G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling.

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.

G protein-coupled receptors (GPCRs) 1 represent the largest family of integral plasma membrane receptors and transduce extracellular signals mediated by light, taste, hormones, and neurotransmitters to the cell interior by coupling to heterotrimeric guanine nucleotide-binding (G) proteins (1)(2)(3). Agonistactivated GPCRs function as guanine nucleotide exchange factors facilitating GDP-for-GTP exchange on the G protein ␣ subunit thus allowing the functional dissociation of the G␣ and G␤␥ subunits, which in turn, regulate the activity of a diverse variety of effector enzymes and ion channels (4). To prevent effector pathway overactivation, mechanisms have evolved to promote the adaptation of GPCR signaling (1)(2)(3). The canonical model for GPCR adaptation involves GRK-mediated GPCR phosphorylation and arrestin protein binding to block receptor/G protein interactions and target receptors for endocytosis (5)(6)(7). The GRK2 crystal structure confirms that GRK2 is composed of three functional domains: a regulator of G protein signaling (RGS) homology (RH) domain, a protein kinase domain, and a carboxyl-terminal G␤␥-binding pleckstrin homology (PH) domain (8 -10). The GRK2 RH domain consists of two discontinuous segments, the first of which forms the ␣-helical bundles characteristic of RGS proteins (8 -10). The GRK2 and GRK3 RH domains specifically interact with G␣ q/11 family proteins in an AlF 4 Ϫ -dependent manner and may function as weak GTPase activating proteins for G␣ q/11 (10). Thus, GRK2 and GRK3 may function as bifunctional regulators of GPCR signaling by modulating both receptor and G protein activity, but the precise mechanism by which this is achieved is not known.
Phosphorylation-independent GRK-mediated desensitization has been reported for both the metabotropic glutamate receptor 1 (mGluR1) and the ␥-aminobutyric acid, type B (GABA) B receptor; for these receptors, it involves an atypical arrestin-independent mechanism (11)(12)(13). For mGluR1a, GRK2 interacts with the activated receptor (14), and expression of the GRK2 RH domain alone (amino acid residues 45-185), lacking both kinase and ␤␥-binding domains, attenuates both intrinsic and agonist-stimulated mGluR1a signaling while retaining receptor-binding activity (12). Mutation of several amino acid residues: Arg-106, Asn-110, and Met-114 to alanine residues (R106A, D110A, and M114A), within the ␣5 helix of the GRK2 RH domain prevents GRK2 binding to G␣ q/11 (15). Therefore, we tested the hypothesis that GRK2 mutants impaired in G␣ q/11 binding are unable to antagonize both basal and agonist-stimulated mGluR1a signaling. We find that G␣ q/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and both agonist treatment and GRK2 overexpression promote the dissociation of this receptor/G␣ q/11 * This work was supported by Canadian Institutes of Health Research (CIHR) Grant MA-15506 (to S. S. G. F.) and National Science Foundation Grants MCB9728179 and MCB0315888 (to R. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ complex. However, receptor/G␣ q/11 complex formation is not altered by GRK2 mutants impaired in G␣ q/11 binding, but these mutants are impaired in their ability to block mGluR1a signaling. Furthermore, a GRK2-D527A mutant that fails to bind mGluR1a does not attenuate mGluR1a signaling, even though the mutant binds G␣ q/11 . Taken together, our observations suggest that phosphorylation-independent blockade of mGluR1a signaling requires the concomitant binding of the GRK2 RH domain to both G␣ q/11 and mGluR1a.

EXPERIMENTAL PROCEDURES
Materials-Human embryonic kidney cells (HEK293) were obtained from American Type Culture Collection. Inositol-free Dulbecco's modified Eagle's medium, minimal essential medium, fetal bovine serum, trypsin, and gentamicin were obtained from Invitrogen. The rabbit polyclonal GRK2 antibody was raised against the peptide sequence, DREARKKAKNKQLGH, corresponding to rat GRK2. Donkey anti-rabbit IgG conjugated to horseradish peroxidase, ECL Western blotting detection reagents, and the protein G-Sepharose beads were obtained from Amersham Biosciences. Quisqualate was purchased from Tocris Cookson Inc. The anti-FLAG monoclonal antibody was purchased from Sigma. [ 3 H]Myo-inositol was acquired from PerkinElmer Life Sciences. The Dowex 1-X8 (formate form) resin with 200 -400 mesh was purchased from Bio-Rad. All other biochemical reagents were purchased from Sigma, Fisher, and VWR Scientific.
Cell Culture and Transfection-HEK293 cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 g/l gentamicin. Cells were transfected with plasmid cDNAs using a modified calcium phosphate method (12). After transfection (18 h), the cells were incubated with fresh medium, allowed to recover for 6 -8 h, and reseeded into either 6-or 24-well dishes, and then grown an additional 18 h prior to experimentation.
Inositol Phosphate Formation-Inositol lipids were radiolabeled by incubating the cells overnight with 1 Ci/ml [ 3 H]myo-inositol in Dulbecco's modified Eagle's medium. Un-incorporated [ 3 H]myo-inositol was removed by washing the cells with HBSS (116 mM NaCl, 20 mM HEPES, 11 mM glucose, 5 mM NaHCO 3 , 4.7 mM KCl, 2.5 mM CaCl 2 , 1.3 mM MgSO 4 , 1.2 mM KH 2 PO 4 , pH 7.4). The cells were pre-incubated for 1 h in HBSS at 37°C and then pre-incubated in 500 l of the same buffer containing 10 mM LiCl for an additional 10 min at 37°C. The cells were then incubated in either the absence or the presence of increasing concentrations (0 -30 M) of quisqualate for 30 min at 37°C. The reaction was stopped on ice by adding 400 l of perchloric acid and then it was neutralized with 500 l of 0.72 M KOH and 0.6 M KHCO 3 . The total [ 3 H]inositol incorporated into the cells was determined by counting the radioactivity present in 50 l of the cell lysate. Total inositol phosphate was purified from the cell extracts by anion exchange chromatography using Dowex 1-X8 (formate form) 200 -400 mesh anion exchange resin. [ 3 H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system.
Co-immunoprecipitation-The cells from 100-mm dishes were washed twice with ice-cold phosphate-buffered saline and were lysed in 400 l of cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X-100 containing protease inhibitors, 20 g/ml leupeptin, 20 g/ml aprotinin, and 20 g/ml phenylmethylsulfonyl fluoride) in either the absence or presence of 100 M GDP, 2 mM MgSO 4 , 30 M aluminum chloride, and 5 mM sodium fluoride. The particulate fraction was removed by centrifugation, and 500 g of supernatant protein was incubated with 5 l of either anti-FLAG or anti-Myc monoclonal antibody and 100 l of 20% protein G-Sepharose beads or 40 l of FLAG-affinity gel for 12-16 h at 4°C. The beads were washed three times with lysis buffer, solubilized in SDS sample buffer, and separated by SDS-PAGE. The proteins were transferred onto nitrocellulose membranes by semidry electroblotting. The membranes were blocked with 10% milk in TBS-T wash buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.0, 0.05% Tween 20) and then incubated with rabbit GRK2, G␣ q/11 -(Santa Cruz Biotechnology) and mGluR1a-specific antibodies (Upstate Biotechnology) diluted 1:1000 in wash buffer containing 3% skim milk. The mem-branes were rinsed three times with wash buffer and then incubated with secondary horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences) diluted 1:2500 in wash buffer containing 3% skimmed milk. Membranes were rinsed twice with TBS-T, twice with TBS, and were incubated with ECL Western blotting detection reagents.

RESULTS AND DISCUSSION
Because expression of the GRK2 RH domain alone effectively attenuates mGluR1a signaling (12), we tested whether GRK2 mutants defective in G␣ q/11 binding (R106A, D110A, and M114A) might be unable to antagonize both basal and agoniststimulated mGluR1a signaling (15). Consistent with our pre- HEK293 cells were transfected with 10 g of pcDNA3 plasmids encoding mGluR1a with and without 10 g of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. The data points represent the mean Ϯ S.D. for three to six independent experiments. The agonist dose-response curves were fit and analyzed using GraphPad Prism software.
GRK2 is abundantly expressed in brain (17), and GRK2 expression in mGluR1a-expressing brain regions (18) is substantially higher than GRK2 expression in HEK293 cells ( Fig.  2A). However, it is suggested that GRK2 RH domain-dependent mGluR1a desensitization occurs merely as the consequence of GRK2 overexpression (19). Therefore, we examined the effect of transfecting HEK293 cells with increasing amounts of GRK2 plasmid cDNA on the V max for mGluR1a-mediated IP formation (Fig. 2B). As illustrated in Fig. 2C, the V max for quisqualate-stimulated IP formation is progressively attenuated as GRK2 expression is increased (Fig. 2C). GRK2-dependent attenuation of mGluR1a signaling is observed at GRK2 expression levels (0.1-1 g of transfected cDNA) that are similar to endogenous GRK2 levels in mGluR1a-expressing regions of rat brain, when compared with endogenous HEK293 cell GRK2 FIG. 2. Wild-type and GRK2 mutant expression level-dependent effects upon agonist-stimulated and basal mGluR1 signaling. A, relative GRK2 expression in 100 g of lysates from HEK293 cells, rat brain cerebellum (Cb) and rat brain cortex (Cx). B, GRK2 immunoblot demonstrating the cDNA transfection-dependent increase in GRK2 expression over basal expression in HEK293 cells. The attenuation of quisqualate-stimulated mGluR1a signaling in the presence of increasing expression levels (0, 0.1, 1, and 10 g) of either GRK2 (C) or GRK2-D110A (D). E, the expression-dependent effects of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A on the V max for mGluR1a-stimulated IP formation. F, the expression-dependent effects of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A on mGluR1a basal IP formation. E and F, HEK293 cells were transfected with 10 g of pcDNA3 plasmids for FLAG-mGluR1a and 0, 0.03, 0.1, 0.3, 1, 3, and 10 g of either GRK2 or GRK2 RH domain mutants. Data points represent the mean Ϯ S.D. for three to six independent experiments. The agonist dose-response curves were fit and analyzed using GraphPad Prism software. expression levels on the same blot (compare Fig 2A with B). In contrast, the V max for quisqualate-stimulated IP formation is inhibited only after transfection of HEK293 cells with 3 and 10 g of GRK2-D110A (Fig. 2, D and E). When GRK2 expression is plotted versus the V max for agonist-stimulated mGluR1a IP formation, an expression (dose)-dependent reduction in the V max for mGluR1a-stimulated IP formation is observed (Fig.  2E). GRK2-D110A, GRK2-R106A, and GRK2-M114A also reduce mGluR1a-stimulated IP formation in an expression-dependent manner but to a far lesser extent than what is observed for wild-type GRK2 (Fig. 2E). Basal mGluR1a IP formation is also attenuated by GRK2 but is unaffected by the expression of GRK2 mutants defective in G␣ q/11 binding (Fig.  2F). The half-maximal effective concentration (EC 50 ) for GRK2 expression-dependent attenuation of agonist-stimulated and basal mGluR1a signaling are identical (EC 50 ϭ 0.9 g cDNA). Thus, GRK2 interactions with G␣ q/11 account for GRK2 phosphorylation-independent adaptation of mGluR1a signaling at expression levels that are physiologically relevant to mGluR1a signaling in vivo.
GRK2, but not GRK2-D110A, GRK2-R106A, and GRK2-M114A, binds to G␣ q/11 in an AlF 4 Ϫ -dependent manner (Fig.  3A). However, the capacity of each of the GRK2 G␣ q/11 binding mutants to associate with mGluR1a is indistinguishable from wild-type GRK2 (Fig 3, B and C). G␣ q/11 is also co-immunoprecipitated as a complex with mGluR1a (Fig. 4A), and mGluR1a/ G␣ q/11 complex formation is reduced 64 Ϯ 12% by the treatment of cell lysates with AlF 4 Ϫ (Fig. 4B). GRK2 overexpression reduces the co-immunoprecipitation of G␣ q/11 with mGluR1a in the absence of AlF 4 Ϫ treatment, and mGluR1a/G␣ q/11 interactions are no longer regulated by AlF 4 Ϫ treatment (Fig. 4, A and  B). In contrast, either GRK2-D110A, GRK2-R106A, or GRK2-M114A expression does not prevent the formation of mGluR1a/ G␣ q/11 complexes, and these complexes retain the capacity to be regulated by AlF 4 Ϫ treatment (Fig. 4, A and B). Similar to AlF 4 Ϫ treatment, agonist promotes the dissociation of mGluR1a/ G␣ q/11 complexes, and GRK2 but not GRK2-D110A expression prevents the co-immunoprecipitation of G␣ q/11 with mGluR1a in the absence of agonist (Fig. 4, C and D). Because it is possible that GRK2-mediated disruption of mGluR1a/G␣ q/11 complexes is simply the consequence of the nonspecific sequestration of G␣ q/11 after GRK2 overexpression, we have examined whether RGS4 overexpression would also disrupt the association of G␣ q/11 with mGluR1a. The overexpression of RGS4, an RGS protein previously shown to regulate mGluR1a/G␣ q/11 signaling (20), did not antagonize G␣ q/11 co-immunoprecipitation with mGluR1a when compared with control (Fig. 4, E and F). Based upon the recently solved crystal structure for GRK2 (9), we hypothesized that residues within the ␣11 helix of the second discontinuous segment of the GRK2 RH domain might be appropriately oriented to interact with the intracellular face of mGluR1a. Consequently, we created a series of mutations (D527A, T528A, A531E, and E532A) within the ␣11 helix domain of the GRK2 RH domain and tested their capacity to attenuate basal and agonist-stimulated mGluR1a signaling. When overexpressed in HEK293 cells, GRK2-T528A, GRK2-A531E, and GRK2-E532A were indistinguishable from wildtype GRK2 in their capacity to attenuate either agonist-stim- Ϫ , as well as the expression of both GRK2 and G␣ q/11 in 100 g of corresponding HEK293 cell lysates. Data shown are representative of four independent experiments. B, co-immunoprecipitation of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A with mGluR1a. A representative immunoblot for wild-type and mutant GRK2 immunoprecipitation with mGluR1a is shown, as well as the expression of both mGluR1a and GRK2 in 100 g of corresponding HEK293 cell lysates. C, densitometric analysis of autoradiographs showing the mean Ϯ S.D. of three independent experiments examining the co-immunoprecipitation of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A with mGluR1a. In these experiments, the data are normalized to GRK2 co-immunoprecipitated with mGluR1a. HEK293 cells were transfected with 10 g of pcDNA3 plasmids encoding mGluR1a with and without 10 g of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. ulated (Fig. 5A) or basal (Fig. 5B) mGluR1a IP formation. In contrast, GRK2-D527A overexpression, at levels comparable with wild-type GRK2, GRK2-T528A, GRK2-A531E, and GRK2-E532A (Fig. 5C), did not attenuate quisqualate-stimulated or basal IP formation when compared with control mGluR1aexpressing HEK293 cells (Fig. 5, A and B). Identical to what was observed for wild-type GRK2, GRK2-D527A and each of the other ␣10 and ␣11 helix RH domain mutants bind to G␣ q/11 in an AlF 4 Ϫ -dependent manner (Fig. 5D). However, only GRK2-D527A exhibited reduced capacity (30 Ϯ 7% versus control) for co-immunoprecipitation with FLAG-mGluR1a from HEK293 cells (Fig. 5E), consistent with the inability of this mutant to attenuate mGluR1a signaling (Fig. 5, A and B). Thus, we con-clude that the phosphorylation-independent desensitization of mGluR1a was not the consequence of the nonspecific sequestration of G␣ q/11 after GRK2 overexpression.
The ability of the GRK2 RH domain to regulate mGluR1a adaptation is consistent with the GRK2 crystal structure-based model for receptor/G␣ q/11 /G␤␥ interactions (9). The GRK crystal structure predicts that G␤␥, G␣ q/11 , and receptor binding to GRK2 can be accommodated simultaneously by distinct vertices of a "triangle" formed by the three-dimensional structure of GRK2 (9). Thus, GRK2 receptor binding may appropriately orient the GRK2 RH domain to regulate mGluR1a/G␣ q/11 interactions. Specifically, our studies suggest that in its inactive conformation (Fig. 6A, R), mGluR1a forms a complex with FIG. 4. Effect of GRK2 on mGluR1a/G␣ q/l1 complexes. A, representative immunoblot demonstrating the co-immunoprecipitation of G␣ q/11 with mGluR1a in the absence and presence of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A. Also shown is G␣ q/11 expression in 100 g of corresponding HEK293 cell lysates, GRK2 co-immunoprecipitated with mGluR1a in the same experiment, and GRK2 expression in 100 g of corresponding HEK293 cell lysates. B, densitometric analysis of autoradiographs showing the mean Ϯ S.D. of six independent experiments examining the co-immunoprecipitation of G␣ q/11 with and without GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A in either the absence or presence of AlF 4 Ϫ . C, representative immunoblot demonstrating the co-immunoprecipitation of G␣ q/11 with mGluR1a prior to and after treatment of cells with 30 M quisqualate for 10 min in the absence and presence of either GRK2 or GRK2-D110A. Cells were transfected with 10 g of pcDNA3 plasmid for mGluR1a with and without 10 g of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. D, densitometric analysis of autoradiographs showing the mean Ϯ S.D. of six independent experiments examining the co-immunoprecipitation of G␣ q/11 in the presence and absence of either GRK2 or GRK2-D110A either prior to or after the treatment of cells with 30 M quisqualate for 10 min. E, representative immunoblot demonstrating the co-immunoprecipitation of G␣ q/11 with mGluR1a prior to and after treatment of cells with 30 M quisqualate for 10 min in the absence and presence of GFP-RGS4. Cells were transfected with 10 g of pcDNA3 plasmid for mGluR1a with and without 10 g of pcDNA3 plasmid for green fluorescent protein (GFP)-tagged RGS4. F, densitometric analysis of autoradiographs showing the mean Ϯ S.D. of three independent experiments examining the co-immunoprecipitation of G␣ q/11 in the presence and absence of GFP-RGS4 either prior to or after the treatment of cells with 30 M quisqualate for 10 min. *, p Ͻ 0.05 versus control in the absence of either AlF 4 Ϫ or agonist, as determined by analysis of variance and posthoc statistical analysis.
GDP-bound G␣ q/11 (Fig. 6A). The ability of mGluR1a to preferentially interact with G␣ q/11 in the GDP-bound state parallels what is observed for small G protein interactions with their cognate guanine nucleotide exchange factors (21). The spontaneous transition of the intrinsically active mGluR1a from an inactive (Fig. 6A, R) to an active (Fig. 6A, R*) conformation both promotes the dissociation of G␣ q/11 from the G␤␥ dimer and recruits GRK2 to bind the activated mGluR1a (14). Membrane translocation of cytosolic GRK2 is facilitated by the association of the GRK2 PH domain with the G␤␥ subunit (22), and GRK2 binding to the receptor is mediated in part by residue 527 within the ␣11 helix of the GRK2 RH domain, in addition to residues localized between amino acid residues 45 and 178 of GRK2 (12).
The GRK2 RH domain fulfills two functions in uncoupling mGluR1a signaling. First, GRK2 disrupts receptor/G␣ q/11 interactions by means of the association of GRK2 with G␣ q/11 at its C site, a surface that requires the ␣5 and ␣6 helices of the GRK2 RH domain (9,10,15). Second, GRK2 binding to the receptor by means of the GRK2 RH domain ␣11 helix prevents the re-association of GDP-bound G␣ q/11 /receptor complexes (Fig. 6C). The GRK2 crystal structure suggests that the C site and the ␣11 helix are spatially distinct and could independently bind mGluR1a and G␣ q/11 (9). Thus, GRK2 bound to the receptor prevents subsequent rounds of receptor-catalyzed GDP for GTP exchange on G␣ q/11 , thereby attenuating mGluR1a signaling. This exclusion of GDP-bound G␣ q/11 from the receptor/GRK2 complex may also require coordinated interactions between GRK2, the receptor, and the G␤␥ subunit to prevent G protein subunit re-association (Fig. 6C).
In agreement with previous observations (11), this mechanism of receptor desensitization does not require ␤-arrestin binding to the receptor. The observation that GRK2-mediated mGluR1a adaptation is impaired by the overexpression of the Ϫ . Lower panel, densitometric analysis of autoradiographs from four independent experiments presented as the mean Ϯ S.D. of G␣ q/11 co-immunoprecipitated with GRK2 mutants in the presence of AlF 4 Ϫ normalized to wild-type GRK2. E, upper panel, representative immunoblot for wild-type GRK2, GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A co-immunoprecipitation with mGluR1a. Lower panel, densitometric analysis of autoradiographs from six independent experiments presented as the mean Ϯ S.D. of mutant GRK2 co-immunoprecipitated with mGluR1a normalized to wild-type GRK2. HEK293 cells were transfected with 10 g of pcDNA3 plasmids encoding mGluR1a with and without 10 g of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. The agonist dose-response curves were fit and analyzed using GraphPad Prism software. *, p Ͻ 0.05 versus control, as determined by analysis of variance and posthoc statistical analysis.
GRK2 PH domain is also consistent with the notion that the binding of endogenous GRK2 to the receptor is essential to attenuate mGluR1a activity (14). Moreover, GRK2 mutants impaired in G␣ q/11 binding do not effectively mediate mGluR1a adaptation at physiologically relevant expression levels by virtue of their inability to promote the dissociation of mGluR1a/ G␣ q/11 complexes. As a consequence, functional GRK2 RH domain interactions with both G␣ q/11 and the receptor, but not GRK2-mediated receptor phosphorylation (12), are essential to mediate the adaptation of both spontaneous and agonist-stimulated mGluR1a signaling. This provides a novel alternative mechanism to the widely accepted paradigm for GPCR adaptation that links GRK-mediated phosphorylation to GPCR desensitization (23). The identification of GRK2 mutants that show a selective loss in either receptor binding or G␣ q/11 bind-ing that upon overexpression do not attenuate mGluR1a signaling strongly support this alternative model for GRK2 phosphorylation-independent desensitization.
Because all GRK isoforms encode RH domains (9), it is possible that RH domains encoded by other GRK family members, in the context of receptor binding, may also exhibit the capacity to modulate GPCR coupling to other G protein subtypes. For example, the phosphorylation-independent regulation of GABA B receptor activity by GRK4 may involve GABA B receptordependent GRK4 RH domain interactions with G␣ i (13). Thus, GRK family RH domains may regulate the adaptation of other GPCRs for which phosphorylation-independent GRK2-mediated desensitization has been reported, such as the parathyroid hormone receptor and endothelin A and B receptors (24,25). In conclusion, the role of GRK2 in regulating GPCR adaptation extends beyond its role as a receptor kinase to involve a novel GTPase-activating protein activity.
FIG. 6. Schematic representation of the proposed model for GRK2 RH domain-mediated mGluR1a adaptation. A, in the inactive state (R), the receptor exists as a complex with the GDP-bound G␣ q/11 heterotrimeric G protein. B, spontaneous transition of the mGluR1a from an inactive to an active (R*) conformation promotes GDP-GTP exchange on the G␣ q/11 subunit, promoting the dissociation of the G protein heterotrimer and facilitating the recruitment of GRK2 to the receptor. This likely allows the association of both the GRK2 RH domain with GTP-bound G␣ q/11 and the GRK2 PH domain with G␤␥. The GRK2 RH domain functions in concert with phospholipase C␤ and RGS proteins to inactivate mGluR1a-stimulated G␣ q/11 signaling. C, interaction between GRK2, the receptor, and the G␤␥ subunit prevents the re-association of GDP-bound G␣ q/11 with the receptor/GRK2/G␤␥ complex, preventing receptor-mediated GDP-GTP exchange on G␣ q/11 , thereby attenuating mGluR1a signaling. PLC␤, phospholipase C␤; P i , free phosphate.