Molecular Organization of the Complex between the Muscarinic M3 Receptor and the Regulator of G Protein Signaling, Gβ5−RGS7

摘要

The complex of the regulator of G protein signaling (RGS), Gβ5-RGS7, can inhibit signalntransduction via theM3 muscarinic acetylcholine receptor (M3R). RGS7 consists of three distinct structuralnentities: the DEP domain and its extension DHEX, the Gγ-like (GGL) domain, which is permanently boundnto Gβ subunit Gβ5, and the RGS domain responsible for the interaction with GR subunits. Inhibition of thenM3R by Gβ5-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the thirdnintracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction betweennthe Gβ5 and DEP domains, which suggested that the Gβ5-RGS7 dimer could alternate between the “open”nand “closed” conformations. Here, we identified point mutations that weaken DEP-Gβ5 binding, pre-nsumably stabilizing the open state, and tested their effects on the interaction ofGβ5-RGS7 with theM3R.Wenfound that these mutations facilitated binding of Gβ5-RGS7 to the recombinant third intracellular loop ofnthe M3R but did not enhance its ability to inhibit M3R-mediated Ca2þ mobilization. This led us to the ideanthat theM3R can effectively induce the Gβ5-RGS7 dimer to open; such amechanism would require a regionnof the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts withnGβ5-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-typenGβ5-RGS7; however, the open mutant of Gβ5-RGS7 was able to inhibit signaling by the truncatednM3R. TheGST fusion of theM3R C-tail could not bind to wild-type Gβ5-RGS7 but could associate with itsnopen mutant as well as with the separated recombinant DEP domain or Gβ5. Taken together, our data arenconsistent with the following model: interaction of the M3R with Gβ5-RGS7 causes the DEP domain andnGβ5 to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop,nthereby inhibiting M3R-mediated signaling.