title: Single-Molecule Analysis of the Supramolecular Organization of the M2 Muscarinic Receptor and the Gαi1 Protein.
abstract: 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.
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: Rabindra V
authors: RV
authors: Shivnaraine
authors: Department of Physics, University of Toronto , Toronto, Ontario M5S 1A7, Canada.|Department of Chemical & Physical Sciences, University of Toronto Mississauga , Mississauga, Ontario L5L 1C6, Canada.
authors: Dennis D
authors: DD
authors: Fernandes
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: Huiqiao
authors: H
authors: Ji
authors: Department of Physics, University of Toronto , Toronto, Ontario M5S 1A7, Canada.|Department of Chemical & Physical Sciences, University of Toronto Mississauga , Mississauga, Ontario L5L 1C6, Canada.
authors: Yuchong
authors: Y
authors: Li
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.|Krembil Research Institute, University Health Network , Toronto, Ontario M5T 2S8, Canada.
authors: Brendan
authors: B
authors: Kelly
authors: Department of Physics, University of Toronto , Toronto, Ontario M5S 1A7, Canada.|Department of Chemical & Physical Sciences, University of Toronto Mississauga , Mississauga, Ontario L5L 1C6, Canada.
authors: Zhenfu
authors: Z
authors: Zhang
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: Yi Rang
authors: YR
authors: Han
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: Fei
authors: F
authors: Huang
authors: Department of Physiology, University of Toronto , Toronto, Ontario M5G 1L7, Canada.
authors: Krishana S
authors: KS
authors: Sankar
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: David N
authors: DN
authors: Dubins
authors: Department of Physiology, University of Toronto , Toronto, Ontario M5G 1L7, Canada.|Institute of Biomedical and Biomaterial Engineering, University of Toronto , Toronto, Ontario M5S 3G9, Canada.
authors: Jonathan V
authors: JV
authors: Rocheleau
authors: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
authors: James W
authors: JW
authors: Wells
authors: Department of Physics, University of Toronto , Toronto, Ontario M5S 1A7, Canada.|Department of Chemical & Physical Sciences, University of Toronto Mississauga , Mississauga, Ontario L5L 1C6, Canada.
authors: Claudiu C
authors: CC
authors: Gradinaru
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D043585:Receptor, Muscarinic M2 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D043585:Receptor, Muscarinic M2; C584114:GNAI1 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go