The M3 muscarinic acetylcholine receptor can signal through multiple G protein families

The M3 muscarinic acetylcholine receptor (M₃R) is a G protein coupled receptor (GPCR) that regulates important physiological processes including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M₃R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M₃R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M₃R. We further show that these interactions are productive as assessed by amplification of classical second messenger signaling events. Our findings demonstrate that the M₃R is more promiscuous with respect to G protein interactions than previously appreciated.