Delineating the conformational landscape of the adenosine A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (F NMR) is used to delineate key functional states of the adenosine A receptor (AR) complexed with heterotrimeric G protein (Gαβγ) in a phospholipid membrane milieu. Analysis of AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.