The Amino-terminal Domain of G-protein-coupled Receptor Kinase 2 Is a Regulatory Gβγ Binding Site

Abstract

G-protein-coupled receptor kinase 2 (GRK2) is activated by free G␤␥ subunits. A G␤␥ binding site of GRK2is localized in the carboxyl-terminal pleckstrin homology domain. This G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits. We report here that truncation of the carboxyl-terminal G␤␥ binding site of GRK2 did not abolish the G␤␥ regulatory activity of GRK2 as determined by the inhibition of a G␤␥-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second G␤␥ binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2 1-185 ) inhibited a G␤␥-stimulated inositol phosphate signal in cells, purified GRK2 1-185 suppressed the G␤␥-stimulated phosphorylation of rhodopsin, and GRK2 1-185 bound directly to purified G␤␥ subunits. The amino-terminal G␤␥ regulatory site does not overlap with the RGS domain of GRK-2 because GRK2 1-53 with truncated RGS domain inhibited G␤␥-mediated signaling with similar potency and efficacy as did GRK2 1-185 . In addition to the G␤␥ regulatory activity, the amino-terminal G␤␥ binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified G␤␥ subunits, strongly suggesting that G␤␥ binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized G␤␥ binding site that regulates GRK2-mediated receptor phosphorylation and inhibits G␤␥-stimulated signaling.Activated G-protein-coupled receptors are switched off by phosphorylation through G-protein-coupled receptor kinases (GRKs) 1 (1). GRKs are modular proteins consisting of at least three structural domains with different functions. The core kinase domain of GRK2 and GRK3, which represents the ␤-adrenergic receptor kinase isozymes, is flanked by an aminoterminal domain, which contains an RGS domain, and a carboxyl-terminal domain, which contains a pleckstrin homology domain (PH domain) (2-4). The activation of GRK2 and GRK3 requires the activation and dissociation of a heterotrimeric G-protein, i.e. the kinases are activated by free G␤␥ subunits (5, 6). A G␤␥ binding site of GRK2 and GRK3 is localized in the carboxyl terminus of the kinase and overlaps the PH domain (7). Truncation of the PH domain of GRK2 generates a kinase with compromised regulation by G␤␥ subunits (7). The carboxyl-terminal G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits (8).Analyzing the G␤␥ regulatory activity of proteins is a means of identifying G␤␥-binding proteins or localizing G␤␥ binding sites of proteins (9 -11). To find out whether the G␤␥ regulatory activity of GRK2 resides entirely in the carboxyl-terminal PH domain, we analyzed the G␤␥-sequestering activity of wildtype GRK2 and of carboxyl-terminal-truncated GRK2 mutants. The capacity of thos...

Raf Kinase Inhibitor Protein (RKIP) Dimer Formation Controls Its Target Switch from Raf1 to G Protein-coupled Receptor Kinase (GRK) 2

“…5 h after seeding, cells were transfected with the indicated cDNAs using the calcium phosphate precipitation method (29). 24 h later, cells were serumstarved.…”

“…After centrifugation (14,000 ϫ g, 4°C, 15 min), the supernatant was incubated with anti-FLAG antibodies bound to Protein A-Sepharose for 2 h at 4°C. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and visualized using the indicated antibodies similarly as described previously (29).…”

“…GRK2 was expressed in Spodoptera frugiperda (Sf9) cells infected with baculoviruses encoding bovine GRK2 and purified via SP-Sepharose and heparin-Sepharose chromatography as described previously (4). Rhodopsin was isolated from rod outer segments (29).…”

Non-visual GRKs: are we seeing the whole picture?

“…This domain contains binding sites for both PtdIns(4,5)P 2 and free Gbg, which play a role in targeting and translocation of these primarily cytosolic GRKs to membranes following GPCR activation [10]. More recently, a second binding site for Gbg-subunits has been identified within the first 53 amino acids of GRK2 [25], which suggests that either the N-or the C-terminal regions might be sufficient to allow GRK2 Box 1. GRKs can regulate GPCRs in a phosphorylation-independent manner Recent studies have provided evidence that certain G-protein-coupled receptor kinases (GRKs) can suppress the interaction between G-proteincoupled receptors (GPCRs) and G proteins in a phosphorylationindependent manner.…”

“…More recently, the N-terminal RGS-like domain has been shown to bind to activated GTP-bound Ga q/11 [22], which not only blocks the interaction of Ga q/11 with phospholipase C (PLC), but might also assist the correct targeting of GRK2 and GRK3 before receptor phosphorylation [57]. A second Gbg binding site has recently been mapped to the N-terminus of GRK2 [25] (dark green), which, given the almost complete sequence homology between the two kinases in this region, also suggests a similar site for GRK3. The GRK4-6 subfamily is predominantly membrane associated.…”