title: Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR.
abstract: Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Rajan
authors.initials: R
authors.lastname: Lamichhane
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Liu
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Goran
authors.initials: G
authors.lastname: Pljevaljcic
authors.affiliation: Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089;
authors.forename: Kate L
authors.initials: KL
authors.lastname: White
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Edwin
authors.initials: E
authors.lastname: van der Schans
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089;
authors.forename: Vsevolod
authors.initials: V
authors.lastname: Katritch
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089; stevens@usc.edu wuthrich@scripps.edu millar@scripps.edu.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland stevens@usc.edu wuthrich@scripps.edu millar@scripps.edu.
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; stevens@usc.edu wuthrich@scripps.edu millar@scripps.edu.
authors.forename: David P
authors.initials: DP
authors.lastname: Millar
mesh_terms: D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry*; D006801:Humans; D056004:Molecular Dynamics Simulation; D011412:Propanolamines / Q000737:chemistry*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; C089187:cyanine dye 3; C026777:ICI 118551
keywords: agonists and inverse agonists; conformational polymorphism; phospholipid nanodiscs; signal transduction mechanisms; single-molecule fluorescence spectroscopy