G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

Abstract

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 Gαq/11 and inhibiting Gαq stimulation of the effector phospholipase Cβ. The binding site for Gαq/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 Gαq/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 Gαq/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 Gαq/11 binding defects impair recruitment to the plasma membrane by activated Gαq and regulation of Gαq-stimulated phospholipase Cβ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The Gα binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices α3 and α4, α5 and α6, and α7 and α8. The adenomatous polyposis coli (APC) binding site of axin involves residues on α helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the Gαq/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 α5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.