Intramolecular interaction between the DEP domain of RGS7 and the Gβ₅ subunit

The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Gα subunits and exhibits GAP activity. The GGL domain permanently associates with Gβ₅. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gβ₅. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gβ₅-RGS7, recombinant Gβγ complexes, and monomeric Gβ₅ and Gβ₁ subunits. Coimmunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gβ₅ and YFP-RGS7, indicating that the DEP-Gβ₅ interaction is dynamic. In transfected cells, R7BP had no effect on the Gβ₅/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gβ₅. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gβ₅ interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca²⁺ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gβ₅-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gβ₅ subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on G _q-mediated signaling than the open conformation.