Single-molecule analysis of ligand efficacy in β2AR-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 signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the beta(2) adrenergic receptor (beta(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 differentially affect TM6 motions to regulate the rate at which GDP-bound beta(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 beta(2)AR-G(s) species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand-and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.