Cloning and characterization of a novel regulator of G protein signalling in human platelets

Cellular Signalling 14 (2002) 595 – 606 www.elsevier.com/locate/cellsig

Cloning and characterization of a novel regulator of G protein signalling in human platelets Alison W. Gagnon1, David L. Murray*, Robert J. Leadley 2 Cardiovascular Drug Discovery, Aventis Pharmaceuticals, 500 Arcola Road, Collegeville, PA 19426, USA Received 15 September 2001; accepted 13 November 2001

Abstract In an effort to understand the modulation of G protein-coupled receptor (GPCR)-mediated signalling in platelets, we sought to identify which regulators of G protein signalling proteins (RGSs) are present in human platelets. Using degenerate oligonucleotides, we performed RT-PCR with human platelet and megakaryocytic cell line RNA. In addition to confirming the presence of several known RGS transcripts, we found a novel RGS domain-containing transcript in platelet RNA. Northern blot analysis of multiple human tissues indicates that this transcript is most abundantly expressed in platelets compared to other tissues examined. Full-length cloning of this novel RGS, which we now term RGS18, demonstrates that this transcript is predicted to encode a 235-amino acid protein that is most closely related to RGS5 (46% identity) and that has
30 – 40% identity to other RGS proteins. RGS18 is expressed in platelet, leukocyte, and megakaryocyte cell lines and binds to endogenous Gai1, Gai2, Gai3, and Gaq but not Gaz, Gas or Ga12 in vitro. D 2002 Elsevier Science Inc. All rights reserved. Keywords: RGS; GTP-binding proteins; Signal transduction; Gi; Gq; Platelets

1. Introduction Signalling through G protein-coupled receptors (GPCRs), such as thrombin, thromboxane A2, and ADP, is in part responsible for platelet activation events such as fibrinogen receptor exposure, granule secretion, and aggregation [1]. Multiple intracellular signalling pathways have been implicated in platelet activation events, although the exact sequence of events and host of intracellular signalling molecules remains undefined. In an effort to identify proteins that might modulate upstream events in GPCR-mediated intracellular signalling in platelets, we sought to identify which members of a recently identified family of

Abbreviations: RGS, regulator of G protein signalling; GPCR, G protein-coupled receptor; GAP, GTPase activating protein * Corresponding author. Present address: Pfizer Central Research, Mail Code MS8220-2132, Eastern Point Road, Groton, CT 06340, USA. Tel.: +1-860-715-4109; fax: +1-860-715-2469. E-mail address: [email protected] (D.L. Murray). 1 Present address: Proteome, A Division of Incyte Genomics, Suite 435M, 100 Cummings Center, Beverly, MA 01915, USA. 2 Present address: Cardiovascular Department, Pfizer PGRD, 2800 Plymouth Road, Ann Arbor, MI 48105, USA.

proteins known as regulators of G protein signalling (RGSs) are present in platelets. RGSs are a family of proteins that function to dampen signals generated upon stimulation of cell-surface G protein-coupled receptors (GPCRs). RGSs were first identified in genetic screens of yeast [2,3] and the nematode Caenorhabditis elegans [4] based on their ability to modulate behavioural responses. Mammalian homologues of these lower eukaryotic RGSs were quickly identified [5 – 9]. The hallmark of this family is a highly homologous 120-amino acid region termed an RGS domain. Currently, there are more than 30 mammalian proteins or partial sequences that contain a putative RGS domain [10]. RGS proteins are thought to regulate GPCR signalling by interacting with the alpha subunits of heterotrimeric GTPbinding proteins. Heterotrimeric G proteins act as molecular switches in GPCR-mediated signal transduction controlling the rate and extent of activation of the effector (for a review of heterotrimeric G proteins, see Ref. [11]). RGS proteins attenuate signalling through GPCRs by acting as GTPase activating proteins (GAPs) for the alpha subunit [9,12,13]. Structural studies indicate that RGSs bind to the transition state of the alpha subunit thereby stabilizing it, accelerating GTP hydrolysis that limits the time the Ga subunit spends in its active state [14,15]. So far, RGSs have been identified

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which interact with and activate members of the Gai family (Gai1, Gai2, Gai3, Gaz, Gao, and Ga t), Gaq/11, and Ga12/13 but not Gas [10]. In addition to their GAP activity, RGSs may also block signalling by acting as effector antagonists [16,17]. RGS proteins have been identified in a variety of cell types and tissues that profoundly alter many GPCRstimulated intracellular effectors, including regulation of adenylyl cyclase [18], MAP kinase activity [6,17], inositol phosphate and Ca2 + signalling [16 – 19], K + channel conductance [20], and visual signal transduction [21,22]. Since several of these signalling cascades are involved in platelet activation, we thought it likely that one or more members of the RGSs superfamily might be present in platelets and be responsible for regulating signalling pathways critical for platelet activation. In platelets, receptors for ADP, thromboxane A2, and thrombin couple to heterotrimeric GTP-binding proteins that transduce the signals to intracellular effectors, resulting in inhibition of adenylyl cyclase, activation of phospholipase C, and mobilization of intracellular calcium [1]. In an effort to understand better the regulation of G protein signalling in human platelets, in the present study we employed an RT-PCR strategy with degenerate oligonucleotides to amplify RGS transcripts from human platelet RNA and several megakaryocytic cell lines. In addition to identifying several known RGSs in these cells, we identified a novel RGS domain-containing protein in human platelets. This RGS, which we now call RGS18, is abundantly expressed in platelets, with much lower expression in other tissues, primarily those of the haematopoetic system. In vitro RGS18 binds to endogenous Gai1/2/3 and Gaq but not to Gaz, Gas or Ga12 in platelet lysates treated with GDP + AlF4  but not GDP alone. Since platelet aggregation requires activation of receptor coupled to Gaq and /or one or more forms of Gai, RGS18 may be responsible in part for regulation of pathways important to platelet activation.

2. Methods 2.1. Miscellaneous Reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted. Oligonucleotides and peptides were produced by the Core Biotechnologies Department at Rhone Poulenc-Rorer Pharmaceuticals. Cell culture reagents were obtained from Gibco/BRL (Rockville, MD). Cell lines were obtained from ATCC (Manassas, VA). 2.2. Preparation of platelets, leukocytes, and cell lines HEL and Meg-01 cells were maintained in RPMI supplemented with 10% foetal bovine serum, 0.3 mg/ml L-glutamine, and 100 U/ml penicillin G/100 mg/ml streptomycin sulfate. DAMI cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% heat-inactivated horse serum, 0.3 mg/ml L-glutamine, and 100 U/ml penicillin

G/100 mg/ml streptomycin sulfate. Human platelets were obtained from Interstate Blood Bank (Memphis, TN) in concentrate form and used on the day after collection. For functional studies, fresh platelets were drawn from consenting donors using 0.38% citrate as the anticoagulant. For both, platelets were spun at 120  g for 15 min to retrieve the platelet-rich plasma (PRP). Prostaglandin E1 (0.5 mg/ml) was added to PRP and platelets were pelleted at 800  g for 15 min. Platelets were washed once in Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl26H20, 5.5 mM glucose, 11.9 mM NaHCO3, 0.36 mM NaH2PO4H2O, 10 mM HEPES pH, 7.35) plus 0.35% human serum albumin (this was reduced to 0.1% for protein lysates to reduce albumin carry over) and 0.25 mg/ml prostaglandin E1. Platelets were pelleted at 800  g and resuspended in the appropriate buffer (Trizol for RNA, lysis buffer for protein lysates). White blood cells were obtained fresh from consenting donors and isolated during platelet preparation. Briefly, after removal of PRP from platelet isolation, the buffy coat was removed and brought up to 15 ml with platelet-poor plasma and diluted 1:1 with PBS. Fifteen milliliters was carefully layered on top of 15 ml of Histopaque and spun at 400  g for 30 min at RT. Human leukocytes were isolated, diluted 1:1 in PBS and spun at 400  g for 10 min and resuspended in the appropriate lysis buffer (Trizol or protein lysis buffer). 2.3. PCR cloning strategy Four degenerate oligonucleotides were synthesized, which were previously used for isolating RGS transcripts and are designed in regions of high homology flanking the RGS domain of several members of the RGS superfamily [4]. Total RNA was prepared using Trizol Reagent (Gibco/ BRL) according to manufacturer’s instructions. Singlestrand cDNA was prepared by reverse transcription of total RNA using Superscript II (Gibco/BRL), 2 –5 mg of total RNA and 160 ng of random hexamer (Roche Biochemicals, Indianapolis, IN) following the manufacturer’s instructions. PCR was performed with Taq polymerase according to the manufacturer’s instructions (Gibco/BRL). To increase the yield of colonies, a second round of amplification was performed using 1– 5 ml of the initial PCR reaction product. The resulting purified PCR reactions were subcloned into pCR2.1 using the TA cloning kit (Invitrogen, Carlsbad, CA). Automated plasmid purification and sequencing was performed on 50 colonies when two rounds of amplification were performed, 25 when one was perfomed. The identity of each PCR product was determined by comparing sequence data to that of known RGSs using Seqman and Megalign programs of the Lasergene software and BESTFIT, GAP, and PILEUP of the GCG program. 2.4. Full-length cloning The novel PCR product was compared to the LIFESEQ proprietary Incyte database of ESTs. Only one match was

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found. Our collaboration with Incyte allowed us to purchase this thyroid library EST, which we grew and sequenced. 5 0 RACE was carried out with the antisense primer (5 0-CGCTAGGGCCTTAGACTCCTTGCTTCTTCC-3 0) from the 30 untranslated region, using Marathon Ready cDNA from human bone marrow and human peripheral blood leukocyte coupled with the Advantage cDNA polymerase (Clontech, Palo Alto, CA) following the manufacturer’s instructions. 2.5. Tissue distribution For Northern blots of human platelets, human leukocytes and the megakaryocytic cells lines, 10 mg of total RNA was run on a 1.5% agarose/5.8% formaldehyde gel in 1  MOPS buffer (20 mM 3-N-Morpholinopropanesulfonic acid, pH 7.0, 5 mM sodium acetate, 1 mM EDTA). RNA was transferred in 20  SSC (0.3 mM NaCl, 0.3 mM sodium citrate) to nylon membrane using the Turboblotter System (Schleicher and Schull, Keene, NH) and UV crosslinked to the membrane (Stratalinker, Stratagene, La Jolla, CA). A multiple tissue Northern with 1 mg of poly(A) + RNA from each of 12 different tissues was purchased from Clontech. Both of these blots were prehybridized in 10 ml ExpressHyb solution (Clontech) at 68 C for 1 h. For each blot, approximately 25 ng of the 1300 base pair (bp) Incyte clone EST encoding the 30 end of the coding region and the 30 untranslated region was radiolabelled with 32P-a(dCTP) and the High Prime Kit (Roche Biochemicals) and purified on a microspin S-200 column (Amersham/Pharmacia, Piscataway, NJ). The two labelled probes were mixed and added to 20 ml of ExpressHyb. Each blot was hybridization with 10 ml of this probe mix for 2 h at 68 C and washed at high stringency as instructed by Clontech. This process ensured that the blots were each hybridized with probes of the same specific activities and would facilitate comparison of the results. Blots were stripped with 0.5% SDS at 100 C and probed with a b-actin probe supplied by Clontech. 2.6. Production of polyclonal antisera, Western blotting, and cell lysate preparation Two peptides were selected to make polyclonal antisera, KLIHGSGEETSKEAKIR from the amino-terminal portion of RGS18 and QRPTNLRRRSRSFTCNEFQ from the carboxy-terminal region. These peptides were synthesized in-house by Rhone Poulenc-Rorer Pharmaceuticals Core Biotechnology Department and conjugated to KLH for injection into rabbits. Custom polyclonal antisera were produced from the conjugated peptides by Rockland Immunochemicals (Gilbertsville, PA) according standard procedures. The specificity of the antisera was characterized using platelet lysates. Typically, antisera were used in Western blotting at 1:500 (3NRGS-12) or 1:1000 (5NRGS-13) dilutions in 5% Carnation nonfat dry milk in TBST

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(20 mM Tris – HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20). For peptide inhibition studies, antisera was incubated in 100 mM Tris, pH 7.5 plus complete protease inhibitor cocktail (Roche Biochemicals) in the absence or presence of 100 mg/ml of the immunizing peptide or an unrelated peptide overnight at 4 C. This was then diluted to the working concentration in blocking buffer (5% milk in TBST) and used in blotting. Whole cell lysates for western blotting were made from platelets, leukocytes, and the three megakaryocyte cell lines in 50 mM HEPES, pH 8.0, 6 mM MgCl2, 300 mM NaCl, 1 mM DTT, 1% Triton X-100, and complete protease inhibitor cocktail. Fifty micrograms of each lysate was run on 15% reducing SDS-PAGE, transferred to 0.2 mM nitrocellulose, and blotted as above. For detection of RGS10 in cell lysates, SDS-PAGE was carried out as above except a 1:500 dilution of anti-RGS10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was blocked with 2% BSA in TBST. Bound antibody was detected with goat antirabbit IgG coupled to HRP (RGS18) (BioRad, Hercules, CA) or mouse antigoat IgG-coupled to HRP (RGS10) (Pierce, Rockford, IL) and the SuperSignal Pico ECL detection kit (Pierce). 2.7. Expression and purification of GST fusion proteins In order to create an in-frame GST fusion protein in pGEX-5X-1, oligonucleotides were synthesized from the 50-most and 30-most coding regions with an in-frame BamHI site on the 50 primer and a XhoI site after the termination codon in the 30 primer and used in RT-PCR of platelet RNA. The resultant PCR amplification product was fully sequenced for fidelity, subcloned into pGEX-5X-1, and transformed into competent BL-21(DE3) (Novagen, San Diego, CA). Expression and purification of the resultant fusion protein was carried out as described in the manufacturer’s directions for batchwise purification of fusion proteins. Typically, we grew a 1-l culture and used 2 ml of a 50% slurry of glutathione – Sepharose 4B to purify the fusion protein. 2.8. G protein alpha subunit binding assays Determination of G protein alpha subunit binding specificity of RGS18 was carried out as in Beadling et al. [23] with minor modifications. Washed platelets from Day 2 concentrate were lysed in 50 mM HEPES, pH 8.0, 300 mM NaCl, 1 mM DTT, 6 mM MgCl2, 1% Triton X-100, and complete EDTA-free protease inhibitor cocktail (Roche Biochemicals). Protein determination was done using Bradford Assay (BioRad) using BSA as a standard and lysates were adjusted to 1 mg/ml in lysis buffer prior to use. Cell lysates (450 ml) were activated with 30 mM GDP or 30 mM GDP plus 30 mM AlCl3 and 100 mM NaF for 30 min at 30 C. Following the incubation, lysates were quickly spun in an microfuge to pellet actin that became insoluble upon activation. Lysates were transferred to fresh tubes and

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incubated with 20 ml of a 50% slurry of GST – RGS18 coupled to glutathione – Sepharose 4B beads (typically
10 mg RGS18 protein) for 1 h at 4 C. The beads were washed twice in 1 ml of wash buffer (50 mM HEPES, pH 8.0, 300 mM NaCl, 1 mM DTT, 6 mM MgCl2, 0.025% C12E10, and protease inhibitors). Bound protein was eluted in two rounds of boiling in reducing Laemmli buffer and subjected to SDS-PAGE on 12% gels, transferred to 0.45 mM nitrocellulose, and blotted with antisera against Gai1/2 (Calbiochem, San Diego, CA), Gai3/O (Calbiochem), Gaz (Santa Cruz Biotechnology), Gaq/11 (Santa Cruz Biotechnology), Ga12 (Santa Cruz Biotechnology) or Gas (Calbiochem and Santa Cruz Biotechnology). Bound antibody was detected as above.

3. Results

Table 1 Identification of RGS isoforms by degenerate RT-PCR of total RNA from human platelets and three megakaryocytic cell lines Plt RNA RGS2 RGS10 RGS16 RGS4 Novel RGS Vector/? ND

3 1 4 17 12/2 11

DAMI RNA

HEL RNA

MEG RNA

1

1

3

33

37

20 3

1 4/4 7

7/2 3

14/0 10

Degenerate oligonucleotide primers designed against conserved regions of the RGS domain were used to amplify RNA from human platelets (Plt), DAMI, HEL, and MEG-01 cells as described in Methods. Fifty colonies were picked and plasmid DNA was isolated and subjected to sequence analysis. The resultant sequence data from each colony was compared to the coding regions of known RGSs and tabulated. ‘‘vector’’= religated plasmids with no insert. ‘‘?’’= artifactual products of the PCR reaction with no homology to known genes. ‘‘ND’’= not determined, when sequencing reactions failed, we did not obtain the identity of those colonies.

3.1. RT-PCR of RGSs from human platelets and megakaryocytic cell lines 3.2. Full-length cloning of the novel platelet RGS A RT-PCR strategy was employed to identify which RGS family members are expressed in human platelets. We synthesized degenerate primers based on highly homologous regions in several of the previously identified RGS proteins [4] and used them to amplify total RNA from human platelets, and three megakaryocytic cell lines, DAMI, HEL, and MEG-01 cells. The resultant
240-bp PCR product was blunt-ligated to pCR2.1 and transformed into Escherichia coli. Fifty colonies from each PCR reaction were picked, grown, and the plasmid was isolated from each and sequenced. Analysis of the resulting sequence data by comparison to known RGS proteins was performed and the results are summarized in Table 1. The most predominant amplification product in all three megakaryocytic cell lines is RGS16, an RGS first identified in mouse retina [24] but also present in lymphocytes and many other tissues [25,26]. In contrast to the cell lines however, the most predominant PCR product from platelet RNA encodes a novel RGS domain that is not present in public domain databases. A single colony out of the 50 examined in DAMI cells also encoded this novel RGS domain. This novel RGS domain displays 30 –46% homology to the known RGSs, with the highest degree of homology to RGS5. Additionally, platelets contain transcript for RGS10, and MEG-01 cells contain RGS4. Not surprisingly all tissues tested contain the ubiquitously expressed RGS2.

In order to identify the full-length sequence of this partial clone, we employed electronic searches of EST databases. No identical hits were found in the public domain EST databases. However, one hit was found in the LIFESEQ proprietary Incyte database, an EST from a human thyroid library, the 50 end of which displays identity to the last 72 bp of our partial platelet clone. Since the library was primed with oligo(dT), we thought it likely that this EST might potentially contain the carboxy-terminal region and 30 untranslated domain of our novel transcript. This clone was purchased from Incyte and analysed by restriction analysis and sequencing. The Incyte clone contains a cDNA insert of
1300 bp, which has identity to our PCR product at the 50 end and a stretch of poly(A) + residues at the 30 end. When translated, this EST contains an open reading frame contiguous with that of our novel PCR product. The fact that homology to other known RGS domain-containing proteins continued beyond the 72-bp overlap supported our belief that this EST represented the 30 end of our novel PCR product. To further confirm that these two cDNAs are in fact from the same transcript, RT-PCR analysis of platelet RNA was performed with a sense primer designed against near 50 sequence information from our platelet PCR product and antisense primers chosen at three different sites within the Incyte EST beyond the region of overlap with our clone.

Fig. 1. Nucleotide and deduced amino acid sequence of RGS18. Panel A: Schematic representation of the full-length cloning of the novel platelet RGS. A schematic of the cDNA for RGS18 is depicted on the bottom, with the boxed region representing the predicted open reading frame and the single lines the 50 and 30 untranslated regions. The relative locations of the initial RT-PCR product, the Incyte EST cDNA and the 50 RACE amplification product are shown above. Panel B: Nucleotide and deduced amino acid sequence of RGS18. The 50 and 30 untranslated regions are given in lower case letters, the predicted amino acid sequence in single letter abbreviations in uppercase letters. The initial RT-PCR product from platelet RNA is shown in boldface type. The 50 and 30 ends of the Incyte EST cDNA are depicted by the diamonds (^). The oligonucleotide sequence of the primer in the far 30 untranslated region used for 50 RACE is underlined. This sequence has been deposited in GENBANK database (the nucleotide and predicted amino acid sequence has been submitted to GENBANK, Accession No. AF268036).

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PCR products of the expected size were obtained from each of these reactions confirming that our novel clone and the Incyte EST cDNA are in fact part of a contiguous transcript in platelet RNA (data not shown). Using sequence information from the 30 untranslated region of the Incyte clone, a 50 RACE strategy was undertaken to isolate the entire coding region of this novel RGS. Since platelets do not contain abundant levels of high quality RNA, we chose to use cDNA from human bone marrow and peripheral blood leukocytes specifically designed for 50 RACE. Preliminary Northern blot data demonstrated that this novel RGS transcript is present at low levels in both these tissues. 50 RACE was performed using the Advantage PCR kit with this 30 untranslated region primer (shown underlined in Fig. 1, panel B). The PCR reaction products were subcloned into pCR2.1 and analysed by restriction mapping. The longest inserts (
2 kb) were chosen for sequence analysis. These inserts contained the entire predicted open reading frame and a short stretch of the 50 untranslated region. Sequence data were compiled from the 50 RACE strategy, the Incyte EST cDNA, and the initial PCR fragment and assembled. A schematic of the relative positions of each of these overlapping clones is shown in Fig. 1, panel A. The entire nucleotide and predicted amino acid sequence of the novel RGS from platelets, which we now term RGS18, are shown Fig. 1, panel B. The cDNA for RGS18 is 2144 bp in length and encodes a protein of 235 amino acids. Scanning for protein sequence motifs, using the Prosite database, confirms that this protein contains a RGS domain (residues 86 through 202), as well as putative consensus sites for phosphorylation by several protein kinases. Residues 213 to 216 form a consensus site for phosphorylation by cAMP- and cGMP-dependent protein kinases (shown underlined in Fig. 1, panel B). Four potential sites for protein kinase C phosphorylation and five for casein kinase II phosphorylation are present (residues 28 –30, 33 –35, 63 –65, and 92– 94 for PKC and residues 28 –31, 33 – 36, 76– 79, 92– 95, and 220 –223 for CK II). Amino acids 221 – 224 in the carboxy-terminus of the protein encode a putative CAAX motif that may act as a site of modification by fatty acylation and serve to regulate the activity of RGS18.

3.3. Homology comparison of RGS18 The predicted amino acid sequence of RGS18 was compared to that of several other closely related RGS proteins. Fig. 2 depicts an alignment of RGS18 with RGS4, RGS5, RGS16, RGS2, RGS1, and RGS10 that was generated using the Pileup program from GCG. Shaded areas represent amino acids that are conserved between RGS18 and at least two other RGSs. The region of the RGS domain that was initially isolated by RT-PCR is shown by a line above the amino acids. RGS18 is most homologous to human RGS5 (47% identity), followed by

rat RGS8 (44%), hRGS2 (41%), hRGS4 and hRGS16 (40%), hRGS1 (37%), hRGS10 and hRGS3 (36%), and hRGS13 (34%). RGS18 displays between 20% and 30% identity to other known RGS proteins. Recently, the sequence of a novel RGS, which they have termed RGS17, from a chicken dorsal root ganglion cDNA library was reported, which is distinct from our platelet RGS18 (31% amino acid identity) [27].

3.4. Tissue distribution of RGS18 in human tissues In order to determine the relative tissue distribution of RGS18, we hybridized two Northern blots with a 30 untranslated region probe from RGS18. One contained platelet and megakaryocytic cell line RNA and the other is a commercially available human multiple tissue Northern. Each blot had one lane of human leukocyte RNA to facilitate comparison. The RGS18 probe hybridizes to a major species at
2.75 kb and minor species at
4.2 kb in platelet RNA and to a lesser extent in DAMI, HEL, and MEG-01 cells (Fig. 3, panel A). Human leukocyte RNA also expresses both of these transcripts at levels equal to or slightly less than that in MEG-01 cells. Since complete separation of platelets and leukocytes is difficult, whenever expression levels of transcripts in platelet RNA are evaluated, one must be concerned with the level of contamination of the platelet RNA by the more abundant leukocyte RNA. The fact that leukocytes have such a low expression level of RGS18 compared to platelet RNA indicates that this is not a concern for this transcript. Results from the human multiple tissue Northern blot are shown in Fig. 3, panel B. Overall, the hybridization signal was much lower than that seen with the Northern blot above and required much longer exposures. The platelet Northern blot was exposed for 6 h versus 6 days for the human multiple tissue Northern. On the multiple tissue blot, the most intense band was consistently in the leukocyte lane, followed by spleen, then heart and liver, and very low levels in skeletal muscle, colon, kidney, small intestine, placenta, and lung. The presence of two mRNA species on the Northern blots indicates that this transcript, like many others, is subject to alternative splicing and/or differential polyadenylation. These Northern blots were repeated at least twice to confirm these results. Using these data for comparison of the expression levels of RGS18, it appears that the level of expression of this message in platelets greatly exceeds that in leukocytes, which express RGS18 in excess of the other tissues examined. In support of this conclusion, we used leukocyte and bone marrow cDNA in the degenerate RT-PCR strategy for mining RGS transcripts, as was done for platelets, and none of the colonies sequenced contained RGS18. As would be predicted based on previous studies, the most abundant amplification products in leukocytes and bone marrow in these studies were RGS2 and RGS16 (data not shown).

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Fig. 2. Alignment of RGS18 with other RGS family members. The predicted amino acid sequence of RGS18 was aligned with six other RGS protein sequences using the PILEUP program of GCG. Amino acids that are conserved between RGS18 and at least two other RGSs are shaded. The solid line above the sequence indicates the conserved RGS domain that was amplified by PCR. The boxed regions indicate the peptide sequences that were synthesized for production of peptide-directed antisera.

3.5. Western blot analysis of RGS18 expression in human platelets Polyclonal anti-RGS18 antisera were generated against two peptides, one in the amino-terminus and the other in the carboxy-terminus of RGS18. The location and sequences of these peptides is shown in the boxed regions of Fig. 2. These regions were selected due to their divergence from similar regions of the other known RGSs. The resulting sera were tested in Western blots with platelet lysates for reactivity and specificity. Both antisera react with a
30-kDa band in platelet lysates, which correlates well with the predicted size (27.5 kDa). Preincubation of each antisera with its corresponding immunizing peptide but not the other peptide ablates antibody reactivity with the 30 kDa protein, indicating that each antibody is indeed specific for RGS18 (Fig. 4, panel A). Antisera 5NRGS-13 (aminoterminal) have a higher titer than antisera 3NRGS-12

(carboxy-terminal) and was used for further evaluation of protein expression levels. Western blots were run with lysates from platelets, leukocytes, DAMI, HEL, and MEG-01 cells to compare the relative protein expression levels of RGS18. Fig. 4, Panel B shows the results of a representative experiment. As would be predicted based on the Northern data, RGS18 protein is more abundant in platelets than in leukocytes, DAMI, HEL, or MEG-01 cells. Although an immunoreactive band in the MEG-01 lane is not visible in this figure, longer exposures do indeed demonstrate the expression of RGS18 in these cells. Since we had detected the presence of RGS10 RNA, we were interested in determining RGS10 protein expression levels in platelets. Western blotting of platelet, leukocyte, and megakaryocyte cell line lysates with an anti-RGS10 antibody, indicates that RGS10 is almost equivalently expressed in platelets, leukocytes, and DAMI cells, with lower levels of expression in the other two megakaryocytic

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Fig. 3. Tissue distribution of RGS18 by Northern blotting. Panel A: A Northern blot of 10 mg of total RNA from human platelets, human leukocytes, DAMI, HEL, and MEG-01 cells probed with a 30 untranslated region probe of RGS18 as described in Methods. This blot was exposed to Kodak BioMax MR film for 6 h at  70 C. Panel B: Hybridization of a human multiple tissue Northern with the same RGS18 probe. This blot was exposed to Kodak BioMax film for 6 days at  70 C. Migration of molecular weight standards on each gel is shown on the left. After stripping, each blot was hybridized with a b-actin probe for normalization, shown below the corresponding blot.

cell lines (Fig. 4, panel B). Taken together, these data indicate that RGS18 and RGS10 are both expressed in platelets and that RGS18 but not RGS10 is preferentially expressed in platelets versus leukocytes. To our knowledge, this is the first demonstration of the expression of RGS10 in platelets.

3.6. Binding of RGS18 to endogenous G protein alpha subunits in human platelets In order to determine the target alpha subunits of RGS18, we performed binding experiments using GST-tagged RGS18 and lysates of human platelets. A similar method

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Fig. 4. Western blotting of platelet, leukocyte, and megakaryocyte cell line lysates. Panel A: Specificity of anti-RGS18 antisera. Nitrocellulose strips containing 50 mg of platelet lysate ran on 15% SDS-PAGE were incubated with a 1:500 dilution of antisera 3NRGS-12 or a 1:1000 dilution of 5NRGS-13. An immunoreactive band that migrates at
30 kDa is detected by both antisera (first lane for each blot). Identical strips were also probed with antisera that had been preincubated with the corresponding immunizing peptide or with an unrelated peptide. Migration of the 30-kDa molecular weight standard is shown on the left, migration of RGS18 on the right. Panel B: Detection of RGS18 expression in platelets, leukocytes, and the megakaryocytic cell lines. Lysates (50 mg) from human platelets, leukocytes, DAMI, HEL, and MEG-01 cells were run on 15% SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies against RGS18 and RGS10. The top blot depicts reactivity of each lysate with the anti-RGS18 antisera and the bottom blot with the antiRGS10 antisera. As seen above, RGS18 co-migrates with the 30-kDa molecular weight marker. RGS10, a much smaller protein, migrates close to the 21-kDa molecular weight marker.

has been used to determine the binding specificity of RGS16 using lysates from Jurkat cells [23]. Previous work has demonstrated that RGS proteins bind G protein alpha subunits only when the alpha subunit is in its transition state or ‘‘activated state’’ [28]. This transition state can be mimicked by treating alpha subunits with AlF4  . For these experiments, platelet lysates were treated with GDP, which holds the alpha subunit in the GDP-bound ‘‘in-activate’’ state, or GDP + AlF4  , which mimics the transition state of the Ga subunit. Recombinant GST-RGS18 bound to Sepharose 4 beads was used to affinity purify any G protein alpha subunits

that bind to RGS18 in each of these lysates. A panel of G protein alpha subunit-specific antibodies was used to detect which alpha subunits bind to RGS18. Fig. 5 shows the results of a representative experiment. RGS18 binds little if any alpha subunit in the inactive GDP-bound state (middle lane of each panel). In contrast, in lysates that have been treated with GDP + AlF4  , RGS18 binds a significant amount of alpha subunit detected by antibodies directed against Gai1/2, Ga0/i3, and Gaq/11. This interaction appears to be specific since RGS18 does not bind Gaz, Ga12, or Gas. Neither GST nor Sepharose 4B alone binds to any of these alpha subunits in

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Fig. 5. Determination of the Ga subunit specificity of RGS18. Platelet lysates were treated with GDP or GDP + AlF4  as indicated and incubated with GST-RGS18 coupled to Sepharose 4B as described in Methods. Bound proteins were subjected to 12% SDS-PAGE and transferred to nitrocellulose and detected with antisera against Gai1/ai2, Gao/i3, Gaq/11, Gaz, Ga12, or Gas. The lane labelled ‘‘Lysate’’ is 35 mg or 7.7% of the input lysate in each of the reactions run alongside as a control for the reactivity of each antisera with platelet lysate.

either GDP or GDP + AlF4  treated platelet lysates (data not shown). The binding selectivity of RGS18 is consistent with that found for other RGS proteins that selectively bind to members of the Gai family and/or Gaq family [10]. 4. Discussion Recently, the complexity of G protein-coupled receptor signalling pathways, beyond the classical view of receptor, G protein, and effector, has been increased through the identification of additional mediators that modulate various aspects of the cascade, including RGSs that regulate the activity of heterotrimeric G proteins. In an effort to better understand GPCR-mediated signalling in human platelets, we initiated studies to examine which isoforms of the RGS family exist in platelets. Although platelets are anucleate cells, they contain small amounts of residual RNA presumably carried over from the megakaryocyte. Degenerate RT-PCR analysis is a good method to study transcript expression in platelets, due to the minimal requirement of RNA. Using this method, we isolated a novel RGS, RGS18, from platelet RNA. Platelets also contain transcripts for other previously known RGS proteins including RGS2, RGS16, and RGS10. In contrast to the platelet RGS expression profile, the megakaryocytic cell lines preferentially express RGS16. These cells probably differ from platelets in their expression profile of RGSs due to their undifferentiated and leukemic state. These differences in expression levels are not unexpected since expression of RGS1 and RGS2 is regulated by mitogens in lymphocytes [7,8] and expression of RGS16 is regulated by IL-1 in

lymphocytes [23] and the tumour suppressor, p53 in a colon carcinoma cell line [25]. Although these megakaryocytic cell lines have many features of the megakaryocytic lineage (i.e., expression of GPIIb/IIIa) [29 –31] and have proved useful for identification of platelet-specific genes, they are not ideal models of platelet signal transduction. Most importantly, these cells do not have intact signalling pathways that stimulate binding of soluble fibrinogen to GPIIb/IIIa, indicating that their signal transduction pathways or complement of signalling proteins differs from platelets [32]. The most abundant RGS isoform in human bone marrow and peripheral blood leukocyte RNA we detected by degenerate RT-PCR was RGS2 (data not shown), which further points out the distinction between platelets and other haematopoetic cells. The fact that RT-PCR detects distinct transcript profiles in different tissues indicates that these primers have minimal bias for one RGS isoform over others and that the proportional expression we see by this method probably reflects relative RNA expression levels. Although we did not detect RGS18 in human leukocyte RNA by degenerate RT-PCR, we were concerned that this transcript was not in fact amplified from platelet RNA but from contaminating white blood cell RNA. Since platelets contain so little RNA, a small amount of white blood cell contamination could lead to a disproportionate contamination of the platelet RNA by leukocyte RNA. Northern blot analysis of human platelet and leukocyte RNA indicates that this is not the case, since platelet RNA expresses significantly higher levels of RGS18 than leukocytes. Examination of the expression of RGS18 by Northern and Western blotting in a wide variety of human tissues indicates that RGS18 is most abundantly expressed in platelets, followed

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by leukocytes, and then other tissues of the haematopoetic system. Northern blotting demonstrates very low level expression in other tissues as well but whether these levels translate to significant expression of the protein remains to be determined. Full-length cloning of RGS18 was achieved by combining the sequence information from the initial PCR reaction, an overlapping Incyte EST cDNA, and the cDNA product from a 50 RACE strategy. RGS18 has very short carboxy and amino terminal domains flanking the internal RGS domain and appears not to contain functional domains for scaffolding (i.e., PH, Dbl, GGL, or DEP). It does however have one putative CAAX motif that might serve as a site of acylation, and permit membrane anchoring. RGS18 also contain several consensus sites for phosphorylation by the enzymes cAMP/cGMP-dependent protein kinase, protein kinase C, and casein kinase II. This indicates the potential for regulation of RGS18 by other signalling cascades. Recently, a phylogenetic analysis of this family has been performed and demonstrates that the RGS superfamily can be divided it into at least six subfamilies (A through F) [33]. RGS18 would most probably be a member of Subfamily B, since it is most closely related to these RGSs, and like RGS18, Subfamily B members characteristically contain short amino- and carboxy-terminal domains. RGS18 does contain a highly conserved asparagine residue at position 152 (relative position 128 in RGS4), which is conserved in three of the six families. Structural studies of RGS4 indicate that this residue is critical for GAP activity and stabilization of the transition state of Ga [14,34]. Subfamily B is a diverse group and only one amino acid, a Ser residue (position 103 in RGS4), is conserved between all the members of Family B. However, the corresponding residue in RGS18 (position 127) is a glycine, which calls into question whether RGS18 is in fact a member of Subfamily B. Seven of eight human RGSs of Family B appear to be clustered on chromosome 1, perhaps due to gene duplication events [33]. It will be interesting to determine if RGS18 is also localized on this chromosome. As additional members of the RGS superfamily are identified, and as more information is gained about the functionality of each RGS, the defining characteristics of the various RGS subfamilies will become more distinct. Platelets are known to express a variety of Ga subunits. Previous work has shown that platelets contain members of the Gai family, Gai1, Gai2, Gai3, Gaz, Ga12/13, Ga16, Gas [1], and Gaq but not Ga11 [35,36]. In vitro Ga subunit binding specificity indicates that RGS18 probably regulates both Gai- and Gaq-linked pathways. This Ga subunit specificity is in line with what has been demonstrated for other RGSs that typically interact with members of the Gai and/or Gaq family. Despite the fact that Gaz is abundantly expressed in platelets [37], RGS18 does not appear to interact with this alpha subunit. The sequence of mouse RGS18 has recently been reported and was found to bind Gaq and Gai, but not Ga12, Ga13, or Gas, which is consistent with what we have

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observed for human RGS18 [38]. Interestingly, RGS10, which we found expressed in platelets, at both the RNA and protein levels has been reported to interact with Gaz as well as Gai3 [39]. Like Gaz, RGS10 is expressed in both platelets and brain (this work and Ref. [40]). It is likely that, due to their somewhat distinct Ga preference, RGS10 and RGS18 might serve to regulate different signalling pathways in platelets. In platelets, aggregation appears to be dependent upon concomitant activation of both Gai- and Gaq-coupled receptors [41,42]. The platelet agonists, thrombin, thromboxane A2, and ADP are all linked to signalling pathways via these Ga subunits. Platelets of Gaq-deficient mice are not responsive to a variety of agonists, and as a result these mice display prolonged bleeding times and are susceptible to haemorrhage [43]. These studies indicate that signalling through Gaq is critical for a variety of platelet activators. Due to the ubiquitous expression of Gaq, these mice also display other deleterious phenotypes, including ataxia, making Gaq a poor target for antiplatelet therapy. Because of its potential to regulate the G protein-mediated pathways in platelets that are critical for platelet activation and the fact that it is enriched in platelets over tissues, RGS18 or an as yet unknown protein that regulates RGS18 might make a good target for therapies aimed at regulating platelet activation. Future studies aimed at deciphering which GPCRs and signalling pathways are regulated by RGS18 and RGS10 in platelets will give us a better understanding of the role that these RGSs play in platelet activation events.

Acknowledgments We acknowledge the contribution to these studies from several members of the Core Biotechnology Group at Aventis Pharmaceuticals. We thank Joseph Bruno and Amy Barber for automated plasmid purification and DNA sequencing and Richard Howk for oligonucleotides and peptide synthesis and conjugation to KLH.

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