RGS3 Is a GTPase-Activating Protein for Gia and Gqa and a Potent Inhibitor of Signaling by GTPase-Deficient Forms of Gqa and G11a

Many Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTPase activity of Gia and Gqa-subunits [i.e., behave as GTPase-activating proteins (GAPs)] and several act as Gqa-effector antagonists. RGS3, a structurally distinct RGS member with a unique N-terminal domain and a C-terminal RGS domain, and an N-terminally truncated version of RGS3 (RGS3CT) both stimulated the GTPase activity of Gia (except Gza) and Gqa but not that of Gsa or G12a. RGS3 and RGS3CT had Gqa GAP activity similar to that of RGS4. RGS3 impaired signaling through Gq-linked receptors, although RGS3CT invariably inhibited better than did full-length RGS3. RGS3 potently inhibited GqaQ209Land G11aQ209L-mediated activation of a cAMP-response element-binding protein reporter gene and GqaQ209L induced inositol phosphate production, suggesting that RGS3 efficiently blocks Gqa from activating its downstream effector phospholipase C-b. Whereas RGS2 and to a lesser extent RGS10 also inhibited signaling by these GTPase-deficient G proteins, other RGS proteins including RGS4 did not. Mutation of residues in RGS3 similar to those required for RGS4 Gia GAP activity, as well as several residues N terminal to its RGS domain impaired RGS3 function. A greater percentage of RGS3CT localized at the cell membrane than the full-length version, potentially explaining why RGS3CT blocked signaling better than did full-length RGS3. Thus, RGS3 can impair Gi(but not Gz-) and Gq-mediated signaling in hematopoietic and other cell types by acting as a GAP for Gia and Gqa subfamily members and as a potent Gqa subfamily effector antagonist. A variety of hormones, neurotransmitters, and physical stimuli trigger intracellular responses by binding to seven transmembrane receptors. These receptors link to downstream signaling pathways by activating heterotrimeric G proteins and, as such, are designated G protein-coupled receptors (GPCRs). In their inactive state heterotrimeric G proteins are composed of three subunits: a, b, and g (see reviews by Bourne et al., 1991; Hepler and Gilman, 1992; Gudermann et al., 1995; Neer, 1995). There are 23 a-subunits divided into four major subfamilies based on primary sequence homology and common downstream effectors termed Gsa, Gia, Gqa, and G12/13a. There are five different b-subunits and 10 different g-subunits. Upon ligand binding a GPCR stimulates the a-subunit of a heterotrimeric G protein to exchange GDP for GTP. In the GTP-bound form, Ga dissociates from Gbg, each of which can activate downstream effectors. Signaling is halted when the GTP-bound Ga-subunits hydrolyze GTP to GDP, which results in reassembly with Gbg-subunits to form inactive heterotrimers. Recent genetic and biochemical experiments have revealed the existence of a novel family of proteins termed Regulators of G protein Signaling (RGS) that act as GTPase-activating proteins (GAPs) for the Gia and Gqa subfamilies (De Vries et al., 1995; Berman et al., 1996b; Dohlman et al., 1996; Druey et al., 1996; Hunt et al., 1996; Koelle and Horvitz, 1996; Watson et al., 1996). Recently, p115 RhoGEF, which contains a highly diverged RGS domain, was shown to be a G12a GAP (Kozasa et al., 1998); however, no Gsa GAP has been shown to exist. Many RGS proteins bind tightly to the GDP-AlF4 activated forms of Gia and Gqa, a conformation that mimics This work was supported in part by a grant from the Deutscher Akademischer Austauschdienst (A.S.) and the Fogarty International Center, National Institutes of Health (Bethesda, MD). 1 Present address: Department of Integrative Biology, Pharmacology and Physiology, University of Texas-Houston Medical School, 6431 Fannin, MSB 4.109, Houston, TX 77225. 2 Present address: Department of Pharmacology/Toxicology, University of Western Ontario, Medical Sciences Building, London, Ontario, N6A5C1 Canada. ABBREVIATIONS: GPCR, G protein-coupled receptors; GAP, GTPase-activating proteins; RGS, regulators of G protein signaling; IP3, inositol triphosphate; GnRH, gonadotropin-releasing hormone; PCR, polymerase chain reaction; CREB, cAMP-response element binding; GST, glutathione S-transferase; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; LPA, lysophosphatidic acid; HA, hemagglutinin. 0026-895X/00/040719-10$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 12961/852446 Mol Pharmacol 58:719–728, 2000 Printed in U.S.A. 719 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from the transition state in the GTPase reaction, and thereby accelerate the intrinsic rate at which the Ga-subunits hydrolyze GTP (Berman et al., 1996a, b; Hunt et al., 1996; Watson et al., 1996; Hepler et al., 1997; Popov et al., 1997). Analysis of crystals of RGS4 complexed with Gia1-GDP-AlF4 2 revealed that the 120-amino acid RGS domain (also referred to as the RGS box) forms a four-helix bundle that directly contacts the three “switch regions” in Gia1 (Tesmer et al., 1997). These regions undergo the greatest conformational change during GTP hydrolysis, and specific amino acids in RGS4 appear to stabilize them in a transition state facilitating the hydrolysis reaction. The specificity of RGS4 protein for the Gia and Gqa subfamilies likely relies on the structure of the switch regions. Based on the RGS4-Gia1 and Gsa crystal structures, the failure of RGS4 to bind Gsa is secondary to specific amino acids in Gsa and RGS4 that disrupt the interaction by steric overlap, charge repulsion, and creations of small cavities at the interface (Sunahara et al., 1997; Tesmer et al., 1997). The failure of RGS4 to act as a GAP for G12a is more easily explained because amino acid differences in the G12a switch regions would disrupt the surface and charge complementarity of the interface observed between RGS4 and Gia1 (Tesmer et al., 1997). Several studies have indicated that the RGS protein RGS3 impairs Giand Gq-mediated signaling. RGS3 inhibited interleukin-8 induced mitogen-activated protein kinase activation (Druey et al., 1996) and inositol triphosphate (IP3) production in response to signaling through the gonadotropinreleasing hormone (GnRH) receptor (Neill et al., 1997), Giand Gq-linked signaling pathways, respectively. A truncated form of RGS3 (RGS3CT) impaired Gqand Gi-mediated signaling as well as Gs-triggered signaling, whereas a fulllength version inhibited only Gi-mediated signaling (Chatterjee et al., 1997a). In contrast, expression of a full-length RGS3 in a human mesangial cell line partially blocked an endothelin-1-induced calcium flux, a Gq-mediated response (Dulin et al., 1999). The present study explored the relative effectiveness of RGS3, RGS3CT, and RGS4 in modulating Gq-mediated signaling. We provide information concerning the relative GAP activity of RGS3, RGS3CT, and RGS4 for Gqa, as well as Gia, Gza, Gsa, and G12a. RGS3 emerges as a potent inhibitor of Gq-mediated signaling by acting not only as a Gq GAP but also as an antagonist of GTP-bound Gq signaling. Experimental Procedures Plasmids. To make His6-RGS3 and His6-RGS3CT (amino acids 314–520) polymerase chain reaction (PCR) fragments generated from RcCMV-RGS3 were inserted into the NdeI/XhoI sites of the bacterial expression vector pET15b (Novagen, Madison, WI) in frame with the hexahistidine tag. To make glutathione S-transferase (GST)-RGS3, a PCR product generated from RcCMV-RGS3 was directionally cloned in the BamHI and EcoRI sites of the bacterial expression vector pGEX2T. To make FLAG-RGS3NT (RGS3 1–313), FLAG-RGS3CT (314–520), and FLAG-RGS3, the appropriate PCR products were subcloned into pFLAGCMV-2. FLAG-RGS3 E419A/ N420A (EN mutant), FLAG-RGS3 R499A/F500A (RF mutant), FLAG-RGS3 K350A/K353A/liter356A (KKL mutant), and FLAGRGS3 E386A/E387A (EE mutant) were created by site-directed mutagenesis of pFLAGCMV-2 RGS3 (Stratagene, La Jolla, CA). Expression vectors for the beta-adrenergic receptor, GqaQ209L and G11aQ209L, were kindly provided by Dr. S. Gutkind (National Institutes of Health, Bethesda, MD). The expression vectors for RGS1, RGS2, RGS3, and RGS4 have been previously described (Druey et al., 1996). Dr. P. Casey (Duke University, Durham, NC) and Dr. J. Gunzburg (Institut Curie, Paris, France) kindly provided the RGS10 and RGS14 expression vectors, respectively. The RGS5 expression vector was created by PCR with known sequence information and subcloned in-frame with a hemagglutinin (HA)-tag into pCRIII. The cAMP-response element binding (CREB)-b-galactosidase reporter plasmid was kindly provided by Dr. R. Cone (Vollum Institute, OR). The pFA2-Elk1, pFR-luc, and pSRE-luciferase plasmids were purchased (Stratagene). Purification of Recombinant Proteins. The His-tagged recombinant RGS protein expressions were performed in Escherichia coli BL21(DE3) by induction with 0.5 mM isopropylthio-b-galactoside at 30°C for 2 h. The recombinant proteins were batch purified under nondenaturing conditions with NiNTA beads (Qiagen, Santa Clara, CA) and eluted with an imidazole gradient. The purified protein fractions were dialyzed against the wash buffer and stored at 270°C. To make the GST fusion proteins, the appropriate constructs were transformed into E. coli BL21(DE3) pLysS, and induced with 0.5 mM isopropylthio-b-galactoside for 2 h at 30°C. Recombinant protein purification was carried out in ice-cold phosphate-buffered saline (PBS)/1% Triton X-100 with glutathione-Sepharose beads (Pharmacia, Piscataway, NJ). After purification the GST-fusion protein was stored on the beads at 4°C or eluted and kept at 270°C. Immunocytochemistry. HEK 293T cells were grown on a cover slip in a 10-cm plate [Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS)] until they were 50% confluent. Transfection with pFLAGCMV-2 RGS3, pFLAGCMV-2 RGS3CT, or empty vector was performed with calcium phosphate. The medium was changed 8 h after transfection, and cells were harvested 2 days later. The cover slips were washed with PBS, covered with 50% acetone/ 50% methanol, and kept at 4°C. After 1 h the liquid was removed, and the cover slips were air dried. Blocking of nonspecific binding sites was performed for 2 h at room temperature with PBS containing 10% FCS and 2% bovine serum albumin (BSA). Then the slides were incubated with mouse anti-FLAG monoclonal antibody (1:1000) in 2% BSA in PBS for 2 h at room temperature. After the sample was washed with PBS for 10 min, the slides were incubated for 2 h with fluorescein isothiocyanate-conjugated affinity-purified goat antimouse Ig 1:1000 in PBS containing 2% BSA. Then, cover slips were washed four times with PBS, air dried, and mounted on slides. Measurement of GAP Activity. Measurements of kcat for hydrolysis of GTP for Gza and G12a were determined as described (Berman et al., 1996b). Direct measurement of the kcat for GTP hydrolysis by Gqa required the use of the mutant GqaR183C, which is based on the analogous mutation in Gsa, R174A (Freissmuth and Gilman, 1989), and Gia, R178C (Kleuss et al., 1994). Although this mutation in Gia markedly reduces its kcat for GTP hydrolysis, the mutant protein retains its responsiveness to RGS proteins (Chediac and Ross, 1999). The method used for Gza hydrolysis of GTP is a modification of that previously described (Berman et al., 1996b). In this study similar methods were used for Gia to approximate as closely as possible the conditions for Gqa, Gza, and G12a. Briefly, G protein a-subunits were loaded with [gP]GTP (5–10 mM, Amersham, Cleveland, OH) in the presence of 50 mM HEPES (pH 7.4), 0.1 mg/ml BSA, 1 mM dithiothreitol, and either 5 mM EDTA and 0.05% C12E10 (for Gia) or 10 mM free Mg, 30 mM (NH4)2SO4, 4% glycerol, and 5.5 mM 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS; for Gqa). The loading reactions were performed for 20 min at 30°C for Gia or 2 h at 20°C for Gqa. After incubation, free [g P]GTP and [P]orthophosphate were removed by chromatography on Sephadex 25 containing 50 mM HEPES (pH 7.4), 1 mM CHAPS, 1 mM dithiothreitol, 18 mg/ml BSA, and either 0.1% octylglucoside plus 5 mM EDTA (Gia) or 10 mM free Mg 21 (Gqa). Hydrolysis of bound [g P]GTP was initiated by addition of 1 mM nonradioactive GTP, 10 mM MgCl2 (for Gia), and RGS protein or buffer. Reaction temperatures for Gia and Gqa were 4 and 20°C, respectively. Aliquots were removed at the 720 Scheschonka et al. at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from indicated times and added to 5% (w/v) Norit (Norit Americas Inc., Atlanta, GA) in 50 mM NaH2P04. After the sample was centrifuged at 1500 rpm for 10 min, aliquots of supernatant containing Pi were counted by liquid scintillation spectrometry. Assessment of Reporter Gene Activity. HEK 293T cells were plated in 10-cm dishes and transfected using calcium phosphate when the cells were 50% confluent. For Gq-mediated signaling, HEK 293T were transfected with constructs that direct the expression of the muscarinic type 1 (M1) receptor (2 mg/plate), FLAG-RGS3 or HA-RGS4, and CREB b-galactosidase reporter plasmid (2 mg/plate) receptor. In some experiments 0.5 mg of a cytomegalovirus-luciferase plasmid (Promega) was used to monitor the transfection efficiency. pcDNA was used to normalize the total amount of DNA used per plate. The medium was replaced 8 h later, and 48 h after transfection the cells were stimulated for 6 h with 1 mM carbachol (Sigma, St. Louis, MO) and then harvested. For Gs-mediated signaling, HEK293T cells were transfected with constructs that direct the expression of the beta-adrenergic receptor (2 mg/plate), CREB-bgalactosidase (1 mg/plate), and FLAG-RGS3 or HA-RGS4. Fortyeight hours after transfection, the cells were stimulated for 6 h with 10 mM isoproterenol (Sigma) and harvested. For GqaQ209Land G11aQ209L-mediated signaling, HEK 293T cells were transfected with constructs that direct the expression of CREB-b-galactosidase (1 mg/plate), GqaQ209L or G11aQ209L (0.5 mg/plate), and different RGS protein expression vectors. The cells were harvested 24 h after transfection. The pelleted cells from the various signaling assays were lysed in 100 ml of reporter lysis buffer (Promega) for 20 min on ice. After the sample was centrifuged, 10 ml of the supernatant were tested for b-galactosidase activity with galactan chemiluminescent substrate (Tropix, Bedford, MA) or luciferase activity with a luciferase substrate (Promega). Data were normalized by protein concentration (Bradford assay, Bio-Rad, Hercules, CA) or by the activity levels of a control reporter gene. The expression levels of various RGS proteins were confirmed by immunoblotting for the appropriate epitope, HA or FLAG. Western Blotting. The HS-Sultan, Molt-4, Jurkat, COS-7, PC12, RAMOS, HeLa, and K562 cell lines were obtained from the American Tissue Culture Collection (Rockville, MD). All the lymphoid cell lines were maintained in RPMI 1640 supplemented with 5 to 10% FCS, and the nonlymphoid cells were maintained in DMEM plus 10% FCS. Cell lysates of various cell lines were obtained by adding 1 3 10 cells to a solution containing 150 mM NaCl, 50 mM Tris (pH 7.5), 5 mM EDTA, and 1% Nonidet P-40, along with a cocktail of protease inhibitors for 20 min on ice. The detergentinsoluble material was removed by microcentrifugation for 10 min at 4°C. In some experiments, cells were lysed in hypotonic buffer (20 mM Tris-HCl, pH 7.5, with protease inhibitors), sonicated, subjected to a low-speed spin to remove the nuclei, and fractionated into a membrane-enriched and -depleted fraction by centrifugation at 52,000 rpm for 30 min. A total of 50 to 100 mg of protein (Bio-Rad assay) from each sample were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to pure nitrocellulose. Membranes were blocked with 3% BSA in TTBS (Tris-HCl, NaCl, Tween 20) for 1 h and then incubated with an appropriate dilution of the primary antibody in 1.5% BSA and 0.05% sodium azide in TTBS overnight. The blots were washed twice with TTBS before the addition of a biotinylated goat-anti rabbit Ig (DAKO, Carpinteria, CA) diluted 1:5000 in TTBS containing 10% FCS. After a 1-h incubation, the blot was washed twice with TTBS and then incubated with streptavidin conjugated to horseradish peroxidase (DAKO). The signal was detected by enhanced chemiluminescence following the recommendations of the manufacturer (Amersham). The antisera against RGS3 were used at a 1:400, and the mouse monoclonal antibodies were reactive with FLAG or HA (Covance, Richmond, CA) at a 1:1000 dilution. The RGS3 antiserum used in this study was prepared against recombinant RGS3 in rabbits and recognized recombinant RGS3, transfected RGS3, and a band of similar mobility in cellular lysates. Another rabbit antiserum raised against a conserved peptide in RGS2 and RGS3 also recognized recombinant RGS3, transfected RGS3, and the same bands as did the first antiserum (data not shown). Inositol Phosphate Production. COS-7 cells were transfected with LipofectAMINE (1:8) after serum starvation for 24 h. Twentyfour hours after transfection, the culture medium was replaced with inositol-free DMEM containing 5% FCS and 1 mM sodium pyruvate for 2 h. Next, 2 mCi/ml of myo-[2-H]inositol (Amersham) were added, and 15 min later, 10 mM LiCl was added. The cells were incubated for an additional 14 h and washed with phosphate-buffered saline, Fig. 1. Expression and intracellular localization of RGS3. A, Different cell lysates were analyzed by immunoblotting with an anti-RGS3 antiserum (lanes 1–16). Hs-Sultan cells were stimulated with LPA (10 M) for the indicated times. Molecular mass markers and the origin of the cell lysates are indicated. B, Localization of RGS3 and RGS3CT. Cos-7 cells were transfected with constructs directing expression of FLAG-RGS3 or FLAGRGS3CT, or with a control plasmid. Immunofluorescent staining with a FLAG antibody is shown. RGS3 Inhibits Ga Signaling 721 at A PE T Jornals on O cber 8, 2017 m oharm .aspeurnals.org D ow nladed from and then 0.5 ml of 20 mM formic acid was added to each well. After an incubation period of 30 min, the supernatant was collected and a second extraction was performed. Each 1-ml extract was neutralized to pH 7.5 with 7.5 mM HEPES and 150 mM KOH. The supernatants were centrifuged for 2 min at 15,000g and collected, and each was loaded onto to a 0.5-ml Dowex AG-X8 column (Bio-Rad), which had been previously washed with 2 ml of 1 M NaOH and 2 ml 1 M formic acid and five washes of 5 ml of water. After the sample was loaded, the column was washed with 5 ml of water, 5 ml of 5 mM borax, and 60 mM sodium formate. The columns were eluted with 3 ml of 0.9 M ammonium formate and 0.1 M formic acid. To 10 ml of CytoScint, 0.2 ml of each elution was added and the sample was analyzed via scintillation counting.