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Originally published In Press as doi:10.1074/jbc.M314090200 on February 4, 2004

J. Biol. Chem., Vol. 279, Issue 16, 16614-16620, April 16, 2004
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G Protein-coupled Receptor Kinase 2 Regulator of G Protein Signaling Homology Domain Binds to Both Metabotropic Glutamate Receptor 1a and G{alpha}q to Attenuate Signaling*

Gurpreet K. Dhami{ddagger}§, Lianne B. Dale{ddagger}, Pieter H. Anborgh{ddagger}, Katharine E. O'Connor-Halligan||, Rachel Sterne-Marr||, and Stephen S. G. Ferguson{ddagger}**

From the {ddagger}Cell Biology Research Group, Robarts Research Institute, §Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5K8, Canada and ||Biology Department, Siena College, Loudonville, New York 12211

Received for publication, December 23, 2003 , and in revised form, January 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 G{alpha}q/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. G{alpha}q/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/G{alpha}q/11 complex. However, these mGluR1a/G{alpha}q/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in G{alpha}q/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds G{alpha}q/11 in an -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 G{alpha}q/11 and mGluR1a. This serves to coordinate GRK2 interactions with G{alpha}q/11 and to disrupt receptor/G{alpha}q/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 (1-3). Agonist-activated GPCRs function as guanine nucleotide exchange factors facilitating GDP-for-GTP exchange on the G protein {alpha} subunit thus allowing the functional dissociation of the G{alpha} and G{beta}{gamma} 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 (1-3). 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 (5-7). 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{beta}{gamma}-binding pleckstrin homology (PH) domain (8-10). The GRK2 RH domain consists of two discontinuous segments, the first of which forms the {alpha}-helical bundles characteristic of RGS proteins (8-10). The GRK2 and GRK3 RH domains specifically interact with G{alpha}q/11 family proteins in an -dependent manner and may function as weak GTPase activating proteins for G{alpha}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 {gamma}-aminobutyric acid, type B (GABA)B receptor; for these receptors, it involves an atypical arrestin-independent mechanism (11-13). 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 {beta}{gamma}-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 {alpha}5 helix of the GRK2 RH domain prevents GRK2 binding to G{alpha}q/11 (15). Therefore, we tested the hypothesis that GRK2 mutants impaired in G{alpha}q/11 binding are unable to antagonize both basal and agonist-stimulated mGluR1a signaling. We find that G{alpha}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{alpha}q/11 complex. However, receptor/G{alpha}q/11 complex formation is not altered by GRK2 mutants impaired in G{alpha}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{alpha}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{alpha}q/11 and mGluR1a.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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. [3H]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.

Plasmid Construction—The FLAG-mGluR1a and GRK2 RH domain mutant constructs were described previously (12, 14, 15). Amino-terminal myc-tagged wild-type GRK2, GRK2-R106A, GRK2-D110, GRK2-M114A, GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A mutants were constructed by PCR amplification and mutagenesis of bovine GRK2 mutant cDNA and inserted into the BamHI/PmlI site of pcDNA3-GRK2. The sequence integrity of each of the mutants was confirmed by automated DNA sequencing.

Cell Culture and Transfection—HEK293 cells were grown at 37 °Cin Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/µl 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 µCi/ml [3H]myo-inositol in Dulbecco's modified Eagle's medium. Un-incorporated [3H]myo-inositol was removed by washing the cells with HBSS (116 mM NaCl, 20 mM HEPES, 11 mM glucose, 5 mM NaHCO3, 4.7 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.2 mM KH2PO4, pH 7.4). The cells were pre-incubated for 1 h in HBSS at 37 °C and then pre-incubated in 500 µl 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 µM) of quisqualate for 30 min at 37 °C. The reaction was stopped on ice by adding 400 µl of perchloric acid and then it was neutralized with 500 µl of 0.72 M KOH and 0.6 M KHCO3. The total [3H]inositol incorporated into the cells was determined by counting the radioactivity present in 50 µl 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. [3H]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 µl of cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X-100 containing protease inhibitors, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 20 µg/ml phenylmethylsulfonyl fluoride) in either the absence or presence of 100 µM GDP, 2 mM MgSO4, 30 µM aluminum chloride, and 5 mM sodium fluoride. The particulate fraction was removed by centrifugation, and 500 µg of supernatant protein was incubated with 5 µl of either anti-FLAG or anti-Myc monoclonal antibody and 100 µl of 20% protein G-Sepharose beads or 40 µl 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 semi-dry 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{alpha}q/11-(Santa Cruz Biotechnology) and mGluR1a-specific antibodies (Upstate Biotechnology) diluted 1:1000 in wash buffer containing 3% skim milk. The membranes 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Because expression of the GRK2 RH domain alone effectively attenuates mGluR1a signaling (12), we tested whether GRK2 mutants defective in G{alpha}q/11 binding (R106A, D110A, and M114A) might be unable to antagonize both basal and agonist-stimulated mGluR1a signaling (15). Consistent with our previous studies (11, 12, 14), wild-type GRK2 expression attenuated the maximum velocity (Vmax) for quisqualate-stimulated inositol phosphate (IP) formation to 20 ± 1% of control levels in human embryonic kidney (HEK293) cells (Fig. 1A). GRK2 mutants defective in G{alpha}q/11 binding (D110A, R106A, and M114A) exhibit a diminished capacity to reduce the Vmax for quisqualate-stimulated IP formation when compared with wild-type GRK2 (Fig. 1B). In contrast, GRK2 mutants (E84A and K90A) with substitutions of amino acid residues within the GRK2 RH domain {alpha}4 helix, a helix not essential for G{alpha}q/11 interactions (15), are indistinguishable from wild-type GRK2 in their ability to antagonize mGluR1a signaling (Fig. 1B). Residual mGluR1a signaling observed in cells expressing GRK2-D110A is not attributable to GRK2 kinase activity, because the ability of a kinase-deficient GRK2-D110A/K220R double-point mutant to attenuate mGluR1a signaling is indistinguishable from GRK2-D110A (compare Fig. 1A with B). mGluR1a exhibits substantial agonist-independent basal activity (12, 14, 16) and GRK2, GRK2-E84A, and GRK2-K90A expression significantly attenuates basal mGluR1a activity to 34 ± 7%, 27 ± 4%, and 35 ± 9% of control levels, respectively (Fig. 1C). However, GRK2 G{alpha}q/11 binding mutant overexpression has no effect on basal mGluR1a activity (Fig. 1C). Thus, GRK2 interactions with G{alpha}q/11 are essential for regulating both spontaneous and agonist-stimulated mGluR1a activity.



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  		FIG. 1.
Effect of GRK2 RH domain mutants on agonist-stimulated and basal mGluR1a activity. A, quisqualate dose-responses for FLAG-mGluR1a-stimulated IP formation in HEK293 cells in the presence of GRK2 and the GRK2-D110A, GRK2-R106A, and GRK2-M114A RH domain mutants. B, agonist dose-responses for FLAG-mGluR1a-stimulated IP formation in the presence of GRK2-D110A/K220R, GRK2-E84A, and GRK2-K90A. For comparison, control and GRK2 quisqualate dose-response curves are the same as those presented in A. C, the effect of GRK2, GRK2-D110A, GRK2-R106A, GRK2-M114A, GRK2-D110A/K220R, GRK2-E84A, and GRK2-K90A upon basal mGluR1 activity in HEK293 cells. HEK293 cells were transfected with 10 µg of pcDNA3 plasmids encoding mGluR1a with and without 10 µg of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. The data points represent the mean ± 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 Vmax for mGluR1a-mediated IP formation (Fig. 2B). As illustrated in Fig. 2C, the Vmax 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 µg of transfected cDNA) that are similar to endogenous GRK2 levels in mGluR1a-expressing regions of rat brain, when compared with endogenous HEK293 cell GRK2 expression levels on the same blot (compare Fig 2A with B). In contrast, the Vmax for quisqualate-stimulated IP formation is inhibited only after transfection of HEK293 cells with 3 and 10 µg of GRK2-D110A (Fig. 2, D and E). When GRK2 expression is plotted versus the Vmax for agonist-stimulated mGluR1a IP formation, an expression (dose)-dependent reduction in the Vmax 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{alpha}q/11 binding (Fig. 2F). The half-maximal effective concentration (EC50) for GRK2 expression-dependent attenuation of agonist-stimulated and basal mGluR1a signaling are identical (EC50 = 0.9 µg cDNA). Thus, GRK2 interactions with G{alpha}q/11 account for GRK2 phosphorylation-independent adaptation of mGluR1a signaling at expression levels that are physiologically relevant to mGluR1a signaling in vivo.



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  		FIG. 2.
Wild-type and GRK2 mutant expression level-dependent effects upon agonist-stimulated and basal mGluR1 signaling. A, relative GRK2 expression in 100 µg 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 µg) of either GRK2 (C) or GRK2-D110A (D). E, the expression-dependent effects of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A on the Vmax 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 µg of pcDNA3 plasmids for FLAG-mGluR1a and 0, 0.03, 0.1, 0.3, 1, 3, and 10 µg of either GRK2 or GRK2 RH domain mutants. Data points represent the mean ± S.D. for three to six independent experiments. The agonist dose-response curves were fit and analyzed using GraphPad Prism software.

 
GRK2, but not GRK2-D110A, GRK2-R106A, and GRK2-M114A, binds to G{alpha}q/11 in an -dependent manner (Fig. 3A). However, the capacity of each of the GRK2 G{alpha}q/11 binding mutants to associate with mGluR1a is indistinguishable from wild-type GRK2 (Fig 3, B and C). G{alpha}q/11 is also co-immunoprecipitated as a complex with mGluR1a (Fig. 4A), and mGluR1a/G{alpha}q/11 complex formation is reduced 64 ± 12% by the treatment of cell lysates with (Fig. 4B). GRK2 overexpression reduces the co-immunoprecipitation of G{alpha}q/11 with mGluR1a in the absence of treatment, and mGluR1a/G{alpha}q/11 interactions are no longer regulated by 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{alpha}q/11 complexes, and these complexes retain the capacity to be regulated by treatment (Fig. 4, A and B). Similar to treatment, agonist promotes the dissociation of mGluR1a/G{alpha}q/11 complexes, and GRK2 but not GRK2-D110A expression prevents the co-immunoprecipitation of G{alpha}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{alpha}q/11 complexes is simply the consequence of the nonspecific sequestration of G{alpha}q/11 after GRK2 overexpression, we have examined whether RGS4 overexpression would also disrupt the association of G{alpha}q/11 with mGluR1a. The overexpression of RGS4, an RGS protein previously shown to regulate mGluR1a/G{alpha}q/11 signaling (20), did not antagonize G{alpha}q/11 co-immunoprecipitation with mGluR1a when compared with control (Fig. 4, E and F).



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  		FIG. 3.
Co-immunoprecipitation of GRK2 with mGluR1a and G{alpha}q/11. A, representative immunoblot for co-immunoprecipitation of G{alpha}q/11 with GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A in the presence of , as well as the expression of both GRK2 and G{alpha}q/11 in 100 µg 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 µg of corresponding HEK293 cell lysates. C, densitometric analysis of autoradiographs showing the mean ± 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 µg of pcDNA3 plasmids encoding mGluR1a with and without 10 µg of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs.

 



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  		FIG. 4.
Effect of GRK2 on mGluR1a/G{alpha}q/l1 complexes. A, representative immunoblot demonstrating the co-immunoprecipitation of G{alpha}q/11 with mGluR1a in the absence and presence of GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A. Also shown is G{alpha}q/11 expression in 100 µg of corresponding HEK293 cell lysates, GRK2 co-immunoprecipitated with mGluR1a in the same experiment, and GRK2 expression in 100 µg of corresponding HEK293 cell lysates. B, densitometric analysis of autoradiographs showing the mean ± S.D. of six independent experiments examining the co-immunoprecipitation of G{alpha}q/11 with and without GRK2, GRK2-D110A, GRK2-R106A, and GRK2-M114A in either the absence or presence of . C, representative immunoblot demonstrating the co-immunoprecipitation of G{alpha}q/11 with mGluR1a prior to and after treatment of cells with 30 µM quisqualate for 10 min in the absence and presence of either GRK2 or GRK2-D110A. Cells were transfected with 10 µg of pcDNA3 plasmid for mGluR1a with and without 10 µg of pcDNA3 plasmids for either wild-type or mutant GRK2 constructs. D, densitometric analysis of autoradiographs showing the mean ± S.D. of six independent experiments examining the co-immunoprecipitation of G{alpha}q/11 in the presence and absence of either GRK2 or GRK2-D110A either prior to or after the treatment of cells with 30 µM quisqualate for 10 min. E, representative immunoblot demonstrating the co-immunoprecipitation of G{alpha}q/11 with mGluR1a prior to and after treatment of cells with 30 µM quisqualate for 10 min in the absence and presence of GFP-RGS4. Cells were transfected with 10 µg of pcDNA3 plasmid for mGluR1a with and without 10 µg of pcDNA3 plasmid for green fluorescent protein (GFP)-tagged RGS4. F, densitometric analysis of autoradiographs showing the mean ± S.D. of three independent experiments examining the co-immunoprecipitation of G{alpha}q/11 in the presence and absence of GFP-RGS4 either prior to or after the treatment of cells with 30 µM quisqualate for 10 min. *, p < 0.05 versus control in the absence of either or agonist, as determined by analysis of variance and posthoc statistical analysis.

 
Based upon the recently solved crystal structure for GRK2 (9), we hypothesized that residues within the {alpha}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 {alpha}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 wild-type GRK2 in their capacity to attenuate either agonist-stimulated (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 mGluR1a-expressing HEK293 cells (Fig. 5, A and B). Identical to what was observed for wild-type GRK2, GRK2-D527A and each of the other {alpha}10 and {alpha}11 helix RH domain mutants bind to G{alpha}q/11 in an -dependent manner (Fig. 5D). However, only GRK2-D527A exhibited reduced capacity (30 ± 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 conclude that the phosphorylation-independent desensitization of mGluR1a was not the consequence of the nonspecific sequestration of G{alpha}q/11 after GRK2 overexpression.



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  		FIG. 5.
Effect of mutations in the {alpha}10 and {alpha}11 helices of GRK2 RH domain on mGluR1a signaling. A, quisqualate dose-responses for FLAG-mGluR1a-stimulated IP formation in HEK293 cells in the presence of wild-type GRK2 and the {alpha}10 and {alpha}11 GRK2 RH domain mutants GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A. B, the effect of GRK2, GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A on basal mGluR1 activity in HEK293 cells. C, lysates (100 µg) from HEK293 cells demonstrating equivalent GRK2, GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A overexpression. D, upper panel, representative immunoblot demonstrating the co-immunoprecipitation of G{alpha}q/11 with GRK2, GRK2-D527A, GRK2-T528A, GRK2-A531E, and GRK2-E532A in the presence of . Lower panel, densitometric analysis of autoradiographs from four independent experiments presented as the mean ± S.D. of G{alpha}q/11 co-immunoprecipitated with GRK2 mutants in the presence of 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 ± S.D. of mutant GRK2 co-immunoprecipitated with mGluR1a normalized to wild-type GRK2. HEK293 cells were transfected with 10 µg of pcDNA3 plasmids encoding mGluR1a with and without 10 µg 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 < 0.05 versus control, as determined by analysis of variance and posthoc statistical analysis.

 
The ability of the GRK2 RH domain to regulate mGluR1a adaptation is consistent with the GRK2 crystal structure-based model for receptor/G{alpha}q/11/G{beta}{gamma} interactions (9). The GRK crystal structure predicts that G{beta}{gamma}, G{alpha}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{alpha}q/11 interactions. Specifically, our studies suggest that in its inactive conformation (Fig. 6A, R), mGluR1a forms a complex with GDP-bound G{alpha}q/11 (Fig. 6A). The ability of mGluR1a to preferentially interact with G{alpha}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{alpha}q/11 from the G{beta}{gamma} 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{beta}{gamma} subunit (22), and GRK2 binding to the receptor is mediated in part by residue 527 within the {alpha}11 helix of the GRK2 RH domain, in addition to residues localized between amino acid residues 45 and 178 of GRK2 (12).



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  		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{alpha}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{alpha}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{alpha}q/11 and the GRK2 PH domain with G{beta}{gamma}. The GRK2 RH domain functions in concert with phospholipase C{beta} and RGS proteins to inactivate mGluR1a-stimulated G{alpha}q/11 signaling. C, interaction between GRK2, the receptor, and the G{beta}{gamma} subunit prevents the re-association of GDP-bound G{alpha}q/11 with the receptor/GRK2/G{beta}{gamma} complex, preventing receptor-mediated GDP-GTP exchange on G{alpha}q/11, thereby attenuating mGluR1a signaling. PLC{beta}, phospholipase C{beta}; Pi, free phosphate.

 
The GRK2 RH domain fulfills two functions in uncoupling mGluR1a signaling. First, GRK2 disrupts receptor/G{alpha}q/11 interactions by means of the association of GRK2 with G{alpha}q/11 at its C site, a surface that requires the {alpha}5 and {alpha}6 helices of the GRK2 RH domain (9, 10, 15). Second, GRK2 binding to the receptor by means of the GRK2 RH domain {alpha}11 helix prevents the re-association of GDP-bound G{alpha}q/11/receptor complexes (Fig. 6C). The GRK2 crystal structure suggests that the C site and the {alpha}11 helix are spatially distinct and could independently bind mGluR1a and G{alpha}q/11 (9). Thus, GRK2 bound to the receptor prevents subsequent rounds of receptor-catalyzed GDP for GTP exchange on G{alpha}q/11, thereby attenuating mGluR1a signaling. This exclusion of GDP-bound G{alpha}q/11 from the receptor/GRK2 complex may also require coordinated interactions between GRK2, the receptor, and the G{beta}{gamma} subunit to prevent G protein subunit re-association (Fig. 6C).

In agreement with previous observations (11), this mechanism of receptor desensitization does not require {beta}-arrestin binding to the receptor. The observation that GRK2-mediated mGluR1a adaptation is impaired by the overexpression of the 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{alpha}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{alpha}q/11 complexes. As a consequence, functional GRK2 RH domain interactions with both G{alpha}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{alpha}q/11 binding 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 GABAB receptor activity by GRK4 may involve GABAB receptor-dependent GRK4 RH domain interactions with G{alpha}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.


   FOOTNOTES
 
* 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. Back

Recipient of a CIHR doctoral training award. Back

** Holder of a Canada Research Chair in Molecular Neuroscience, recipient of Premier's Research Excellence Award, and Career Investigator of the Heart and Stroke Foundation of Ontario. To whom correspondence should be addressed: Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, Ontario N6A 5K8, Canada. Tel.: 519-663-3825; Fax: 519-663-3314; E-mail: ferguson{at}robarts.ca.

1 The abbreviations used are: GPCR, G protein-coupled receptor; G, guanine nucleotide-binding; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; RH, RGS homology; PH, pleckstrin homology; GABAB, {gamma}-aminobutyric acid, type B; mGluR, metabotropic glutamate receptor; HEK293 cells, human embryonic kidney cells; TBS, Tris-buffered saline; IP, inositol phosphate. Back


   ACKNOWLEDGMENTS
 
We thank J. Tesmer for sharing GRK2 co-ordinates and for constructive discussions, S. Nakanishi for providing the cDNA for mGluR1a, T. Dobransky for affinity-purifying the GRK2 antibody, as well as J. Cilente and M. Ragusa for assistance in the preparation of GRK2 constructs.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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