Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

Significance The family of G-protein–coupled receptors (GPCRs) is the largest receptor family, and each member detects specific ligands, which in turn, activate selected members of one or more G-protein families. We developed a broadly applicable assay to study the specificity of GPCR–G-protein interaction in a subtype selective manner. We were able to quantitatively measure both association and dissociation rates of G proteins from agonist-activated GPCRs. We found that the stability of the receptor–G-protein complex is an inherent property of both interaction partners independent on the agonist used. Furthermore, its lifetime correlated closely with the ability of the receptor to activate the corresponding G-protein subtype. G-protein–coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR–G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein–M3–R interactions but rather the affinities of Gq and Go proteins to M3–Rs, their GPCR–G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.