G protein-coupled receptor kinase 2/Gαq/11 interaction -: A novel surface on a regulator of G protein signaling homology domain for binding Gα subunits

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding Galpha(q/11) and inhibiting Galpha(q) stimulation of the effector phospholipase Cbeta. The binding site for Galpha(q/11) resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha(q/11) binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to Gaq/11. These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing Gaq/11 binding defects impair recruitment to the plasma membrane by activated Galpha(q) and regulation of Galpha(q)-stimulated phospholipase Cbeta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The Galpha binding sites on RGS4 and RGS9, called the "A' site, is localized to the loops between helices alpha3 and alpha4, alpha5 and alpha6, and alpha7 and alpha8. The adenomatous polyposis coli (APQ binding site of axin involves residues on a helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the Gaq/11 binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.