Single-molecule analysis of ligand efficacy in β(2)AR-G protein activation

G protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signaling responses remain poorly understood. In Class A GPCRs, receptor activation and G protein coupling entail outward movements of transmembrane segment 6 (TM6). Using single-molecule Fluorescence Resonance Energy Transfer (smFRET) imaging, we examine TM6 motions in the β(2) adrenergic receptor (β(2)AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G(s) heterotrimer. We show that partial and full agonists affect TM6 motions in a manner that differentially regulates the rate at which GDP-bound β(2)AR-G(s) complexes are formed and the efficiency of nucleotide exchange leading to G(s) activation. These data also reveal transient nucleotide-bound β(2)AR-G(s) species distinct from known structures and single-molecule perspectives on the allosteric link between ligand and nucleotide binding pockets that shed new light on the G protein activation mechanism.