Two-step structural changes in M3 muscarinic receptor activation rely on the coupled G protein cycle

Abstract

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation–deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gβγ subunits. We also find that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle. © 2023, The Author(s).