Evidence for precoupling of inactive M3 muscarinic receptors and Gq Protein

Abstract

Two models have been proposed to explain how G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins to transduce physiological signals. One model suggests that GPCRs and G proteins collide with each other randomly after receptor activation and that binding is transient. An alternative model suggests that GPCRs and G proteins are bound to each other (precoupled) before receptor activation. We have studied interactions between GPCRs and G proteins using fluorescence recovery after photobleaching (FRAP) and avidin-mediated crosslinking in HEK 293 cells. We have previously shown that immobile CFP-labeled α2A-adrenoreceptors (C-α2ARs) do not decrease the mobility of the G proteins that they activate, consistent with a collision-coupling model. Here we show that immobile CFP-labeled M3 muscarinic receptors (C-M3Rs) decrease the lateral mobility of citrine-labeled Gαq. C-M3Rs failed to decrease the mobility of venus-labeled GαoA. Conversely, the C-M4Rs (which activate Gi/o proteins) failed to decrease the mobility of Gq-citrine. Slowing of Gαq-citrine by immobile C-M3R was unaffected by an agonist (carbachol) or an inverse agonist (atropine), and thus did not depend on activation of the receptor. Slowing of Gαq-citrine by immobile C-M3Rs was enhanced by carbachol when nucleotides were depleted, as predicted by the ternary complex model of G protein coupling. These results suggest that inactive M3Rs precouple with Gq proteins, and that different coupling models apply to different GPCR-G protein pairs.Supported by grants GM078319 from the NIH and MCB0620024 from the NSF.