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J. Biol. Chem., Vol. 277, Issue 28, 25266-25272, July 12, 2002

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Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor Signaling by G Protein-coupled Receptor Kinase 2^*

Gurpreet Kaur Dhami^§, Pieter H. Anborgh, Lianne B. Dale, Rachel Sterne-Marr^¶, and Stephen S. G. Ferguson^§**

From the  Cell Biology Research Group, the John P. Robarts Research Institute, Departments of ^§ Pharmacology and Toxicology,  Physiology, and ^** Medicine, University of Western Ontario, Ontario N6A 5K8, Canada and the ^¶ Biology Department, Siena College, Loudonville, New York 12211

Received for publication, April 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, -arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Glutamate is the principal excitatory transmitter within the vertebrate central nervous system and is essential in the regulation of brain functions such as memory and learning as well as neural development (1, 2). The receptors for glutamate are divided into two types: ionotropic and metabotropic. The ionotropic receptors are subdivided into N-methyl-D-asparate, amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate type cation channels. The metabotropic glutamate receptors (mGluRs)¹ are members of the G protein-coupled receptor (GPCR) superfamily and consist of eight distinct subtypes (1-4). The mGluR subtypes can be divided into three groups based on their sequence homology, pharmacology, and G protein coupling specificity. Group I receptors (mGluR1 and mGluR5) are coupled via the heterotrimeric G protein G_q to the activation of phospholipase C, resulting in the formation of both inositol triphosphate and diacylglycerol, as well as increases in intracellular Ca²⁺ concentrations (1-4).

The overactivation of group I mGluRs has been implicated in glutamate-mediated neurotoxicity associated with acute brain ischemia and neurotrauma (5-7). The feedback mechanism that protects against overstimulation is desensitization, a process that is initiated shortly following GPCR activation by an agonist (8, 9). GPCR desensitization involves multiple mechanisms, the most rapid of which is thought to be receptor phosphorylation by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) (8, 9). For many GPCRs, GRK phosphorylation promotes receptor binding of proteins called arrestins that function to sterically interdict receptor G protein coupling (8, 9).

Similar to what is observed for other GPCRs, the desensitization of group I mGluRs is also mediated by both second messenger-dependent protein kinases (e.g. protein kinase C) and GRKs (10-13). Co-expression of either GRK2 or GRK5 with mGluR1a results in the attenuation of both constitutive and agonist-stimulated mGluR1a activity in human embryonic kidney (HEK) 293 cells (12). A similar effect is also observed in primary cell cultures of Purkinje cells, where GRK4 was demonstrated to be essential for mediating mGluR1a desensitization (13). The attenuation of mGluR1a signaling by GRK2, GRK4, and GRK5 was interpreted as being associated with mGluR1a phosphorylation (12, 13). In particular, the expression of the C-terminal domain of GRK2 to inhibit GRK2 membrane translocation through its association with the subunit of the heterotrimeric G protein blocks GRK2-mediated mGluR1a phosphorylation and augmented mGluR1a signaling (12). However, inconsistent with the idea that phosphorylation and -arrestins contribute to mGluR1a desensitization, -arrestin binding was not required for mGluR1a desensitization, and the expression of a kinase-deficient GRK2-K220R mutant attenuated mGluR1a signaling (12, 14).

In the present study, we examined whether GRK2-mediated phosphorylation of serine and threonine residues localized within the extremely long C-terminal tail (C-tail) of mGluR1a contributed to mGluR1a G protein uncoupling by GRK2. In addition, we tested whether GRK2 plays a role in the attenuation of the activity of an mGluR1 alternative splice variant (mGluR1b) that lacks an extended C-tail. We found that GRK2 contributed to the desensitization of both mGluR1a and mGluR1b. However, GRK2-mediated phosphorylation was not required for effective inactivation of either mGluR1a or mGluR1b signaling, and the last 333 amino acids of the mGluR1a C-terminal tail were not required for the GRK2 binding. Furthermore, we found that expression of the N-terminal domain of GRK2, which exhibits homology to the regulator G protein signaling (RGS) proteins, associated with the receptor and was sufficient to reduce both constitutive and agonist-stimulated mGluR1a signaling. Taken together, our observations suggest that GRK2-mediated phosphorylation is not required for GRK2-dependent regulation of mGluR1a activity but rather involves the interaction of the GRK2 RGS homology domain with the receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- HEK 293 cells were obtained from the American Tissue Culture Collection. Inositol-free Dulbecco's modified Eagle's medium, minimal essential medium, fetal bovine serum, trypsin, and gentamicin were from Invitrogen. The rabbit polyclonal GRK2 antibody was raised against the peptide sequence DRLEARKKAKNKQLGH, which corresponds to rat GRK2 (12). Donkey anti-rabbit IgG conjugated to horseradish peroxidase, ECL Western blotting detection reagents, and the protein G-Sepharose beads were from Amersham Biosciences. [³²P]Orthophosphate was from ICN. 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 C-tail truncation mutants of FLAG-mGluR1a were engineered using standard PCR techniques by introducing a stop codon following alanine residue 1149 (1149), glycine residue 1049 (1049), threonine residue 1000 (1000), threonine residue 953 (953), and proline residue 866 (866). Hemagglutinin (HA)-tagged GRK2 (1-190) and GRK2 (45-185) were constructed by PCR amplification of bovine GRK2 cDNA and inserted into the BamHI/EcoRI site of pcDNA3-HA (15). The sequence integrity of each of the mutants was confirmed by automated DNA sequencing.

Cell Culture and Transfection-- HEK 293 cells were grown at 37 °C in minimal essential medium supplemented with 10% fetal bovine serum, 100 µg/µl gentamicin. The cells were transfected with plasmid cDNAs using a modified calcium phosphate method as described previously (12). Following transfection (~18 h), the cells were incubated with fresh medium, allowed to recover for 6-8 h, and then grown an additional 18 h prior to experimentation.

Inositol Phosphate Formation-- HEK 293 cells transiently transfected with 5 µg of FLAG-mGluR1 constructs and 5 µg of either empty plasmid vector or plasmid vector containing GRK2 constructs were seeded into 24-well dishes. Inositol lipids were radiolabeled by incubating the cells overnight with 1 µCi/ml [³H]myo-inositol in inositol-free Dulbecco's modified Eagle's medium. Unincorporated [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 preincubated for 1 h in HBSS 37 °C and then preincubated 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 quisqualate for 30 min at 37 °C. The reaction was stopped on ice by adding 500 µl of perchloric acid and then neutralized with 500 µl of 0.72 M KOH, 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) anion exchange resin with 200-400 mesh. [³H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system.

Co-immunoprecipitation Experiments-- HEK 293 cells were transiently transfected with 5 µg of FLAG-mGluR1 constructs and 5 µg of either empty plasmid vector or plasmid vector containing GRK2 constructs. 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 aprotonin, and 20 µg/ml phenylmethylsulfonyl fluoride). The particulate fraction was removed by centrifugation, and 500 µg of supernatant protein was incubated in the presence of 5 µl of anti-FLAG monoclonal antibody and 100 µl of 20% slurry protein G-Sepharose beads at 4 °C. The beads were subsequently washed three times with lysis buffer and were solubilized in 3× SDS sample buffer, and the immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose as described previously (12). GRK2 was detected by immunoblotting with a rabbit GRK2 antibody diluted 1:1000, and HA-tagged GRK2 mutants were detected with mouse monoclonal anti-HA antibody diluted 1:1000 in wash buffer (TBS-Tween: 150 mM NaCl, 10 mM Tris-HCl, pH 7.0, 0.05% Tween 20) containing 3% skim milk. The nitrocellulose membranes were rinsed three times with wash buffer and then incubated with secondary horseradish peroxidase-conjugated donkey anti-rabbit IgG diluted 1:2500 in wash buffer containing 3% skim milk. The nitrocellulose membranes were then rinsed twice with TBS-Tween, rinsed twice with Tris-buffered saline, and incubated with ECL Western blotting detection reagents.

Whole Cell Phosphorylation-- Whole cell phosphorylation experiments were performed as described previously (16). In brief, HEK 293 cells transfected with 5 µg of FLAG-tagged mGluR constructs either alone or together with 5 µg of GRK2 or empty pcDNA3 expression vector were reseeded in six-well dishes. The intracellular ATP pools were labeled with [³²P]orthophosphate (100 µCi/ml) in phosphate-free HEPES-buffered salt solution for 1 h at 37 °C. Subsequently, the cells were incubated in either the absence or the presence of 10 µM quisqualate for 10 min at 37 °C. The cells were solubilized in radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 10 mM NaF, 10 mM Na₂ pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.1 mM phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, pH 7.4). The FLAG-tagged mGluRs were immunoprecipitated using an anti-FLAG monoclonal antibody and subsequently subjected to SDS-PAGE followed by autoradiography. The extent of receptor phosphorylation was quantitated by densitometry of the resulting autoradiographs.

Receptor Expression-- Cells expressing FLAG-tagged mGluR1 were labeled on ice with an anti-FLAG antibody (1:500) for 45 min. The cells were washed with cold phosphate-buffered saline and subsequently labeled with a goat anti-mouse IgG antibody conjugated to FITC (1:500) for 45 min on ice. The cells were harvested, and cell surface immunofluorescence was assessed by flow cytometry as described previously (17).

Data Analysis-- For phosphorylation data, the data represent the means ± S.D. The data points plotted for the dose-response curves represent the means ± S.E. for the number of separate experiments indicated. Dose-response and time course data were curve fit and analyzed using GraphPad Prism. The statistical significance was determined using unpaired two-tailed t test.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Phosphorylation of mGluR1a Truncation Mutants and mGluR1b-- Previous studies investigating the role of GRKs in the regulation mGluR1a signaling indicated that GRK-mediated phosphorylation contributes to mGluR1a desensitization (12, 13). There are 63 serine and threonine residues localized within the C-tail domain of mGluR1a that may serve as potential sites for GRK-mediated phosphorylation (Fig. 1A) (12). To determine which regions of the mGluR1a C-tail might be phosphorylated by GRK2, we examined the phosphorylation of a series of mGluR1a C-tail truncation mutants and an mGluR1 splice variant, mGluR1b, for which 312 amino acid residues of the mGluR1a C-tail are replaced by 20 amino acid residues (Fig. 1A). In HEK 293 cells, FLAG epitope-tagged mGluR1a C-tail deletion mutants lacking the final 50 (FLAG-mGluR1a-1149), 150 (FLAG-mGluR1a-1049), and 333 (FLAG-mGluR1a-866) amino acids were expressed at the cell surface at levels that were comparable with wild-type FLAG-mGluR1a (Fig. 1B). In contrast, the cell surface expression of FLAG-mGluR1a-1000 and FLAG-mGluR1a-953 was severely impaired (Fig. 1B), with the majority of immunostaining localized to the endoplasmic reticulum (data not shown). These results were consistent with the previous findings of Chan et al. (18), who demonstrated that the deletion of amino acid residues 975-1098 in mGluR1a unmasked an endoplasmic reticulum retention signal. Consequently, our initial experiments focused on the phosphorylation of FLAG-mGluR1a, FLAG-mGluR1a-1149, FLAG-mGluR1a-1049, FLAG-mGluR1a-866, and FLAG-mGluR1b.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Relative cell surface expression of mGluR1a, mGluR1b, and mGluR1a truncation mutants in HEK 293 cells. A, schematic representation of mGluR1a and mGluR1b extracellular, transmembrane, and intracellular domains. Serine and threonine residues localized to the intracellular domains of mGluR1a and mGluR1b are shown in black. The asterisks identify serine and threonine residues that conform to putative consensus sites for protein kinase C-dependent phosphorylation. The open circles represent any other amino acid residue. The arrow marks the point where alternative splicing replaces the mGluR1a C-terminal tail with the 20 amino acid residues encoded by mGluR1b. The sites at which the mGluR1a C-terminal tail has been truncated to yield the mGluR1a-866, mGluR1a-953, mGluR1a-1000, mGluR1a-1049, and mGluR1b-1149 are marked by bars. B, shown is the relative cell surface expression of FLAG epitope-tagged mGluR1a, mGluR1a-866, mGluR1a-953, mGluR1a-1000, mGluR1a-1049, mGluR1b-1149, and mGluR1b. Cell surface expression was determined by flow cytometry and represents the mean cell surface fluorescence. The mean fluorescence value for FLAG-mGluR1a was 407 ± 66 and is equivalent to ~1.5-2 pmol of receptor protein/mg of whole cell protein. The data represent the means ± S.D. for four independent experiments. *, p < 0.05 versus mGluR1a.

We found that the removal of the last 150 amino acids (including 29 serine and threonine residues) from the mGluR1a C-tail did not alter the pattern of constitutive and agonist-stimulated mGluR1a phosphorylation in either the absence or the presence of overexpressed GRK2 (Fig. 2). However, we found that in either the presence or the absence of overexpressed GRK2, FLAG-mGluR1b did not serve as a substrate for phosphorylation and that the removal of the last 333 amino acids from the C-terminal tail resulted in a complete loss of mGluR1a phosphorylation (Fig. 2).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of mGluR1a, mGluR1a-866, mGluR1a-1049, mGluR1b-1149, and mGluR1b in the presence and the absence of GRK2 in HEK 293 cells. A, shown is a representative autoradiograph demonstrating the whole cell phosphorylation of FLAG-mGluR1a, FLAG-mGluR1a-866, FLAG-mGluR1a-1049, FLAG-mGluR1b-1149, and FLAG-mGluR1b treated either with or without 10 µM quisqualate for 10 min in the presence and absence of GRK2. B, the densitometric analysis of the autoradiograms showing the means ± S.D. of four independent experiments. In these experiments the data are normalized to FLAG-mGluR1a phosphorylation in the absence of agonist treatment and the absence of GRK2. In all of the experiments, HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNA encoding FLAG-mGluR1 constructs with (+) and without () 5 µg of pcDNA3 plasmid cDNA encoding GRK2.

Phosphorylation-independent mGluR1a and mGluR1b Desensitization-- Because FLAG-mGluR1b and FLAG-mGluR1a-866 do not serve as substrates for phosphorylation in either the presence or the absence of GRK2, we tested whether GRK2 still contributes to the attenuation of mGluR1 activity. The activation of FLAG-mGluR1a, FLAG-mGluR1a-866, and FLAG-mGluR1b with increasing concentrations of the Group I mGluR agonist quisqualate results in similar increases in inositol phosphate formation with half-maximal effective concentration (EC₅₀) values of 57, 141, and 56 nM, respectively (Table I). Co-expression of GRK2 with wild-type FLAG-mGluR1a results in a 60% reduction in quisqualate-stimulated inositol phosphate formation in HEK 293 cells (Fig. 3A). Despite the fact that FLAG-mGluR1a-866 and FLAG-mGluR1b are not phosphorylated, GRK2 overexpression effectively reduces FLAG-mGluR1a-866- and FLAG-mGluR1b-mediated inositol phosphate formation by 57 and 74%, respectively (Fig. 3, B and C). We demonstrated previously that GRK2 is co-precipitated by FLAG-mGluR1a (12). In the present study, we find that FLAG-mGluR1a-866 and FLAG-mGluR1b co-precipitate GRK2 as effectively as the wild-type receptor (Fig. 4). Taken together, these results indicate that phosphorylation is not an absolute prerequisite for the attenuation of mGluR1a signaling by GRK2 and that neither receptor phosphorylation by GRK2 nor an extended C-tail is required for GRK2 association with mGluR1 alternatively spliced isoforms.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   GRK2-mediated attenuation of mGluR1 receptor-stimulated inositol phosphate formation in HEK 293 cells. Shown is FLAG-mGluR1a-stimulated (A), FLAG-mGluR1a-866-stimulated (B), and FLAG-mGluR1b-stimulated (C) inositol phosphate formation in response to increasing concentrations of quisqualate for 30 min at 37 °C in either the absence (open circles) or the presence (closed circles) of GRK2. The data points represent the means ± S.E. for four independent experiments. HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNA encoding FLAG-mGluR1 constructs with 5 µg of either empty plasmid vector or pcDNA3 plasmid cDNA encoding GRK2.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Co-immunoprecipitation of GRK2 with FLAG-mGluR1a, FLAG-mGluR1a-866, and mGluR1b from HEK 293 cell lysates. A, immunoblot of GRK2 protein co-immunoprecipitated (IP) in the absence of agonist with anti-FLAG antibody from lysates of HEK 293 transfected with either vector alone, FLAG-mGluR1a alone, FLAG-mGluR1a and GRK2, FLAG-mGluR1a-866 and GRK2, mGluR1b and GRK2, or GRK2 alone. B, immunoblot of GRK2 expression in HEK 293 lysates from the corresponding lanes in A. The data are representative immunoblots from three independent experiments. In all of the experiments HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNAs that encode mGluR1a, mGluR1a-866, or mGluR1b along with 5 µg of either empty vector or pcDNA3 GRK2.

Phosphorylation-independent Regulation of mGluR1 Activity by GRK2-- The overall topology of GRK family members is relatively conserved and can be subdivided into three functional domains: 1) a modestly conserved N-terminal domain that is proposed to mediate receptor interactions, 2) a central catalytic domain, and 3) a highly variable C-terminal domain that is required for membrane targeting of the kinases (19). The C-terminal domain (Ct) of GRK2 encodes a pleckstrin homology domain that is essential for interactions with both phosphatidylinositol-4,5-bisphosphate and the subunit of the heterotrimeric G protein and is involved in the targeting of the kinase to the plasma membrane in response to receptor activation (Fig. 5A) (19). In contrast, the ~190 amino acid residues that comprise the GRK2 N-terminal domain exhibit sequence homology with the RGS box of RGS proteins (Fig. 5A) (20, 21). Carman et al. (21) recently demonstrated that the GRK2 RGS domain effectively inhibits G_q-mediated activation of phospholipase C. Consequently, we have examined the effect of co-expressing the GRK2-Ct, a catalytically inactive GRK2-K220R mutant, and GRK2 N-terminal domain constructs on FLAG-mGluR1a-866 and FLAG-mGluR1b signaling.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of GRK2 mutant expression on mGluR1 receptor-stimulated inositol phosphate formation in HEK 293 cells. A, schematic representation of GRK2 functional domains. Shown are the N-terminal RGS homology domain, the central catalytic domain, and the C-terminal pleckstrin homology domain. The location of lysine residue 220, which is essential for GRK2 catalytic activity, is identified with an arrow, and the -binding domain is identified with a line. Also shown are the N-terminal and C-terminal domain expression constructs. B and C, shown is FLAG-mGluR1a-866-stimulated (B) and FLAG-mGluR1b-stimulated (C) inositol phosphate formation in response to increasing concentrations of quisqualate for 30 min at 37 °C in either the absence (open circles) or the presence of either GRK2-K220R (closed circles) or GRK2-Ct (closed squares). The data points represent the means ± S.E. for three independent experiments. HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNA encoding FLAG-mGluR1a-866 and FLAG-mGluR1b with 5 µg of either empty plasmid vector GRK2-K220R or GRK2-Ct.

Previously, we demonstrated that the co-expression of the GRK2-Ct potentiated mGluR1a signaling, whereas co-expression of a GRK2-K220R attenuates mGluR1a signaling (12). In the present study, we find that the expression of GRK2-K220R is as effective as wild-type GRK2 at attenuating inositol phosphate formation in response to agonist activation of either FLAG-mGluR1a-886 or FLAG-mGluR1b (Fig. 5, B and C). The maximal activation of inositol phosphate formation by either FLAG-mGluR1a-886 or FLAG-mGluR1b is reduced 57 and 55%, respectively, in the presence of GRK2-K220R. In contrast, the co-expression of the GRK2-Ct potentiates mGluR1a-886- and FLAG-mGluR1b-stimulated inositol phosphate formation in response to increasing concentrations of quisqualate by 31 and 22%, respectively (Fig. 5, B and C). The expression of two GRK2 N-terminal constructs, GRK2 (1-190) and GRK2 (45-185), encoding the GRK2 RGS domain also effectively attenuate FLAG-mGluR1a-, FLAG-mGluR1a-886-, and FLAG-mGluR1b-stimulated inositol phosphate formation (Fig. 6, A-C). GRK2 (1-190) reduces FLAG-mGluR1a-, FLAG-mGluR1a-886-, and FLAG-mGluR1b-stimulated inositol phosphate formation by 49, 47, and 61%, respectively, and GRK2 (45-185) reduces FLAG-mGluR1a-, FLAG-mGluR1a-886-, and FLAG-mGluR1b-stimulated inositol phosphate formation by 58, 52, and 49%, respectively. Similar to what we have observed previously for wild-type GRK2 and GRK2-K220R (12), expression of both GRK2 (1-190) and GRK2 (45-185) effectively reduces the constitutive activity of FLAG-mGluR1a, as well as reduces the constitutive activity of FLAG-mGluR1a-886 and FLAG-mGluR1b (Fig. 6D). Furthermore, we find that both GRK2 (1-190) and GRK2 (45-185) can be co-immunoprecipitated from HEK 293 cells with FLAG-mGluR1a (Fig. 7). Taken together, these observations indicate that the association of GRK2 N terminus with mGluR1a and mGluR1b is sufficient to mediate the maximal attenuation of mGluR1 signaling.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effect of the expression of the GRK2 N-terminal domain on mGluR1 receptor-stimulated inositol phosphate formation in HEK 293 cells. A-C, shown is FLAG-mGluR1a-stimulated (A), FLAG-mGluR1a-866-stimulated (B), and FLAG-mGluR1b-stimulated (C) inositol phosphate formation in response to increasing concentrations of quisqualate for 30 min at 37 °C in either the absence (open circles) or presence of either GRK2 (45-185) (closed circles) or GRK2 (1-190) (closed squares). D, shows the effect of GRK2 (45-185) and GRK2 (1-190) expression on basal mGluR1a-, mGluR1a-866-, and mGluR1b-stimulated inositol phosphate formation. The data points represent the means ± S.E. for three independent experiments. HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNA encoding FLAG-mGluR1a, FLAG-mGluR1a-866 and FLAG-mGluR1b with and without 5 µg of either empty plasmid vector, GRK2 (45-185), or GRK2 (1-190).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Co-immunoprecipitation of GRK2 (45-185) and GRK2 (1-190) with mGluR1a from HEK 293 cell lysates. Immunoblot (IB) of HA epitope-tagged GRK2 (45-185) and GRK2 (1-190) protein co-immunoprecipitated (IP) with anti-FLAG antibody from lysates of HEK 293 transfected with FLAG-mGluR1a and GRK2 (45-185) (lane 1), FLAG-mGluR1a and GRK2 (1-190) (lane 2), empty vector and GRK2 (45-185) (lane 3), or empty vector and GRK2 (1-190) (lane 4). Shown in lanes 5-7 are HA antibody immunoblots of lysates from cells transfected with mGluR1a alone (lane 5), mGluR1a with GRK2 (45-185) (lane 6), and mGluR1a with GRK2 (1-190) (lane 7). The data are representative immunoblots from three independent experiments. In all of the experiments HEK 293 cells were transfected with 5 µg of pcDNA3 plasmid cDNAs encoding FLAG-mGluR1a along with 5 µg of either empty vector, HA-GRK2 (45-185), or HA-GRK2 (1-190).

GPCR desensitization represents an important regulatory mechanism by which acute and/or chronic receptor overstimulation is avoided. For many GPCRs desensitization correlates GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins (8, 9). However, this simple correlation does not necessarily hold for all GPCRs. For instance, unlike what is observed for many GPCRs, -arrestins do not contribute to the attenuation of mGluR1 signaling (12). Therefore, we have investigated whether GRK-mediated uncoupling of mGluR1a responses requires the phosphorylation of serine and threonine residues localized within the extended mGluR1a C-tail domain. We find that GRK2 regulation of mGluR1 activity occurs via a phosphorylation-independent mechanism that does not require the C-tail domain of mGluR1a. Moreover, we find that effective attenuation of mGluR1a- and mGluR1b-stimulated phospholipase C activity is mediated by the GRK2 N-terminal domain. The GRK2 N-terminal domain exhibits ~20% identity to the RGS domain encoded by members of the family of RGS proteins, suggesting that the combined association of the GRK2 N terminus with mGluR1 and G_q may dampen mGluR1 signaling in the absence of phosphorylation (20, 21).

The desensitization of several other G_q-coupled GPCRs including the ₁-adrenergic, angiotensin II type 1A, thromboxane A2, m1 muscarinic acetylcholine, endothelin A, endothelin B, thrombin, and parathyroid hormone receptors is regulated by GRKs (21-27). These studies have reported a variety of effects of GRK overexpression on G_q-coupled GPCR signaling, many of which have been interpreted to involve GRK-mediated receptor phosphorylation. In particular, the effectiveness of different GRK2 isoforms to mediate angiotensin II type 1A, endothelin A, and endothelin B receptor desensitization is correlated with increased agonist-stimulated receptor phosphorylation following the overexpression of different GRK subtypes (23, 25). However, more recent studies indicate that, at least for some G_q-coupled GPCRs, GRK-mediated receptor phosphorylation may not be absolutely required for GRK-dependent receptor desensitization. For example, wild-type GRK2 and GRK2-K220R are equally effective inhibitors of parathyroid receptor signaling (24), and an angiotensin II type 1A receptor mutant that is not phosphorylated in response to agonist activation desensitizes normally (28). Consistent with these observations, we find that phosphorylation of mGluR1a is not required for GRK2-dependent uncoupling of a C-tail truncated mGluR1a mutant. Furthermore, a naturally occurring mGluR1b splice variant does not serve as a substrate for phosphorylation, but this receptor variant is effectively uncoupled from phospholipase C by the expression of GRK2. Taken together, these observations suggest that GRK-mediated receptor phosphorylation and desensitization are dissociable, indicating that GRK2 kinase activity may be secondary to the role of GRK2 in attenuating GPCR signal transduction.

In studies demonstrating the inhibitory effects of GRK2-K220R overexpression on GPCR desensitization, the dampening of GPCR signaling was interpreted to occur solely as the consequence of a direct interaction between the GPCR and the catalytically inactive GRK2 (23, 24). However, in an eloquent study by Carman et al. (21), GRK2 and GRK3 were demonstrated to possess selective, albeit weak, GTPase activating protein activity toward G_q/11 and to inhibit G_q-mediated activation of phospholipase C activity in response to receptor activation. This inhibition of G_q-mediated activation of phospholipase C involved the association of the N-terminal domains of GRK2 and GRK3 with G_q (21). The N-terminal domains of GRK2 and GRK3 exhibit 20 and 30% homology, respectively, with the RGS box found in RGS proteins but lack conservation of key residues required for RGS/G protein interactions and GTPase activating protein activity (21, 29). Despite this fact, similar to what has been reported for p115 Rho-GEF, GRK2 and GRK3 may function as atypical RGS proteins (30).

The data presented here suggest that in addition to functioning as a GAPase activating protein for G_q/11 family G proteins, the N-terminal domain, specifically amino acid residues 45-185, targets GRK2 to bind mGluR1 family receptors. Consequently, it appears that a series of protein-protein interactions must occur either simultaneously or in series for GRK2 to effectively antagonize mGluR1 signaling. Not only is the GRK2 C-terminal domain essential for the membrane targeting of the kinase, but the kinase is localized to its target via the association of the N-terminal domain with both the receptor and the  subunit of the heterotrimeric G protein. It is likely that this coordinated series of events leading to the inactivation of mGluR1 signaling is also required for the desensitization of other GPCRs. The expression of the GRK2 N terminus mediates the partial desensitization of endothelin receptors but was not as effective as the wild-type kinase in promoting the desensitization of these receptors (23). In addition, Palczewski et al. (31) reported that antibodies raised against peptides corresponding to N-terminal domain residues 17-34 of rhodopsin kinase block rhodopsin kinase-mediated desensitization of rhodopsin. However, the corresponding amino acid residues in GRK2 do not appear to be required for GRK2 interactions with mGluR1a. Nevertheless, the ability of the GRK2 N-terminal domain to both associate with a GPCR and regulate the activity of G_q/11 family G proteins may be akin to the ability of the N-terminal 33 amino acid residues of RGS4 to provide anchorage and receptor selectivity to RGS4 (32-34).

In summary, despite the fact that mGluR1 bears no sequence homology to with either the rhodopsin/₂-adrenergic or the secretin/glucagon classes of GPCRs, GRK-dependent desensitization remains a conserved regulatory mechanism for this unique class of GPCRs. We find that although GRK2 plays a central role in regulating the activity of mGluR1, this regulation is independent of its function as a receptor kinase. Instead GRK-mediated mGluR1 desensitization departs from the conventional model of GRK-mediated phosphorylation followed by -arrestin binding and involves the binding of the GRK2 N-terminal domain to the receptor and G_q subunit. Based on the observation that the N-terminal domain of GRK2 is sufficient to mediate mGluR1a and mGluR1b desensitization, we propose that the targeted association of the GRK2 RGS domain with the complex formed between mGluR1 family members and G_q proteins allows GRK2 to effective regulate receptor/G protein activity in the absence of phosphorylation. It is possible that the interactions between GRK2 and the receptor, G_q and G occur either simultaneously or independently. Further investigation will be required to dissociate these possibilities.

	FOOTNOTES

^* This work was supported by Canadian Institutes of Health Research Grant MA-15506 (to S. S. G. F.), National Science Foundation Grant MCB9728179 (to R. S. M.), and funds from the Southeastern Pennsylvania Affiliate of the American Heart Association (to R. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a McDonald Scholarship from the Heart and Stroke Foundation of Canada, the Premier's Research Excellence Award, and the Canada Research Chair in Molecular Neuroscience. To whom correspondence should be addressed: Robarts Research Inst., 100 Perth Dr., P.O. Box 5015, London, ON N6A 5K8, Canada. Tel.: 519-663-3825; Fax: 519-663-3789; E-mail: ferguson@rri.ca.

Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M203593200

	ABBREVIATIONS

The abbreviations used are: mGluR, metabotropic glutamate receptor; C-tail, C-terminal tail; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; HEK, human embryonic kidney; HA, hemagglutinin; Ct, C-terminal domain.

	REFERENCES

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