Instructor, Molecular & Cellular Physiology
Honors & Awards
Interdisciplinary Scholar, Stanford Neurosciences Institute (2018-2020)
Dean's Fellow, Stanford School of Medicine, Stanford University (2016-2017)
Allostery of atypical modulators at oligomeric G protein-coupled receptors.
2021; 11 (1): 9265
Many G protein-coupled receptors (GPCRs) are therapeutic targets, with most drugs acting at the orthosteric site. Some GPCRs also possess allosteric sites, which have become a focus of drug discovery. In the M2 muscarinic receptor, allosteric modulators regulate the binding and functional effects of orthosteric ligands through a mix of conformational changes, steric hindrance and electrostatic repulsion transmitted within and between the constituent protomers of an oligomer. Tacrine has been called an atypical modulator because it exhibits positive cooperativity, as revealed by Hill coefficients greater than 1 in its negative allosteric effect on binding and response. Radioligand binding and molecular dynamics simulations were used to probe the mechanism of that modulation in monomers and oligomers of wild-type and mutant M2 receptors. Tacrine is not atypical at monomers, which indicates that its atypical effects are a property of the receptor in its oligomeric state. These results illustrate that oligomerization of the M2 receptor has functional consequences.
View details for DOI 10.1038/s41598-021-88399-x
View details for PubMedID 33927236
Ligand-Induced Coupling between Oligomers of the M-2 Receptor and the G(i1 )Protein in Live Cells
2018; 115 (5): 881–95
Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M2 muscarinic receptor and of Gi1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M2 and mCherry-M2) and G proteins (i.e., eGFP-Gαi1β1γ2 and mCherry-Gαi1β1γ2) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and Gi1 can exist as homo-oligomers. Oligomerization of differently tagged Gi1 decreased upon the activation of co-expressed wild-type M2 receptor by an agonist. Measurements on a tagged M2 receptor (M2-mCherry) and eGFP-Gαi1β1γ2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M2(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gαi1β1γ2, indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M2 receptor on the one hand and of Gi1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.
View details for DOI 10.1016/j.bpj.2018.08.001
View details for Web of Science ID 000443570800016
View details for PubMedID 30131171
View details for PubMedCentralID PMC6127463
Allosteric regulation and oligomerization of muscarinic cholinergic receptors
AMER CHEMICAL SOC. 2017
View details for Web of Science ID 000429556701358
Single-Molecule Analysis of the Supramolecular Organization of the M-2 Muscarinic Receptor and the G alpha(i1) Protein
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2016; 138 (36): 11583-11598
G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected by GTPγS. A complex of M2R and Gi1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of Gi1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gαi1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gαi1 was predominantly dimeric. The oligomeric nature of Gi1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gαi1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-Gi1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of Gi1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.
View details for DOI 10.1021/jacs.6b04032
View details for Web of Science ID 000383410700031
View details for PubMedID 27494760
Allosteric modulation in monomers and oligomers of a G protein-coupled receptor
The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.
View details for DOI 10.7554/eLife.11685
View details for Web of Science ID 000387459600001
View details for PubMedID 27151542
View details for PubMedCentralID PMC4900804