J Biol Chem, Vol. 274, Issue 48, 34483-34492, November 26, 1999

Selective Regulation of G_q/11 by an RGS Domain in the G Protein-coupled Receptor Kinase, GRK2^*

Christopher V. Carman, Jean-Luc Parent, Peter W. Day, Alexey N. Pronin, Pamela M. Sternweis, Philip B. Wedegaertner, Alfred G. Gilman, Jeffrey L. Benovic^§, and Tohru Kozasa^¶

From the Departments of Biochemistry & Molecular Pharmacology and Microbiology & Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the  Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein  subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF₄-dependent binding to G protein  subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF₄. This revealed the specific ability of bovine brain G_q/11 to bind to both GRK2 and GRK3 in an AlF₄-dependent manner. In contrast, G_s, G_i, and G_12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain G_q/11 could also bind to an N-terminal construct of GRK2, while no binding of G_q/11, G_s, G_i, or G_12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified G_q revealed significant binding of both G_q GDP/AlF₄ and G_q(GTPS), but not G_q(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active G_q(R183C) but not wild type G_q. In vitro analysis revealed that GRK2 possesses weak GAP activity toward G_q that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited G_q-mediated activation of phospholipase C- both in vitro and in cells, possibly through sequestration of activated G_q. These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

G protein-coupled receptors (GPCRs)¹ reside at the plasma membrane where they receive diverse extracellular stimuli, in the form of light, odorants, neurotransmitters, and hormones. This information is translated into intracellular signals when agonist-bound GPCRs activate exchange of GTP for GDP on the  subunit of heterotrimeric G proteins. Activated, GTP-bound G (G(GTP)) then dissociates from G and each of these G protein components go onto regulate downstream effector molecules. In general the intracellular signal is limited by the presence of the extracellular stimuli and by the intrinsic GTPase activity of G. However, in order to selectively modulate the appropriate magnitude and duration of signals in diverse cellular contexts, several ubiquitous mechanisms are utilized to regulate these signaling cascades both at the level of the GPCR and at the level of the G protein.

At the level of the GPCR, agonist-specific loss of receptor responsiveness involves a family of G protein-coupled receptor kinases (GRK1-6). GRKs phosphorylate the agonist-activated form of GPCRs which in turn promotes the high-affinity binding of a second family of proteins termed arrestins (1). These interactions function to uncouple the GPCR from further G protein activation and to promote clathrin-mediated internalization of the receptor (1). Initiation of this process is controlled by GRKs, which are, in turn, regulated by a variety of molecules including the activated GPCRs themselves, G subunits, PIP₂, PKC, calmodulin, and caveolin (1-3). The overall topology of GRKs includes a somewhat conserved catalytic domain of ~270 residues which is flanked by N- and C-terminal regulatory domains. The C terminus is highly variable (~100-230 residues) and has the general function of mediating membrane localization. For example, GRK2 and GRK3 possess a C-terminal plecktrin homology domain which binds to both PIP₂ and free G promoting membrane recruitment and subsequent receptor phosphorylation (2). Interestingly, the ability of GRK2 and GRK3 to bind to G has also been implicated as playing a direct role in the regulation of G protein signaling via the sequestration of free G (4-6). The ~190 residue N terminus of GRKs is modestly conserved and has been suggested to contain receptor binding determinants (7). Recently, calmodulin (8), PIP₂ (9), and caveolin (3) have also been shown to interact with the N terminus. However, the overall structure and function of this domain has remained largely uncharacterized.

At the level of the G protein, regulation occurs through intrinsic GTPase activity possessed by the G subunits which hydrolyze bound GTP and promote rebinding of G. This process has recently been found to be modulated by a ubiquitous family of proteins termed regulators of G protein signaling (RGS), which serve as GTPase-activating proteins (GAPs) that accelerate the rate of GTP hydrolysis and thereby limit the half-life of the activated species (10, 11). RGS proteins share a ~120-residue region of homology termed an RGS domain which folds into an -helical module that binds preferentially to the transition state of G (12). This preferential binding to the transition state, which can be mimicked in vitro by the addition of GDP/AlF₄ (13), compared with the active state, which can be stably generated in vitro by addition of GTPS, is thought to serve as the driving force for acceleration of GTPase activity (14, 15).

At least 18 RGS proteins have been identified. In general, these RGS proteins interact with the  subunits of the G_i and G_q families (10, 11, 16). In addition, a small collection of proteins including GRKs (17), axin (18), D-AKAP (19), and p115 Rho-GEF (20, 21) have been identified as having somewhat less conserved RGS domains. Recently, one of these atypical RGS proteins, p115 Rho-GEF, was shown to function as a selective GAP for G_12/13 suggesting that sequence differences in these RGS proteins may correlate with different preferences for G protein-binding partners (20, 21). To date, no functionality has been attributed to any of the other atypical RGS domains. Given that GRK2 and GRK3 represent well characterized components of GPCR regulation that are already known to bind to G subunits, we explored the possibility that these GRKs may interact with G subunits. These experiments revealed selective and high affinity binding of activated G_q/11 to GRK2 and GRK3, an interaction that may function to regulate phospholipase C- (PLC-) activity in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Hemagglutinin (HA)-specific monoclonal and polyclonal antibodies were from Roche Molecular Biochemicals and Babco, respectively. ECL reagents were from Pierce while Fugene^TM was from Life Technologies, Inc. Polymerase II (pol II)-specific polyclonal antibody was from Santa Cruz. G_q/11-, G_i-, G_s-, G_12/13-, and G_common-specific polyclonal antibodies were generously provided by Dr. D. Manning. CNBr-activated Sepharose 4B was from Amersham Pharmacia Biotech. ProBlott was purchased from Applied Biosystems. Phosphatidylinositol 3,4-phosphate (PIP₂) and phosphatidylethanolamine were from Sigma. [-³²P]ATP, myo-[³H]inositol, and [³H]PIP₂ were from NEN Life Science Products Inc. Most other reagents were from sources previously described (3, 20).

Protein Expression and Purification-- GRK2 and GRK3 were overexpressed in and purified from Sf9 insect cells as described previously (22). Purified GST, GST-GRK2(1-178), GST-GRK2(45-178), GST-GRK5 (1-200), and GST-GRK6 (1-192) fusion proteins and urea stripped rod outer segments were prepared as described previously (8, 23). Sf9-expressed G_q, G_q(R183C), and G₁₂ (24), as well as Escherichia coli expressed myristoylated wild type G_i and hexahistadine-tagged G_s and RGS4 were purified as described previously (25, 26). M₁ muscarinic cholinergic receptor (M₁AChR), PLC-1, and PLC-2 were purified from Sf9 cells and reconstituted into phospholipid vesicles along with purified G_q and G_1/2 as described previously (27).

Gel Electrophoresis and Immunoblotting-- SDS-PAGE was performed using standard methods (28). Following electrophoresis, proteins were electroblotted onto polyvinylidene difluoride for peptide sequence analysis or nitrocellulose for immunoblotting. Immunoblotting was performed using G_q/11-, G_s-, G_i-, G_12/13-, -tubulin-, actin-, GRK2-, G_common-, HA-, or EE-specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution). Immunoblots were visualized by ECL following the manufacturer's guidelines.

Synthesis of GRK-coupled Affinity Resins-- Two g of CNBr-activated Sepharose 4B was hydrated and successively washed with 5 × 25 ml of 1 mM HCl. One mg of GRK2, GST-GRK2(1-178), GRK3, GST-GRK5(1-200), GST-GRK6(1-192), or GST was dialyzed against 3 × 500 ml of coupling buffer (0.1 M NaHCO₃, pH 8.6, 500 mM NaCl). Dialyzed proteins (or an equal volume of coupling buffer for mock) were mixed with a 1-ml bed volume of CNBr-activated Sepharose 4B and rocked overnight at 4 °C. Resins were then washed with 5 × 20 ml of coupling buffer and residual unreacted sites were blocked by incubation with 0.1 M Tris-HCl, pH 8.0, for 2 h at 4 °C. Resins were then washed with 2 × 20 ml of buffer A (20 mM Hepes, pH 7.4, 5 mM EDTA, 0.02% Triton X-100) containing 500 mM NaCl followed by 2 × 20 ml of buffer A containing 150 mM NaCl and finally adjusted to 50% bed volume. Coupling efficiencies ranged from 85 to 95%.

Identification of GRK-binding Proteins-- Fresh bovine calf brain was stripped of connective tissue and minced in ~1 ml of homogenization buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 5 mM benzamidine, 5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.2% Triton X-100) per mg of tissue using a Brinkman Polytron (14,000 rpm, 30 s). The homogenate was centrifuged at 45,000 × g for 20 min and the resulting supernatant at 300,000 × g for 60 min. The final supernatant was aliquoted and stored at 70 °C until use. 250-µl aliquots (~125 µg) of GRK-, GST-GRK-, GST-, or mock-coupled resins (50% bed volume) were incubated with 10 ml of the soluble brain extract (~10 mg/ml total protein) and 10 ml of buffer B (20 mM Tris-HCl, pH 8.0, 2 mM MgSO₄, 6 mM -mercaptoethanol, 100 mM NaCl, 0.05% Lubrol, and 5% glycerol) with 100 µM GDP in the absence or presence of AlF₄ (5 mM sodium fluoride and 30 µM AlCl₃) for ~12 h at 4 °C. The incubation mixture was then centrifuged at 1000 × g for 1 min and the pellet washed four times with buffer B containing 100 µM GDP in the absence or presence of AlF₄. Bound proteins were released from the pelleted resin by addition of 150 µl of SDS sample buffer followed by boiling for 10 min. The eluted proteins were then subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane. A specific ~42-kDa protein band was identified by Ponceau-S staining, excised, and subjected to peptide sequence analysis. Alternatively, proteins were transferred to nitrocellulose membrane and subjected to immunoblot analysis.

Purified G/GRK Binding Assay-- 0.5-5.0 µg of purified GRK2, GRK3, GST-GRK2(1-178), GST-GRK2(45-178), GST-GRK5(1-200), GST-GRK6(1-192), or GST immobilized on either CNBr-activated Sepharose 4B or glutathione-agarose beads were combined at 4 °C with 0.1-200 nM purified G_q, G_q (R183C), G_s, G_i1, or G₁₂ in buffer B containing 100 µM GDP in the absence or presence of AlF₄. For binding curve experiments fixed amounts of G_q and GRK2 affinity column were incubated in various volumes of binding buffer to produce the desired G_q concentrations. For some experiments, G was preincubated in buffer B with either 1 mM GTP, 1 mM GTPS, 1 mM GDP or 1 mM GDP/AlF₄ at 25 °C for 2 h prior to addition to binding reactions. Samples were incubated at 30 °C for 60 min and chilled on ice for 5 min. The resins were then pelleted in a microcentrifuge for 10 s, washed three times with 400 µl of the appropriate binding buffers, and boiled with 50 µl of SDS sample buffer. Samples were subjected to 10% SDS-PAGE and immunoblotting using G-specific antibodies.

Phosphorylation Assay-- Phosphorylation reactions contained, in a total volume of 20 µl, 30 nM GRK2 or GRK3, 200 nM G_q, 100 µM [-³²P]ATP (5 cpm/fmol), 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 7.5 mM MgCl₂ in the absence or presence of one or more of the following: 100 µM GDP, AlF₄, 400 nM rhodopsin, or 60 nM G. Reactions were incubated at 37 °C for 0-60 min, stopped with SDS sample buffer, and subjected to 10% SDS-PAGE and autoradiography.

GTPase Assay-- G GTPase activity was determined in solution using a single turnover assay essentially as described previously (20, 29). Briefly, GTP-loaded G_q(R183C), G_s, G_i1, G_o, and G₁₂ were generated by incubating G proteins in the presence of [-³²P]GTP followed by gel filteration on G-25 Sephadex. Next, G([-³²P]GTP) was incubated in the absence or presence of RGS4 (100 nM), GRK2 (300 nM), GST-GRK2(1-178) (500 nM), GST (500 nM), or control buffer. Reactions were quenched with 9 volumes of 5% (w/v) Norit A charcoal in 50 mM NaH₂PO₄. The charcoal was pelleted and the ³²P_i-containing supernatant was counted. Alternatively, the steady state GTPase activity was measured in phospholipid vesicles reconstituted with M₁AChR and heterotrimeric G_q as described previously (27, 29). Briefly, the vesicles were equilibrated for 5 min at 20 °C in the absence or presence of carbachol (1 mM) and RGS4 (50 nM), GRK2 (300 nM), GST-GRK2(1-178) (500 nM), or GST (500 nM) in buffer containing GTP (4 µM). The experiment was initiated by addition of [-³²P]GTP (10⁶ cpm) followed by incubation at 30 °C. Reactions were quenched and quantitated as above.

In Vitro Inositol Phosphate Assay-- Determination of G_q-mediated activation of PLC-1 and PLC-2 activity was performed in vitro essentially as described previously (30, 31). Briefly, lipid substrate was prepared by combining PIP₂ and phosphatidylethanolamine (1:10) with [³H]PIP₂ (5,000-10,000 cpm per assay) followed by sonication. Lipids were then combined with purified PLC-1 or PLC-2 in the absence or presence of purified G_q(GDP)/AlF₄ (150 nM) and 6 µM RGS4, GST-GRK2(1-178), or GST on ice. The incubation contained 50 µM PIP₂, 50 mM Na-Hepes, pH 7.2, 3 mM EGTA, 0.2 mM EDTA, 0.83 mM MgCl₂, 20 mM NaCl, 30 mM KCl, 1 mM dithiothreitol, 0.1% ultrapure albumin (bovine), 0.16% sodium cholate, and 1.5 mM CaCl₂ in a total volume of 60 µl. Reactions were initiated by raising the temperature to 30 °C for 0-15 min and quenched by addition of 200 µl of 10% trichloroacetic acid and 100 µl of bovine serum albumin (10 mg/ml) and placing the reactions on ice. [³H]inositol 1,4,5-phosphate (IP₃) (supernatant) was separated from unhydrolyzed [³H]PIP₂ (pellet) by centrifugation at 2,000 × g for 10 min. Released [³H]IP₃ was quantified by liquid scintillation counting.

Cell Culture and Transfection-- COS-1 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO₂. COS-1 and HEK293 cells grown to 75-95% confluence were transfected with either 20 µg (100-mm plate) or 3 µg (12-well plate) of total DNA using Fugene^TM according to the manufacturer's instructions.

Immunoprecipitation-- 100-mm plates of COS-1 or HEK293 cells were co-transfected with pcDNA3-GRK2 and pcDNA3-HA-G_q, pcDNA3-HA-G_q(R183C), pcDNA3-HA-G_s, pcDNA3-HA-G_s(R201C), pcDNA3-EE-G_i2, or pcDNA3-EE-G_i2(R179C) and in some experiments pcDNA3-M₃AChR. At 24 h after transfection, cells were rinsed with ice-cold phosphate-buffered saline and harvested by addition of 1 ml of buffer-C (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 5 mM MgCl₂, 0.7% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine, and 10 µg/ml each of leupeptin, pepstatin A, and aprotinin). For cells co-transfected with pcDNA3-M₃AChR, cells were incubated for 0-60 min at 37 °C in the absence or presence of 100 µM carbachol prior to harvesting. Cells were scraped and homogenized with two 15-s bursts with a Brinkman Polytron (2500 rpm) and lysates were centrifuged at 4 °C for 10 min at maximum speed in a microcentrifuge and the supernatant removed. For immunoprecipitation, 100 µl of supernatant was incubated with 4 µg of either GRK2- or pol II-specific polyclonal antibodies for 30 min at 4 °C followed by addition of 50 µl of 50% protein A-agarose pre-equilibrated in buffer C and an additional 60-min incubation at 4 °C. Samples were then centrifuged for 10 s in a microcentrifuge and the pellets were washed three times with 1 ml of buffer C each for 30 min at 4 °C. Bound proteins were eluted by addition of 50 µl of SDS sample buffer followed by boiling for 10 min. Initial supernatants, as well as elutions from immunoprecipitation reactions, were subjected to 10% SDS-PAGE and immunoblotting using GRK2- and HA-specific monoclonal antibodies.

Inositol Phosphate Determination in Cells-- Measurement of inositol phosphate production in cells was essentially as described previously (32). Briefly, HEK293 cells were seeded at a density of 80,000 cells per well in 12-well plates and transfected with the thromboxane A₂- receptor (TXA₂R), M₃AChR, or vector (pcDNA3) and a variety of GRK or RGS constructs (pcDNA3-GRK2, pcDNA3-GRK2(K220R), pcDNA3-HA-GRK2(45-178), pcDNA3-HA-GRK2(468-689), pcDNA3-GRK3, pcDNA3-GRK3(K220R), pcDNA3-GRK5, pcDNA3-GRK6, pB6-RGS4, and pB6-GAIP). The following day, cells were labeled for 18-24 h with myo-[³H]inositol at 4 µCi/ml in Dulbecco's modified Eagle's medium (high glucose without inositol). After labeling, cells were washed once in phosphate-buffered saline and incubated in pre-warmed Dulbecco's modified Eagle's medium (high glucose, without inositol) containing 0.5% bovine serum albumin, 20 mM Hepes, pH 7.5, and 20 mM LiCl for 10 min. Cells were then stimulated for 10 min with 100 nM U46619 (TXA₂R) or 100 µM carbachol (M₃AChR). The reactions were terminated by removing the stimulation media and adding 0.8 ml of 0.4 M perchloric acid to the cells. Samples were harvested in Eppendorf tubes and 0.4 ml of 0.72 N KOH, 0.6 M KHCO₃ was added. Tubes were vortexed and centrifuged for 5 min at maximum speed in a microcentrifuge. Total inositol phosphates were separated on Dowex AG1-X8 columns, and quantitated by liquid scintillation counting. Alternatively, HEK293 cells were co-transfected with pcDNA3-HA-G_q or pcDNA3-HA-G_q(R183C) (instead of the GPCR constructs) and various GRK or RGS constructs as stated above. For these experiments inositol phosphate measurement was as described above with the exception that these cells were not stimulated with agonist.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Homology between the N Terminus of GRKs and RGS Domains-- Whereas the central catalytic and C-terminal domains of GRKs have been well characterized, the overall structure and function of the ~190 residue N-terminal domain has remained relatively uncharacterized (1, 2). Interestingly, Siderovski et al. (17) identified sequence homology between RGS domains and an ~120 residue region in the N terminus of GRKs through a BLAST search of the NCBI protein data base (17). Indeed, both GRK2 and GRK3 (residues 51-173) are ~20% identical and ~30% similar to various RGS domains (Fig. 1). This compares with an average of 44% identity (~54% similarity) shared among various RGS proteins. Importantly, the majority of the conserved hydrophobic residues shown to make up the hydrophobic core of the RGS domain (12, 15) are shared throughout the GRK family (Fig. 1 and data not shown). This suggests that the N terminus of GRKs may have a three-dimensional topology that is similar to RGS domains. Residues thought to be critical for G binding and GAP activity in most RGS proteins are only partially conserved by GRK2 and GRK3 (Fig. 1) (12, 15). However, p115 Rho-GEF, a new member of the RGS family that serves as a GAP for G_12/13, also exhibits only partial conservation of these residues compared with other RGS proteins (Fig. 1) (20, 21).

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Alignment of GRK2 and GRK3 N termini with RGS domains. Top, overall topology of GRK2 and GRK3 is shown as a hatched bar (GRK2/3). The N-terminal RGS domain of ~120 residues is shaded. The central catalytic domain of ~270 residues is shown in light gray, whereas the C-terminal plecktrin homology domain of ~100 residues is shown in dark gray. Black bars above and below GRK2/3 indicate regions shown previously to contain critical binding determinants for G (1, 2) and caveolin (3). Bottom, GRK2 and GRK3 (residues 51-173) were aligned with the RGS domains of RGS12 (residues 712-830), RGS14 (64-182), RGS2 (80-197), RGS4 (59-176), GAIP (87-204), and p115 Rho-GEF (p115) (45-170). The predicted secondary structure is represented by black bars labeled 1-9 for each of the -helices in this structure (12). Hydrophobic residues thought to be largely involved in forming the hydrophobic core of this structure (12, 15) are shown in gray. Residues in RGS4 shown to contact G (12, 15) are designated . Residues in GRK2 shown to be critical caveolin binding determinants (3) are designated .

Finally, it is noteworthy that residues previously defined as conserved caveolin binding determinants in GRKs (residues 60-73 in GRK2 and GRK3 (3)) fall within -helix 3 of the putative RGS domain (Fig. 1). Interestingly, several RGS proteins including RGS2 and RGS12 possess significant sequence similarity to GRKs within the caveolin-binding region suggesting that these RGS proteins may possibly interact with caveolin.

Binding of Bovine Brain Extracts to GRKs in the Absence or Presence of AlF₄-- Based on the identified GRK/RGS sequence homology discussed above we speculated that GRKs may bind to G subunits in an AlF₄-dependent fashion. To test this hypothesis an affinity column containing covalently bound GRK2 was generated and 0.2% Triton X-100 solubilized bovine brain extract was passed over it in the presence of either GDP or GDP/AlF₄. After extensive washing, bound proteins were eluted with SDS sample buffer, subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and stained with Ponceau-S. This experiment revealed the presence of an ~55 kDa AlF₄-independent band that was identified by immunoblotting as tubulin in agreement with previous studies demonstrating GRK2/tubulin interactions (33, 34). In addition, a ~45-kDa AlF₄-independent band was identified by immunoblotting as actin in agreement with previous studies demonstrating GRK/actin interactions (35).² Interestingly, a ~42-kDa protein was identified that bound to the GRK2 column (but not to mock or GST control columns) in a strictly AlF₄-dependent fashion (Fig. 2A). This band was excised and directly microsequenced. This yielded the sequence TLES(I/M)MAXXL with the fifth cycle detecting both isoleucine and methionine. A subsequent data base search with this sequence suggests that the 42-kDa band represents a mixture of G_q (¹MTLESIMACCL¹¹) and G₁₁ (¹MTLESMMACCL¹¹), two G proteins that are highly related in sequence (88% similarity) and function (36).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Binding of soluble bovine brain extract to GRK affinity columns in the absence or presence of AlF4. Covalently bound GRK, GST-GRK, GST, and mock affinity columns were prepared and combined with a soluble bovine brain extract in the absence or presence of AlF4 as described under "Experimental Procedures." After washing the columns extensively, bound proteins were eluted by boiling with SDS sample buffer and then subjected to SDS-PAGE. A, brain proteins eluted from GRK2-, GST-, and mock-coupled affinity resins from experiments performed in the absence () or presence of AlF4 were visualized by Ponceau-S staining. Total brain extract (Ext) and molecular weight standards (Std) are shown on the right. Identity of specific bands including GRK2, tubulin, actin, and Gq/11, as well as an unidentified protein of ~70 kDa are shown on the right. B, experiments identical to those shown above were subjected to immunoblotting with Gq/11-, Gs-, Gi-, and G12/13-specific antibodies (indicated on right). C, experiments similar to those shown above (A) were performed using GST-GRK5(1-200), GST-GRK2(1-178), and GST-GRK2(469-689) affinity columns and were immunoblotted with Gq/11-specific antibodies.

In order to further analyze the specificity of G protein binding to the GRK2 affinity column, experiments identical to those described above were performed and analyzed by Western blotting. Immunoblotting with G_q/11-, G_s-, G_i-, and G_12/13-specific antibodies confirmed the identification of the 42-kDa AlF₄-dependent band as G_q/11 and suggested specificity in that G_s, G_i, and G_12/13 binding was not detected despite their presence in the extract (Fig. 2B). Given the well characterized sequence (93% similarity) and functional similarity between GRK2 and GRK3 (1, 2), we also generated a GRK3 affinity column and incubated it with the bovine brain extract. This experiment revealed that GRK3, like GRK2, can bind to G_q/11 but not G_s-, G_i-, and G_12/13 in an AlF₄-dependent fashion (data not shown). In order to establish that GRK/G_q/11 binding was dependent on the GRK RGS domain, experiments were also performed using either GRK2 N-terminal (GST-GRK2(1-178)) or C-terminal (GST-GRK2(468-689)) affinity columns. These studies revealed the specific AlF₄-dependent binding of bovine brain G_q/11 (Fig. 2C), but not other G proteins (data not shown), to GST-GRK2(1-178), which contains the GRK2 RGS domain. In contrast, G_q/11 did not bind to GST-GRK2(468-689) which contains the GRK2 plecktrin homology domain. Interestingly, the N-terminal domains of GRK5 (GST-GRK5(1-200)) (Fig. 2C and data not shown) and GRK6 (GST-GRK6(1-192)) (data not shown) did not bind to G_q/11, G_s, G_i, or G_12/13. However, this blotting analysis is not exhaustive and it remains possible that the N-terminal domain of other GRKs interact with distinct G subunits that remain to be identified.

GRK2 and GRK3 Binding to Purified G_q-- In order to more thoroughly examine GRK/G interactions, we performed experiments with purified G_q. Initially, 100 nM purified G_q was combined with GRK2, GST-GRK2(1-178), GST-GRK2(45-178), GST-GRK2(468-689), GRK3, GST-GRK5(1-200), GST-GRK6(1-192), GST, and mock affinity columns. These experiments demonstrated binding of nearly 100% of the purified G_q to the GRK2, GST-GRK2(1-178), GST-GRK2(45-178), and GRK3 affinity columns in a strictly AlF₄-dependent manner (data not shown). G_q binding to all other columns was <5% and was unaffected by the presence of AlF₄ (data not shown). Identical experiments were then performed using purified G_s, G_i1, and G₁₂. Unlike G_q these other purified G proteins did not exhibit significant binding to any of the affinity columns in either the absence or presence of AlF₄ (data not shown). Thus, experiments with purified G_q demonstrated the same selectivity with respect to GRKs (i.e. preferential binding to GRK2 and GRK3) that was observed in experiments with bovine brain G_q/11 (Fig. 2).

In order to approximate the strength of the GRK2/G_q interaction, fixed amounts of G_q and GRK2 affinity resin were incubated in various volumes of binding buffer in the absence or presence of AlF₄. This approach allowed for direct comparison of the amounts of G_q bound to GRK2 at different G_q concentrations. These studies revealed that, even at relatively low concentrations, nearly 100% of the G_q bound to GRK2 in the presence of AlF₄ (Fig. 3). In contrast, only ~5% of G_q(GDP) bound to GRK2. These results suggest that G_q binds to GRK2 with high affinity.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   GRK2/Gq binding in the absence or presence of AlF4. 1.5 pmol of purified Gq(GDP) was incubated with 6.25 pmol of GRK2 (covalently coupled to CNBr-activated Sepharose) in a total volume from 50 µl to 15 ml in the absence or presence of AlF4 as described under "Experimental Procedures." Bound Gq was eluted from the affinity column with SDS, subjected to SDS-PAGE, and immunoblotting with a Gq/11-specific antibody. A representative immunoblot of Gq (0.1-30 nM) binding to GRK2 or GST columns is shown along with standards representing 25, 50, and 100% of the Gq loaded into the binding experiments (% Load).

Binding of Transition and Active States of G_q to GRK2-- Addition of AlF₄ to inactive G(GDP) produces a stable conformation that is thought to represent the transition state produced during hydrolysis of G(GTP) to G(GDP) (13). Many molecules, such as the effector adenylyl cyclase, do not appear to discriminate between the active state (G(GTP) or G(GTPS)) and the transition state (G(GDP/AlF₄)) of the G subunit. However, RGS proteins are unique in that they exhibit preferential binding to G_q(GDP/AlF₄) (12, 14, 15). This preference for the transition state is thought to enable RGS proteins to stabilize this conformation and thus promote GTP hydrolysis (12, 14, 15). To determine if GRK2 binding to G_q is also selective for the G_q transition state we compared GRK2 binding to 3 nM G_q(GDP), G_q(GDP/AlF₄) and G_q(GTPS). Interestingly, significant GRK2 binding was observed to both the G_q(GDP/AlF₄) (~95% binding) and G_q(GTPS) (~45% binding), whereas G_q(GDP) failed to demonstrate significant binding (Fig. 4). When a similar analysis was performed using G_q(R183C), a GTPase-deficient mutant, both GTP and GTPS forms bound to GRK2 to a similar extent (35-40% binding), whereas the GDP/AlF₄ form again bound more extensively (~90% binding) (Fig. 4). Because, the loading of GTP or GTPS onto G_q proteins in the absence of an activated GPCR is significantly less efficient than that of AlF₄ (29, 30), the observed binding of GRK2 to the GTP- and GTPS-bound forms of G_q is likely underestimated. Nevertheless, GRK2 appears to bind extensively to both the "active" and "transition" states of G_q. This binding profile is in contrast to other RGS proteins which, when analyzed in a similar fashion, bound preferentially to the transition state of G_i/o or G_q (14, 15, 37-39).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Binding of GRK2 to both active and transition states of Gq. Purified Gq or Gq(R183C) (3 nM) were preincubated with 1 mM GTP, 1 mM GTPS, 1 mM GDP, or 1 mM GDP/AlF4 and then combined with GRK2 affinity resin (1 µg of GRK2) as described under "Experimental Procedures." After binding the samples were washed and proteins were eluted with SDS. Samples were subjected to SDS-PAGE and Western blotting with a Gq/11-specific antibody. Binding was quantitated by densitometry, and plotted as a percent of the total Gq or Gq(R183C) loaded. All values are mean ± S.E. from three separate experiments.

Function of GRK2/G_q Interaction-- Given the ability of RGS proteins to serve as GAPs for G proteins, we next investigated whether GRK2 may serve as a GAP for G_q. To test this possibility we initially utilized a single turnover assay, which involves pre-loading of [-³²P]GTP onto G in the absence of MgSO₄ to slow hydrolysis. Unfortunately, GDP dissociation, and therefore GTP loading, in the absence of activated GPCR is particularly inefficient compared with the k_cat for GTP hydrolysis for G_q precluding use of the single turnover assay (30). However, the k_cat for GTP hydrolysis of G_q(R183C) is significantly reduced allowing GTP loading to occur more efficiently (29). Moreover, it was recently shown that the GTPase activity of G_q(R183C) can be promoted by RGS4 in a single turnover assay (29). Thus, we utilized this assay to monitor the GTPase activity of G_q(R183C) in the absence or presence of purified RGS4, GRK2, GST-GRK2(1-178), or GST alone. While 100 nM RGS4 promoted rapid GTP hydrolysis releasing up to 5 fmol of P_i, GRK2 and GST-GRK2(1-178) at concentrations up to 500 nM failed to enhance GTP hydrolysis (Fig. 5A). We also performed single turnover GTPase assays on wild type G_i1, G_o, G_s, G₁₂, and G₁₃ in the absence or presence of RGS4 (100 nM) and GRK2 (100 nM). As previously shown, RGS4 significantly promoted the GTPase activity of G_i1, and G_o while having no effect on either G_s or G₁₂ (Ref. 26, data not shown). In contrast, GRK2 failed to effect the GTPase activity of any of these G proteins (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Regulation of Gq GTPase activity by GRK2. A, the Gq GTPase activity was determined in solution by incubating Gq(R183C)([-32P]GTP) in the absence or presence of 100 nM RGS4 (), 300 nM GRK2 (), 500 nM GST-GRK2(1-178) (), or control buffer () for 0-15 min as described under "Experimental Procedures." Reactions were quenched and pelleted and the 32Pi-containing supernatant was counted and plotted as a function of time. B, steady-state carbachol-stimulated Gq GTPase activity was measured with phospholipid vesicles reconstituted with M1AChR and heterotrimeric Gq in the presence of [-32P]GTP and in the absence or presence of 50 nM RGS4 (), 300 nM GRK2 (), 500 nM GST-GRK2(1-178) (), or control buffer () for 0-12 min as described under "Experimental Procedures." Reactions were quenched and quantitated as above. The amount of 32Pi generated was plotted as a function of time. All values are averages of duplicate reactions from representative experiments.

It was previously demonstrated that while RGS2 does not serve as a GAP for G_i in single turnover GTPase assays, it is an effective GAP in the presence of purified heterotrimeric G_i and the G_i-coupled M₂AChR (29). Thus, we reconstituted heterotrimeric G_q and G_q-coupled M₁AChR into phospholipid vesicles allowing measurement of agonist-promoted steady-state GTPase activity of G_q. Addition of carbachol produced a steady-state rate of G_q-mediated [-³²P]GTP hydrolysis of ~5 fmol/min (basal activity). The basal activity was not significantly altered by addition of buffer control (Fig. 5B) or GST (500 nM) (~7 fmol/min). Addition of RGS4 (50 nM), however, produced an ~33-fold increase in the rate of GTP hydrolysis (~166 fmol/min) (Fig. 5B). Addition of GRK2 (300 nM) or GST-GRK2(1-178) (500 nM) produced more modest enhancements of GTPase activity of ~7-fold (~34 fmol/min) and ~9-fold (~46 fmol/min), respectively. Thus, it appears that GRK2 may have a weak ability to function as a GAP for G_q. The fact that the GRK2-dependent GAP activity was only ~25% that of RGS4 and required concentrations up to 10-fold greater than that of RGS4 raises the question of whether this activity is important under physiological conditions. However, the fact that this GRK2 GAP activity is apparent only in the presence of an activated GPCR suggests the possibility that receptors could have a critical role in potentiating GRK2-dependent GAP activity in cells. Perhaps in the presence of other GPCRs, GRK2 may serve as a more efficient GAP for G_q. Indeed it has been demonstrated that the ability of RGS2 to inhibit G_q-mediated signals in cells is highly dependent on the nature of the receptors that are being stimulated (40). The authors of this study suggested that regulatory selectivity may be conferred by specific receptor-RGS complexes. Moreover, it has been well established that the kinase activity of GRK2 can be stimulated by binding to an activated GPCR (41). Thus, further investigation of the role of receptors in modulating GRK/G interactions seem warranted.

The primary role of activated G_q in cells is stimulation of PLC- which hydrolyses PIP₂ (and other phosphoinositides) to the second messengers IP₃ (and other inositol phosphates) and diacylglycerol (36). Given the high affinity binding that GRK2 possesses for activated G_q (Figs. 2-4), we speculated that even in the absence of significant GAP activity, GRK2 may be able to regulate PLC- activity simply by competing for binding to activated G_q. To test this, purified PLC-1 was combined with phospholipid vesicles containing [³H]PIP₂ and G_q in the absence or presence of AlF₄ and in the presence of 0-3 µM RGS4, GST-GRK2(1-178), or GST. Addition of AlF₄ produced substantial stimulation of PLC-1 activity (~6-fold) in the presence of G_q (Fig. 6). While addition of GST or buffer control had no effect on this activity, RGS4 and GST-GRK2(1-178) substantially inhibited PLC-1 activity (Fig. 6). Similar results were observed with PLC-2 (data not shown). Thus, GRK2 is effective in inhibition of G_q promoted activation of PLC-.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Regulation of Gq activation of PLC-1 in vitro. Measurement of Gq-mediated activation of PLC-1 was performed by combining [3H]PIP2 with purified PLC-1 in the absence (basal, ) or presence of purified Gq(GDP/AlF4) (150 nM) and 0-3 µM RGS4 (), GST-GRK2(1-178)(), or GST() as described under "Experimental Procedures." Reactions were quenched by addition of 10% trichloroacetic acid and bovine serum albumin and [3H]IP3 was separated from unhydrolyzed [3H]PIP2 by centrifugation. [3H]IP3 production was quantified by liquid scintillation counting and plotted against the concentration of added proteins. All values are averages of duplicate reactions from a representative experiment.

These data suggest that, even in the absence of significant GAP activity, GRK/G_q binding may be able to regulate G protein signaling simply by sequestration of activated G_q. This is particularly interesting in light of extensive previous studies that the C terminus of GRK2 and GRK3 can sequester free G subunits and thereby inhibit their signaling in cells (4-6). Thus, the possibility exists that these GRKs may be able to concomitantly sequester both components of the bifurcating G_q signal (i.e. G_q(GTP) and free G). Alternatively, if G_q/11 and G binding to GRKs are mutually exclusive, this would provide a mechanism for complex regulation of GRK2 and GRK3 activity.

Another possible consequence of GRK2/G_q and GRK3/G_q interactions would be phosphorylation of G_q. Indeed, members of the G_q family have previously been shown to be regulated by tyrosine phosphorylation and by PKC-mediated serine phosphorylation (42, 43). In order to test whether GRK2 or GRK3 phosphorylate G_q, these GRKs were combined with [-³²P]ATP and G_q(GDP) in the absence or presence of AlF₄ for 0 to 60 min at 37 °C. These experiments failed to produce any detectable phosphorylation of G_q by these GRKs (data not shown). Additional experiments were performed in the presence of G and/or light-activated rhodopsin in order to test the possibility that these GRK activators might be required for G_q phosphorylation. These experiments also failed to produce detectable GRK-mediated G_q phosphorylation (data not shown). Thus, G_q does not appear to be a substrate for GRK2 or GRK3.

GRK2/G_q Interaction in Intact Cells-- In order to determine if the GRK/G_q interaction occurs in intact cells we co-expressed GRK2 and either G_q or G_q(R183C) in COS-1 cells. This particular mutation (R183C) in G_q nearly abolishes GTPase activity trapping the GTP-bound active state of G_q but does not directly confer the active state. Thus, a portion of the expressed G_q will accumulate in the active state over time due to basal stimulation of GPCRs during cell culture. Presumably, acute GPCR stimulation should drive further accumulation of G_q(R183C) trapped in this active, GTP-bound state. Therefore, COS-1 cells were transfected with GRK2 and either HA-G_q or HA-G_q(R183C) cDNAs. Immunoblotting of lysates with an HA-specific antibody revealed that the total expression of HA-G_q and HA-G_q(R183C) was similar (Fig. 7). Moreover, GRK2 expression levels were also similar in cells co-expressing HA-G_q and those co-expressing HA-G_q(R183C) (data not shown). For immunoprecipitation, lysates were incubated with either GRK2- or pol II (control)-specific polyclonal antibodies. Subsequent blotting of these immunoprecipitation reactions with a GRK2-specific monoclonal antibody revealed that the GRK2 polyclonal antibody effectively immunoprecipitated GRK2 in both cells co-expressing HA-G_q and those expressing HA-G_q(R183C), while the pol II antibody did not precipitate GRK2 (data not shown). Immunoblotting the immunoprecipitates with an HA-specific monoclonal antibody to detect G_q revealed that only a small amount (~1%) of wild type G_q co-precipitated with GRK2 whereas a significant amount (~20%) of G_q(R183C) co-immunoprecipitated with GRK2 (Fig. 7). Similar results were obtained using HEK293 cells (data not shown). Importantly, this experiment is in agreement with our in vitro data (Fig. 4) and underscores the ability of GRK2 to bind tightly to the active state of G_q as opposed to other RGS proteins which appear to require the transition state for significant binding (12, 14, 15, 37-39).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Co-immunoprecipitation of activated Gq and GRK2 from COS-1 cells. COS-1 cells co-expressing GRK2 and either HA-Gq or HA-Gq(R183C) were harvested and lysed as described under "Experimental Procedures." Immunoprecipitation (IP) from cell extracts was performed by incubating with either GRK2- or pol II (Pol)-specific polyclonal antibodies followed by incubation with protein A-agarose. Immunopreciptitated proteins were eluted from protein A-agarose with SDS sample buffer and elutions and initial cell extracts (20% of total used in immunoprecipitations) were subjected to SDS-PAGE and immunoblotting using an HA-specific monoclonal antibody. A representative Western blot is shown.

In order to examine the effect of GPCR stimulation on GRK2/G_q interaction, COS-1 cells were co-transfected with the M₃AChR, GRK2, and either HA-G_q or HA-G_q(R183C). Incubation of these cells with 100 µM carbachol for 20 min enhanced the amount of G_q(R183C) co-immunoprecipitated by nearly 2-fold, while stimulation for longer periods (up to 60 min) did not produce further enhancement (data not shown). In contrast, wild type HA-G_q co-immunoprecipitation was not significantly enhanced by carbachol although this may be a consequence of GTP hydrolysis over the duration of the immunoprecipitation (~3-4 h). These studies support the idea that under physiological conditions GPCR stimulation may promote GRK2/G_q interaction. We next examined the selectivity of these interactions by co-expressing GRK2 with HA-G_s, HA-G_s(R201C) (a GTPase deficient mutant), EE-G_i, or EE-G_i(R179C) (a GTPase-deficient mutant), in HEK293 cells and performed immunoprecipitation experiments analogous to those described above. Here, neither the wild type nor GTPase-deficient mutants of G_s or G_i were co-immunoprecipitated with GRK2 (data not shown) demonstrating that the selectivity that we observed in vitro occurs in intact cells.

Finally, given the ability of GRK2 to bind to activated G_q in intact cells (Fig. 7) along with the regulatory function of GRK2 toward PLC- shown in vitro (Fig. 6), we examined whether GRKs may regulate PLC- activity in intact cells. Initially, we took advantage of the functional properties of G_q(R183C). We found that expression of HA-G_q(R183C) in HEK293 cells, in the absence of specific GPCR stimulation, generated a significantly elevated production (~10-fold) of inositol phosphates as compared with cells transfected with vector control or wild type HA-G_q (data not shown). To examine the effect of GRKs on inositol phosphate production, cells were co-transfected with HA-G_q(R183C) and either vector (100% control), GRK2, GRK2(K220R), HA-GRK2(45-178), GRK3, GRK3(K220R), GRK5, GRK6, RGS4, or GAIP. Importantly, levels of HA-G_q(R183C) expression, as assessed by Western analysis, were similar regardless of the nature of the co-transfected DNA (data not shown). These experiments reveal, as predicted, that RGS4 and GAIP expression both lead to a significant (~40%) inhibition of PLC- activity (Fig. 8A). Expression of GRK2 or GRK3 lead to a similar inhibition of PLC- activity (~40%), while the catalytically inactive versions of these kinases ((GRK2(K220R) and GRK3(K220R)) were equally effective at blunting PLC- activity (~45% inhibition) (Fig. 8A). This demonstrates that this inhibition does not require phosphorylation activity. Alternatively, expression of full-length GRK5 had no effect on PLC- activity, while GRK6 exhibited a small (~15%) inhibition (Fig. 8A) in general agreement with the in vitro GRK selectivity demonstrated above. Since a GRK2 RGS domain construct (GST-GRK2(45-178)) can bind G_q in vitro (data not shown), we also generated an HA-tagged pcDNA3 minigene construct containing this domain (residues 45-178). Co-expression of the GRK2 RGS construct produced a dramatic (~65%) inhibition of PLC-1 activity (Fig. 8A). Given that PLC- activity is dependent on overexpressed HA-G_q(R183C) in this system, the level of competing GRK constructs would seem to be critical. Thus, we believe that the enhanced effectiveness of the GRK2 RGS domain construct is likely a consequence of a higher molar expression of this relatively small construct compared with the full-length GRK2 and GRK3 constructs. Taken together, these data demonstrate that the RGS domains of GRK2 and GRK3 can effectively inhibit G_q(R183C)-stimulated PLC- activity.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Inhibition of Gq signaling by RGS and GRK constructs in HEK293 cells. A, HEK293 cells expressing HA-Gq(R183C) along with vector (control) or the indicated GRK or RGS constructs were metabolically labeled with myo-[3H]inositol and the total [3H]inositol phosphates produced were isolated as described under "Experimental Procedures." Total [3H]inositol phosphates were quantitated by liquid scintillation counting, expressed as a percent of control and plotted against the indicated experimental conditions (i.e. co-expressed constructs). B, HEK293 cells expressing TXA2R along with vector (control) or the indicated GRK or RGS constructs were metabolically labeled with myo-[3H]inositol and then stimulated for 10 min with 100 nM U46619. Total [3H]inositol phosphate production was measured as described under "Experimental Procedures" and plotted as above. All values are mean ± S.E. from three to eight separate experiments.

Given that the approach used above provides a direct stimulation of PLC- activity via G_q(R183C), we can state with relative certainty that the inhibition observed above is mediated at the level of the G, as opposed to, for example, the GPCR. Having established this, we were next interested in examining a more physiologically relevant system involving receptor-stimulated activation of PLC-. To accomplish this, HEK293 cells were co-transfected with cDNA for TXA₂R, a G_q-coupled GPCR, along with all of the GRK and RGS constructs described above. Stimulation of TXA₂R-containing cells with the agonist U46619 (100&