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.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546 -565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546 -565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546 -565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.Cell surface localized G protein-coupled receptors (GPCRs) 1 detect a large variety of extracellular stimuli and initiate numerous intracellular signaling pathways. Proper regulation of GPCR signaling is maintained in part by a process known as desensitization (1), which refers to the turning off of a GPCRinitiated signaling event in the continuous presence of agonist. A key mechanism of GPCR desensitization is phosphorylation of agonist-occupied receptor by the G protein-coupled receptor kinases (GRKs) (2, 3). This phosphorylation promotes binding of arrestin proteins to the GPCR and results in subsequent uncoupling of the GPCR from G protein.To function properly GRKs need to be localized to the cytoplasmic face of the plasma membrane (PM) where their GPCR substrates are located. Different members of the GRK family utilize a variety of mechanisms to bind to cellular membranes (2, 3). GRK2 and GRK3 contain well characterized C-terminal pleckstrin homology domains (4) that mediate translocation of the kinases from the cytoplasm to the PM in response to GPCR activation (5, 6). The pleckstrin homology domains allow PM binding by interacting with both phospholipids and free G protein ␤␥ subunits. In contrast to GRK2/3, other GRK family members exhibit a more constitutive association with cellular membranes. GRK1 and GRK7 contain a C-terminal CAAX motif, which directs covalent modification by a farnesyl or gerany...