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• W2122340660 abstract "Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR γ-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cγ (PLCγ). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5′-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR γ-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation. Platelets play an essential role in wound healing by forming thrombi that plug holes in the walls of damaged blood vessels. To achieve this, platelets express a diverse array of cell surface receptors and signaling proteins that induce rapid platelet activation. In this study we show that two platelet glycoprotein receptors that signal via an immunoreceptor tyrosine-based activation motif (ITAM) or an ITAM-like domain, namely the collagen receptor complex glycoprotein VI (GPVI)-FcR γ-chain and the C-type lectin-like receptor 2 (CLEC-2), respectively, support constitutive (i.e. agonist-independent) signaling in a cell line model using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay that can detect low level activation of phospholipase Cγ (PLCγ). Constitutive and agonist signaling by both receptors is dependent on Src and Syk family kinases, and is inhibited by G6b-B, a platelet immunoglobulin receptor that has two immunoreceptor tyrosine-based inhibitory motifs in its cytosolic tail. Mutation of the conserved tyrosines in the two immunoreceptor tyrosine-based inhibitory motifs prevents the inhibitory action of G6b-B. Interestingly, the inhibitory activity of G6b-B is independent of the Src homology 2 (SH2)-domain containing tyrosine phosphatases, SHP1 and SHP2, and the inositol 5′-phosphatase, SHIP. Constitutive signaling via Src and Syk tyrosine kinases is observed in platelets and is associated with tyrosine phosphorylation of GPVI-FcR γ-chain and CLEC-2. We speculate that inhibition of constitutive signaling through Src and Syk tyrosine kinases by G6b-B may help to prevent unwanted platelet activation. The GPVI-FcR γ-chain complex is the major signaling receptor for collagen on platelets and megakaryocytes (1Clemetson J.M. Polgar J. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 2Nieswandt B. Watson S.P. Blood. 2003; 102: 449-461Crossref PubMed Scopus (909) Google Scholar). Cross-linking of GPVI leads to Src-dependent phosphorylation of a tandem YXXL sequence on the FcR γ-chain known as an immunoreceptor tyrosine-based activation motif (ITAM). 4The abbreviations used are:ITAMimmunoreceptor tyrosine-based activation motifPLCγ2phospholipase γ2CLEC-2C-type lectin-like receptor 2ITIMimmunoreceptor tyrosine-based inhibitory motifSH2Src homology domain 2PECAM-1platelet/endothelial cell adhesion molecule-1NFATnuclear factor of activated T-cellsmAbmonoclonal antibodyWTwild typeSHIPSH2-domain-containing inositol 5′-phosphataseGPglycoproteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol In turn, this leads to recruitment and activation of the tyrosine kinase Syk through its tandem SH2 domains. Syk initiates a signaling cascade that generates a linker for activation of T (LAT) cell-dependent signalosome, which mediates activation of phospholipase Cγ2 (PLCγ-2) (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 4Watson S.P. Asazuma N. Atkinson B. Berlanga O. Best D. Bobe R. Jarvis G. Marshall S. Snell D. Stafford M. Tulasne D. Wilde J. Wonerow P. Frampton J. Thromb. Haemostasis. 2001; 86: 276-288Crossref PubMed Scopus (119) Google Scholar). This pathway initiates a rise in intracellular Ca2+ and activation of protein kinase C leading to platelet aggregation. immunoreceptor tyrosine-based activation motif phospholipase γ2 C-type lectin-like receptor 2 immunoreceptor tyrosine-based inhibitory motif Src homology domain 2 platelet/endothelial cell adhesion molecule-1 nuclear factor of activated T-cells monoclonal antibody wild type SH2-domain-containing inositol 5′-phosphatase glycoprotein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol CLEC-2 is a 32-kDa C-type lectin-like receptor that functions as a platelet receptor for the snake venom toxin rhodocytin and the lymphatic endothelial marker, podoplanin (5Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar, 6Suzuki-Inoue K. Kato Y. Inoue O. Kaneko M.K. Mishima K. Yatomi Y. Yamazaki Y. Narimatsu H. Ozaki Y. J. Biol. Chem. 2007; 282: 25993-26001Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 7Christou C.M. Pearce A.C. Watson A.A. Mistry A.R. Pollitt A.Y. Fenton-May A.E. Johnson L.A. Jackson D.G. Watson S.P. O'Callaghan C.A. Biochem. J. 2008; 411: 133-140Crossref PubMed Scopus (88) Google Scholar). Like glycoprotein (GP) VI, CLEC-2 signals via sequential activation of Src and Syk tyrosine kinases leading to activation of PLCγ2 (5Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar). The regulation of PLCγ2 by CLEC-2 is distinct from that of GPVI in that it uses a single YXXL sequence and is only partially dependent on the adapter SLP-76 (5Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar, 8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). G6b is a recently identified member of the immunoglobulin superfamily that exists in several splice variants (9de Vet E.C. Aguado B. Campbell R.D. J. Biol. Chem. 2001; 276: 42070-42076Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). G6b-B is the only one of these variants to contain both a transmembrane region and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that support binding to the two SH2 domain-containing protein tyrosine phosphatases, SHP1 and SHP2 (9de Vet E.C. Aguado B. Campbell R.D. J. Biol. Chem. 2001; 276: 42070-42076Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10Senis Y.A. Tomlinson M.G. Garcia A. Dumon S. Heath V.L. Herbert J. Cobbold S.P. Spalton J.C. Ayman S. Antrobus R. Zitzmann N. Bicknell R. Frampton J. Authi K.S. Martin A. Wakelam M.J. Watson S.P. Mol. Cell Proteomics. 2007; 6: 548-564Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). ITIMs are defined by the consensus sequence (L/I/V/S)-X-Y-X-X-(L/V) and are commonly found in pairs separated by 15 to 30 amino acid residues (11Vivier E. Daeron M. Immunol. Today. 1997; 18: 286-291Abstract Full Text PDF PubMed Scopus (327) Google Scholar, 12Ravetch J.V. Lanier L.L. Science. 2000; 290: 84-89Crossref PubMed Scopus (1070) Google Scholar). The second ITIM in G6b-B is located ∼20 amino acids downstream of the first ITIM and has a slightly different sequence to the above, TXYXXV. G6b-B is the third ITIM-containing protein to be identified on platelets (10Senis Y.A. Tomlinson M.G. Garcia A. Dumon S. Heath V.L. Herbert J. Cobbold S.P. Spalton J.C. Ayman S. Antrobus R. Zitzmann N. Bicknell R. Frampton J. Authi K.S. Martin A. Wakelam M.J. Watson S.P. Mol. Cell Proteomics. 2007; 6: 548-564Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13Newland S.A. Macaulay I.C. Floto A.R. de Vet E.C. Ouwehand W.H. Watkins N.A. Lyons P.A. Campbell D.R. Blood. 2007; 109: 4806-4809Crossref PubMed Scopus (55) Google Scholar). The other two platelet ITIM receptors are platelet/endothelial cell adhesion molecule-1 (PECAM-1) and TREM-like transcript-1 (14Washington A.V. Quigley L. McVicar D.W. Blood. 2002; 100: 3822-3824Crossref PubMed Scopus (51) Google Scholar, 15Washington A.V. Schubert R.L. Quigley L. Disipio T. Feltz R. Cho E.H. McVicar D.W. Blood. 2004; 104: 1042-1047Crossref PubMed Scopus (92) Google Scholar, 16Barrow A.D. Astoul E. Floto A. Brooke G. Relou I.A. Jennings N.S. Smith K.G. Ouwehand W. Farndale R.W. Alexander D.R. Trowsdale J. J. Immunol. 2004; 172: 5838-5842Crossref PubMed Scopus (81) Google Scholar). ITIM-containing receptors were originally identified by their ability to inhibit signaling by ITAM receptors, as demonstrated by the selective inhibition of the B-cell receptor when cross-linked by surface immunoglobulin to Fcγ receptor IIb (FcγRIIb) (17Bijsterbosch M.K. Klaus G.G. J. Exp. Med. 1985; 162: 1825-1836Crossref PubMed Scopus (159) Google Scholar, 18Daeron M. Latour S. Malbec O. Espinosa E. Pina P. Pasmans S. Fridman W.H. Immunity. 1995; 3: 635-646Abstract Full Text PDF PubMed Scopus (389) Google Scholar). However, it is now recognized that ITIM-containing receptors can also generate stimulatory signals or inhibit activation by G protein-coupled receptors. For example, PECAM-1 has been reported to induce integrin activation in T cells (19Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M. Zwartkruis F.J. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (366) Google Scholar), whereas it causes mild inhibition of platelet activation by the ITAM and ITAM-like receptors, GPVI and CLEC-2, and by the G protein-coupled receptor agonist, thrombin (20Dhanjal T.S. Ross E.A. Auger J.M. McCarty O.J. Hughes C.E. Senis Y.A. Buckley C.D. Watson S.P. Platelets. 2007; 18: 56-67Crossref PubMed Scopus (30) Google Scholar, 21Jones K.L. Hughan S.C. Dopheide S.M. Farndale R.W. Jackson S.P. Jackson D.E. Blood. 2001; 98: 1456-1463Crossref PubMed Scopus (116) Google Scholar, 22Cicmil M. Thomas J.M. Leduc M. Bon C. Gibbins J.M. Blood. 2002; 99: 137-144Crossref PubMed Scopus (132) Google Scholar, 23Patil S. Newman D.K. Newman P.J. Blood. 2001; 97: 1727-1732Crossref PubMed Scopus (141) Google Scholar). The latter action is similar to that of G6b-B, which upon cross-linking by a specific antibody inhibits activation of platelets by the GPVI-specific agonist, collagen-related peptide, and the G protein-coupled receptor agonist ADP (13Newland S.A. Macaulay I.C. Floto A.R. de Vet E.C. Ouwehand W.H. Watkins N.A. Lyons P.A. Campbell D.R. Blood. 2007; 109: 4806-4809Crossref PubMed Scopus (55) Google Scholar). In contrast, the ITIM receptor, TREM-1, which is present on platelet intracellular granules, is translocated to the surface of activated platelets and reinforces platelet activation (16Barrow A.D. Astoul E. Floto A. Brooke G. Relou I.A. Jennings N.S. Smith K.G. Ouwehand W. Farndale R.W. Alexander D.R. Trowsdale J. J. Immunol. 2004; 172: 5838-5842Crossref PubMed Scopus (81) Google Scholar). In this study we have used a sensitive NFAT reporter assay that monitors activation of PLCγ in DT40 B cells to investigate the effect of G6b-B on signaling by the GPVI-FcR γ-chain complex and CLEC-2. The NFAT reporter assay monitors weak, sustained signaling events and gives robust detection of activation of GPVI by collagen and of CLEC-2 by rhodocytin and podoplanin (7Christou C.M. Pearce A.C. Watson A.A. Mistry A.R. Pollitt A.Y. Fenton-May A.E. Johnson L.A. Jackson D.G. Watson S.P. O'Callaghan C.A. Biochem. J. 2008; 411: 133-140Crossref PubMed Scopus (88) Google Scholar, 8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Tomlinson M.G. Calaminus S.D. Berlanga O. Auger J.M. Bori-Sanz T. Meyaard L. Watson S.P. Thromb. Haemostasis. 2007; 5: 2274-2283Crossref Scopus (51) Google Scholar). The availability of variants of the DT40 cell line deficient in key signaling proteins allows dissection of the signaling pathways used by the above receptors. Here we show that expression of the GPVI-FcR γ-chain complex and CLEC-2 in DT40 cells leads to the generation of both constitutive and agonist-induced signals that are inhibited by G6b-B. This effect is dependent on the two ITIMs in the cytosolic tail of G6b-B, although it is independent of the two SH2-domain containing tyrosine phosphatases, SHP1 and SHP2, and of the inositol lipid 5′-phosphatase, SHIP. Significantly, we also provide evidence for constitutive signaling through Src and Syk-dependent kinases in platelets, thereby raising the possibility that inhibition of this by G6b-B could help to prevent unwanted platelet activation in the vasculature. Antibodies and Reagents—Anti-human GPVI monoclonal antibody (mAb) 204-11 has been described previously (25Moroi M. Mizuguchi J. Kawashima S. Nagamatsu M. Miura Y. Nakagaki T. Ito K. Jung S.M. Thromb. Haemostasis. 2003; 89: 996-1003Crossref PubMed Scopus (22) Google Scholar). Anti-Syk polyclonal Ab (pAb) (26Burkhardt A.L. Stealey B. Rowley R.B. Mahajan S. Prendergast M. Fargnoli J. Bolen J.B. J. Biol. Chem. 1994; 269: 23642-23647Abstract Full Text PDF PubMed Google Scholar) was kindly provided by J. B. Bolen (DNAX, CA). Anti-phospho-Syk (Tyr525/526) pAb and anti-Myc mAb were purchased from Cell Signaling Technology (New England Biolabs UK Ltd., Herts, UK). T7-Tag mAb was purchased from Novagen (Nottingham, UK). Anti-phosphotyrosine mAb 4G10, anti-FcR γ-chain pAb, and normal rabbit IgG were purchased from Upstate Biotechnology (Milton Keynes, UK). Anti-human CLEC-2 mAb was purchased fromR&D Systems Inc. (Minneapolis, MN). Anti-PECAM-1 mAb AB468 was from Autogen-Bioclear (Wiltshire, UK). Horseradish peroxidase-conjugated donkey anti-rabbit secondary Ab and enhanced chemiluminescence reagents (ECL) were purchased from Amersham Biosciences. Mouse IgG1 monoclonal was purchased from Abcam (Cambridge, UK). Fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody was purchased from Sigma. Collagen was obtained from Nycomed Austria GmbH (Linz, Austria). Rhodocytin was purified from the venom of Calloselasma rhodostoma (27Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The Src kinase inhibitors used were, PP1, purchased from BioSource Europe (Nivelles, Belgium); PP2, purchased from Calbiochem (Nottingham, UK); and PD0173952, a gift from Pfizer Global Research and Development (Ann Arbor, MI). The Syk kinase inhibitor, R406, was a kind gift of Dr. D. Simmons (Cellzome UK Ltd., Cambridge). FcR γ-chain “knock-out” mice were bred as heterozygotes as described (28Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (396) Google Scholar). 10 mm Pervanadate was freshly prepared on the day for use by mixing sodium orthovanadate and hydrogen peroxide in phosphate-buffered saline to final concentrations 10 mm, then left for 5 min at room temperature and kept on ice. Other reagents were from previously described sources (6Suzuki-Inoue K. Kato Y. Inoue O. Kaneko M.K. Mishima K. Yatomi Y. Yamazaki Y. Narimatsu H. Ozaki Y. J. Biol. Chem. 2007; 282: 25993-26001Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Tomlinson M.G. Calaminus S.D. Berlanga O. Auger J.M. Bori-Sanz T. Meyaard L. Watson S.P. Thromb. Haemostasis. 2007; 5: 2274-2283Crossref Scopus (51) Google Scholar). Constructs—The human pRc/GPVI, pEF6/FcR γ-chain, pEF6/CLEC-2, and mutant CLEC-2 (Y7F) expression plasmids have been previously described (8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The human pcDNA3/-G6b-B (kindly given by Prof R. D. Campbell, Oxford, UK) has been described (9de Vet E.C. Aguado B. Campbell R.D. J. Biol. Chem. 2001; 276: 42070-42076Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and the human pcDNA3/PECAM-1 (kindly given by C. D. Buckley, Birmingham, UK) has also been described (29Newton J.P. Buckley C.D. Jones E.Y. Simmons D.L. J. Biol. Chem. 1997; 272: 20555-20563Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The NFAT luciferase reporter containing three copies of the distal NFAT site from the interleukin-2 promoter has been described (30Shapiro V.S. Mollenauer M.N. Greene W.C. Weiss A. J. Exp. Med. 1996; 184: 1663-1669Crossref PubMed Scopus (69) Google Scholar). Making Myc-tagged FcR γ-Chain—The c-Myc epitope tag (EQKLISEEDL) was fused at the amino terminus of FcR γ-chain by using pEF6/FcR γ-chain as a template and the primers: forward (5′-GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG CTG GGA GAG CCT CAG CTC-3′) and reverse (5′-CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC GGC CGC TGC TTG TTC AAC-3′), and subcloned into pEF6. Site-directed Mutagenesis of G6b-B—Site-directed mutagenesis of G6b-B was performed by a QuikChange® Site-directed Mutagenesis Kit (Stratagene, Cambridge, UK). The primers G6b-B-Y211F-forward (5′-CCG AGC CTG CTC TTT GCG GAT CTG GAC-3′) and G6b-B-Y211F-reverse (5′-GTC CAG ATC CGC AAA GAG CAG GCT CGG-3′) were used for tyrosine to phenylalanine mutation in G6b-B (Y211F) using wild-type human pcDNA/G6b-B as a template. The primers G6b-B-Y237F-forward (5′-GAT GCC TCC ACC ATC TTT GCA GTT GTA GTT TG-3′) and G6b-B-Y237F-reverse (5′-CAA ACT ACA ACT GCA AAG ATG GTG GAG GCA TC-3′) were used for tyrosine to phenylalanine mutation in G6b-B (Y237F) using wild-type human pcDNA/G6b-B as a template and they were also used for tyrosine to phenylalanine mutation in G6b-B (Y211F/Y237F) using mutant pcDNA/G6b-B (Y211F) as a template. All sequences were verified by sequencing. Cell Culture—Wild-type (WT), Syk-deficient (31Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar), SHP1 and SHP2 double-deficient (32Maeda A. Kurosaki M. Ono M. Takai T. Kurosaki T. J. Exp. Med. 1998; 187: 1355-1360Crossref PubMed Scopus (171) Google Scholar) (kindly donated by L. Meyaard, Utrecht, The Netherlands), SHIP-deficient (33Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) (kindly donated by D. K. Newman, Milwaukee, WI) DT40 chicken B cells were grown in RPMI supplemented with 10! fetal bovine serum, 1! chicken serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μm β-mercaptoethanol, and 20 mm glutamine. Luciferase Assay—The NFAT reporter assay was performed as described (8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 24Tomlinson M.G. Calaminus S.D. Berlanga O. Auger J.M. Bori-Sanz T. Meyaard L. Watson S.P. Thromb. Haemostasis. 2007; 5: 2274-2283Crossref Scopus (51) Google Scholar). The indicated amount of DNA of each construct and 15 μg of NFAT-luciferase reporter construct were transfected by electroporation at 350 V and 500 microfarads into 2 × 107 cells of WT, Syk-deficient, and SHP1/SHP2-deficient DT40 cells. Twenty hours after transfection, live cells were counted by trypan blue exclusion, and samples divided for luciferase assay (2 × 106 cells/ml), flow cytometry (5 × 105 cells/sample), and Western blotting (1 × 106 cells/sample). Collagen was used at 10 μg/ml and rhodocytin was used at 50 nm. Luciferase activity was measured with a Centro LB960 microplate luminometer (Berthold Technologies, Germany). All results were compared with basal in mock-transfected cells. Flow Cytometry—Cell surface expression of transfected cells was analyzed by flow cytometry using 1 μg/ml, GPVI mAb, PECAM-1 mAb, and Myc mAb to detect CLEC-2 and FcR γ-chain, T7-Tag mAb to detect G6b-B, or mouse IgG followed by staining with 4 μg/ml fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody, and assessed on a FACScalibur (Becton Dickinson, San Jose, CA). Data were analyzed using CellQuest software. Human and Mouse Platelets—Washed preparations of human and mouse platelets were prepared as previously described (5Suzuki-Inoue K. Fuller G.L. Garcia A. Eble J.A. Pohlmann S. Inoue O. Gartner T.K. Hughan S.C. Pearce A.C. Laing G.D. Theakston R.D. Schweighoffer E. Zitzmann N. Morita T. Tybulewicz V.L. Ozaki Y. Watson S.P. Blood. 2006; 107: 542-549Crossref PubMed Scopus (382) Google Scholar, 8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Platelets were resuspended in a modified Tyrodes-HEPES buffer at concentrations of 4 × 108/ml (human) or 2 × 108/ml (mouse). Platelets were prewarmed to 37 °C for 5 min and incubated with inhibitors or solvent controls for up to 10 min. Immunoprecipitation and Western Blotting—Transfected cells (1 × 108/ml) were incubated for 30 min in RPMI at 37 °C before stimulating. After stimulation, transfected cells or platelets were lysed with ice-cold 2× lysis buffer (2! Triton X-100, 2! dodecyl maltoside, 4 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 2 μg/ml pepstatin, 10 mm sodium orthovanadate, pH 7.5) and insoluble material was removed by centrifugation. For immunoprecipitation, lysates were precleared with protein A (G)-Sepharose beads for 30 min at 4 °C and mixed with 2 μg of the indicated antibodies and protein A-Sepharose beads (protein G-Sepharose). The mixture was rotated for 2 h at 4 °C. Whole cell lysates or immunoprecipitated lysates were added to 2× Laemmli sample buffer. Samples were separated by SDS-PAGE on 10 or 4–12! BisTris gels (Invitrogen) and transferred to polyvinylidene difluoride membrane. Western blotting was carried out as described previously (24Tomlinson M.G. Calaminus S.D. Berlanga O. Auger J.M. Bori-Sanz T. Meyaard L. Watson S.P. Thromb. Haemostasis. 2007; 5: 2274-2283Crossref Scopus (51) Google Scholar). Statistical Analysis—Experiments were performed on at least three occasions and results are shown as mean ± S.E. with the exception of the representative Western blots and flow cytometry histograms. Statistical significance was determined using Student's t test. Constitutive Signaling of GPVI-FcR γ-Chain in DT40 Cells—We set out to extend our previous characterization of the GPVI-FcR γ-chain signaling pathway in the DT40 B cell line (24Tomlinson M.G. Calaminus S.D. Berlanga O. Auger J.M. Bori-Sanz T. Meyaard L. Watson S.P. Thromb. Haemostasis. 2007; 5: 2274-2283Crossref Scopus (51) Google Scholar) by testing whether activation of GPVI by collagen is dependent on its associated FcR γ-chain. Activation was monitored in the absence and presence of collagen and compared with mock-transfected cells that had been allowed to settle on fibronectin using a NFAT-luciferase reporter assay. Adhesion to fibronectin alone did not induce NFAT activation (not shown). As anticipated, collagen stimulated NFAT activation in DT40 cells expressing both GPVI and an NH2-terminal Myc-tagged version of FcR γ-chain, whereas it had no effect when either subunit was transfected on its own (Fig. 1A and not shown). The Myc-tagged version of FcR γ-chain supports a similar level of activation to that of the wild-type protein (not shown) and has the advantage that it can be used to monitor surface expression by flow cytometry, which is not altered by expression of GPVI (Fig. 1A, panel ii). In contrast, GPVI was not expressed on the surface of DT40 cells in the absence of the FcR γ-chain (not shown), as is the case for platelets (34Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). These results are consistent with a model in which cross-linking of GPVI by collagen leads to tyrosine phosphorylation of the FcR γ-chain and activation of the tyrosine kinase Syk. These studies further revealed the unexpected observation that expression of the Myc-tagged or wild-type FcR γ-chain caused a significant increase in luciferase activity that approached almost 10! of the response to collagen (Fig. 1, A, panel i and B, panel i). Furthermore, the constitutive signal from the FcR γ-chain was not altered by co-expression with GPVI (Fig. 1A, panel i). Thus the increase in luciferase activity that was observed in the absence of a stimulatory agonist reflects signaling by the ITAM-containing protein. To investigate constitutive signaling in further detail, DT40 cells were transfected with varying concentrations of the FcR γ-chain plasmid and the level of NFAT activation monitored. Increasing levels of transfection of FcR γ-chain led to a corresponding increase in both NFAT activation (Fig. 1B, panel i) and FcR γ-chain expression as shown by Western blotting (Fig. 1B, panel ii). Furthermore, Western blotting of GPVI-FcR γ-chain-transfected cells demonstrated an increase in tyrosine phosphorylation of a band of 72 kDa that comigrates with Syk, along with constitutive tyrosine phosphorylation of FcR γ-chain (Fig. 1C, panel i). Confirmation that the 72-kDa protein corresponds to Syk was confirmed using a phosphospecific antibody (phospho-Syk Tyr525/526) that binds to the activated tyrosine kinase (not shown). Tyrosine phosphorylation of Syk and FcR γ-chain were inhibited in the presence of the Src family kinase inhibitor PD0173952 (Fig. 1C, panel i, and not shown). These results demonstrate that the FcR γ-chain generates constitutive signals in DT40 cells that lead to tyrosine phosphorylation of Syk and NFAT activation. Stimulation of GPVI-FcR γ-chain-transfected cells with collagen leads to a further increase in tyrosine phosphorylation of Syk (Fig. 1C, panel ii), consistent with the activation of Syk by the collagen receptor complex, as described in earlier studies. Constitutive Signaling of CLEC-2 in DT40 Cells—The above studies were extended to CLEC-2, which is a recently identified platelet glycoprotein receptor that mediates activation through a single YXXL motif in its cytosolic tail. CLEC-2 is a receptor for the snake venom toxin, rhodocytin, and the lymphatic marker podoplanin. Both agonists induce activation of CLEC-2-transfected DT40 cells, with the response being dependent on the conserved tyrosine in the cytoplasmic YXXL motif (7Christou C.M. Pearce A.C. Watson A.A. Mistry A.R. Pollitt A.Y. Fenton-May A.E. Johnson L.A. Jackson D.G. Watson S.P. O'Callaghan C.A. Biochem. J. 2008; 411: 133-140Crossref PubMed Scopus (88) Google Scholar, 8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). In the present study, we have confirmed that rhodocytin stimulates NFAT activation in CLEC-2-transfected DT40 cells, with the level of response increasing with the level of surface expression of CLEC-2 (Fig. 2A, panels i and ii). Moreover, as is the case with the FcR γ-chain, expression of CLEC-2 was sufficient to increase NFAT activity in the absence of agonist stimulation, with the level of response increasing in parallel with that of CLEC-2 (Fig. 2A, panels i and ii). The level of constitutive activity approached between 10 and 20! the response to rhodocytin. Constitutive signaling by CLEC-2 is abolished following mutation of the tyrosine in the CLEC-2 YXXL sequence to a phenylalanine (Y7F) (Fig. 2B, panel i), as is the case for stimulation by rhodocytin (8Fuller G.L. Williams J.A. Tomlinson M.G. Eble J.A. Hanna S.L. Pohlmann S. Suzuki-Inoue K. Ozaki Y. Watson S.P. Pearce A.C. J. Biol. Chem. 2007; 282: 12397-12409Abstract Full Text Fu" @default.
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• W2122340660 title "G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2" @default.
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