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 G␣ q/ll and inhibiting G␣ q stimulation of the effector phospholipase C␤. The binding site for G␣ q/ll 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 G␣ q/ll 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 G␣ q/ll . 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 G␣ q/ll binding defects impair recruitment to the plasma membrane by activated G␣ q and regulation of G␣ q -stimulated phospholipase C␤ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G␣ binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices ␣3 and ␣4, ␣5 and ␣6, and ␣7 and ␣8. The adenomatous polyposis coli (APC) binding site of axin involves residues on ␣ helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G␣ q/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its ␣5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.

Roles of Phosphorylation-dependent and -independent Mechanisms in the Regulation of M1 Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 in Hippocampal Neurons

“…DISCUSSION We have previously shown using the IP 3 biosensor eGFP-PH PLC␦ that expression of GRK2 in hippocampal neurons leads to an almost complete suppression of M 1 mACh receptor-mediated IP 3 signaling and that this suppression is independent of kinase activity, because the catalytically inactive K220R GRK2 mutant also completely inhibited signaling (18). In common with a number of other groups (14,15,19,21), we proposed that the suppression of signaling is a consequence of the direct interaction of the N-terminal RH domain of GRK2 with GTPbound G␣ q/11 . In the present study we have examined whether the catalytic activity of GRK2 acts as an alternative or additional mechanism for GRK2-mediated suppression of M 1 mACh receptor signaling.…”

“…To determine whether GRK2-mediated receptor phosphorylation is required for M 1 mACh receptor desensitization, we introduced a single point mutation D110A to create both GRK2 and K220R GRK2 mutants, which are incapable of binding G␣ q/11 (19). When transiently transfected, expression levels of the GRK2, D110A GRK2, K220R GRK2, and D110A,K220R GRK2 constructs were similar in HEK293 cells (data not shown).…”

“…More recent studies have shown that the N-terminal RH domain of GRK2 is responsible for inhibiting G␣ q/11 -coupled signaling to PLC (14,15). Indeed, the key amino acids involved in this interaction have been mapped to the ␣5 helix of the N-terminal domain of GRK2 (19). Mutation of several individual amino acid residues within this region prevents G␣ q binding to GRK2 (19) and restores the ability of the G␣ q/11 -coupled mGlu1a receptor to signal through PLC (21).…”

“…GRK2-mediated, phosphorylation-independent receptor regulation has now been demonstrated not only in recombinant systems but also for endogenous receptors in cell lines (17) and in primary, cultured hippocampal neurons (18). Phosphorylation-independent receptor regulation by GRK2 is mediated through a specific interaction of GTP-loaded G␣ q/11 with the RGS homology (RH) domain situated at the N terminus of GRK2 (14,15,19). Mutational and crystallographic analyses have identified specific amino acid residues within the RH domain that mediate G␣ q binding that map almost exclusively to the ␣5 helix of the RH domain of GRK2 (19 -21).…”

Functional consequences of Gα13 mutations that disrupt interaction with p115RhoGEF

“…However, the a 13 / p115RhoGEF pair is not the only case in which a Gly to Ser mutation at this Ga position fails to affect the interaction; a recent report demonstrated that such a Gly to Ser mutant of a q , a q G188S, retains interaction with the RH domain of GRK2 even though it loses interaction with RGS2 (Sterne-Marr et al, 2003).…”