4 microM, with the best molecule binding with a K(i) of 9 nM (95% confidence interval 7-10 nM). Five of these molecules were inverse agonists. The high hit rate, the high affinity of the most potent molecule, the discovery of unprecedented chemotypes among the new inhibitors, and the apparent bias toward inverse agonists among the docking hits, have implications for structure-based approaches against GPCRs that recognize small organic molecules.

  View details for DOI 10.1073/pnas.0812657106

  View details for Web of Science ID 000265506800075

  View details for PubMedID 19342484

  View details for PubMedCentralID PMC2672528

• A Microsecond Time Scale Molecular Dynamics Simulation of B2AR in a Membrane Romo, T. D., Grossfield, A., Pitman, M. C., Deupi, X., Cordomi, A., Kobilka, B. CELL PRESS. 2009: 340A

  View details for Web of Science ID 000426353300757

• CRYSTAL STRUCTURES OF THE beta(2)-ADRENERGIC RECEPTOR 40th Erice Course on From Molecules to Medicine - Structure of Biological Macromolecules and Its Relevance in Combating New Diseases and Bioterrorism Weis, W. I., Rosenbaum, D. M., Rasmussen, S. G., Choi, H., Thian, F. S., Kobilka, T. S., Yao, X., Day, P. W., Parnot, C., Fung, J. J., Ratnala, V. R., Kobilka, B. K., Cherezov, V., Hanson, M. A., Kuhn, P., Stevens, R. C., Edwards, P. C., Schertler, G. F., Burghammer, M., Sanishvili, R., Fischetti, R. F., Masood, A., Rohrer, D. K. SPRINGER. 2009: 217–230

  View details for Web of Science ID 000266237200014

• Understanding the ligand-receptor-G protein ternary complex for GPCR drug discovery. Methods in molecular biology (Clifton, N.J.) Ratnala, V. R., Kobilka, B. 2009; 552: 67-77

  Abstract

  Understanding the ternary complex between G protein-coupled receptors (GPCRs), cognate G proteins, and their ligands is an important landmark for drug discovery. Yet, little is known about the specific interactions between GPCRs and G proteins. For a better perspective on the ternary complex dynamics, we adapted a beta(2)-adrenergic receptor(beta(2)AR)-tetGs(alpha) reconstitution system and found evidence that for efficient coupling of the beta(2)AR to Gs does not require specific interactions between the betagamma-subunits and the beta(2)AR. Our results demonstrate that specific interactions between betagamma and the beta(2)AR are not required for G protein activation but likely serve to anchor Gs(alpha) to the plasma membrane. Our results also suggests that the advantages of analysis of G protein activation by using beta(2)AR receptor-tetGs(alpha) system in vitro at the close proximity of the receptor may constitute a simple screening system that avoids false positives and potentially adapted to screen drugs for other GPCRs.

  View details for DOI 10.1007/978-1-60327-317-6_5

  View details for PubMedID 19513642

• Structural insights into G-protein-coupled receptor activation CURRENT OPINION IN STRUCTURAL BIOLOGY Weis, W. I., Kobilka, B. K. 2008; 18 (6): 734-740

  Abstract

  G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two beta-adrenergic receptors have defined some of the conformational changes associated with activation.

  View details for DOI 10.1016/j.sbi.2008.09.010

  View details for Web of Science ID 000262064300014

  View details for PubMedID 18957321

• Signaling from beta 1-and beta 2-adrenergic receptors is defined by differential interactions with PDE4 Richter, W., Day, P., Agrawal, R., Bruss, M. D., Granier, S., Wang, Y. L., Rasmussen, S. G., Homer, K., Wang, P., Lei, T., Patterson, A. J., Kobilka, B., Conti, M. FEDERATION AMER SOC EXP BIOL. 2008

  View details for Web of Science ID 000208467804672

• New G-protein-coupled receptor crystal structures: insights and limitations TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B., Schertler, G. F. 2008; 29 (2): 79-83

  Abstract

  G-protein-coupled receptors (GPCRs) constitute a large family of structurally similar proteins that respond to a chemically diverse array of physiological and environmental stimulants. Until recently, high-resolution structural information was limited to rhodopsin, a naturally abundant GPCR that is highly specialized for the detection of light. Non-rhodopsin GPCRs for diffusible hormones and neurotransmitters have proven more resistant to crystallography approaches, possibly because of their inherent structural flexibility and the need for recombinant expression. Recently, crystal structures of the human beta(2) adrenoceptor have been obtained using two different approaches to stabilize receptor protein and increase polar surface area. These structures, together with the existing structures of rhodopsin, represent an important first step in understanding how GPCRs work at a molecular level. Much more high-resolution information is needed for this important family of membrane proteins, however: for example, the structures of GPCRs from different families, structures bound to ligands having different efficacies, and structures of GPCRs in complex with G proteins and other signaling molecules. Methods to characterize the dynamic aspects of the GPCR architecture at high resolution will also be important.

  View details for DOI 10.1016/j.tips.2007.11.009

  View details for Web of Science ID 000253599000005

  View details for PubMedID 18194818

• Signaling from beta(1)- and beta(2)-adrenergic receptors is defined by differential interactions with PDE4 EMBO JOURNAL Richter, W., Day, P., Agrawal, R., Bruss, M. D., Granier, S., Wang, Y. L., Rasmussen, S. G., Horner, K., Wang, P., Lei, T., Patterson, A. J., Kobilka, B., Conti, M. 2008; 27 (2): 384-393

  Abstract

  Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

  View details for DOI 10.1038/sj.emboj.7601968

  View details for Web of Science ID 000253408600009

  View details for PubMedID 18188154

  View details for PubMedCentralID PMC2196435

• GPCR engineering yields high-resolution structural insights into beta(2)-adrenergic receptor function SCIENCE Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Yao, X., Weis, W. I., Stevens, R. C., Kobilka, B. K. 2007; 318 (5854): 1266-1273

  Abstract

  The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

  View details for DOI 10.1126/science.1150609

  View details for Web of Science ID 000251086600034

  View details for PubMedID 17962519

• High-resolution crystal structure of an engineered human beta(2)-adrenergic G protein-coupled receptor SCIENCE Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. 2007; 318 (5854): 1258-1265

  Abstract

  Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

  View details for DOI 10.1126/science.1150577

  View details for Web of Science ID 000251086600033

  View details for PubMedID 17962520

  View details for PubMedCentralID PMC2583103

• Crystal structure of the human beta(2) adrenergic G-protein-coupled receptor NATURE Rasmussen, S. G., Choi, H., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fischetti, R. F., Schertler, G. F., Weis, W. I., Kobilka, B. K. 2007; 450 (7168): 383-U4

  Abstract

  Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

  View details for DOI 10.1038/nature06325

  View details for Web of Science ID 000250918600046

  View details for PubMedID 17952055

• A monoclonal antibody for G protein-coupled receptor crystallography NATURE METHODS Day, P. W., Rasmussen, S. G., Parnot, C., Fung, J. J., Masood, A., Kobilka, T. S., Yao, X., Choi, H., Weis, W. I., Rohrer, D. K., Kobilka, B. K. 2007; 4 (11): 927-929

  Abstract

  G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  View details for DOI 10.1038/NMETH1112

  View details for Web of Science ID 000250575700017

  View details for PubMedID 17952087

• N-ethylmaleimide-sensitive factor regulates beta(2) adrenoceptor trafficking and signaling in cardiomyocytes MOLECULAR PHARMACOLOGY Wang, Y., Lauffer, B., von Zastrow, M., Kobilka, B. K., Xiang, Y. 2007; 72 (2): 429-439

  Abstract

  Recycling of G protein-coupled receptors determines the functional resensitization of receptors and is implicated in switching beta2 adrenoceptor (beta2AR) G protein specificity in cardiomyocytes. The human beta2AR carboxyl end binds to the N-ethylmaleimide-sensitive factor (NSF), an ATPase integral to membrane trafficking machinery. It is interesting that the human beta2AR (hbeta2AR) carboxyl end pulled down NSF from mouse heart lysates, whereas the murine one did not. Despite this difference, both beta2ARs exhibited substantial agonist-induced internalization, recycling, and Gi coupling in cardiomyocytes. The hbeta2AR, however, displayed faster rates of agonist-induced internalization and recycling compared with the murine beta2AR (mbeta2AR) and a more profound Gi component in its contraction response. Replacing the mbeta2AR proline (-1) with a leucine generated a gain-of-function mutation, mbeta2AR-P417L, with a rescued ability to bind NSF, faster internalization and recycling than the mbeta2AR, and a significant enhancement in Gi signaling, which mimics the hbeta2AR. Selective disruption of the mbeta2AR-P417L binding to NSF inhibited the receptor coupling to Gi. Mean-while, inhibiting NSF with N-ethylmaleimide blocked the mbeta2AR recycling after agonist-induced endocytosis. Expressing the NSF-E329Q mutant lacking ATPase activity inhibited the mbeta2AR coupling to Gi in cardiomyocytes. Our results revealed a dual regulation on hbeta2AR trafficking and signaling by NSF through direct binding to cargo receptor and its ATPase activity and uncovered an unprecedented role for the receptor binding to NSF in regulating G protein specificity that has diverged between mouse and human beta2ARs.

  View details for DOI 10.1124/mol.107.037747

  View details for Web of Science ID 000248282900023

  View details for PubMedID 17510209

• Conformational complexity of G-protein-coupled receptors TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B. K., Deupi, X. 2007; 28 (8): 397-406

  Abstract

  G-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human beta2 adrenergic receptor.

  View details for DOI 10.1016/j.tips.2007.06.003

  View details for Web of Science ID 000248898900007

  View details for PubMedID 17629961

• Structure and conformational changes in the C-terminal domain of the beta(2)-adrenoceptor - Insights from fluorescence resonance energy transfer studies JOURNAL OF BIOLOGICAL CHEMISTRY Granier, S., Kim, S., Shafer, A. M., Ratnala, V. R., Fung, J. J., Zare, R. N., Kobilka, B. 2007; 282 (18): 13895-13905

  Abstract

  The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.

  View details for DOI 10.1074/jbc.M611904200

  View details for Web of Science ID 000246060300079

  View details for PubMedID 17347144

• A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Whorton, M. R., Bokoch, M. P., Rasmussen, S. G., Huang, B., Zare, R. N., Kobilka, B., Sunahara, R. K. 2007; 104 (18): 7682-7687

  Abstract

  G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.

  View details for DOI 10.1073/pnas.0611448104

  View details for Web of Science ID 000246239400068

  View details for PubMedID 17452637

  View details for PubMedCentralID PMC1863461

• G protein coupled receptor structure and activation BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Kobilka, B. K. 2007; 1768 (4): 794-807

  Abstract

  G protein coupled receptors (GPCRs) are remarkably versatile signaling molecules. The members of this large family of membrane proteins are activated by a spectrum of structurally diverse ligands, and have been shown to modulate the activity of different signaling pathways in a ligand specific manner. In this manuscript I will review what is known about the structure and mechanism of activation of GPCRs focusing primarily on two model systems, rhodopsin and the beta(2) adrenoceptor.

  View details for DOI 10.1016/j.bbamem.2006.10.021

  View details for Web of Science ID 000245972500005

  View details for PubMedID 17188232

  View details for PubMedCentralID PMC1876727

• Distinct PDE4D splice variants regulate beta-adrenergic signaling in neonatal mouse cardiac myocytes Experimental Biology 2007 Annual Meeting Richter, W., Day, P., Wang, Y., Bruss, M., Wang, P., Kobilka, B., Conti, M. FEDERATION AMER SOC EXP BIOL. 2007: A997–A997

  View details for Web of Science ID 000245708702131

• Organization of beta-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes JOURNAL OF CELL BIOLOGY Shcherbakova, O. G., Hurt, C. M., Xiang, Y., Dell'Acqua, M. L., Zhang, Q., Tsien, R. W., Kobilka, B. K. 2007; 176 (4): 521-533

  Abstract

  The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). beta(1) and beta(2)ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of beta(1) and beta(2)ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The beta(1)ARs are enriched within these zones, whereas beta(2)ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by beta(1) and beta(2)ARs in vivo.

  View details for DOI 10.1083/jcb.200604167

  View details for Web of Science ID 000244348300013

  View details for PubMedID 17296797

  View details for PubMedCentralID PMC2063986

• Counting low-copy number proteins in a single cell SCIENCE Huang, B., Wu, H., Bhaya, D., Grossman, A., Granier, S., Kobilka, B. K., Zare, R. N. 2007; 315 (5808): 81-84

  Abstract

  We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.

  View details for DOI 10.1126/science.1133992

  View details for Web of Science ID 000243259100039

  View details for PubMedID 17204646

• Activation of G Protein Coupled Receptors MECHANISMS AND PATHWAYS OF HETEROTRIMERIC G PROTEIN SIGNALING Deupi, X., Kobilka, B. 2007; 74: 137-166

  Abstract

  G protein-coupled receptors (GPCRs) mediate responses to hormones and neurotransmitters, as well as the senses of sight, smell, and taste. These remarkably versatile signaling molecules respond to structurally diverse ligands. Many GPCRs couple to multiple G protein subtypes, and several have been shown to activate G protein-independent signaling pathways. Drugs acting on GPCRs exhibit efficacy profiles that may differ for different signaling cascades. The functional plasticity exhibited by GPCRs can be attributed to structural flexibility and the existence of multiple ligand-specific conformational states. This chapter will review our current understanding of the mechanism by which agonists bind and activate GPCRs.

  View details for DOI 10.1016/S0065-3231(07)74004-4

  View details for Web of Science ID 000250106300004

  View details for PubMedID 17854657

• PDZ-domain arrays for identifying components of GPCR signaling complexes TRENDS IN PHARMACOLOGICAL SCIENCES Day, P., Kobilka, B. 2006; 27 (10): 509-511

  Abstract

  Many G-protein-coupled receptors (GPCRs) modulate the activity of multiple effectors. Yet, despite this apparent promiscuity, signaling in the context of differentiated cells is often highly specific. This specificity is attributable to the formation of cell-type-specific signaling complexes that are held together by scaffolding proteins, many of which contain one or more PDZ domains. Identifying the set of potential interactions among GPCRs, other signaling molecules and these scaffolding proteins is essential for understanding physiological signaling processes. A recent article describes an elegantly simple PDZ-domain array that can identify potential interacting partners of GPCRs and other signaling molecules.

  View details for DOI 10.1016/j.tips.2006.08.003

  View details for Web of Science ID 000241258000001

  View details for PubMedID 16904197

• PHYS 338-Quantitating low-copy-number proteins in a single cell by direct counting Zare, R. N., Huang, B., Wu, H., Bhaya, D., Grossman, A. R., Kobilka, B. K. AMER CHEMICAL SOC. 2006

  View details for Web of Science ID 000207781609055

• Differential targeting and function of alpha(2A) and alpha(2C) adrenergic receptor subtypes in cultured sympathetic neurons NEUROPHARMACOLOGY Brum, P. C., Hurt, C. M., Shcherbakova, O. G., Kobilka, B., Angelotti, T. 2006; 51 (3): 397-413

  Abstract

  Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

  View details for DOI 10.1016/j.neuropharm.2006.03.032

  View details for Web of Science ID 000240811900001

  View details for PubMedID 16750543

• Coupling ligand structure to specific conformational switches in the beta(2)-adrenoceptor NATURE CHEMICAL BIOLOGY Yao, X., Parnot, C., Deupi, X., Ratnala, V. R., Swaminath, G., Farrens, D., Kobilka, B. 2006; 2 (8): 417-422

  Abstract

  G protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human beta2-adrenoceptor (beta2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the beta2-AR.

  View details for DOI 10.1038/nchembio801

  View details for Web of Science ID 000239146800010

  View details for PubMedID 16799554

• Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization LAB ON A CHIP Huang, B., Wu, H. K., Kim, S., Kobilka, B. K., Zare, R. N. 2006; 6 (3): 369-373

  Abstract

  Polydimethylsiloxane (PDMS) surfaces can be functionalized with biotin groups by adding biotinylated phospholipids to the PDMS prepolymer before curing. The addition of beta-D-dodecyl-N-maltoside (DDM) in the solution blocks non-specific protein binding on these functionalized PDMS surfaces. We characterize the surface by measuring fluorescently labeled streptavidin binding. Single molecule tracking shows that the phospholipids are not covalently linked to PDMS polymer chains, but the surface functionalization is not removed by washing. We demonstrate the immobilization of biotinylated antibodies and lectins through biotin-avidin interactions.

  View details for DOI 10.1039/b515840k

  View details for Web of Science ID 000235993800005

  View details for PubMedID 16511619

• Effect of targeted deletions of beta(1)- and beta(2)-adrenergic-receptor subtypes on heart rate variability AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Ecker, P. M., Lin, C. C., Powers, J., Kobilka, B. K., Dubin, A. M., Bernstein, D. 2006; 290 (1): H192-H199

  Abstract

  Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling.

  View details for DOI 10.1152/ajpheart.00032.2005

  View details for Web of Science ID 000234148200023

  View details for PubMedID 16113068

• Differential cardioprotective/cardiotoxic effects mediated by ss-adrenergic receptor subtypes AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Bernstein, D., Fajardo, G., Zhao, M. M., Urashima, T., Powers, J., Berry, G., Kobilka, B. K. 2005; 289 (6): H2441-H2449

  Abstract

  Recent data suggest that beta-adrenergic receptor subtypes couple differentially to signaling pathways regulating cardiac function vs. cardiac remodeling. To dissect the roles of beta1- vs. beta2-receptors in the pathogenesis of cardiomyopathy, doxorubicin was administered to beta1, beta2, and beta1/beta2 knockout (-/-) and wild-type mice. Expression and activation of MAPKs were measured. Wild-type and beta1-/- mice showed no acute cardiovascular effects, whereas beta2-/- mice all died within 30 min. The additional deletion of the beta1-receptor (beta1/beta2-/-) totally rescued this toxicity. beta2-/- mice developed decreased contractile function, hypotension, QTc prolongation, and ST segment changes and a 20-fold increase in p38 MAPK activity not seen in the other genotypes. The MAPK inhibitor SB-203580 rescued beta2-/- mice from this acute toxicity. The enhanced toxicity in beta2-/- mice was also recapitulated in wild-type mice with the beta2-selective antagonist ICI-118,551, although the rescue effect of the beta1-deletion was not recapitulated using the beta1-selective antagonist metoprolol or the nonselective beta-antagonist propranolol. These data suggest that beta2-adrenergic receptors play a cardioprotective role in the pathogenesis of cardiomyopathy, whereas beta1-adrenergic receptors mediate at least some of the acute cardiotoxicity of anthracyclines. Differential activation of MAPK isoforms, previously shown in vitro to regulate beta-agonist as well as doxorubicin cardiotoxicity, appears to play a role in mediating the differential effects of these beta-adrenergic receptor subtypes in vivo.

  View details for DOI 10.1152/ajpheart.00005.2005

  View details for Web of Science ID 000233176600023

  View details for PubMedID 16040722

• Using synthetic lipids to stabilize purified beta(2) adrenoceptor in detergent micelles ANALYTICAL BIOCHEMISTRY Yao, Z. P., Kobilka, B. 2005; 343 (2): 344-346

  View details for DOI 10.1016/j.ab.2005.05.002

  View details for Web of Science ID 000231163900022

  View details for PubMedID 16005425

• Probing the beta(2) adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., Deupi, X., LEE, T. W., Zhu, W., Thian, F. S., Kobilka, T. S., Kobilka, B. 2005; 280 (23): 22165-22171

  Abstract

  The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

  View details for Web of Science ID 000229557900061

  View details for PubMedID 15817484

• Mass spectrometric analysis of agonist effects on posttranslational modifications of the beta-2 adrenoceptor in mammalian cells BIOCHEMISTRY Trester-Zedlitz, M., Burlingame, A., Kobilka, B., von Zastrow, M. 2005; 44 (16): 6133-6143

  Abstract

  Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine > dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.

  View details for DOI 10.1021/bi0475469

  View details for Web of Science ID 000228678900018

  View details for PubMedID 15835901

• Phosphodiesterase 4D is required for beta(2) adrenoceptor subtype-specific signaling in cardiac myocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xiang, Y., Naro, F., Zoudilova, M., Jin, S. L., Conti, M., Kobilka, B. 2005; 102 (3): 909-914

  Abstract

  beta adrenoceptor (betaAR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, beta1AR activates the conventional Gs/cAMP pathway, whereas beta2AR sequentially activates both the Gs and Gi pathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by beta2AR in neonatal cardiac myocytes, while having little or no effect on beta1AR signaling. Although beta2AR activation leads to an increase in cAMP production, the cAMP generated does not have access to the protein kinase A-dependent signaling pathways by which the beta1AR regulates the contraction rate. However, this restricted access is lost in the presence of PDE4 inhibitors or after ablation of PDE4D. These results not only suggest that PDE4D is an integral component of the beta2AR signaling complex, but also underscore the critical role of subcellular cAMP regulation in the complex control of receptor signaling. They also illustrate a mechanism for fine-tuned betaAR subtype signaling specificity and intensity in the cardiac system.

  View details for DOI 10.1073/pnas.0405263102

  View details for Web of Science ID 000226436000069

  View details for PubMedID 15644445

  View details for PubMedCentralID PMC544184

• Toward understanding GPCR dimers NATURE STRUCTURAL & MOLECULAR BIOLOGY Parnot, C., Kobilka, B. 2004; 11 (8): 691-692

  View details for DOI 10.1038/nsmb0804-691

  View details for Web of Science ID 000222930600007

  View details for PubMedID 15280880

• The state of GPCR research in 2004 NATURE REVIEWS DRUG DISCOVERY Bartfai, T., Benovic, J. L., Bockaert, J., Bond, R. A., Bouvier, M., Christopoulos, A., Civelli, O., Devi, L. A., George, S. R., Inui, A., Kobilka, B., Leurs, R., Neubig, R., Pin, J. P., QUIRION, R., Roques, B. P., Sakmar, T. P., Seifert, R., Stenkamp, R. E., Strange, P. G. 2004; 3 (7): 574-626

  View details for DOI 10.1038/nrd1458

  View details for Web of Science ID 000222388900019

• Agonist binding: A multistep process MOLECULAR PHARMACOLOGY Kobilka, B. 2004; 65 (5): 1060-1062

  View details for Web of Science ID 000220951600002

  View details for PubMedID 15102933

• Protecting the myocardium: A role for the beta 2 adrenergic receptor in the heart CRITICAL CARE MEDICINE Patterson, A. J., Zhu, W. Z., Chow, A., Agrawal, R., Kosek, J., Xiao, R. P., Kobilka, B. 2004; 32 (4): 1041-1048

  Abstract

  The sympathetic nervous system enhances cardiac muscle function by activating beta adrenergic receptors (betaARs). Recent studies suggest that chronic betaAR stimulation is detrimental, however, and that it may play a role in the clinical deterioration of patients with congestive heart failure. To examine the impact of chronic beta1AR and beta2AR subtype stimulation individually, we studied the cardiovascular effects of catecholamine infusions in betaAR subtype knockout mice (beta1KO, beta2KO).Prospective, randomized, experimental study.Animal research laboratory.beta1KO and beta2KO mice and wild-type controls.The animals were subjected to 2 wks of continuous infusion of the betaAR agonist isoproterenol. Analyses of cardiac function and structure were performed during and 3 days after completion of the infusions. Functional studies included graded exercise treadmill testing, in vivo assessments of left ventricular function using Mikro-Tip catheter transducers, right ventricular pressure measurements, and analyses of organ weight to body weight ratios. Structural studies included heart weight measurements, assessments of myocyte ultrastructure using electron microscopy, and in situ terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining to quantitate myocyte apoptosis.We found that isoproterenol-treated beta2KO mice experienced greater mortality rates (p =.001, chi-square test using Fisher's exact method) and increased myocyte apoptosis at 3- and 7-day time points (p =.04 and p =.0007, respectively, two-way analysis of variance).The results of this study suggest that in vivo beta2AR activation is antiapoptotic and contributes to myocardial protection.

  View details for DOI 10.1097/01.CCM.0000120049.43113.90

  View details for Web of Science ID 000220872700021

  View details for PubMedID 15071399

• Plasmon-waveguide resonance studies of ligand binding to the human beta(2)-adrenergic receptor BIOCHEMISTRY Devanathan, S., Yao, Z. P., Salamon, Z., Kobilka, B., Tollin, G. 2004; 43 (11): 3280-3288

  Abstract

  Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.

  View details for DOI 10.1021/bi035825a

  View details for Web of Science ID 000220276700030

  View details for PubMedID 15023079

• Sequential binding of agonists to the beta(2) adrenoceptor - Kinetic evidence for intermediate conformational states JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., Xiang, Y., LEE, T. W., Steenhuis, J., Parnot, C., Kobilka, B. K. 2004; 279 (1): 686-691

  Abstract

  The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.

  View details for DOI 10.1074/jbc.M310888200

  View details for Web of Science ID 000187555300082

  View details for PubMedID 14559905

• The PDZ-binding motif of the beta(2)-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xiang, Y., Kobilka, B. 2003; 100 (19): 10776-10781

  Abstract

  beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.

  View details for DOI 10.1073/pnas.1831718100

  View details for Web of Science ID 000185415300038

  View details for PubMedID 12954981

  View details for PubMedCentralID PMC196879

• Myocyte adrenoceptor signaling pathways SCIENCE Xiang, Y., Kobilka, B. K. 2003; 300 (5625): 1530-1532

  Abstract

  Adrenoceptors (ARs), members of the G protein-coupled receptor superfamily, form the interface between the sympathetic nervous system and the cardiovascular system, with integral roles in the rapid regulation of myocardial function. However, in heart failure, chronic catecholamine stimulation of adrenoceptors has been linked to pathologic cardiac remodeling, including myocyte apoptosis and hypertrophy. In cardiac myocytes, activation of AR subtypes results in coupling to different G proteins and induction of specific signaling pathways, which is partly regulated by the subtype-specific distribution of receptors in plasma membrane compartments containing distinct complexes of signaling molecules. The Connections Maps of the Adrenergic and Myocyte Adrenergic Signaling Pathways bring into focus the specific signaling pathways of individual AR subtypes and their relevant functions in vivo.

  View details for Web of Science ID 000183333100040

  View details for PubMedID 12791980

• Genetic manipulation of mice for analysis of beta-adrenergic receptor pharmacology and physiology Experimental Biology 2003 Annual Meeting Angelotti, T., Desai, K., Kobilka, B. FEDERATION AMER SOC EXP BIOL. 2003: A210–A210

  View details for Web of Science ID 000181733101013

• Identification of an allosteric binding site for ZN(2+) on the beta(2) adrenergic receptor JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., LEE, T. W., Kobilka, B. 2003; 278 (1): 352-356

  Abstract

  The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.

  View details for DOI 10.1074/jbc.M206424200

  View details for Web of Science ID 000180255700047

  View details for PubMedID 12409304

• Efficient adenylyl cyclase activation by a beta(2)-adrenoceptor-G(i)alpha(2) fusion protein BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Seifert, R., Wenzel-Seifert, K., Arthur, J. M., Jose, P. O., Kobilka, B. K. 2002; 298 (5): 824-828

  Abstract

  The G-protein G(i)alpha can activate adenylyl cyclase (AC), but the relevance of this AC activation is unknown. We used receptor-G protein co-expression and receptor-G protein fusion proteins to investigate G(i)alpha(2) regulation of AC in Sf9 cells. G(i)alpha(2) was fused to the beta(2)-adrenoceptor (beta(2)AR), a preferentially G(s)-coupled receptor, or the formyl peptide receptor (FPR), a G(i)-coupled receptor. The FPR co-expressed with, or fused to, G(i)alpha(2), reduced AC activity. In contrast, the beta(2)AR fused to G(i)alpha(2) was a highly efficient AC activator, while the beta(2)AR co-expressed with G(i)alpha(2) was not. Agonist efficiently stimulated incorporation of [alpha-32P]GTP azidoanilide into beta(2)AR-G(i)alpha(2). We explain AC activation by beta(2)AR-G(i)alpha(2) by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2) from interacting with the inhibitory G(i)alpha site of AC. The postulated beta(2)AR/AC interaction brings G(i)alpha(2) into close proximity of the G(s)alpha site of AC, enabling G(i)alpha(2) to activate AC.

  View details for Web of Science ID 000179374500030

  View details for PubMedID 12419329

• Differential cardioprotective/cardiotoxic effects of beta adrenergic receptor subtypes in isolated cardiac myocytes and fibroblasts American-Heart-Association Abstracts From Scientific Sessions Fajardo, G., Zhao, M. M., Powers, J., Kobilka, B., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2002: 123–23

  View details for Web of Science ID 000179142700656

• Abnormal cardiac function associated with sympathetic nervous system hyperactivity in mice AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Brum, P. C., Kosek, J., Patterson, A., Bernstein, D., Kobilka, B. 2002; 283 (5): H1838-H1845

  Abstract

  alpha(2A)-Adrenergic receptors (ARs) in the midbrain regulate sympathetic nervous system activity, and both alpha(2A)-ARs and alpha(2C)-ARs regulate catecholamine release from sympathetic nerve terminals in cardiac tissue. Disruption of both alpha(2A)- and alpha(2C)-ARs in mice leads to chronically elevated sympathetic tone and decreased cardiac function by 4 mo of age. These knockout mice have increased mortality, reduced exercise capacity, decreased peak oxygen uptake, and decreased cardiac contractility relative to wild-type controls. Moreover, we observed significant abnormalities in the ultrastructure of cardiac myocytes from alpha(2A)/alpha(2C)-AR knockout mice by electron microscopy. Our results demonstrate that chronic elevation of sympathetic tone can lead to abnormal cardiac function in the absence of prior myocardial injury or genetically induced alterations in myocardial structural or functional proteins. These mice provide a physiologically relevant animal model for investigating the role of the sympathetic nervous system in the development and progression of heart failure.

  View details for DOI 10.1152/ajpheart.01063.2001

  View details for Web of Science ID 000178625800012

  View details for PubMedID 12384461

• Functional immobilization of a ligand-activated G-protein-coupled receptor CHEMBIOCHEM Neumann, L., Wohland, T., Whelan, R. J., Zare, R. N., Kobilka, B. K. 2002; 3 (10): 993-998

  Abstract

  G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest family of receptors in the human genome and constitute the largest class of targets for drug discovery. To facilitate studies of GPCR activation and interactions with other proteins, we developed a simple method to immobilize a functional, detergent-solubilized GPCR on gold and glass surfaces. The beta(2) adrenergic receptor (beta(2)AR), a prototypical GPCR, was purified and labeled with a reporter fluorophore at a conformationally sensitive site. The detergent-soluble fluorescent beta(2)AR was immobilized through its amino-terminal FLAG epitope on a surface layered with biotinylated bovine serum albumin, avidin, and biotinylated M1 antibody. Agonist activation of the beta(2)AR was monitored in real time by fluorescence microscopy. This approach will make it possible to study conformational dynamics of single immobilized receptors and to generate arrays of functional GPCRs for novel high-throughput screening strategies.

  View details for Web of Science ID 000178555900009

  View details for PubMedID 12362365

• Heterozygous alpha(2A)-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, C. M., Wilson, M. H., MacMillan, L. B., Kobilka, B. K., Limbird, L. E. 2002; 99 (19): 12471-12476

  Abstract

  Genetic manipulation of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) in mice has revealed the role of this subtype in numerous responses, including agonist-induced hypotension and sedation. Unexpectedly, alpha(2)-agonist treatment of mice heterozygous for the alpha(2A)-AR (alpha(2A)-AR(+/-)) lowers blood pressure without sedation, indicating that more than 50% of alpha(2A)-AR must be activated to evoke sedation. We postulated that partial activation of alpha(2A)-AR in wild-type alpha(2A)-AR(+/+) animals could be achieved with partial agonists, agents with variable ability to couple receptor occupancy to effector activation, and might elicit one versus another pharmacological response. In vitro assays reveal that moxonidine is a partial agonist at alpha(2A)-AR. Although moxonidine was developed to preferentially interact with imidazoline binding sites, it requires the alpha(2A)-AR to lower blood pressure because we observe no hypotensive response to moxonidine in alpha(2A)-AR-null (alpha(2A)-AR(-/-)) mice. Moreover, we observe that moxonidine lowers blood pressure without sedation in wild-type mice, consistent with the above hypothesis regarding partial agonists. Our findings suggest that weak partial agonists can evoke response-selective pathways and might be exploited successfully to achieve alpha(2A)-AR pharmacotherapy where concomitant sedation is undesirable, i.e., in treatment of depression or attention deficit hyperactivity disorder, in suppression of epileptogenesis, or enhancement of cognition. Furthermore, rigorous physiological and behavioral assessment of mice heterozygous for particular receptors provides a general strategy for elucidation of pathways that might be selectively activated by partial agonists, thus achieving response-specific therapy.

  View details for DOI 10.1073/pnas.122368499

  View details for Web of Science ID 000178187000086

  View details for PubMedID 12205290

  View details for PubMedCentralID PMC129469

• The PDZ binding motif of the beta(1) adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes JOURNAL OF BIOLOGICAL CHEMISTRY Xiang, Y., Devic, E., Kobilka, B. 2002; 277 (37): 33783-33790

  Abstract

  Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).

  View details for DOI 10.1074/jbc.M204136200

  View details for Web of Science ID 000177959100035

  View details for PubMedID 12097326

• Caveolar localization dictates physiologic signaling of beta(2)-adrenoceptors in neonatal cardiac myocytes JOURNAL OF BIOLOGICAL CHEMISTRY Xiang, Y., Rybin, V. O., Steinberg, S. F., Kobilka, B. 2002; 277 (37): 34280-34286

  Abstract

  There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.

  View details for DOI 10.1074/jbc.M201644200

  View details for Web of Science ID 000177959100098

  View details for PubMedID 12097322

• Analysis of bimolecular interactions using a miniaturized surface plasmon resonance sensor ANALYTICAL CHEMISTRY Whelan, R. J., Wohland, T., Neumann, L., Huang, B., Kobilka, B. K., Zare, R. N. 2002; 74 (17): 4570-4576

  Abstract

  A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found to be 2.1 fmol, which corresponds to a surface concentration of 21 pg/mm2. Simple surface immobilization procedures relying on biotin/avidin or glycoprotein/lectin chemistry have been explored. Equilibrium dissociation constants for the binding of the FLAG peptide to its monoclonal antibody (M1) and for the binding of concanavalin A to a glycoprotein have been determined. The close agreement of these measurements with values obtained by surface fluorescence microscopy and fluorescence correlation spectroscopy helps to validate the use of this device. Thus, this sensor shows promise as an inexpensive, portable, and accurate tool for bioanalytical applications in laboratory and clinical settings.

  View details for DOI 10.1021/ac025669y

  View details for Web of Science ID 000177862800039

  View details for PubMedID 12236371

• beta AR signaling required for diet-induced thermogenesis and obesity resistance SCIENCE Bachman, E. S., Dhillon, H., Zhang, C. Y., Cinti, S., Bianco, A. C., Kobilka, B. K., Lowell, B. B. 2002; 297 (5582): 843-845

  Abstract

  Excessive caloric intake is thought to be sensed by the brain, which then activates thermogenesis as a means of preventing obesity. The sympathetic nervous system, through beta-adrenergic receptor (betaAR) action on target tissues, is likely the efferent arm of this homeostatic mechanism. To test this hypothesis, we created mice that lack the three known betaARs (beta-less mice). beta-less mice on a Chow diet had a reduced metabolic rate and were slightly obese. On a high-fat diet, beta-less mice, in contrast to wild-type mice, developed massive obesity that was due entirely to a failure of diet-induced thermogenesis. These findings establish that betaARs are necessary for diet-induced thermogenesis and that this efferent pathway plays a critical role in the body's defense against diet-induced obesity.

  View details for Web of Science ID 000177192800053

  View details for PubMedID 12161655

• The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region - Studies using chimeric human and fly receptors JOURNAL OF BIOLOGICAL CHEMISTRY NISHI, S., Nakabayashi, K., Kobilka, B., Hsueh, A. J. 2002; 277 (6): 3958-3964

  Abstract

  Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.

  View details for DOI 10.1074/jbc.M109617200

  View details for Web of Science ID 000173813900023

  View details for PubMedID 11723133

• Isoflurane and nociception - Spinal alpha(2A) adrenoceptors mediate antinociception while supraspinal alpha(1) adrenoceptors mediate pronociception ANESTHESIOLOGY Kingery, W. S., Agashe, G. S., Guo, T. Z., Sawamura, S., Davies, M. F., Clark, J. D., Kobilka, B. K., Maze, M. 2002; 96 (2): 367-374

  Abstract

  The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal alpha2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which alpha adrenoceptors mediate this effect.After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the alpha1 and alpha2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three alpha2-adrenoceptor subtypes (alpha2A, alpha2B, and alpha2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane.Both DbetaH-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The alpha1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The alpha2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal alpha2 adrenoceptors. Null mice for the alpha2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane.The authors suggest that, at clinically effective concentrations, isoflurane can modulate nociception via three different mechanisms: (1) a pronociceptive effect requiring descending spinal pathways, brainstem noradrenergic nuclei, and supraspinal alpha1 adrenoceptors; (2) an antinociceptive effect requiring descending noradrenergic neurons and spinal alpha2A adrenoceptors; and (3) an antinociceptive effect mediated within the spinal cord for which no role for adrenergic mechanism has been found.

  View details for PubMedID 11818770

• Allosteric modulation of beta(2)-adrenergic receptor by Zn2+ MOLECULAR PHARMACOLOGY Swaminath, G., Steenhuis, J., Kobilka, B., LEE, T. W. 2002; 61 (1): 65-72

  Abstract

  Zn(2+) is abundant in the brain, where it plays a role in the function of a number of enzymes, structural proteins, and transcription factors. Zn(2+) is also found in synaptic vesicles and is released into synapses achieving concentrations in the range of 100 to 300 microM [Proc Natl Acad Sci USA 1997;94:13386-13387; Mol Pharmacol 1997;51:1015-1023]. Therefore, Zn(2+) may play a physiological role in regulating the function of postsynaptic channels and receptors. We characterized the effect of Zn(2+) on the functional properties of the beta2-adrenergic receptor (beta2AR). We found that physiological concentrations of Zn(2+) increased agonist affinity and enhanced cAMP accumulation stimulated by submaximal concentrations of the betaAR agonist isoproterenol. These results provide evidence that Zn(2+) released at nerve terminals may modulate signals generated by the beta2AR in vivo.

  View details for Web of Science ID 000173135000009

  View details for PubMedID 11752207

• Use of fluorescence spectroscopy to study conformational changes in the beta(2)-adrenoceptor G PROTEIN PATHWAYS, PT A, RECEPTORS Kobilka, B. K., Gether, U. 2002; 343: 170-182

  View details for Web of Science ID 000171866900011

  View details for PubMedID 11665566

• Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors MOLECULAR PHARMACOLOGY Chruscinski, A., Brede, M. E., Meinel, L., Lohse, M. J., Kobilka, B. K., Hein, L. 2001; 60 (5): 955-962

  Abstract

  beta-Adrenergic receptors (beta-AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, beta-AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking beta(1)- and/or beta(2)-adrenergic receptor subtypes (beta(1)-KO, beta(2)-KO, beta(1)beta(2)-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of beta-receptor stimulation was absent in femoral and pulmonary arteries from beta(1)-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking beta(1)- or beta(2)-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both beta(1)- and beta(2)-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of beta(1)- and beta(2)-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtype-selective beta-receptor antagonists. These studies demonstrate that beta(1)-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, beta(2)-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of beta-adrenergic receptor subtypes may play an important role in redirection of tissue perfusion.

  View details for Web of Science ID 000171985300010

  View details for PubMedID 11641423

• beta-adrenergic receptor subtype-specific signaling in cardiac myocytes from beta(1) and beta(2) adrenoceptor knockout mice MOLECULAR PHARMACOLOGY Devic, E., Xiang, Y., Gould, D., Kobilka, B. 2001; 60 (3): 577-583

  Abstract

  The sympathetic nervous system modulates cardiac contractility and rate by activating beta-adrenergic receptors (beta AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that beta-adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both beta(1) and beta(2) adrenergic receptors are expressed in human and murine hearts. We have examined the effect of beta AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and beta receptor knockout (KO) mice (beta(1)AR-KO, beta(2)AR-KO and beta(1)beta(2)AR-KO mice). Stimulation of the beta(1)AR in beta(2)AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the beta(2)AR in beta(1)AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in beta(1)beta(2)AR-KO myocytes can be attributed to the beta(3)AR. These studies show that all three beta AR subtypes are expressed in neonatal cardiac myocytes, and the beta(1)AR and beta(2)AR couple to distinct signaling pathways.

  View details for Web of Science ID 000170593800022

  View details for PubMedID 11502890

• A genetically engineered cell-based biosensor for functional classification of agents BIOSENSORS & BIOELECTRONICS Aravanis, A. M., DeBusschere, B. D., Chruscinski, A. J., Gilchrist, K. H., Kobilka, B. K., Kovacs, G. T. 2001; 16 (7-8): 571-577

  Abstract

  Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).

  View details for Web of Science ID 000171257900017

  View details for PubMedID 11544051

• Single-molecule spectroscopy of the beta(2) adrenergic receptor: Observation of conformational substates in a membrane protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peleg, G., Ghanouni, P., Kobilka, B. K., Zare, R. N. 2001; 98 (15): 8469-8474

  Abstract

  Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

  View details for Web of Science ID 000169967000049

  View details for PubMedID 11438704

  View details for PubMedCentralID PMC37459

• Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta(2) adrenergic receptor JOURNAL OF BIOLOGICAL CHEMISTRY Ghanouni, P., Gryczynski, Z., Steenhuis, J. J., LEE, T. W., Farrens, D. L., Lakowicz, J. R., Kobilka, B. K. 2001; 276 (27): 24433-24436

  Abstract

  G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

  View details for Web of Science ID 000169800700001

  View details for PubMedID 11320077

• Functional differences between full and partial agonists: Evidence for ligand-specific receptor conformations JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Seifert, R., Wenzel-Seifert, K., Gether, U., Kobilka, B. K. 2001; 297 (3): 1218-1226

  Abstract

  The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.

  View details for Web of Science ID 000169005200048

  View details for PubMedID 11356949

• Agonist-induced conformational changes in the G-protein-coupling domain of the beta(2) adrenergic receptor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghanouni, P., Steenhuis, J. J., Farrens, D. L., Kobilka, B. K. 2001; 98 (11): 5997-6002

  Abstract

  The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the beta(2) adrenergic receptor (beta(2)AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-beta(2)AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the beta(2)AR, which may reflect the different energetics of activation by diffusible ligands.

  View details for PubMedID 11353823

• Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of alpha(2B) adrenoceptors JOURNAL OF NEUROSCIENCE Sawamura, S., Kingery, W. S., Davies, M. F., Agashe, G. S., Clark, J. D., Kobilka, B. K., Hashimoto, T., Maze, M. 2000; 20 (24): 9242-9251

  Abstract

  Although nitrous oxide (N(2)O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N(2)O-induced release of norepinephrine mediates the analgesic action at alpha(2) adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which alpha(2) adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the analgesic and sedative actions of N(2)O were determined. Null mice for each of the three alpha(2) adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N(2)O. Exposure to N(2)O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DbetaH-saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N(2)O. Null mice for the alpha(2B) adrenoceptor subtype exhibited a reduced or absent analgesic response to N(2)O, but their sedative response to N(2)O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and alpha(2B) adrenoceptors in the analgesic, but not the sedative effects of N(2)O. Previously we demonstrated that the analgesic actions of alpha(2) adrenoceptor agonists are mediated by the alpha(2A) subtype; taken together with these data we propose that exogenous and endogenous alpha(2) adrenoceptor ligands activate different alpha(2) adrenoceptor subtypes to produce their analgesic action.

  View details for Web of Science ID 000165976500035

  View details for PubMedID 11125002

• Cell-type specific targeting of the alpha(2c)-andrenoceptor - Evidence for the organization of receptor microdomains during neuronal differentiation of PC12 cells JOURNAL OF BIOLOGICAL CHEMISTRY Hurt, C. M., Feng, F. Y., Kobilka, B. 2000; 275 (45): 35424-35431

  Abstract

  We have previously shown differences in the intracellular targeting of alpha2a (alpha(2A))- and alpha2c (alpha(2C))-adrenoreceptors expressed in the same cell line (von Zastrow, M., Link, R., Daunt, D. , Barsh, G., and Kobilka, B. (1993) J. Biol. Chem. 268, 763-766; Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720). alpha(2A)-Adrenoreceptors reside primarily in the plasma membrane in HEK 293 cells, while co-expressed alpha(2C)-adrenoreceptors are found mainly in an intracellular compartment. Since alpha(2c)-adrenoreceptors are expressed primarily in the brain, we compared the intracellular targeting of alpha(2C)-adrenoreceptors in two neuroendocrine cell lines with the targeting in three epithelial cell lines and one fibroblast cell line. In transiently transfected COS7 cells, and in stably transfected normal rat kidney cells, Madin-Darby canine kidney cells, and Rat1 fibroblasts, a significant proportion of alpha(2C)-adrenoreceptor detected by immunocytochemistry co-localized with markers for both the endoplasmic reticulum and the cis/medial Golgi compartments. In contrast, both PC12 cells and AtT20 cells efficiently targeted alpha(2C)-adrenoreceptors to the plasma membrane. Ligand binding and Western blot analyses indicate that intracellular receptor in normal rat kidney cells is functional and undergoes normal post-translational processing. In PC12 cells the expressed alpha(2C)-adrenoreceptors become concentrated in neurite outgrowths in discrete regions of the plasma membrane having a high density of F-actin following treatment with nerve growth factor. These findings provide evidence for cell-type specific factors that facilitate the targeting of the G protein-coupled receptors to the plasma membrane.

  View details for Web of Science ID 000165422800079

  View details for PubMedID 10906149

• Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region JOURNAL OF BIOLOGICAL CHEMISTRY Nakabayashi, K., Kudo, M., Kobilka, B., Hsueh, A. W. 2000; 275 (39): 30264-30271

  Abstract

  Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.

  View details for Web of Science ID 000089577900049

  View details for PubMedID 10889210

• Allosteric activation of the CaR by L-amino acids PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kobilka, B. 2000; 97 (9): 4419-4420

  View details for Web of Science ID 000086703000004

  View details for PubMedID 10781033

• The effect of pH on beta(2) adrenoceptor function - Evidence for protonation-dependent activation JOURNAL OF BIOLOGICAL CHEMISTRY Ghanouni, P., Schambye, H., Seifert, R., LEE, T. W., Rasmussen, S. G., Gether, U., Kobilka, B. K. 2000; 275 (5): 3121-3127

  Abstract

  The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the beta(2) adrenergic receptor (beta(2)AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified beta(2)AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Galpha(s) by the beta(2)AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of beta(2)AR activation is not abrogated by mutation of Asp(130), which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu(134)) and the alpha(1B) adrenergic receptor (Asp(142)).

  View details for Web of Science ID 000085146500017

  View details for PubMedID 10652295

• The Effect of pH on b2 Adrenoceptor Function. Evidence for protonation-dependent activation J Biol Chem Kobilka, B.K., Ghanouni, P., Schambye, H., Seifert, R., Lee, T.W., Rasmussen, S.G., Gether, U. 2000
• Two functionally distinct alpha(2)-adrenergic receptors regulate sympathetic neurotransmission NATURE Hein, L., Altman, J. D., Kobilka, B. K. 1999; 402 (6758): 181-184

  Abstract

  The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.

  View details for Web of Science ID 000083716400050

  View details for PubMedID 10647009

• ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors NEURON Winder, D. G., Martin, K. C., Muzzio, I. A., Rohrer, D., Chruscinski, A., Kobilka, B., Kandel, E. R. 1999; 24 (3): 715-726

  Abstract

  MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

  View details for Web of Science ID 000083913000025

  View details for PubMedID 10595521

• Restricting the mobility of G(s)alpha: Impact on receptor and effector coupling BIOCHEMISTRY LEE, T. W., Seifert, R., Guan, X. M., Kobilka, B. K. 1999; 38 (42): 13801-13809

  Abstract

  The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.

  View details for Web of Science ID 000083288400005

  View details for PubMedID 10529225

• GPCR-G alpha fusion proteins: molecular analysis of receptor-G-protein coupling TRENDS IN PHARMACOLOGICAL SCIENCES Seifert, R., Wenzel-Seifert, K., Kobilka, B. K. 1999; 20 (9): 383-389

  Abstract

  The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR-Galpha fusion proteins, which are formed by the fusion of GPCR to Galpha. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Galpha and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Galpha enhances coupling efficiency can be used to study aspects of receptor-G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR-Galpha fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

  View details for Web of Science ID 000082561500009

  View details for PubMedID 10462762

• Effects of guanine, inosine, and xanthine nucleotides on beta(2)-adrenergic receptor/G(s) interactions: Evidence for multiple receptor conformations MOLECULAR PHARMACOLOGY Seifert, R., Gether, U., Wenzel-Seifert, K., Kobilka, B. K. 1999; 56 (2): 348-358

  Abstract

  The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.

  View details for Web of Science ID 000081646500013

  View details for PubMedID 10419554

• Abnormal regulation of the sympathetic nervous system in alpha(2A)-adrenergic receptor knockout mice MOLECULAR PHARMACOLOGY Altman, J. D., Trendelenburg, A. U., MacMillan, L., Bernstein, D., Limbird, L., Starke, K., Kobilka, B. K., Hein, L. 1999; 56 (1): 154-161

  Abstract

  alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.

  View details for Web of Science ID 000081240600019

  View details for PubMedID 10385696

• Cardiovascular and metabolic alterations in mice lacking both beta 1-and beta 2-adrenergic receptors JOURNAL OF BIOLOGICAL CHEMISTRY Rohrer, D. K., Chruscinski, A., Schauble, E. H., Bernstein, D., Kobilka, B. K. 1999; 274 (24): 16701-16708

  Abstract

  The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.

  View details for Web of Science ID 000080780400008

  View details for PubMedID 10358009

• Targeted disruption of the beta 2 adrenergic receptor gene JOURNAL OF BIOLOGICAL CHEMISTRY Chruscinski, A. J., Rohrer, D. K., Schauble, E., Desai, K. H., Bernstein, D., Kobilka, B. K. 1999; 274 (24): 16694-16700

  Abstract

  beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.

  View details for Web of Science ID 000080780400007

  View details for PubMedID 10358008

• Examining the efficiency of receptor/G-protein coupling with a cleavable beta(2)-adrenoceptor-G(s alpha) fusion protein EUROPEAN JOURNAL OF BIOCHEMISTRY Seifert, R., Wenzel-Seifert, K., Gether, U., Lam, V. T., Kobilka, B. K. 1999; 260 (3): 661-666

  Abstract

  Reconstitution of high-affinity agonist binding at the beta2-adrenoceptor (beta2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G-protein of adenylyl cyclase, Gsalpha, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem. 255, 369-382]. In a fusion protein of the beta2AR and Gsalpha (beta2AR-Gsalpha), which has only a 1 : 1 stoichiometry of receptor and G-protein, high-affinity agonist binding and agonist-stimulated GTP hydrolysis, guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G-protein interaction, we constructed a fusion protein with a thrombin-cleavage site between beta2AR and Gsalpha (beta2AR-TS-Gsalpha). beta2AR-TS-Gsalpha efficiently reconstituted high-affinity agonist binding, agonist-stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves approximately 70% of beta2AR-TS-Gsalpha molecules in Sf9 membranes. Thrombin cleavage did not impair high-affinity agonist binding and GTP[S] binding but strongly reduced ligand-regulated GTPase activity and AC activity. We conclude that fusion of the beta2AR to Gsalpha promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G-protein. Dilution of Gsalpha in the membrane and release of activated Gsalpha into the cytosol can both prevent cleaved beta2AR-TS-Gsalpha from undergoing multiple activation/deactivation cycles.

  View details for Web of Science ID 000079371200009

  View details for PubMedID 10102993

• Cardiovascular and metabolic alterations in mice lacking both b1- and b2-adrenergic receptors J Biol Chem Kobilka, B.K., Rohrer, D.K., Chruscinski, A., Schauble, E.H., Bernstein, D. 1999; 274 (24): 16701
• Restricting mobility of G(s)alpha relative to the beta(2)-adrenoceptor enhances adenylate cyclase activity by reducing G(s)alpha GTPase activity BIOCHEMICAL JOURNAL Wenzel-Seifert, K., LEE, T. W., Seifert, R., Kobilka, B. K. 1998; 334: 519-524

  Abstract

  The beta2-adrenoceptor (beta2AR) activates the G-protein Gsalpha to stimulate adenylate cyclase (AC). Fusion of the beta2AR C-terminus to the N-terminus of Gsalpha (producing beta2ARGsalpha) markedly increases the efficiency of receptor/G-protein coupling compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein we constructed fusion proteins from which 26 [beta2AR(Delta26)Gsalpha] or 70 [beta2AR(Delta70)Gsalpha] residues of the beta2AR C-terminus had been deleted and compared the properties of these fusion proteins with the previously described beta2ARGsalpha. Compared with beta2ARGsalpha, basal and agonist-stimulated GTP hydrolysis was markedly decreased in beta2AR(Delta70)Gsalpha, whereas the effect of the deletion on binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions did not alter the efficiency of coupling of the beta2AR to Gsalpha as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing beta2AR(Delta70)Gsalpha and beta2AR(Delta26)Gsalpha were higher than in membranes expressing beta2ARGsalpha. These findings suggest that restricting the mobility of Gsalpha relative to the beta2AR results in a decrease in G-protein inactivation by GTP hydrolysis and thereby enhanced activation of AC.

  View details for Web of Science ID 000076087600004

  View details for PubMedID 9729456

• G protein-coupled receptors - II. Mechanism of agonist activation JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Kobilka, B. K. 1998; 273 (29): 17979-17982

  View details for Web of Science ID 000074828500001

  View details for PubMedID 9660746

• Reconstitution of beta(2)-adrenoceptor-GTP-binding-protein interaction in Sf9 cells - High coupling efficiency in a beta(2)-adrenoceptor-G(s alpha) fusion protein EUROPEAN JOURNAL OF BIOCHEMISTRY Seifert, R., LEE, T. W., Lam, V. T., Kobilka, B. K. 1998; 255 (2): 369-382

  Abstract

  In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.

  View details for Web of Science ID 000074954500006

  View details for PubMedID 9716378

• Neuropeptide Y receptor 1 (NPY-Y1) expression in human heart failure and heart transplantation JOURNAL OF THE AUTONOMIC NERVOUS SYSTEM Gullestad, L., Aass, H., Ross, H., Ueland, T., Geiran, O., KJEKSHUS, J., Simonsen, S., Fowler, M., Kobilka, B. 1998; 70 (1-2): 84-91

  Abstract

  Neuropeptide Y (NPY) is a neurotransmitter released from cardiac sympathetic nerve terminals along with catecholamines. It influences vascular tone and cardiac function, probably through the receptor subtype Y1. The present study examined the expression of Y1 in patients with end-stage heart failure and in heart transplant recipients. Y1 mRNA was analyzed in right ventricular endomyocardial biopsies taken from 12 donor hearts prior to implantation (controls), 15 patients with end stage heart failure at time of transplantation, and 16 patients more than 1 year after transplantation. RT-PCR (reverse transcription polymerase chain reaction) was used to detect mRNA for the Y1 receptor, the beta1-adrenergic-receptor, and beta-actin. Y1 mRNA was present in biopsies of all donor hearts, but was observed significantly less frequently in the two patient groups; only 5 out of 15 (P < 0.01) heart failure and 9 out of 16 (P < 0.05) transplant recipients demonstrated visible PCR product. In contrast, mRNA for the beta1-adrenergic receptor and beta-actin were detected by RT-PCR in all samples. Our results provide the first evidence for altered regulation of the neuropeptide Y1 receptor in heart failure and transplant patients, and suggests that loss of signal transduction by this receptor may be adaptive in both groups.

  View details for Web of Science ID 000074669200012

  View details for PubMedID 9686908

• Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor. journal of biological chemistry Seifert, R., Wenzel-Seifert, K., LEE, T. W., Gether, U., Sanders-Bush, E., Kobilka, B. K. 1998; 273 (18): 5109-5116

  Abstract

  The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

  View details for PubMedID 9556548

• Alterations in dynamic heart rate control in the beta(1)-adrenergic receptor knockout mouse AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Rohrer, D. K., Schauble, E. H., Desai, K. H., Kobilka, B. K., Bernstein, D. 1998; 274 (4): H1184-H1193

  Abstract

  beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

  View details for Web of Science ID 000072839800015

  View details for PubMedID 9575921

• Alterations in dynamic heart rate control in the beta1-adrenergic receptor knockout mouse. American journal of physiology. Heart and circulatory physiology Rohrer, D. K., Schauble, E. H., Desai, K. H., Kobilka, B. K., Bernstein, D. 1998; 274 (4): H1184–H1193

  Abstract

  beta1-Adrenergic receptors (beta1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta1-AR gene knockout model to test the hypothesis that beta1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta1-AR knockouts is markedly limited, with peak heart rates 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

  View details for DOI 10.1152/ajpheart.1998.274.4.H1184

  View details for PubMedID 29585980

• Different effects of G(s)alpha splice variants on beta(2)-adrenoreceptor-mediated signaling - The beta(2)-adrenoreceptor coupled to the long splice variant of G(s)alpha has properties of a constitutively active receptor JOURNAL OF BIOLOGICAL CHEMISTRY Seifert, R., Wenzel-Seifert, K., LEE, T. W., Gether, U., Sanders-Bush, E., Kobilka, B. K. 1998; 273 (9): 5109-5116

  Abstract

  The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gsalpha (GsalphaL) binds GDP with lower affinity than the short splice variant (GsalphaS), but the impact of this difference on the interaction of Gsalpha with the beta2AR is not known. We studied the beta2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta2ARGsalphaS and beta2ARGsalphaL) expressed in Sf9 cells. Fusion of the beta2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2AR or Gsalpha. The beta2AR in beta2ARGsalphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2AR in beta2ARGsalphaS. The apparent constitutive activity of the beta2AR in beta2ARGsalphaL may be due to the lower GDP affinity of GsalphaL compared with GsalphaS, i.e. GsalphaL is more often nucleotide-free than GsalphaS and, therefore, more frequently available to stabilize the beta2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

  View details for Web of Science ID 000072310400047

  View details for PubMedID 9478963

• The developmental and physiological consequences of disrupting genes encoding beta 1 and beta 2 adrenoceptors. Advances in pharmacology (San Diego, Calif.) Rohrer, D. K., Bernstein, D., Chruscinski, A., Desai, K. H., Schauble, E., Kobilka, B. K. 1998; 42: 499-501

  View details for PubMedID 9327949

• G protein-coupled receptors: Functional and mechanistic insights through altered gene expression PHYSIOLOGICAL REVIEWS Rohrer, D. K., Kobilka, B. K. 1998; 78 (1): 35-52

  Abstract

  G protein-coupled receptors (GPCRs) comprise a large and diverse family of molecules that play essential roles in signal transduction. In addition to a constantly expanding pharmacological repertoire, recent advances in the ability to manipulate GPCR expression in vivo have provided another valuable approach in the study of GPCR function and mechanism of action. Current technologies now allow investigators to manipulate GPCR expression in a variety of ways. Graded reductions in GPCR expression can be achieved through antisense strategies or total gene ablation or replacement can be achieved through gene targeting strategies, and exogenous expression of wild-type or mutant GPCR isoforms can be accomplished with transgenic technologies. Both the techniques used to achieve these specific alterations and the consequences of altered expression patterns are reviewed here and discussed in the context of GPCR function and mechanism of action.

  View details for Web of Science ID 000071629200002

  View details for PubMedID 9457168

• Insights from in vivo modification of adrenergic receptor gene expression ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY Rohrer, D. K., Kobilka, B. K. 1998; 38: 351-373

  Abstract

  Adrenergic receptors are key targets within the autonomic nervous system, regulating a wide variety of physiological processes. The ability to modify adrenergic receptor expression patterns in vivo has added a powerful new tool to the functional analysis of these receptors. Modification of adrenergic receptor gene expression by overexpression, genetic ablation, or site-specific mutation has added new insight to models of receptor coupling behavior, pharmacology, and subtype-specific physiological function. This review highlights some of the recent advances resulting from such genetic approaches to the study of adrenergic receptors.

  View details for Web of Science ID 000073483100014

  View details for PubMedID 9597159

• Site-specific fluorescence labeling of the beta(2) adrenergic receptor amino terminus ANALYTICAL BIOCHEMISTRY PAROLA, A. L., Lin, S. S., Kobilka, B. K. 1997; 254 (1): 88-95

  Abstract

  A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.

  View details for Web of Science ID A1997YK71500013

  View details for PubMedID 9398350

• Agonists induce conformational changes in transmembrane domains III and VI of the beta(2) adrenoceptor EMBO JOURNAL Gether, U., Lin, S., Ghanouni, P., Ballesteros, J. A., Weinstein, H., Kobilka, B. K. 1997; 16 (22): 6737-6747

  Abstract

  Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

  View details for Web of Science ID A1997YJ20700013

  View details for PubMedID 9362488

  View details for PubMedCentralID PMC1170278

• Echocardiographic evaluation of the roles of beta 1, beta 2 and beta 3 adrenergic receptors in regulating cardiovascular function in knockout mice Desai, K. H., Rohrer, D., Eric, S., Chruscinski, A., Kobilka, B. K., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 1997: 3554–54

  View details for Web of Science ID A1997YC88003536

• Evaluation of the roles of beta 1, beta 2 and beta 3 adrenergic receptors in regulating cardiac and peripheral vascular function in knockout mice. Rohrer, D., Schauble, E., Chruscinski, A., Desai, K. H., Kobilka, B. K., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 1997: 285–85

  View details for Web of Science ID A1997YC88000284

• Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation JOURNAL OF BIOLOGICAL CHEMISTRY Osuga, Y., Hayashi, M., Kudo, M., Conti, M., Kobilka, B., Hsueh, A. J. 1997; 272 (40): 25006-25012

  Abstract

  Gonadotropin receptors are unique members of the seven-transmembrane (TM), G protein-coupled receptor family with a large extracellular (EC) sequence forming the high-affinity ligand binding domain. In a patient with Leydig cell hypoplasia, we identified a mutant LH receptor that is truncated at TM5. This protein retains limited ligand binding ability but cannot mediate cAMP responses. To study interactions between receptor fragments defective in either ligand binding or signal transduction, we co-expressed this truncated receptor together with a chimeric receptor containing the EC region of the FSH receptor and the TM region of the LH receptor. Although the chimeric receptor could not respond to human chorionic gonadotropin in producing cAMP, co-expression with the truncated LH receptor allowed partial restoration of ligand signaling through intermolecular interactions. In addition, co-expression of the same truncated LH receptor with an N-terminally truncated LH receptor that lacked the EC ligand binding domain also partially restored ligand signaling. Further shortening of the TM region in the mutant receptor found in the patient indicated that the EC domain and TM1 were sufficient for interactions with the N terminally truncated receptor. In contrast, co-expression of the N terminally truncated receptor together with cell-associated or soluble EC region of the LH receptor did not allow ligand signaling. Unlike thrombin receptors, co-expression of the anchored EC region of the LH receptor together with the N-terminally truncated receptor did not allow ligand signaling despite moderate levels of human chorionic gonadotropin binding in transfected cells. These studies demonstrate that the co-expression of binding (+)/signaling (-) and binding (-)/signaling (+) receptor fragments partially restores ligand-induced signal generation and indicate the importance of TM1 of the LH receptor in the proper orientation of the EC ligand binding domain.

  View details for Web of Science ID A1997XY97000046

  View details for PubMedID 9312107

• Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors MOLECULAR ENDOCRINOLOGY Osuga, Y., Kudo, M., Kaipia, A., Kobilka, B., Hsueh, A. J. 1997; 11 (11): 1659-1668

  Abstract

  Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.

  View details for Web of Science ID A1997XY29500009

  View details for PubMedID 9328348

• Intracellular trafficking of angiotensin II and its AT(1) and AT(2) receptors: Evidence for selective sorting of receptor and ligand MOLECULAR ENDOCRINOLOGY Hein, L., Meinel, L., Pratt, R. E., Dzau, V. J., Kobilka, B. K. 1997; 11 (9): 1266-1277

  Abstract

  Angiotensin II (Ang II) binds to two different receptor subtypes, AT1 and AT2 receptors. In many cases, receptor stimulation by Ang II is followed by a rapid desensitization of the intracellular signal transduction and a decrease in cell surface receptor number. The present study was designed to examine by immunofluorescence microscopy the cellular trafficking pathways of Ang II and its AT1a and AT2 receptors in human embryonal kidney 293 cells stably expressing these receptor subtypes. Fluorescently labeled Ang II and AT1a receptors were rapidly internalized into endosomes. AT2 receptors were localized in the plasma membrane and did not undergo endocytosis upon agonist stimulation. After removal of agonist, AT1a receptors recycled to the plasma membrane, whereas fluorescently labeled Ang II was targeted to the lysosomal pathway. Even though no further loss of surface receptor was measurable by ligand binding at steady state, fluorescein-Ang II was continuously internalized, and cycling of receptor between endosomal vesicles and the plasma membrane was detected by antibody feeding. These experiments provide evidence for subtype-specific receptor sorting and internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the sequestration of receptors into endosomes is in dynamic equilibrium with receptor cycling to the plasma membrane. Finally, internalization of AT1a receptors and Ang II persists after desensitization mechanisms have attenuated Ca2+ and inositol 1,4,5-trisphosphate signaling.

  View details for Web of Science ID A1997XN62300008

  View details for PubMedID 9259318

• A novel interaction between adrenergic receptors and the alpha-subunit of eukaryotic initiation factor 2B JOURNAL OF BIOLOGICAL CHEMISTRY Klein, U., Ramirez, M. T., Kobilka, B. K., VONZASTROW, M. 1997; 272 (31): 19099-19102

  Abstract

  The alpha-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the alpha2A- and alpha2B-adrenergic receptors in a yeast two-hybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the alpha2A-, alpha2B-, alpha2C-, and beta2-adrenergic receptors, but not the vasopressin (V2) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2Balpha with full-length beta2-adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2Balpha co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2Balpha in 293 cells caused a small (approximately 15%) but significant enhancement of beta2-adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.

  View details for Web of Science ID A1997XP06300002

  View details for PubMedID 9235896

• Adrenergic receptors - From molecular structure to in vivo function TRENDS IN CARDIOVASCULAR MEDICINE Hein, L., Kobilka, B. K. 1997; 7 (5): 137-145

  Abstract

  Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145). © 1997, Elsevier Science Inc.

  View details for Web of Science ID A1997XM99100001

  View details for PubMedID 21235877

• Overexpression of angiotensin AT(1) receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hein, L., Stevens, M. E., Barsh, G. S., Pratt, R. E., Kobilka, B. K., Dzau, V. J. 1997; 94 (12): 6391-6396

  Abstract

  Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the alpha-myosin heavy chain (alphaMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription-PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction.

  View details for Web of Science ID A1997XD84400077

  View details for PubMedID 9177228

  View details for PubMedCentralID PMC21060

• Subtype-specific intracellular trafficking of alpha(2)-adrenergic receptors MOLECULAR PHARMACOLOGY Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., Kobilka, B. K. 1997; 51 (5): 711-720

  Abstract

  The three alpha2-adrenergic receptor subtypes (alpha2a, alpha2b, and alpha2c) are highly homologous G protein-coupled receptors. These receptors all couple to pertussis toxin-sensitive G proteins and have relatively similar pharmacological properties. To further explore functional differences between these receptors, we used immunocytochemical techniques to compare the ability of the three alpha2-receptor subtypes to undergo agonist-mediated internalization. The alpha2a-receptor does not internalize after agonist treatment. In contrast, we observed that the alpha2b-receptor is able to undergo agonist-induced internalization and seems to follow the same endosomal pathway used by the beta2-adrenergic receptor. Attempts to examine internalization of the alpha2c-receptor were complicated by the fact that the majority of the alpha2c receptor resides in the endoplasmic reticulum and cis/media Golgi and there is relatively little cell surface localization. Nevertheless, we were able to detect some internalization of the alpha2c-receptor after prolonged agonist treatment. However, we observed no significant movement of alpha2c-receptor from the intracellular pool to the plasma membrane during a 4-hr treatment of cells with cycloheximide, suggesting that these cells are unable to process alpha2c-receptors in the same way they process the alpha2a or alpha2b subtypes.

  View details for Web of Science ID A1997WY28000003

  View details for PubMedID 9145909

• Cardiovascular indexes in the mouse at rest and with exercise: New tools to study models of cardiac disease AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Desai, K. H., Sato, R., Schauble, E., Barsh, G. S., Kobilka, B. K., Bernstein, D. 1997; 272 (2): H1053-H1061

  Abstract

  Manipulations of the murine genome that alter cardiovascular function have created the need for methods to study cardiovascular physiology in genetically altered animals in vivo. We adapted chronic physiological measurement techniques to the nonanesthetized, nonrestrained murine model, established strain-specific cardiovascular and metabolic norms, and evaluated responses to anesthesia, exercise, and adrenergic stimulation. Anesthesia resulted in alterations in heart rate (HR), blood pressure (BP), and O2 consumption (V(O2)) and CO2 production (V(CO2)) for up to 6 h postoperatively. There were significant interstrain differences in resting values of HR and BP Graded treadmill exercise resulted in linear increases in HR, V(O2), V(CO2), and respiratory exchange ratio (RER) similar to those seen in larger species. Response to beta-adrenergic stimulation showed a classic sigmoidal dose-response curve; however, there was very little tachycardiac response to vagal blockade, indicating low resting vagal tone. This study demonstrates the feasibility of performing chronic cardiovascular measurements in nonanesthetized mice and stresses the importance of allowing for anesthetic recovery and strain variability. Murine cardiovascular responses to exercise can be reliably measured and are qualitatively similar to those in humans.

  View details for Web of Science ID A1997WJ80900057

  View details for PubMedID 9124413

• Structural instability of a constitutively active G protein-coupled receptor - Agonist-independent activation due to conformational flexibility JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Ballesteros, J. A., Seifert, R., SANDERSBUSH, E., Weinstein, H., Kobilka, B. K. 1997; 272 (5): 2587-2590

  Abstract

  Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.

  View details for Web of Science ID A1997WE66700002

  View details for PubMedID 9006889

• Ligand stabilization of the beta(2) adrenergic receptor: Effect of DTT on receptor conformation monitored by circular dichroism and fluorescence spectroscopy BIOCHEMISTRY Lin, S. S., Gether, U., Kobilka, B. K. 1996; 35 (46): 14445-14451

  Abstract

  Treatment of the beta 2 adrenergic receptor with the reducing agent dithiothreitol (DTT) is known to abolish ligand binding to the receptor. Interestingly, the loss of binding can be prevented by preoccupation of the receptor with ligand. It is unclear, however, whether the ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the beta 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified beta 2 receptor in the detergent micelle indicated that the receptor has an alpha-helix content of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The alpha-helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the beta 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the ability of the receptor to bind antagonist. Both changes in receptor binding and fluorescence emission were reversible, as removal of DTT allowed the receptor to restore 70% of ligand binding and return to the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor.

  View details for Web of Science ID A1996VU22400001

  View details for PubMedID 8931540

• The role of beta-1 adrenergic receptors in basal end exercise stimulated heart function: Studies on mice lacking the beta-1 adrenergic receptor gene Rohrer, D. K., Desai, K. H., Schauble, E., Kobilka, B. K., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 1996: 1674–74

  View details for Web of Science ID A1996VN11901670

• Diminished contractile response to isoproterenol in beta-1 adrenergic receptor deficient mice Desai, K. H., Rohrer, D. K., Kobilka, B. K., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 1996: 1675–75

  View details for Web of Science ID A1996VN11901671

• Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop JOURNAL OF BIOLOGICAL CHEMISTRY Kudo, M., Osuga, Y., Kobilka, B. K., Hsueh, A. J. 1996; 271 (37): 22470-22478

  Abstract

  Gonadotropin receptors are members of the seven-transmembrane (TM) receptor family. Several point mutations in TM V and VI and the intracellular loop 3 (i3) have been identified in the luteinizing hormone (LH) receptor gene, leading to constitutive activation of the receptor. Because gonadotropin receptors are highly conserved, we mutated the follicle-stimulating hormone (FSH) receptor at the corresponding amino acids. However, the FSH receptor mutants showed minimal increases in basal cAMP production. Taking advantage of this difference between the two receptors, we designed chimeric receptors with or without a point mutation in the i3 to identify the region in the LH receptor important for its constitutive activation. Introduction of the point mutation into chimeric receptors containing only TM V to VI from the LH receptor led to major increases in ligand-independent cAMP production. Furthermore, a chimeric receptor with only TM V and VI derived from the LH receptor can be rendered constitutively active by the mutation in the i3 from the FSH receptor. These results suggest that interactions between TM V and VI of the FSH receptor are essential for maintaining the receptor in the more constrained state, whereas interactions between these domains of the LH receptor are permissive for constitutively activating mutations in the i3.

  View details for Web of Science ID A1996VG67200031

  View details for PubMedID 8798412

• Cardiovascular regulation in mice lacking alpha(2)-adrenergic receptor subtypes b and c SCIENCE Link, R. E., Desai, K., Hein, L., Stevens, M. E., Chruscinski, A., Bernstein, D., Barsh, G. S., Kobilka, B. K. 1996; 273 (5276): 803-805

  Abstract

  alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.

  View details for Web of Science ID A1996VB42900045

  View details for PubMedID 8670422

• Targeted disruption of the mouse beta 1-adrenergic receptor gene: Developmental and cardiovascular effects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rohrer, D. K., Desai, K. H., Jasper, J. R., Stevens, M. E., Regula, D. P., Barsh, G. S., Bernstein, D., Kobilka, B. K. 1996; 93 (14): 7375-7380

  Abstract

  At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

  View details for Web of Science ID A1996UW79200098

  View details for PubMedID 8693001

• Arrangement of transmembrane domains in adrenergic receptors - Similarity to bacteriorhodopsin JOURNAL OF BIOLOGICAL CHEMISTRY Mizobe, T., Maze, M., Lam, V., Suryanarayana, S., Kobilka, B. K. 1996; 271 (5): 2387-2389

  Abstract

  G protein-coupled receptors (GPCRs) have seven hydrophobic domains, which are thought to span the lipid bilayer as alpha helical transmembrane domains (TMDs). The tertiary structure of GPCRs has not been determined; however, molecular models of GPCRs have generally been based on bacteriorhodopsin, which is functionally unrelated to GPCRs but has a similar secondary structure. We sought to examine the validity of using bacteriorhodopsin as a scaffold for GPCR model building by experimentally determining the orientation of the TMDs of adrenergic receptors in the plasma membrane. In separate experiments, three sequential amino acid residues (Leu-310, Leu-311, Asn-312) in TMD VII of the beta 2 adrenoreceptors were mutated to the amino acids found in the homologous domain of the alpha 2 adrenoceptor (Phe, Phe, Phe). Exchange of Asn-312 and Leu-311 in the beta 2 adrenoceptor resulted in nonfunctional proteins, most likely due to incompatibility of the introduced bulky phenylalanine side chain with adjacent structural domains in the beta 2 adrenoreceptor. This structural incompatibility was "repaired" by replacing the specific beta 2 TMD sequence with an alpha 2 receptor sequence. TMD I and TMD II complemented the Asn-312-->Phe mutation, and TMD III and TMD VI complemented the Leu-311-->Phe mutation. These results indicate that TMDs I, II, III, and VI surround TMD VII in a counter-clockwise orientation analogous to the orientation of TMDs in bacteriorhodopsin.

  View details for Web of Science ID A1996TT48800010

  View details for PubMedID 8576196

• Adrenergic receptor signal transduction Symposium on Structure and Function of 7TM Receptors Kobilka, B. K., Gether, U. MUNKSGAARD. 1996: 171–179

  View details for Web of Science ID A1996BF83Y00011

• FLUORESCENT LABELING OF PURIFIED BETA(2) ADRENERGIC-RECEPTOR - EVIDENCE FOR LIGAND-SPECIFIC CONFORMATIONAL-CHANGES JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Lin, S. S., Kobilka, B. K. 1995; 270 (47): 28268-28275

  Abstract

  The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.

  View details for Web of Science ID A1995TG21000049

  View details for PubMedID 7499324

• BEHAVIORAL AND CARDIOVASCULAR EFFECTS OF DISRUPTING THE ANGIOTENSIN-II TYPE-2 RECEPTOR GENE IN MICE NATURE Hein, L., Barsh, G. S., Pratt, R. E., Dzau, V. J., Kobilka, B. K. 1995; 377 (6551): 744-747

  Abstract

  Angiotensin II, a potent regulator of blood pressure and of water and electrolyte balance, binds to two different G-protein-coupled receptors. The type-1 receptor (AT1) mediates the vasopressive and aldosterone-secreting effects of angiotensin II, but the function of the type-2 receptor (AT2) is unknown, although it is expressed in both adult and embryonic life. To address this question, we have generated mice lacking the gene encoding the AT2 receptor. Mutant mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. Their baseline blood pressure is normal, but they show an increased vasopressor response to injection of angiotensin II. Thus, although the AT2 receptor is not required for embryonic development, it plays a role in the central nervous system and cardiovascular functions that are mediated by the renin-angiotensin system.

  View details for Web of Science ID A1995TB46900060

  View details for PubMedID 7477266

• AMINO AND CARBOXYL-TERMINAL MODIFICATIONS TO FACILITATE THE PRODUCTION AND PURIFICATION OF A G-PROTEIN-COUPLED RECEPTOR ANALYTICAL BIOCHEMISTRY Kobilka, B. K. 1995; 231 (1): 269-271

  View details for Web of Science ID A1995RZ60200039

  View details for PubMedID 8678314

• TARGETED INACTIVATION OF THE GENE ENCODING THE MOUSE ALPHA(2C)-ADRENOCEPTOR HOMOLOG MOLECULAR PHARMACOLOGY Link, R. E., Stevens, M. S., KULATUNGA, M., Scheinin, M., Barsh, G. S., Kobilka, B. K. 1995; 48 (1): 48-55

  Abstract

  alpha 2-Adrenergic receptors (alpha 2-ARs) regulate a wide range of physiological functions and are targets for clinically important antihypertensive and anesthetic agents. Three genes encoding alpha 2-AR subtypes have been cloned in humans and mice, but the physiological significance of each subtype has not been completely characterized. The available agonist and antagonist compounds are not sufficiently subtype selective to allow the unambiguous dissection of these receptors in vivo. As an alternative approach, we have used gene targeting in embryonic stem cells to disrupt the Adra2c gene, which encodes the alpha 2c-AR subtype in mice. Adra2c-/Adra2c- animals do not express a functional alpha 2c-AR transcript, as detected by Northern blotting or reverse transcription-polymerase chain reaction analysis. In addition, these mice have markedly reduced [3H]rauwolscine binding in their caudate putamen and in other brain regions normally expressing Adra2c binding sites. Adra2c-/Adra2c- mice, however, are viable and fertile and appear grossly normal. Expression levels of Adra2a and Adra2b mRNA in brain and kidney are not altered by the Adra2c knockout. These data suggest that up-regulation of Adra2a or Adra2b does not compensate for the Adra2c deficiency and that the receptor encoded by Adra2c is not required for normal mouse development or for survival in a laboratory environment.

  View details for Web of Science ID A1995RK03200007

  View details for PubMedID 7623774

• Adrenergic receptor signal transduction and regulation. Neuropharmacology Hein, L., Kobilka, B. K. 1995; 34 (4): 357-366

  View details for PubMedID 7566466

• NEUROTRANSMITTER RECEPTORS .4. ADRENERGIC-RECEPTOR SIGNAL-TRANSDUCTION AND REGULATION NEUROPHARMACOLOGY Hein, L., Kobilka, B. K. 1995; 34 (4): 357-366

  View details for Web of Science ID A1995RC35200001

• CARDIORESPIRATORY PERFORMANCE OF UNTRAINED MICE Sato, R., Desai, K., Kobilka, B., Barsh, G., Bernstein, D. NATURE PUBLISHING GROUP. 1995: A33–A33

  View details for Web of Science ID A1995QP08200185

• THE CARDIOVASCULAR ROLE OF ALPHA-2B ADRENERGIC-RECEPTORS DETERMINED BY TARGETED GENE DISRUPTION Desai, K., Link, R., Barsh, G., Kobilka, B., Bernstein, D. NATURE PUBLISHING GROUP. 1995: A25–A25

  View details for Web of Science ID A1995QP08200135

• GENETIC MODELS OF HUMAN VASCULAR-DISEASE CIRCULATION Dzau, V. J., Gibbons, G. H., Kobilka, B. K., Lawn, R. M., Pratt, R. E. 1995; 91 (2): 521-531

  Abstract

  The use of genetic models has greatly assisted investigations of the natural history, mechanisms, and potential therapy for human vascular disease. In the past, genetic models of vascular disease were obtained through serendipity and/or selective breeding to obtain inbred lines that express the phenotype of interest. This approach has yielded several valuable models of atherosclerosis and hypertension. In the past several years, the advent of molecular techniques has enabled investigators to produce additional novel genetic models of disease that have further enhanced the study of vascular biology and medicine. Transgenic techniques and the techniques of homologous recombination have allowed researchers to alter the genotype of an animal in a precise manner and to study the resultant change in phenotype. More recently, techniques of in vivo gene transfer have also accelerated and enhanced the development of novel models. The application of these methodologies has resulted in important breakthroughs in our understanding of the pathogenesis and treatment of vascular diseases. In this review, we compare and contrast these technologies along with examples of their use in the studies of vascular biology and medicine.

  View details for Web of Science ID A1995QB42600036

  View details for PubMedID 7805258

• INTRACELLULAR TARGETING AND TRAFFICKING OF THROMBIN RECEPTORS - A NOVEL MECHANISM FOR RESENSITIZATION OF A G-PROTEIN-COUPLED RECEPTOR JOURNAL OF BIOLOGICAL CHEMISTRY Hein, L., Ishii, K., Coughlin, S. R., Kobilka, B. K. 1994; 269 (44): 27719-27726

  Abstract

  The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.

  View details for Web of Science ID A1994PV77100087

  View details for PubMedID 7961693

• MURINE CARDIORESPIRATORY PHYSIOLOGY - IN-VIVO STUDY OF GENETICALLY ALTERED MODELS Desai, K., Sato, R., Kobilka, B., Barsh, G., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 1994: 148–48

  View details for Web of Science ID A1994PN41700829

• ANTAGONIST-DEPENDENT AND ANTAGONIST-INDEPENDENT STEPS IN THE MECHANISM OF ADRENERGIC-RECEPTOR INTERNALIZATION JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Kobilka, B. K. 1994; 269 (28): 18448-18452

  Abstract

  Epitope tagging and immunocytochemical techniques were used to examine the agonist-regulated internalization of human beta 2-adrenergic receptors in 293 cells. In the absence of agonist, receptors tagged with monoclonal antibody remain in the plasma membrane for > 1 h. In the presence of agonist, tagged receptors are endocytosed within 10 min. Endocytosed receptors are located in endosomes and can be recycled to the plasma membrane. In the prolonged presence of agonist, receptor endocytosis continues even after maximum sequestration of surface receptors (measured by radioligand binding to intact cells) has occurred. The process of receptor endocytosis requires cellular ATP and is temperature-dependent. At 4 degrees C, no agonist-induced redistribution of receptors located in the plasma membrane is observed. At 16 degrees C, agonist causes receptors to cluster in and around coated invaginations of the plasma membrane, but receptor endocytosis does not occur. Agonist treatment of cells at 16 degrees C, but not 4 degrees C, predisposes receptors to agonist-independent endocytosis upon warming to 37 degrees C. These studies suggest that: 1) beta 2-adrenergic receptors reside stably in the plasma membrane of untreated cells, while they continuously cycle between the plasma membrane and endosomes in the presence of agonist; 2) agonist regulates an early step in the endocytosis mechanism, which is associated with the redistribution of adrenergic receptors between distinct microdomains of the plasma membrane; and 3) later steps in the endocytosis mechanism do not require agonist and may utilize the same endocytic machinery that mediates the endocytosis of constitutively recycling receptors.

  View details for Web of Science ID A1994NW79800036

  View details for PubMedID 7518433

• PROBING THE PHYSIOLOGICAL SIGNIFICANCE OF ALPHA(2)-ADRENOCEPTOR SUBTYPE DIVERSITY IN GENETICALLY-ENGINEERED MICE Link, R. E., Stevens, M. E., Desai, K., Scheinin, M., Bernstein, D., Barsh, G. S., Kobilka, B. K. SLACK INC. 1994: A331–A331

  View details for Web of Science ID A1994NF02001247

• RESTING AND STRESSED CARDIORESPIRATORY PARAMETERS IN THE MOUSE - NEW TOOLS FOR THE ASSESSMENT OF TRANSGENIC MODELS Desai, K., Kobilka, B., Barsh, G., Bernstein, D. NATURE PUBLISHING GROUP. 1994: A33–A33

  View details for Web of Science ID A1994NG77900184

• THE PEPTIDE PRODUCT OF A 5' LEADER CISTRON IN THE BETA(2) ADRENERGIC-RECEPTOR MESSENGER-RNA INHIBITS RECEPTOR SYNTHESIS JOURNAL OF BIOLOGICAL CHEMISTRY PAROLA, A. L., Kobilka, B. K. 1994; 269 (6): 4497-4505

  Abstract

  The 5' leader region of mammalian beta 2 adrenergic receptor messenger RNAs (mRNA) have a short open reading frame (sORF) preceding the receptor cistron. Mutational inactivation of the sORF start codon increased beta 2 receptor expression and translation 1.9-fold from beta 2 receptor genes transfected into COS-7 cells. sORF inactivation also increased receptor synthesis 2.4-fold in a cell-free expression system that synthesizes functional beta 2 receptor in vitro. Translational initiation at the sORF was demonstrated both in vitro and in transfected COS-7 cells using an epitope-tagged fusion protein. Using the fusion protein as a reporter for initiation at the sORF shows that 5' leader mutations which increase translation of the sORF decrease receptor translation. Mutation analysis of the 5' leader region and peptide coding sequences suggests the peptide itself inhibits beta 2 receptor expression. Consistent with this hypothesis, a synthetic peptide corresponding to the peptide encoded by the beta 2 receptor sORF potently inhibits translation in vitro. Our results suggest that a nonoverlapping cistron in the beta 2 receptor mRNA 5' leader region is translated and the resulting peptide inhibits receptor translation.

  View details for Web of Science ID A1994MW98900091

  View details for PubMedID 8308019

• LINKAGE MAPPING OF ALPHA-2-ADRENERGIC RECEPTOR GENES TO MOUSE CHROMOSOME-2 AND CHROMOSOME-5 MAMMALIAN GENOME Link, R. E., Kobilka, B. K., Barsh, G. S. 1993; 4 (11): 650-655

  Abstract

  alpha-2 adrenergic receptors can be subdivided into three related subtypes which are conserved in humans, rats, and mice. In the mouse, these receptors are encoded by three genes (Adra-2a, Adra-2b, Adra-2c). To gain insight into the evolution of this multigene family and to investigate whether these genes are candidates for previously identified mouse mutations, we have determined the map positions of the Adra-2b and Adra-2c genes. The Adra-2a gene has been previously mapped to mouse Chromosome (Chr) 19 (Oakey et al. Genomics 10, 338-344, 1991). Using segregation among recombinant inbred strains of a single-stranded conformational polymorphism specific for alleles of Adra-2b and Adra-2c, we present map positions for these genes on mouse Chrs 2 and 5, respectively. In the case of Adra-2b, these results have been confirmed by an analysis of somatic cell hybrids. In addition, we generate AKXD recombinant inbred strain distribution patterns for 11 previously defined SSLP microsatellite markers, further refining the haplotype maps for these chromosomes. Finally, several candidate mouse mutations that map close to Adra-2b and Adra-2c are discussed.

  View details for Web of Science ID A1993MF57400005

  View details for PubMedID 8281014

• PRIMARY STRUCTURE OF THE MOUSE BETA(1)-ADRENERGIC RECEPTOR GENE BIOCHIMICA ET BIOPHYSICA ACTA Jasper, J. R., Link, R. E., Chruscinski, A. J., Kobilka, B. K., Bernstein, D. 1993; 1178 (3): 307-309

  Abstract

  The mouse beta 1-adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK-. Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse beta 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.

  View details for Web of Science ID A1993LX80300011

  View details for PubMedID 8395893

• AMINO-ACID SUBSTITUTIONS AT POSITION 312 IN THE 7TH HYDROPHOBIC SEGMENT OF THE BETA(2)-ADRENERGIC RECEPTOR MODIFY LIGAND-BINDING SPECIFICITY MOLECULAR PHARMACOLOGY Suryanarayana, S., Kobilka, B. K. 1993; 44 (1): 111-114

  Abstract

  We previously reported that Asn312 of the beta 2-adrenergic receptor and Asn385 in the homologous position in the 5-hydroxytryptamine1A receptor are important for binding to a class of beta-adrenergic receptor antagonists including propranolol and alprenolol. We proposed that the asparagine may be forming a hydrogen bond with the phenoxy oxygen common to these ligands. To further test this hypothesis we made alanine, threonine, phenylalanine, and glutamine substitutions at position 312 in the beta 2-adrenergic receptor. We observed that substitution with amino acids that permit formation of hydrogen bonds (threonine and glutamine) supported binding to aryloxyalkylamines, whereas substitution with amino acids that cannot form hydrogen bonds (alanine and phenylalanine) did not permit binding to these compounds. We were surprised to find that two of these substitutions led to an increase in affinity for alpha-adrenergic ligands. Substitution with glutamine and threonine at position 312 led to a 11-15-fold increase in affinity for yohimbine and enabled p-aminoclonidine to act as an agonist. These results further emphasize the role of position 312 in the formation of the ligand binding site for multiple ligands.

  View details for Web of Science ID A1993LN99200015

  View details for PubMedID 8101966

• SUBTYPE-SPECIFIC DIFFERENCES IN THE INTRACELLULAR SORTING OF G-PROTEIN-COUPLED RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Link, R., Daunt, D., Barsh, G., Kobilka, B. 1993; 268 (2): 763-766

  Abstract

  We have examined the subcellular distribution of three subtypes of adrenergic receptor by immunocytochemical localization of wild-type and epitope-tagged proteins expressed in Cos-7 and K293 cells. Two subtypes (beta 2 and M alpha 2-10H) are localized in the plasma membrane at steady state in untreated cells, while another subtype (M alpha 2-4H) is found both in the plasma membrane and in a population of intracellular vesicles. Within 15 min following the addition of adrenergic agonists, beta 2 and M alpha 2-10H receptors are differentially sorted; beta 2 receptors are selectively internalized to intracellular vesicles, which are distinct from those containing M alpha 2-4H receptors, while M alpha 2-10H receptors remain in the plasma membrane. Subtype-specific sorting suggests a new class of functional properties that may differentiate the signaling and regulation of homologous G protein-coupled receptors.

  View details for Web of Science ID A1993KG07700003

  View details for PubMedID 7678260

• ENHANCEMENT OF MEMBRANE INSERTION AND FUNCTION IN A TYPE IIIB MEMBRANE-PROTEIN FOLLOWING INTRODUCTION OF A CLEAVABLE SIGNAL PEPTIDE JOURNAL OF BIOLOGICAL CHEMISTRY Guan, X. M., Kobilka, T. S., Kobilka, B. K. 1992; 267 (31): 21995-21998

  Abstract

  The human beta 2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the beta 2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of beta 2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NH2-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.

  View details for Web of Science ID A1992JW71900003

  View details for PubMedID 1331042

• IDENTIFICATION OF INTRAMOLECULAR INTERACTIONS IN ADRENERGIC-RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY Suryanarayana, S., VONZASTROW, M., Kobilka, B. K. 1992; 267 (31): 21991-21994

  Abstract

  Adrenergic receptors are representative of a large family of plasma membrane receptors that interact with G proteins during the process of transmembrane signal transduction. G protein-coupled receptors have a primary structure that is homologous to bacteriorhodopsin and are proposed to have a similar three-dimensional structure; however, it has not yet been possible to examine this hypothesis experimentally. We have used a novel mutagenesis approach to identify intramolecular interactions. Our results indicate that specific amino acids in the seventh hydrophobic segment of alpha 2 and beta 2 adrenergic receptors lie adjacent to the first hydrophobic segment. These studies provide the first experimental evidence defining spatial relationships that exist in the three-dimensional structure of adrenergic receptors.

  View details for Web of Science ID A1992JW71900002

  View details for PubMedID 1331041

• CLONING OF 2 MOUSE GENES ENCODING ALPHA-2-ADRENERGIC RECEPTOR SUBTYPES AND IDENTIFICATION OF A SINGLE AMINO-ACID IN THE MOUSE ALPHA-2-C10 HOMOLOG RESPONSIBLE FOR AN INTERSPECIES VARIATION IN ANTAGONIST BINDING MOLECULAR PHARMACOLOGY Link, R., Daunt, D., Barsh, G., Chruscinski, A., Kobilka, B. 1992; 42 (1): 16-27

  Abstract

  Molecular cloning and ligand binding studies have shown the alpha 2 class of adrenergic receptor (alpha 2-AR) to be a family of at least three related subtypes in humans. These studies have not, however, identified distinct subtype-specific functions for these receptors in vivo. It should be possible to extend the analysis of alpha 2-AR subtype function to the animal level through the use of experimental mammalian embryology in mice. To begin this process, we have isolated two mouse genomic clones encoding alpha 2-AR subtypes and expressed these genes in COS-7 cells for binding studies. Sequence homology and ligand binding data allow the assignment of one clone (M alpha 2-4H) as the mouse homolog of the human alpha 2-C4 subtype. The other clone (M alpha 2-10H) closely resembles the human alpha 2-C10 subtype in sequence but binds with significantly lower affinity to yohimbine and rauwolscine, members of a distinct class of bulky alpha 2-selective antagonists commonly used to evaluate alpha 2-AR function in vivo. To define the domain(s) responsible for this unusual binding property, we constructed a series of M alpha 2-10H/human alpha 2-C10 chimeric receptors. Analysis of these receptors identified a conservative Cys201 to Ser201 change in the fifth transmembrane domain of M alpha 2-10H as being responsible for the low affinity of the mouse receptor for yohimbine.

  View details for Web of Science ID A1992JE03200004

  View details for PubMedID 1353249

• IDENTIFICATION OF A SINGLE AMINO-ACID RESIDUE RESPONSIBLE FOR THE BINDING OF A CLASS OF BETA-ADRENERGIC-RECEPTOR ANTAGONISTS TO 5-HYDROXYTRYPTAMINE1A RECEPTORS MOLECULAR PHARMACOLOGY Guan, X. M., Peroutka, S. J., Kobilka, B. K. 1992; 41 (4): 695-698

  Abstract

  The 5-hydroxytryptamine1A (5-HT1A) receptor can bind certain beta-adrenergic receptor antagonists, such as pindolol, with high affinity. Such pharmacological cross-reactivity suggests a structural similarity in the ligand binding site between the two receptors. To identify this structural entity, we mutated Asn385 in the seventh transmembrane domain of the human 5-HT1A receptor, based on the observation that this residue is conserved in all 5-HT1A and beta-adrenergic receptors of different species but is absent in all other cloned guanine nucleotide-binding protein-coupled receptors. This single point mutation (Asn385 to valine) causes a highly selective decrease in the affinity of pindolol and other aryloxyalkylamines for the mutant receptor (about 100-fold), while producing only minor changes in the binding of other 5-HT agonists and antagonists. The results provide direct evidence that Asn385 is responsible for the high affinity interaction between 5-HT1A receptors and aryloxyalkylamine beta-adrenergic antagonists but is not required for the binding of other chemical classes of ligands.

  View details for Web of Science ID A1992HP40500016

  View details for PubMedID 1349154

• LIGAND-REGULATED INTERNALIZATION AND RECYCLING OF HUMAN BETA-2-ADRENERGIC RECEPTORS BETWEEN THE PLASMA-MEMBRANE AND ENDOSOMES CONTAINING TRANSFERRIN RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Kobilka, B. K. 1992; 267 (5): 3530-3538

  Abstract

  Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.

  View details for Web of Science ID A1992HD15400107

  View details for PubMedID 1371121

• ADRENERGIC-RECEPTORS AS MODELS FOR G PROTEIN-COUPLED RECEPTORS ANNUAL REVIEW OF NEUROSCIENCE Kobilka, B. 1992; 15: 87-114

  View details for Web of Science ID A1992HF92100004

  View details for PubMedID 1575451

• A POINT MUTATION IN THE 7TH HYDROPHOBIC DOMAIN OF THE ALPHA-2 ADRENERGIC-RECEPTOR INCREASES ITS AFFINITY FOR A FAMILY OF BETA-RECEPTOR-ANTAGONISTS JOURNAL OF BIOLOGICAL CHEMISTRY Suryanarayana, S., Daunt, D. A., VONZASTROW, M., Kobilka, B. K. 1991; 266 (23): 15488-15492

  Abstract

  Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.

  View details for Web of Science ID A1991GB09700098

  View details for PubMedID 1678390

• MOLECULAR AND CELLULAR BIOLOGY OF ADRENERGIC-RECEPTORS TRENDS IN CARDIOVASCULAR MEDICINE Kobilka, B. 1991; 1 (5): 189-194

  Abstract

  Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system. Genomic or cDNA clones for 8 types of mammalian adrenergic receptors have been obtained. Much has been learned about the structure and functional properties of the β(2)-adrenergic receptor. Less is known about the functional properties and the physiologic role of the other adrenergic receptors. Further progress in this field may lead to the development of more selective drugs to modify the physiologic processes controlled by these receptors.

  View details for Web of Science ID A1991GD51800003

  View details for PubMedID 21239309

• A SINGLE POINT MUTATION IN THE 7TH HYDROPHOBIC DOMAIN OF THE ALPHA-2-ADRENERGIC RECEPTOR CHANGES ANTAGONIST BINDING-SPECIFICITY TO THAT OF A BETA-RECEPTOR 104TH SESSION OF THE ASSOC OF AMERICAN PHYSICIANS Suryanarayana, S., Daunt, D. A., VONZASTROW, M., Kobilka, B. K. ASSOC AMER PHYSICIANS. 1991: 62–68

  View details for Web of Science ID A1991BV21X00009

  View details for PubMedID 1688262

• THE ROLE OF CYTOSOLIC AND MEMBRANE FACTORS IN PROCESSING OF THE HUMAN BETA-2 ADRENERGIC-RECEPTOR FOLLOWING TRANSLOCATION AND GLYCOSYLATION IN A CELL-FREE SYSTEM JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K. 1990; 265 (13): 7610-7618

  Abstract

  The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.

  View details for Web of Science ID A1990DB30700079

  View details for PubMedID 1692024

• ANALYSIS OF LIGAND-BINDING SPECIFICITY OF RECEPTOR CHIMERAS - RESPONSE SCIENCE Kobilka, B. K., Kobilka, T. S., DANIELS, K. W., Regan, J. W., CARAN, M. G., Lefkowitz, R. J. 1989; 243 (4888): 237-237

  View details for Web of Science ID A1989R736800044

• MOLECULAR-CLONING AND EXPRESSION OF THE CDNA FOR THE HAMSTER ALPHA-1-ADRENERGIC RECEPTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cotecchia, S., Schwinn, D. A., RANDALL, R. R., Lefkowitz, R. J., Caron, M. G., Kobilka, B. K. 1988; 85 (19): 7159-7163

  Abstract

  The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

  View details for Web of Science ID A1988Q358500025

  View details for PubMedID 2845398

• THE GENOMIC CLONE G-21 WHICH RESEMBLES A BETA-ADRENERGIC-RECEPTOR SEQUENCE ENCODES THE 5-HT1A RECEPTOR NATURE FARGIN, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K., Caron, M. G., Lefkowitz, R. J. 1988; 335 (6188): 358-360

  Abstract

  The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.

  View details for Web of Science ID A1988Q137300061

  View details for PubMedID 3138543

• CLONING AND EXPRESSION OF A HUMAN-KIDNEY CDNA FOR AN ALPHA-2-ADRENERGIC RECEPTOR SUBTYPE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Regan, J. W., Kobilka, T. S., YANGFENG, T. L., Caron, M. G., Lefkowitz, R. J., Kobilka, B. K. 1988; 85 (17): 6301-6305

  Abstract

  An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

  View details for Web of Science ID A1988Q047600016

  View details for PubMedID 2842764

• HUMAN BETA-1-ADRENERGIC AND BETA-2-ADRENERGIC RECEPTORS - STRUCTURALLY AND FUNCTIONALLY RELATED RECEPTORS DERIVED FROM DISTINCT GENES TRENDS IN NEUROSCIENCES Frielle, T., Kobilka, B., Lefkowitz, R. J., Caron, M. G. 1988; 11 (7): 321-324

  View details for Web of Science ID A1988N945500010

  View details for PubMedID 2465637

• CHIMERIC ALPHA-2-ADRENERGIC, BETA-2-ADRENERGIC RECEPTORS - DELINEATION OF DOMAINS INVOLVED IN EFFECTOR COUPLING AND LIGAND-BINDING SPECIFICITY SCIENCE Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., Lefkowitz, R. J. 1988; 240 (4857): 1310-1316

  Abstract

  The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.

  View details for Web of Science ID A1988N633900023

  View details for PubMedID 2836950

• FUNCTIONAL-ACTIVITY AND REGULATION OF HUMAN BETA-2-ADRENERGIC RECEPTORS EXPRESSED IN XENOPUS OOCYTES JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K., MacGregor, C., Daniel, K., Kobilka, T. S., Caron, M. G., Lefkowitz, R. J. 1987; 262 (32): 15796-15802

  Abstract

  The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.

  View details for Web of Science ID A1987K886800076

  View details for PubMedID 2824467

• CLONING OF THE CDNA FOR THE HUMAN BETA-1-ADRENERGIC RECEPTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J., Kobilka, B. K. 1987; 84 (22): 7920-7924

  Abstract

  Screening of a human placenta lambda gt11 library has led to the isolation of the cDNA for the human beta 1-adrenergic receptor (beta 1AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human beta 2-adrenergic receptor (beta 2AR). The 2.4-kilobase cDNA for the human beta 1AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian beta AR but only 54% homologous with the human beta 2AR. This suggests that the avian gene encoding beta AR and the human gene encoding beta 1AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with beta 1AR binding. This pattern is quite distinct from the pattern obtained when the beta 2AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical beta 1AR specificity. This contrasts with the typical beta 2 subtype specificity observed when the human beta 2AR cDNA is expressed in this system. Mammalian beta 1AR and beta 2AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

  View details for Web of Science ID A1987L051100026

  View details for PubMedID 2825170

• CLONING, SEQUENCING, AND EXPRESSION OF THE GENE CODING FOR THE HUMAN-PLATELET ALPHA-2-ADRENERGIC RECEPTOR SCIENCE Kobilka, B. K., MATSUI, H., Kobilka, T. S., YANGFENG, T. L., FRANCKE, U., Caron, M. G., Lefkowitz, R. J., Regan, J. W. 1987; 238 (4827): 650-656

  Abstract

  The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.

  View details for Web of Science ID A1987K595500029

  View details for PubMedID 2823383

• AN INTRONLESS GENE ENCODING A POTENTIAL MEMBER OF THE FAMILY OF RECEPTORS COUPLED TO GUANINE-NUCLEOTIDE REGULATORY PROTEINS NATURE Kobilka, B. K., Frielle, T., Collins, S., YANGFENG, T., Kobilka, T. S., FRANCKE, U., Lefkowitz, R. J., Caron, M. G. 1987; 329 (6134): 75-79

  Abstract

  Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.

  View details for Web of Science ID A1987J846700060

  View details for PubMedID 3041227

• DELINEATION OF THE INTRONLESS NATURE OF THE GENES FOR THE HUMAN AND HAMSTER BETA-2-ADRENERGIC RECEPTOR AND THEIR PUTATIVE PROMOTER REGIONS JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K., Frielle, T., Dohlman, H. G., BOLANOWSKI, M. A., Dixon, R. A., Keller, P., Caron, M. G., Lefkowitz, R. J. 1987; 262 (15): 7321-7327

  Abstract

  The beta 2-adrenergic receptor is the first adenylate cyclase-coupled receptor to be cloned. We provide here a detailed characterization of its complete gene in both the human and hamster which reveals several unusual and provocative features. The genes are present in a single copy, are intronless, and are bounded by homologous 18-bp (base pair) direct repeats. These findings suggest that the beta 2-adrenergic receptor may have arisen as a processed gene for another related gene. Genomic Southern blots done at reduced stringency in fact reveal additional weak signals. The human and hamster gene sequences 5' to the principal site of transcription initiation are highly homologous and share many characteristics of promoters for housekeeping genes. Moreover, there is present in the human genome a long (777 bp) open reading frame which is in frame with the beta-adrenergic receptor coding block and which ends only 234 bp 5' to the initiator methionine of the receptor. An unusual cDNA has been found, transcribed from a putative second more 5' promoter which contains the 5' half of the beta-adrenergic receptor as well as 1065-bp 5' to the receptor coding region, including the entire upstream long open reading frame (sufficient to encode a putative protein of Mr approximately 28,000).

  View details for Web of Science ID A1987H414700060

  View details for PubMedID 3034889

• CDNA FOR THE HUMAN BETA-2-ADRENERGIC RECEPTOR - A PROTEIN WITH MULTIPLE MEMBRANE-SPANNING DOMAINS AND ENCODED BY A GENE WHOSE CHROMOSOMAL LOCATION IS SHARED WITH THAT OF THE RECEPTOR FOR PLATELET-DERIVED GROWTH-FACTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kobilka, B. K., Dixon, R. A., Frielle, T., Dohlman, H. G., BOLANOWSKI, M. A., SIGAL, I. S., YANGFENG, T. L., FRANCKE, U., Caron, M. G., Lefkowitz, R. J. 1987; 84 (1): 46-50

  Abstract

  We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

  View details for Web of Science ID A1987F667900010

  View details for PubMedID 3025863

• BETA-ADRENERGIC RECEPTORS AND RHODOPSIN - SHEDDING NEW LIGHT ON AN OLD SUBJECT TRENDS IN PHARMACOLOGICAL SCIENCES Lefkowitz, R. J., Benovic, J. L., Kobilka, B., Caron, M. G. 1986; 7 (11): 444-448

  View details for Web of Science ID A1986F039400011

• CLONING OF THE GENE AND CDNA FOR MAMMALIAN BETA-ADRENERGIC-RECEPTOR AND HOMOLOGY WITH RHODOPSIN NATURE Dixon, R. A., Kobilka, B. K., STRADER, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., BOLANOWSKI, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., SIGAL, I. S., Caron, M. G., Lefkowitz, R. J., Strader, C. D. 1986; 321 (6065): 75-79

  Abstract

  The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

  View details for Web of Science ID A1986C169000052

  View details for PubMedID 3010132