Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β₂AR

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 46; pp. 14254 - 14259
• Main Authors: Lamichhane, Rajan, Liu, Jeffrey J., Pljevaljcic, Goran, White, Kate L., van der Schans, Edwin, Katritch, Vsevolod, Stevens, Raymond C., Wüthrich, Kurt, Millar, David P.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 17.11.2015; National Acad Sciences
• Subjects: Binding Sites; Biological Sciences; Carbocyanines - chemistry; Humans; Molecular Dynamics Simulation; Propanolamines - chemistry; Receptors, Adrenergic, beta-2 - chemistry; signal transduction mechanisms; phospholipid nanodiscs; conformational polymorphism; agonists and inverse agonists; single-molecule fluorescence spectroscopy
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1519626112

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.