G Protein-coupled Receptor Kinase 2/Galpha q/11 Interaction

A NOVEL SURFACE ON A REGULATOR OF G PROTEIN SIGNALING HOMOLOGY DOMAIN FOR BINDING Galpha SUBUNITS*

Rachel Sterne-MarrDagger §, John J. G. Tesmer, Peter W. Day||, RoseAnn P. StracquatanioDagger ||, Jill-Ann E. CilenteDagger , Katharine E. O'ConnorDagger , Alexey N. Pronin||**, Jeffrey L. Benovic||, and Philip B. Wedegaertner||

From the Dagger  Biology Department, Siena College, Loudonville, New York 12211, the  Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, the || Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and ** Senomyx, Inc., La Jolla, California 92037

Received for publication, August 27, 2002, and in revised form, October 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding Galpha q/ll and inhibiting Galpha q stimulation of the effector phospholipase Cbeta . The binding site for Galpha q/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha q/ll binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to Galpha q/ll. These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing Galpha q/ll binding defects impair recruitment to the plasma membrane by activated Galpha q and regulation of Galpha q-stimulated phospholipase Cbeta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The Galpha binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8. The adenomatous polyposis coli (APC) binding site of axin involves residues on alpha  helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the Galpha q/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha 5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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G protein-coupled receptors (GPCRs)1 are a large family of integral membrane proteins that form seven transmembrane helices and couple to heterotrimeric guanine nucleotide (G)-binding proteins on their cytoplasmic surface. They transmit the signals from light and odorant receptors as well as the signals initiated by numerous hormones and neurotransmitters. In their inactive state, heterotrimeric G proteins are complexes of three polypeptide chains (Galpha beta gamma ). Upon activation, GPCRs catalyze the exchange of GTP for GDP on the Galpha subunit resulting in dissociation of the GTP-bound Galpha subunit from the Gbeta gamma dimer (1). Galpha and Gbeta gamma are then free to regulate effectors such as adenylyl cyclase, phospholipase Cbeta (PLCbeta ), cGMP phosphodiesterase, ion channels, Rho family guanine-nucleotide exchange factors (RhoGEF), and activate mitogen-activated protein kinase signal transduction pathways (2-5). One common feature of GPCR signaling is the rapid loss of cellular sensitivity even in the presence of a stimulus. Insensitivity to the extracellular stimulus reflects intracellular events: receptor/G protein uncoupling, G protein inactivation, and receptor sequestration (and receptor degradation), which together act to regulate the duration and/or magnitude of the signaling event (6). One mode of receptor desensitization is initiated by phosphorylation of the activated receptor by a kinase of the G protein-coupled receptor kinase (GRK) family (7). Phosphorylation then promotes binding of the GPCR to a family of proteins called arrestins (8). This occludes Galpha beta gamma interaction with receptor and, in some nonvisual cells, leads to sequestration of the receptor away from the plasma membrane into endocytic vesicles (8-11).

GRKs are found in metazoans and, in mammals, the GRK family has seven members (7, 12). GRKs are serine/threonine kinases with a tripartite modular structure. A central ~350 amino acid kinase domain is closely related by sequence identity to those of cAMP-dependent protein kinases, protein kinase C, and ribosomal S6 kinases (13). At the carboxyl terminus of the catalytic core (14) homology to cAMP-dependent protein kinase predicts a putative "nucleotide gate" (15). The catalytic domain is flanked by an amino-terminal domain of 178 residues and a carboxyl-terminal domain that varies in structure among members of the family. Using distinct mechanisms, the carboxyl-terminal domains of GRKs direct the membrane association of these kinases (16, 17).

The amino-terminal domains of all GRK family members are homologous to the regulator of G-protein signaling (RGS) family of proteins (18). RGS proteins are a multifunctional family of proteins of variable length that share a ~120-amino acid "RGS domain." In this paper, we refer to this domain as the RGS homology (RH) domain. RGS proteins act as GTPase activating proteins (GAPs) for the Galpha i/o (including Galpha t) and Galpha q family of Galpha subunits (19-21) and as antagonists of Galpha /effector interaction (19, 22). In general, these proteins bind preferentially to the GDP-aluminum fluoride (GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>) > GTPgamma S GDP-bound form of Galpha (21). The crystal structures of the RGS4·Galpha i1 complex and the RGS9·Galpha t/i chimera·cGMP phosphodiesterase gamma  complex show that the RGS proteins contact switch regions I, II, and III of Galpha , which are polypeptide loops that undergo conformational changes in the transformation between the GDP-bound (inactive) and the GTP-bound (active) states of the G protein. In these examples, the binding of the RGS protein appears to stabilize the three switch regions in a conformation that preferentially binds the transition state for GTP hydrolysis (23, 24). RH domains can be grouped into five subfamilies based on their evolutionary relatedness: R4, R7, R12, RZ, and RA (axin) families (21). Members of the R4, R7, R12, and RZ families are negative regulators of G protein signaling as described above. The newly described Galpha s-specific RGS, RGS-PX1 (25), likely defines another RGS subfamily as this protein is similarly related to all 5 RGS subfamilies (~24% amino acid identity).

Other families of proteins have RH domains but their roles in regulating heterotrimeric G protein signaling are either distinct from RGS proteins, not well characterized, or do not regulate heterotrimeric G protein signaling. Axin plays a role in the wnt/embryonic development signaling pathway (26) and shares ~30% amino acid identity with RGS proteins of other subfamilies. This RH domain has never been demonstrated to bind or GAP a Galpha subunit (27). Instead the RH domain of axin binds the tumor suppressor protein, APC (28), a downstream target in the wnt signaling pathway. The APC binding site of axin is distinct from the Galpha binding site of RGS proteins. A family of guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) also has RH domains that share <20% identity to the RGS family. p115RhoGEF, PDZRhoGEF, and LARG (leukemia-associated RhoGEF), bind and in some cases serve as GAPs for Galpha 12, Galpha 13, and Galpha q, via their RH domains yet they are also downstream effectors of Galpha 12 and Galpha 13 (29-32). D-AKAP2, dual specificity A kinase anchoring protein 2, binds the regulatory subunit of cAMP-dependent protein kinase and has 2 RH domains. However, no G protein interaction has been reported for this protein (33).

The RH domain of GRK2 is most closely related to that of axin (26% amino acid identity) and RGS12 (24% amino acid identity) and binds to Galpha q and Galpha 11 in an AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-dependent fashion, but not to Galpha s, Galpha i, or Galpha 12/13 (34-36). Whereas all GRKs have putative amino-terminal RH domains, Galpha interaction has only been observed for GRK2 and GRK3. Unlike other Galpha q-binding RGS proteins such as RGS2 (37), RGS3 (38), RGS4 (22), and RGS18 (39), the GRK2 RH domain does not stimulate the GTPase activity of Galpha q in a single turnover GAP assay and only weakly stimulates GTPase when Galpha q is reconstituted with M1 muscarinic receptor and assayed in an agonist-induced steady-state GTPase assay (34). Because the GRK2 RH domain inhibits Galpha q-stimulated PLCbeta activity both in vivo and in vitro yet lacks significant GAP activity in vitro, it has been postulated that GRK2 RGS acts by sequestration of Galpha q. It is also possible that GRK2 is an effector of Galpha q. In this scenario, activation of Galpha q would recruit GRK2 to the site of an activated receptor. To investigate the role of GRK2/Galpha q interaction in the regulation of Gq signaling, we have created mutations in the RH domain of GRK2 that result in altered binding to Galpha q/11. Surprisingly, we found that the surface of GRK2 used to bind Galpha q/11 is distinct from the interaction site utilized by other RGS proteins to bind Galpha subunits and from the site used by axin to bind APC.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials

Human embryonic kidney cells (HEK293) and African Green monkey kidney cells (COS-1) were from the American Tissue Culture Collection. Galpha q/11-specific polyclonal antibodies were generously provided by Dr. D. Manning or purchased from Santa Cruz Antibodies, and EE-specific monoclonal antibody was provided by Dr. H. Bourne. RGS2-GFP (40) and GRK2-(45-178)-GFP were expressed from the plasmid pEGFP (Clontech, Palo Alto, CA) and were generously provided by Dr. S. Heximer and Dr. R. Penn, respectively. myo-[3H]Inositol was from Amersham Biosciences, Dowex AG1-X8 resin was from Bio-Rad, and scintillation fluid was from Packard. Molecular biologicals were from Roche Molecular Biochemicals unless otherwise indicated, immunoblotting detection reagents were from Pierce, and all other biochemicals were from Sigma or Fisher.

Preparation and Mutagenesis of pGEX-GRK2-(45-178) Constructs

Nucleotides encoding residues 45-178 of bovine GRK2 cDNA were amplified by the polymerase chain reaction using primers that incorporated BamHI and EcoRI restriction sites at the 5' and 3' ends of the coding region, respectively. The resulting PCR fragment was subcloned into BamHI and EcoRI sites of the glutathione S-transferase fusion protein vector, pGEX-2T (Amersham Biosciences) to generate pGEX-GRK2-(45-178). Sequential PCR (41) was used to produce the E78K, V83A, and D160K derivatives of pGEX-GRK2-(45-178), and Quik-Change mutagenesis (Stratagene) was used to generate all other mutations. The GRK2 portion of each construct was sequenced to verify that only the intended mutation had occurred.

Purification of GST-GRK2 Fusion Proteins

GST-GRK2 fusion proteins were expressed and purified by modifications of the procedures of Smith and Johnson (42) and Frangioni and Neel (43). Briefly, 40-ml cultures in Luria broth containing 5 µg/ml carbenicillin were grown at 37 °C to an optical density of 0.5, fusion protein expression was induced by the addition of isopropyl-1-thio-beta -D-galactopyranoside to 0.5 mM, and incubation was continued for 3 h at 25 °C. Cells were pelleted, washed in STE (20 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8), and frozen at -70 °C. Pellets were resuspended on ice in STE containing 100 µg/ml lysozyme and incubated on ice for 15 min before the addition of beta -mercaptoethanol to 10 mM, phenylmethylsulfonyl fluoride to 100 µM, leupeptin to 1 µg/ml, benzamidine to 20 µg/ml, and Sarkosyl to 1.5%. Lysates were sonicated in 10-s bursts followed by 15-s rest periods to reduce viscosity. Insoluble protein was removed by centrifugation at 12,000 rpm for 10 min at 4 °C and Triton X-100 was added to a final concentration of 2%. The lysate was adjusted to 25 mg/ml protein as determined by the Bradford assay using gamma -globulin as a standard (Bio-Rad) and fusion proteins were bound to glutathione-agarose beads (3 ml of packed beads/100 mg of protein) by mixing for 1 h at 4 °C. The beads were washed once with STE, 1.5% Sarkosyl, 2% Triton X-100, three times with STE, and stored in STE, 25% glycerol, 10 mM beta -mercaptoethanol at -20 °C. To determine the amount of GRK2 associated with glutathione-agarose beads, fusion proteins were eluted in 50 mM Tris-HCl, pH 8, 10 mM glutathione, 10 mM beta -mercaptoethanol at room temperature for 1 h. Bradford assays were then carried out on the eluates.

GST-GRK2 Pull-down Assays with Bovine Brain Galpha q/11

Bovine brain extract was used as a source of Galpha q/11 for in vitro binding assays. For 1-ml binding assays, 8 µg of fusion protein was incubated overnight at 4 °C with 200 µg of brain extract protein, prepared as described by Carman et al. (34), in 20 mM Tris-HCl, pH 8, 2 mM MgSO4, 6 mM beta -mercaptoethanol, 100 mM NaCl, 0.05% C12E10, 5% glycerol, 100 µM GDP in the absence or presence of 30 µM aluminum chloride and 5 mM sodium fluoride (AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>). Glutathione-agarose beads were washed 4 times with the buffer described above (in the absence or presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> as appropriate), proteins were eluted with SDS-PAGE sample buffer, and separated on 12% polyacrylamide gels. The proteins were transferred to nitrocellulose, probed with anti-Galpha q/11-specific polyclonal antibodies, incubated with peroxidase-conjugated secondary antibody, and Galpha q/11 was visualized by chemiluminescence using SuperSignal West Pico (Pierce).

Rhodopsin Phosphorylation

COS-1 cells were grown at 37 °C to 50-90% confluence on 10-cm dishes in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells were transfected with 10 µg of total DNA (pcDNA3 alone, pcDNA3-GRK2, or pcDNA3-mutant GRK2) using FuGENE 6 (Roche Molecular Biochemicals) following the manufacturer's recommendations. The cells were harvested after 48 h, washed twice in ice-cold phosphate-buffered saline, and lysed in 1 ml of buffer (20 mM HEPES, pH 7.2, 150 mM NaCl, 10 mM EDTA, 0.02% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 100 µg/ml benzamidine) by Polytron homogenization (two 15-s bursts at 2500 rpm). Lysates were centrifuged for 10 min at 40,000 × g to remove particulate matter and supernatants were then assayed.

To test for GRK activity, lysates containing wild type (WT) or mutant GRK2 protein were assayed for their ability to phosphorylate light-activated rhodopsin. Two microliters of COS-1 cell lysate were incubated with 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 100 µM ATP, ~1 µCi of [gamma -32P]ATP, and ~3.5 µM rhodopsin for 10 min at 30 °C in room light. Reactions were quenched by addition of SDS sample buffer followed by 30 min incubation at room temperature. Rhodopsin was separated by electrophoresis on a 10% SDS-polyacrylamide gel, and gels were fixed in 0.7 M trichloroacetic acid, 0.14 M 5'-sulfosalicylic acid for 10 min to remove unincorporated radionucleotide, washed twice in 50% ethanol, 16% acetic acid for 10 min, dried, and then subjected to autoradiography. Rhodopsin bands were excised and counted in a liquid scintillation counter. Repeated measures analysis of variance was used to test the statistical significance.

Homology Modeling

A homology model of the RH domain of GRK2 (residues 42-178) was based on the structure of the RH domain of axin (PDB code 1EMU), which is the closest homolog based on a BLAST (44) search of the protein data bank (26% identity within residues 64-174 of GRK2). The GRK2 model was built manually using the program O (45) by choosing appropriate and reasonable rotamers for nonidentical residues (46). In regions that appeared to have higher sequence identity with other RH domains of known structure, the GRK2 model was adjusted locally according to those models. The alpha 5/alpha 6 loop of the GRK2 RH domain has no obvious sequence homology to RH domains of known structure and was ultimately modeled based on the axin structure because the fit of the side chains appeared to be reasonable and because the alpha 5/alpha 6 loops of GRK2 and axin are identical in length (the loop is one amino acid shorter in the RGS family of proteins). The overall model was refined in O to idealize its stereochemistry.

In Vivo Inositol Phosphate Determination

GRK2 Mutants-- To measure in vivo synthesized inositol phosphate (IP), 3.3 × 105 COS-1 cells were plated on 6-cm dishes in DMEM (Mediatech, Herndon, VA) containing penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum. After 24 h, cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) with 1 µg of total DNA at a ratio of 3:1 pcDNA3-HA-Galpha q-R183C:pcDNA3-GRK2 (or mutant derivatives of GRK2). Following a 24-h incubation, transfected cells were replated (~7 × 104 cell/well) in triplicate on 24-well plates and incubated in complete DMEM. The media was removed and cells were labeled with myo-[3H]inositol (Amersham Biosciences) for 13-18 h in DMEM, without sodium pyruvate, with high glucose, with L-glutamine, and with pyridoxine hydrochloride. In early experiments, labeling was carried out in inositol-free DMEM (Invitrogen), whereas later experiments utilized complete DMEM. Cells were washed in the same media lacking radiolabel but containing 5 mM LiCl for 1 h at 37 °C. The media was removed and cells were lysed with 0.75 ml of 20 mM formic acid for 30 min at 4 °C before 0.1 ml of 3% ammonium hydroxide was added. Inositol was separated from IP by sequential elution from 1-ml Dowex AG1-X8 (100-200 mesh) columns. The inositol fraction was eluted with 0.18% ammonium hydroxide, whereas IPs were eluted with 4 M ammonium formate, 0.2 M formic acid. The inositol and IP fractions were mixed with Ultima Gold and Ultima Flo AF scintillation fluid (Packard), respectively, and subjected to scintillation counting. To compare experiments with differing levels of myo-[3H]inositol incorporation, IP production was determined as a fraction, IP/(IP + inositol), and plotted relative to the control Galpha q-R183C-stimulated IP production. Statistical significance was assessed using repeated measures analysis of variance with a Dunnett's post-test.

Galpha q-G188S Mutant-- IP accumulation experiments shown in Fig. 7 were carried out as described above except that HEK293 cells were utilized. In addition, 250 ng of plasmids encoding Galpha q-R183C or Galpha q-R183C/G188S were transfected with increasing amounts of pcDNA3-GRK2, pcDNA3-RGS2, and pB6-GAIP plasmids as indicated in the figure. pcDNA3 vector was used as carrier DNA such that 1 µg of DNA was transfected in each well of the 6-well plate. An unpaired t test was used to assess statistical significance.

Confocal Microscopy-- HEK293 cells were transfected in 6-well plates with the indicated amounts of expression plasmids for GRK2-(45-178)-GFP, RGS2-GFP, and/or Galpha q using FuGENE 6 reagent (Roche Molecular Biochemicals). After 24 h, transfected cells were replated onto glass coverslips and grown for an additional 24 h before fixing in 3.7% formaldehyde for 20 min. Cells were washed with phosphate-buffered saline and then incubated in blocking buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 2.5% nonfat milk). Coverslips were then incubated in blocking buffer containing a 1:100 dilution of anti-Galpha q polyclonal antibody (Santa Cruz) for 1 h. Following washes with blocking buffer, cells were incubated in a 1:100 dilution of Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 30 min. The coverslips were washed and mounted on glass slides with Prolong Antifade reagent. Representative images were recorded by confocal microscopy at the Kimmel Cancer Center Bioimaging Facility using a Bio-Rad MRC-600 laser scanning confocal microscope running CoMos 7.0a software and interfaced to a Zeiss Axiovert 100 microscope with Zeiss Plan-Apo 63× 1.40 NA oil immersion objective. Dual-labeled samples were analyzed using simultaneous excitation at 488 and 568 nm. Images of "x-y" sections through the middle of a cell were recorded.

    RESULTS
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Identification of GRK2 RGS Domain Mutants That Are Defective in Binding to Galpha q/11-- For a growing list of Gq-coupled receptors, it has been reported that desensitization can occur in a GRK2-dependent but phosphorylation-independent fashion. For example, hormone-mediated PLCbeta activation via the metabotropic glutamate receptor (mGluR1a) (47), the parathyroid hormone receptor (48), the thromboxane A2 receptor (34), the endothelin receptor (49), and the angiotensin II-1A receptor (50), is inhibited by overexpression of kinase-deficient GRK2-K220R. The parathyroid receptor and mGluR1a interact with full-length GRK2 (47, 48) and the RH domain of GRK2 co-immunoprecipitates with mGluR1a (51). For the mGluR1a (51), the endothelin receptor (49), and the thromboxane A2 receptor (34), overexpression of the RH domain of GRK2 inhibits Gq-stimulated phosphoinositide hydrolysis. Thus, phosphorylation-independent regulation of these receptors may be due either to GRK2/receptor interaction or to GRK2·Galpha q complex formation (or both). As a first step to determine the extent to which Galpha q binding by GRK2 regulates Gq-coupled receptor signaling, we used site-directed mutagenesis to create GRK2 mutants that are defective in Galpha q binding.

The three-dimensional structures of several RH domains have been determined by x-ray crystallography (RGS4, RGS9, axin, PDZRhoGEF, and p115RhoGEF) (23, 24, 52-54), and solution NMR (GAIP and RGS4) (55, 56). Together these studies show that these RH domains share a very similar fold (two four-helix bundles, see Fig. 1, B and C). The x-ray structures of two of these proteins, RGS4 and RGS9, were determined both alone and in complex with their Galpha ligands. The structures demonstrate that the RGS protein contacts all three switch regions of the Galpha subunit and that the RH domain does not undergo a large conformational change in tertiary structure upon binding the Galpha subunit. Crystal structures in combination with mutational analyses have identified amino acids in the R4 family that contact the Galpha subunit (23, 24, 57, 58). For RGS4 and RGS9, Galpha contact sites are primarily localized to the loops between alpha  helices, alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8 (see Fig. 1, A and B, for details). This has been designated the "A" site (59). Galpha i residues important for RGS4 interaction are also conserved in other Galpha subunits including Galpha q. Furthermore, mutation of an RGS4 residue in the alpha 3/alpha 4 loop critical to the RGS4/Galpha i1 interaction (E87K) prevents RGS4/Galpha q interaction (60). Therefore, it is presumed that the RGS4/Galpha q interface mimics the RGS4/Galpha i1 interaction.


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Fig. 1.   Protein binding surfaces of RH domains. A, structural alignment of the RH domain of GRKs with RGS, axin, and p115RhoGEF family members. An initial multiple sequence alignment of the RH domains was generated by ClustalW (72). Subsequently, RH domains of known structure were superimposed within the program O (73) and were used to verify and adjust the sequence alignment. Sequences with a known crystal or NMR structure are labeled with an asterisk. Boundaries of alpha  helices within the domains are depicted as blue boxes above the alignment. In p115RhoGEF and presumably LARG, the lengths of helices 7 and 8 differ from other RGS proteins and their locations are indicated by the orange helices below the alignment. Residues that were mutated (usually to alanine, see text) in GRK2 but do not alter binding to Galpha q-GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> have backgrounds colored gold, whereas those that decrease binding to Galpha q-GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> or increase binding to Galpha q-GDP are colored white on top of a purple background (this work, see text). Residues of RGS4 and RGS9 that contact the switch regions of Galpha i1 and the Galpha i/t chimera, respectively, are yellow (23, 24), whereas contacts of RGS9 with the alpha  helical domain of the Galpha i/t chimera are teal (24). Axin contacts with the APC peptide are green (52). Residues in RGS2 that, when converted to their equivalents in RGS4, enhance GAP activity toward Galpha i (74) are indigo. Mutations in p115RhoGEF that decrease Galpha 13 GAP activity are hot pink (53). Dashed boxes indicate regions that are structurally heterogeneous among the known structures. All sequences are human unless otherwise indicated. Accession numbers for the sequences used in the alignment are Q15835 (GRK1), P21146 (GRK2), P26818 (GRK3), P32298 (GRK4), P34947 (GRK5), P43250 (GRK6), NP_631948 (GRK7), P41220 (RGS2), P49799 (RGS4), O46469 (RGS9), NP_005864 (RGS19/GAIP), AAC51624 (axin), BAA20834 (PDZRhoGEF), NP_004697 (P115RhoGEF), and NP_056128 (LARG). B, structure of the RH domain from RGS4. Residues that contact Galpha i are drawn with yellow carbons atoms. The nine alpha -helices of the canonical RH domain are labeled alpha 1-alpha 9 (23). Nitrogen atoms are colored blue and oxygen atoms red. C, structure of axin bound to the APC peptide (52). Residues that contact the APC peptide are drawn with green carbon atoms. The APC peptide is drawn as a black coil. D, homology model of the RH domain of GRK2. The model was built using the structures of axin and GAIP as a guide (see "Experimental Procedures"). The gold and purple color scheme described above applies to parts B-D of this figure.

The first residues of GRK2 targeted for mutagenesis were those that are identical or similar to residues in RGS4 known to contact Galpha i1 (GRK2 residues Asp160 and Lys164 corresponding to RGS4 residues Asp163 and Arg167, respectively) (Fig. 1A). A construct that encodes a GST fusion protein bearing the RH domain of GRK2 (amino acids 45-178) was used as a template for mutagenesis. Purified WT and mutant GST fusion proteins on glutathione-agarose beads were incubated with bovine brain lysates in the presence of GDP or GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. Beads were pelleted and the binding of Galpha q/11 was assessed by immunoblotting with Galpha q/11-specific antibody. WT, D160K, and K164A bound similarly to Galpha q/11 in an AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-dependent fashion (data not shown). Further mutagenesis of residues in the putative loops between alpha 3 and alpha 4 and between alpha 7 and alpha 8 (H75A/L76A, E77A, E78K, K80A, V83A, and N156A) was carried out without identifying any residues important for Galpha q/11 binding. Furthermore, double mutants such as E78K/D160K, V83A/D160K, and D160K/K164A retained the ability to bind Galpha q/11 in the presence of AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (data not shown). Thus, unlike RGS4 and RGS9 binding to Galpha i and Galpha t, respectively, residues in the alpha 3/alpha 4 and alpha 7/alpha 8 loops did not appear to be critical for Galpha q/11 binding by GRK2.

Binding of axin to its RH domain ligand, an alpha  helix from APC, utilizes residues within the alpha 3, alpha 4, and alpha 5 helices (52). This region, which has been designated the "B" site (59), was targeted in the next round of mutagenesis. Whereas mutations in alpha 4 (E84A, E87A, and K90A) and a double mutant in alpha 5 (V103A/C104A) had no effect on binding, alpha 5 substitutions R106A and D110A resulted in diminished binding to Galpha q/11 (Fig. 2).


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Fig. 2.   Identification of eight GRK2 RH domain mutants with altered binding to Galpha q/11. Upper panel, glutathione-agarose beads bearing GST fusion proteins, either WT (GST-GRK2-(45-178)) or GST-GRK2-(45-178) substituted at one of eight single amino acid positions, were incubated with bovine brain extract in the presence (+) or absence (-) of aluminum fluoride (AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>). Bound Galpha q/11 was visualized by immunoblotting. Lower panel, GST fusion proteins used in the GST pull-down assay above were separated by SDS-PAGE and visualized by Coomassie staining.

We then compiled a structural alignment to compare the RH domain of GRKs to RH domains whose structures have been solved (Fig. 1A) and used the known three-dimensional structures of RH domains that had the highest sequence identity to the GRK2 RH domain (axin and GAIP) to model residues 42-175. The model was then used to predict residues that might be close to Arg106 and Asp110 in three-dimensional space. Substitutions F109I, M114A, and E116A exhibited the greatest diminution in the binding to Galpha q/11 in the presence of GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, whereas L117A and V137A had a lesser effect (Fig. 2). E107A, Q133A, and K139A while spatially near R106A and D110A, had no observable effect on binding Galpha q/11. Interestingly, K115A showed no reproducible decrease in GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-dependent binding, but a dramatic increase in the ability to bind GDP-bound Galpha q/11. Mutations with the greatest effect map to the alpha 5 helix and the beginning of the alpha 5/alpha 6 loop (and to the alpha 6/alpha 7 loop to a much lesser extent) and form a continuous surface (assuming that the axin-based model is a good representation of the GRK2 RH domain).

GRK Mutants with Galpha q Binding Defects Are Not Impaired in in Vitro Receptor Phosphorylation-- All amino acids selected for mutagenesis were predicted by our homology model to be exposed at the surface of the RH domain. However, it is possible that some of these residues are in fact buried and substitution to alanine might cause alteration in the tertiary structure. To test whether single amino acid mutations in the RH domain altered another function of the NH2-terminal domain, we looked at the ability of Galpha q-binding mutants to phosphorylate activated rhodopsin. The NH2-terminal domain of GRKs is thought to be involved in the activation of the kinase domain by agonist-stimulated receptors. An antibody directed at residues 17-34 of GRK1 blocks rhodopsin, but not peptide, phosphorylation (61). Likewise, E7A-GRK1 and E5A-GRK2 mutants are defective in rhodopsin, but not peptide, phosphorylation (62). To test the integrity of the NH2-terminal domain of Galpha q-binding mutants, the R106A, D110A, M114A, K115A, and V137A mutations were introduced into an expression vector encoding full-length GRK2 (pcDNA3-GRK2). Lysates from COS-1 cells transfected with WT or mutant GRK2 were assayed in a rhodopsin phosphorylation assay. We did not observe any statistically significant defects in the ability of Galpha q-binding mutants of GRK2 to phosphorylate light-activated rhodopsin (Fig. 3).


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Fig. 3.   Galpha q/11 binding-defective mutants phosphorylate rhodopsin. Upper panel, lysates were prepared from COS-1 cells transfected with full-length WT or mutant GRK2 constructs and equal volumes were used in a kinase assay with light-activated rhodopsin as a substrate. An autoradiograph representative of three separate experiments is shown. Middle panel, levels of WT or mutant GRK2 present in each lysate were compared by immunoblotting of equal volumes of lysate. Lower panel, the kinase activity in WT and mutant GRK2 lysates was quantified and mean ± S.E. for the three separate experiments are displayed.

GRK Mutants with Galpha q Binding Defects Are Not Recruited to Plasma Membrane by Activated Galpha q-- A constitutively active mutant of Galpha q, Galpha q-Q209L, is able to promote plasma membrane recruitment of a GFP fusion protein containing the RH domain of GRK2, GRK2-(45-178)-GFP.2 The ability to induce plasma membrane localization of GRK2-(45-178)-GFP is specific to Galpha q because other activated Galpha subunits fail to recruit GRK2-(45-178)-GFP to plasma membranes.2 We thus used this assay to examine interactions between Galpha q-Q209L and a Galpha q binding-defective mutant of GRK2 in cultured cells (Fig. 4). When expressed in HEK293 cells, the GFP-tagged RH domain of GRK2 is localized in the nucleus and throughout the cytoplasm (Fig. 4A). Likewise, GRK2-(45-178)-GFP bearing the D110A mutation is diffusely localized throughout the nucleus and cytoplasm when expressed alone (Fig. 4D). Co-expression of Galpha q-Q209L and GRK2-(45-178)-GFP results in their co-localization at cellular plasma membranes (Fig. 4, B and C). However, D110A-GRK2-(45-178)-GFP remains in the nucleus and cytoplasm when co-expressed with Galpha q-Q209L (Fig. 4, E and F). This defect in Galpha q-Q209L-induced plasma membrane recruitment of D110A-GRK2-(45-178)-GFP parallels the failure of this mutant to bind Galpha q/11 in the in vitro binding assay. When R106A-GRK2-(45-178)-GFP was co-expressed with Galpha q-Q209L in similar experiments, this mutant demonstrated consistent but weak plasma membrane localization (data not shown).


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Fig. 4.   Galpha q-Q209L induces plasma membrane recruitment of GRK-(45-178)-GFP but not D110A-GRK2-(45-178)-GFP. HEK293 cells were transfected with 0.02 µg of WT GRK2-(45-178)-GFP (A-C), D110A-GRK2-(45-178)-GFP (D-F), along with 1 µg of pcDNA3 (A and D) or 1 µg of pcDNA3 containing EE epitope-tagged Galpha q-Q209L (B, C, E, and F). Subcellular localization was determined by confocal microscopy. GRK2-(45-178)-GFP (A and B) and D110A-GRK2-(45-178)-GFP (D and E) were visualized by GFP fluorescence, whereas Galpha q- Q209L (C and F) was visualized using an anti-alpha q polyclonal antibody followed by an Alexa 594-conjugated anti-rabbit antibody. Representative micrographs are shown. More than 100 cells were examined in at least five separate experiments. Bar, 10 µm.

Galpha q Binding Mutants of GRK2 Are Defective in the Regulation of Galpha q-R183C-stimulated PLCbeta Activity in Vivo-- To determine whether the binding defects are manifested in full-length GRK2, we tested the ability of GRK2 mutants to regulate Galpha q-R183C activation of PLCbeta in vivo. COS-1 cells were transfected with Galpha q-R183C alone, or co-transfected with Galpha q-R183C and WT or mutant GRK2, and inositol phosphate production was determined. WT GRK2 reduced Galpha q-R183C-stimulated inositol phosphate production by ~48% (p < 0.01, Fig. 5). Likewise, the K115A mutant, which binds both the GDP- and GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> complexed forms of Galpha q, was equally effective (53% reduction) as WT GRK2 at preventing PLCbeta activation (p < 0.01). In contrast, the R106A, D110A, and M114A derivatives had no statistically significant effect on Galpha q-R183C-stimulated PLC activity. The V137A mutant, which exhibited only a modest binding defect in the in vitro binding assay, only inhibited PLCbeta activation by 27% (p < 0.05). In general, the ability of full-length WT or mutant GRK2 to inhibit Galpha q-R183C-stimulated inositol phosphate production in vivo reflects the propensity of the analogous GST-GRK2-(45-178) fusion protein to bind brain Galpha q/11 in vitro.


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Fig. 5.   Inhibition of Galpha q-R183C-stimulated inositol phosphate accumulation by WT but not mutant GRK2. COS-1 cells, transfected with Galpha q-R183C alone or in combination with full-length GRK2 or mutant derivative, were labeled with myo-[3H]inositol for 18 h. Inositol was separated from inositol phosphates as described under "Experimental Procedures" and quantified by scintillation counting. Shown are mean ± S.E. for four to 10 experiments performed in triplicate. Differences between Galpha q-R183C alone and Galpha q-R183C + GRK2-WT, GRK2-K115A, or GRK2-V137A were statistically significant (p < 0.05). Differences between Galpha q-R183C alone and Galpha q-R183C + GRK2-R106A, GRK2-D110A or GRK2-M114A were not statistically significant.

GRK2 Binds RGS-resistant Galpha q-G188S Mutant-- Because the RH domain of GRK2 preferentially binds to the transition state of Galpha q/11, at least one of the three switch regions is expected to be involved in their interaction. However, because Galpha q binds a surface of the GRK2 RH domain that is distinct from RGS4/Galpha interface, the Galpha q contribution to the GRK2/Galpha q interface is likely to be distinct as well. To test this hypothesis, we evaluated the ability of the RGS-resistant mutant, Galpha q-G188S, to interact with GRK2. An RGS-resistant Galpha mutant was first described for the Galpha subunit of the Saccharomyces cerevisiae trimeric G protein, Gpa1p (63). gpa1sst mutants are supersensitive to the mating pheromone, alpha -factor, because of a failure to bind the RGS protein, Sst2p. The binding defect is because of a Gly right-arrow Ser substitution in switch I of the Galpha subunit. Gly right-arrow Ser substitutions in mammalian Galpha subunits Galpha i, Galpha o, and Galpha q also result in resistance to RGS proteins of the R4 and R7 subfamilies (63, 64).

We first looked at the ability of the RGS-resistant mutant of Galpha q to recruit GRK2-GFP to the plasma membrane. HEK293 cells were transiently transfected with Galpha q, Galpha q-R183C, or Galpha q-R183C/G188S and either GRK2-(45-178)-GFP or RGS2-GFP. A previous report demonstrated that co-expression of an activated mutant of Galpha q promoted the plasma membrane localization of RGS2-GFP, whereas expression of RGS2-GFP alone resulted in nuclear and cytoplasmic localization, with very weak plasma membrane localization (40). When co-transfected with Galpha q, GRK2-GFP was found throughout the cell, and RGS2-GFP displayed prominent nuclear staining with some cytoplasmic and faint plasma membrane localization (Fig. 6). In contrast, when transfected with the R183C GTPase mutant of Galpha q, GFP-GRK2 was partially recruited to the plasma membrane (Fig. 6), as also observed with expression of Galpha q-Q209L (Fig. 4B), and GFP-RGS2 was strongly recruited to the plasma membrane (Fig. 6). However, GFP-RGS2 and GFP-GRK2 differed when assayed for plasma membrane recruitment upon co-expression with the RGS-resistant mutant of Galpha q. When co-expressed with Galpha q-R183C/G188S, GRK2-GFP was recruited to the plasma membrane to the same extent as when co-expressed with Galpha q-R183C. In contrast, RGS2-GFP was only faintly detected at the plasma membrane, and instead, was localized to the nucleus and cytoplasm (Fig. 6). Thus, the G188S mutation of Galpha q prevents RGS2 but not GRK2 binding.


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Fig. 6.   Galpha q-R183C/G188S recruits GRK2 RH domain, but not RGS2, to the plasma membrane. HEK293 cells were co-transfected with 0.25 µg of pcDNA3 containing hemagglutinin-tagged versions of Galpha q, Galpha q-R183C, or Galpha q-R183C/G188S, and either 0.025 µg of GRK2 (45-178)-GFP (left panel) or 0.25 µg of RGS2-GFP (right panel) as indicated. Either 0.725 or 0.5 µg of pcDNA3 was included to adjust total transfected DNA to 1 µg. Cells on coverslips were fixed in formaldehyde and mounted on glass slides as described under "Experimental Procedures." The localization of GFP-tagged proteins was visualized by confocal microscopy. Only cells expressing plasma membrane localized Galpha q, Galpha q-R183C, or Galpha q-R183C/G188S (not shown), as determined by using an anti-alpha q polyclonal antibody followed by an Alexa 594-conjugated anti-rabbit antibody, were chosen to identify subcellular localization of the GFP-tagged proteins. Representative micrographs are shown. More than 100 cells were examined in at least five experiments. Bar, 10 µm.

We next looked at the ability of GRK2 to attenuate signaling by the RGS-resistant mutant. HEK293 cells were co-transfected with Galpha q-R183C or Galpha q-R183C/G188S and increasing amounts of RGS2, GAIP, or GRK2 (full-length) cDNAs, and inositol phosphate production was measured. GRK2, RGS2, and GAIP each inhibited the Galpha q-stimulated PLCbeta activity in a dose-dependent fashion with the inhibition being 89, 71, and 54%, respectively, at the highest level of DNA transfected (Fig. 7). Immunoblotting suggested that Galpha q expression was similar in all experiments and that increasing the level of GRK2, RGS2, and GAIP cDNA increased the level of expression (data not shown). Whereas GAIP inhibited Galpha q-R183C-stimulated PLCbeta activity by 54%, it was much less effective at inhibiting Galpha q-R183C/G188S-stimulated IP production (19% decrease, p < 0.05). Likewise, RGS2 showed diminished ability to regulate IP production stimulated by the RGS-resistant mutant (31% decrease) relative to its ability to decrease the Galpha q-R183C-stimulated PLCbeta activity (71%, p < 0.05). In stark contrast, GRK2 inhibited PLCbeta activity stimulated by both Galpha q-R183C (89% decrease) and Galpha q-R183C/G188S (95% decrease) to a similar extent. Thus, unlike RGS proteins RGS2 and GAIP, GRK2 can tolerate substitution of Ser for the conserved Gly in the switch I region.


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Fig. 7.   Galpha q-R183C/G188S-stimulated inositol phosphate production is sensitive to GRK2 but resistant to RGS2 and GAIP. HEK293 cells were transfected with Galpha q-R183C (A) or Galpha q-R183C/G188S (B) alone or with increasing amounts of full-length GRK2 (black-square), GAIP (black-triangle), or RGS2 (black-diamond ). Cells were labeled with myo-[3H]inositol and inositol was separated from inositol phosphates as described under "Experimental Procedures" and quantified by scintillation counting. The experiment shown is representative of three independent experiments and displayed as the average of triplicates ± S.D.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Through extensive mutational analysis, we have identified eight residues in the GRK2 RH domain (Arg106, Phe109, Asp110, Met114, Lys115, Glu116, Leu117, and Val137) that, when mutated, alter binding to Galpha q/11. The binding defects attributed to these mutations are not likely to be due to misfolding of the RH domain because we were unable to detect any differences between the ability of WT and GRK2 mutants to carry out receptor phosphorylation, a function that requires an intact NH2-terminal domain. With the exception of K115A, all of the mutants exhibit diminished binding to GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-bound Galpha q/11. Mutations with the most dramatic effects map to the COOH-terminal half of the alpha 5 helix and the NH2-terminal region of the alpha 5/alpha 6 loop (Fig. 1, A and D). The GRK2 binding surface is distinct from both the binding interface used by RGS4 and RGS9 to bind Galpha subunits (alpha 3/alpha 4, alpha 5/alpha 6, and alpha 7/alpha 8 loops) and the surface used by axin to bind APC helical peptide (alpha  helices 3, 4, and 5). Comparison of the Galpha binding site on GRK2 with the Galpha contact sites on RGS4/RGS9 suggests that these sites do not overlap (Fig. 1, B-D). Although RGS4 and RGS9 also utilize the alpha 5/alpha 6 loop, the GRK2 contact sites are in the amino terminus of that loop whereas the RGS4/RGS9 contact sites are concentrated toward the carboxyl terminus. Therefore, our experiments identify a novel Galpha binding site on the RH domain. Consistent with the nomenclature of Zhong and Neubig (59) we propose that this surface be termed the "C" site.

Galpha q binding-defective mutants not only fail to bind Galpha q/11 in an in vitro pull-down assay, but they are also defective in cell-based assays. First, unlike the WT GRK2 RH domain fusion protein GRK2-(45-178)-GFP that co-localizes with activated Galpha q at the plasma membrane, the Galpha q binding-defective mutant D110A-GRK2-(45-178)-GFP fails to be recruited to the plasma membrane and instead remains in the nucleus and cytoplasm. Likewise, whereas full-length WT GRK2 inhibits Galpha q-R183C-stimulated PLCbeta activity by ~50%, Galpha q binding-defective mutants GRK2-R106A, GRK2-D110A, and GRK2-M114A have no affect on PLCbeta activity. Thus, the failure to bind to Galpha q in vitro has the expected in vivo ramifications: these GRK2 mutants fail to bind to activated Galpha q at the plasma membrane and fail to regulate the activity of this Galpha subunit.

One mutant, K115A, has a particularly interesting phenotype. WT GRK2 prefers binding to the transition state over the GDP-bound form of Galpha q (Fig. 1) (34). Whereas undiminished in its capacity to bind Galpha q/11-GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, the mutant has a greatly enhanced ability to bind Galpha q/11-GDP. One hypothesis is that binding by the K115A mutant traps Galpha q/11-GDP in a Galpha q/11-GDP·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>-like conformation. Another idea is that Lys115 may normally play a negative role in preventing GRK2 from binding the GDP-bound form of Galpha q/11. In this scenario, the Lys right-arrow Ala substitution would relieve that inhibition. An alternative hypothesis is that the K115A mutation allows recognition of some feature of the GDP-bound state that is not accessible to the WT protein.

Switch regions I, II, and III constitute most of the buried surfaces on Galpha subunits in the RGS4/Galpha i1 and RGS9/Galpha t interfaces (23, 24). Because the RH domain interface in the GRK2/Galpha q interaction is novel, we propose that some aspect of the Galpha q interaction surface is distinct from the surfaces utilized by Galpha i1 and Galpha t in their RGS4 and RGS9 interactions. In support of this hypothesis, we found that the RGS-resistant G188S mutant of Galpha q is not refractory to the GRK2 RH domain. The Galpha q-R183C/G188S mutant recruits GFP-GRK2-(45-178) to the plasma membrane and full-length GRK2 effectively blocks Galpha q-R183C/G188S-stimulated PLCbeta activity. The analogous residue in Galpha i1, Gly183, sits in close proximity to RGS4 Glu83 in the RGS4/Galpha i1 co-crystal and is surrounded by other switch I residues that make hydrogen bonds and ionic contacts with RGS4. Substitution of the Gly183 with almost any residue would be predicted to alter the tertiary structure and decrease complementarity to the RGS4 interface. Because Galpha q-R183C/G188S does not inhibit GRK2 interaction, we speculate either that Gly188 is not located in the GRK2 interface or that minor changes in the Galpha q and/or GRK2 tertiary structure can accommodate the Galpha q Gly right-arrow Ser substitution within the interface.

Of the eight residues whose alterations cause Galpha q binding defects, six are strictly conserved in GRK3 but not in other members of the GRK family (Fig. 1). This clearly explains why GRK2 and GRK3, but not GRK1, GRK4, GRK5, nor GRK6, bind Galpha q. Surprisingly, four residues that make important contributions to Galpha q binding by GRK2 are conserved in other RGS proteins. For example, the Arg106 position is a basic residue in RGS2, RGS3, RGS4, RGS5, RGS8, RGS13, RGS16, RGS17, RGS18, RGS19, and RGS20. Phe109, a residue that is exposed to the solvent in RGS4, is Phe or Tyr in most RGS proteins. Asp110 is an asparagine or glutamate in RGS4, RGS5, RGS8, RGS16, RGS17, RGS18, GAIP, and RGS20. Finally, Glu116 is glutamate, aspartate, or glutamine in RGS2, RGS3, RGS4, RGS5, RGS8, RGS16, and RGS18. Thus, four of the presumed GRK2 contact residues are each conserved in RGS2, RGS4, RGS5, RGS8, RGS16, and RGS18. RGS proteins are modular and the family members are sometimes categorized based on presence or absence of protein interaction domains in the full-length polypeptide (20). RGS2, RGS4, RGS5, RGS8, RGS16, and RGS18 are all members of the "small" RGS subfamily and contain only short sequences outside the RGS domain. Because RGS2, RGS4, and RGS16 can bind Galpha q and because the GRK2/Galpha q interface is contiguous with the RGS4/Galpha i1 and RGS9/Galpha t interface, perhaps residues in the alpha 5 helix and NH2-terminal portion of the alpha 5/alpha 6 loop may play a role in binding of Galpha q by other RGS family members.

Residues in the RGS alpha 5 helix may be conserved because they bind other regulatory ligands. Indeed, RGS4 binds phosphatidylinositol 3,4,5-trisphosphate (PIP3) but a double mutant K112E/K113E lacks this capability (65). In our alignment (Fig. 1A), RGS4 Lys112 corresponds to GRK2 Arg106; therefore, a Galpha q contact site in GRK2 corresponds to a PIP3 contact site in RGS4. PIP3 inhibits the GAP activity toward Galpha i1 of RGS1, RGS10, and GAIP, but not that of RGS16. Furthermore, many RGS proteins have putative calmodulin (CaM) binding sites that map to a region spanning the COOH terminus of alpha 4 helix and the NH2-terminal half of the alpha 5 helix. CaM binds to RGS1, RGS2, RGS4, RGS10, RGS16, and GAIP in a calcium-dependent fashion, but does not alter the GAP activity of RGS4 (65). However, CaM does reverse the inhibitory effect of PIP3 on GAP activity. Lys112 and Lys113 of RGS4 represent the COOH terminus of the consensus CaM binding site, yet the K112E/K113E double mutant does not alter CaM binding to RGS4. Because of the proximity of the putative PIP3- and CaM-binding sites to the C site of the GRK2 RH domain, it would be interesting to test whether PIP3 (or other phosphatidylinositides) or CaM affect binding to Galpha q by RGS family proteins. Incidentally, GRK2 binds to phosphatidylinositides and phosphatidylserine (66-70) and to CaM with low affinity (71), but none of the these ligand-binding sites are located within its RH domain.

It has recently been shown that LARG, in addition to binding Galpha 12 and Galpha 13, can also bind Galpha q, a characteristic that distinguishes it from its close relatives, p115RhoGEF and PDZRhoGEF (32). The RH domain of RhoGEF family members shares only a small number of residues implicated in Galpha interaction by RGS4 and RGS9. Likewise the alpha 5, alpha 5/alpha 6 loop region of LARG does not bear similarity to the Galpha q binding region of GRK2 (see alignment in Fig. 1A). Thus, the Galpha q interaction site on this RH domain-containing protein appears distinct from the C site.

In summary, extensive mutational analysis of GRK2 shows that Galpha q/11 binding to the RH domain occurs at a novel Galpha binding site that we have called a C site. This, in combination with the inability of the switch I mutant Galpha q-G188S to affect GRK2 RH domain association, suggests that GRK2 binding occurs on a distinct surface of Galpha q/11 as well. Interestingly, several mutations in the alpha 5 helix that inhibit Galpha q/11 binding do not impair rhodopsin phosphorylation, suggesting that the region of the GRK2 NH2-terminal domain necessary for receptor phosphorylation is distinct from the C site of the RH domain. Further studies are necessary to map residues of the GRK2 NH2 terminus that are necessary for receptor phosphorylation and to define features of Galpha q that are required for GRK2 interaction.

    ACKNOWLEDGEMENTS

We thank Drs. Dave Manning and Henry Bourne for providing Galpha q/11-specific and EE antibodies, respectively, and Drs. Scott Heximer, Ray Penn, and Chris Carman for providing RGS2-GFP, GRK2-GFP, and GST-GRK2(RH)-K164A constructs. R. S. M. thanks Dr. Richard Neubig for helpful suggestions, Dr. Ray Penn for statistical advice, and Siena undergraduates Erin Twiss, Carlos Gonzalez, Billy Robinson, Jay Kubik, and Mike Ragusa for their contributions to this work. R. S. M. also thanks Dr. Ken Helm for sharing equipment and reagents, Betsey Harvey for support, and Drs. Jim Angstadt, Tom Coohill, and Doug Fraser for encouragement. Finally, we thank the reviewer for helpful comments.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB9728179 and an American Heart Association Southeastern Pennsylvania Affiliate Beginning grant-in-aid (to R. S. M.), American Heart Association Texas Affiliate Beginning Grant-in-aid 0060118Y and a Welch Foundation Chemical Research Grant F-1487 (to J. J. G. T.), a fellowship from the American Heart Association Pennsylvania-Delaware Affiliate (to P. W. D.), National Institutes of Health Grants GM44944 and GM47417 (to J. L. B.) and GM56444 and GM628884, and a grant from the Pew Scholars Program in the Biomedical Sciences (to P. B. W.).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.

§ To whom correspondence should be addressed: Siena College, Biology Dept., 123 Morrell Science Center, 515 Loudon Rd., Loudonville, NY 12211. Tel.: 518-783-2462; Fax: 518-783-2986; E-mail: sternemarr@siena.edu.

Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208787200

2 P. W. Day and P. B. Wedegaertner, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; PLCbeta , phospholipase Cbeta ; GRK, GPCR kinase; RGS, regulator of G protein signaling; GAP, GTPase activating protein; RH, RGS homology; AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, aluminum fluoride; GFP, green fluorescent protein; WT, wild type; GST, glutathione S-transferase; GEF, guanine-nucleotide exchange factor; APC, adenomatous polyposis coli; LARG, leukemia-associated RhoGEF; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; CaM, calmodulin; PIP3, phosphatidylinositol 3,4,5-trisphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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