The complex of the regulator of G protein signaling (RGS), Gβ5-RGS7, can inhibit signal transduction via the M3 muscarinic acetylcholine receptor (M3R). RGS7 consists of three distinct structural entities: the DEP domain and its extension DHEX, the G-like (GGL) domain, which is permanently bound to Gβ subunit Gβ5, and the RGS domain responsible for the interaction with Gα subunits. Inhibition of the M3R by Gβ5-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the third intracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction between the Gβ5 and DEP domains, which suggested that the Gβ5-RGS7 dimer could alternate between the "open" and "closed" conformations. Here, we identified point mutations that weaken DEP-Gβ5 binding, presumably stabilizing the open state, and tested their effects on the interaction of Gβ5-RGS7 with the M3R. We found that these mutations facilitated binding of Gβ5- RGS7 to the recombinant third intracellular loop of the M3R but did not enhance its ability to inhibit M3R-mediated Ca2+ mobilization. This led us to the idea that the M3R can effectively induce the Gβ5-RGS7 dimer to open; such a mechanism would require a region of the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts with Gβ5-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-type Gβ5-RGS7; however, the open mutant of Gβ5-RGS7 was able to inhibit signaling by the truncated M3R. The GST fusion of the M3R C-tail could not bind to wild-type Gβ5-RGS7 but could associate with its open mutant as well as with the separated recombinant DEP domain or Gβ5. Taken together, our data are consistent with the following model: interaction of the M3R with Gβ5-RGS7 causes the DEP domain and Gβ5 to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop, thereby inhibiting M3R-mediated signaling.
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