The C-type Lectin Receptors CLEC-2 and Dectin-1, but Not DC-SIGN, Signal via a Novel YXXL-dependent Signaling Cascade
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m609558200
ISSN1083-351X
AutoresGemma L.J. Fuller, Jennifer A. E. Williams, Michael G. Tomlinson, Johannes A. Eble, Sheri L. Hanna, Stefan Pöhlmann, Katsue Suzuki‐Inoue, Yukio Ozaki, Steve P. Watson, Andrew C. Pearce,
Tópico(s)Complement system in diseases
ResumoThe two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cγ. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters. The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cγ. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters. The C-type lectin superfamily of transmembrane proteins consists of at least 70 members in the human genome (1Drickamer K. Fadden A.J. Biochem. Soc. Symp. 2002; 69: 59-72Crossref PubMed Scopus (112) Google Scholar). The superfamily can be divided into "classical" C-type lectins, which contain a carbohydrate recognition domain and bind sugars in a calcium-dependent manner, and the "nonclassical" C-type lectin-like proteins, which contain a C-type lectin-like domain, homologous to a carbohydrate recognition domain, but lacks the consensus sequence for binding sugars and calcium (2Drickamer K. Curr. Opin. Struct. Biol. 1999; 9: 585-590Crossref PubMed Scopus (520) Google Scholar). Protein ligands for a number of classical and nonclassical C-type lectin receptors have been described.C-type lectin-like receptor 2 (CLEC-2) 6The abbreviations used are: CLEC-2, C-type lectin-like receptor 2; SH, Src homology; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; PLCγ, phospholipase Cγ; WT, wild type; IL, interleukin; FWD, forward; REV, reverse; NFAT, nuclear factor of activated T cells. 6The abbreviations used are: CLEC-2, C-type lectin-like receptor 2; SH, Src homology; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; PLCγ, phospholipase Cγ; WT, wild type; IL, interleukin; FWD, forward; REV, reverse; NFAT, nuclear factor of activated T cells. is a type II transmembrane protein and a nonclassical C-type lectin (3Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (184) Google Scholar). The C-type lectin-like domain in CLEC-2 is supported by a 41-amino acid neck region, a single transmembrane domain, and 31-amino acid cytoplasmic domain (3Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (184) Google Scholar). CLEC-2 mRNA has been identified in liver and in blood cells, mostly of myeloid origin, including monocytes, granulocytes, and dendritic cells (3Colonna M. Samaridis J. Angman L. Eur. J. Immunol. 2000; 30: 697-704Crossref PubMed Scopus (184) Google Scholar). Recently, we have identified expression of CLEC-2 in platelets and have shown that it functions as a receptor for the snake venom toxin rhodocytin (also known as aggretin), which elicits powerful platelet activation (4Suzuki-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 (370) Google Scholar). Rhodocytin, however, also binds to several other platelet receptors (5Chung C.H. Peng H.C. Huang T.F. Biochem. Biophys. Res. Commun. 2001; 285: 689-695Crossref PubMed Scopus (34) Google Scholar, 6Huang T.F. Liu C.Z. Yang S.H. Biochem. J. 1995; 309: 1021-1027Crossref PubMed Scopus (90) Google Scholar), making it unclear whether CLEC-2 is sufficient to mediate activation alone and thereby hampering analysis of the mechanism of activation.The cytosolic domain of CLEC-2 contains a single tyrosine residue within a YXXL motif, a consensus sequence for phosphorylation by Src family kinases in immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ITAMs have the sequence YXX(L/I)X6–12YXX(L/I), and ITIMs have the sequence (L/I/V)XYXX(L/I/V). Phosphorylation of the two tyrosine residues within an ITAM leads to recruitment of the tyrosine kinases Syk and Zap-70 via their tandem Src homology 2 (SH2) domains, leading to cellular activation (7Iwashima M. Irving B.A. van Oers N.S. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar, 8Koyasu S. Tse A.G. Moingeon P. Hussey R.E. Mildonian A. Hannisian J. Clayton L.K. Reinherz E.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6693-6697Crossref PubMed Scopus (67) Google Scholar). Phosphorylated ITIMs binds to the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, or the lipid phosphatases SHIP1 and SHIP2, in most cases leading to cellular inhibition (9Unkeless J.C. Jin J. Curr. Opin. Immunol. 1997; 9: 338-343Crossref PubMed Scopus (126) Google Scholar).Signaling by ITAM receptors, such as the platelet collagen receptor complex, GPVI/FcR γ-chain, or the B and T cell antigen receptors, is mediated via members of the Src, Syk, Tec, Vav, SLP-76/BLNK, and PLCγ families of signaling proteins (reviewed in Refs. 10Watson S.P. Auger J.M. McCarty O.J. Pearce A.C. J. Thromb. Haemostasis. 2005; 3: 1752-1762Crossref PubMed Scopus (326) Google Scholar, 11Lin J. Weiss A. J. Cell Sci. 2001; 114: 243-244Crossref PubMed Google Scholar, 12Wang L.D. Clark M.R. Immunology. 2003; 110: 411-420Crossref PubMed Scopus (67) Google Scholar). The specific members of each family that mediate ITAM signaling are cell-dependent. For example, SLP-76 is used by the T cell receptor (13Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) and the platelet collagen receptor GPVI (14Gross B.S. Lee J.R. Clements J.L. Turner M. Tybulewicz V.L. Findell P.R. Koretzky G.A. Watson S.P. J. Biol. Chem. 1999; 274: 5963-5971Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), although B cells use the homologous protein BLNK (15Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (231) Google Scholar).We have shown that activation of platelets by rhodocytin is critically dependent on the tyrosine kinase Syk and many of the proteins that participate in ITAM signaling in platelets (4Suzuki-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 (370) Google Scholar). This has led us to propose that the snake venom toxin signals through a similar pathway to that of ITAM receptors, with Syk being recruited via the phosphorylated YXXL sequence in the cytosolic tail of the lectin-like receptor. A similar coupling to Syk has been proposed for a second C-type lectin receptor, Dectin-1, which mediates activation of dendritic cells by zymosan (16Rogers N.C. Slack E.C. Edwards A.D. Nolte M.A. Schulz O. Schweighoffer E. Williams D.L. Gordon S. Tybulewicz V.L. Brown G.D. Reis E.S.C. Immunity. 2005; 22: 507-517Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). A third YXXL-containing member of the C-type lectin superfamily, DC-SIGN, has also been reported recently to signal to PLCγ in dendritic cells, although the role of Syk in signaling by this receptor is not known (17Caparros E. Munoz P. Sierra-Filardi E. Serrano-Gomez D. Puig-Kroger A. Rodriguez-Fernandez J.L. Mellado M. Sancho J. Zubiaur M. Corbi A.L. Blood. 2006; 107: 3950-3958Crossref PubMed Scopus (181) Google Scholar).The aim of this study was to characterize the mechanism of CLEC-2 signaling in platelets and in two hematopoietic-derived cell line model systems and to compare this to signaling by Dectin-1 and DC-SIGN. The results demonstrate that signaling by CLEC-2 is completely dependent on the cytoplasmic YXXL motif and requires both SH2 domains of Syk. The signaling pathway activated by CLEC-2 involves Src, Syk, and Tec family kinases and PLCγ, but it is distinct from that of ITAM signaling in that it has a partial rather than absolute dependence on the SLP-76/BLNK family of adapter proteins. Dectin-1 signals in a similar way to CLEC-2, whereas the mechanism of signaling by DC-SIGN is distinct. The results demonstrate that some but not all lectin receptors signal through a single YXXL motif leading to activation of PLCγ.EXPERIMENTAL PROCEDURESAntibodies and Reagents—Polyclonal goat α-human CLEC-2, α-mouse Dectin-1, and normal goat IgG were purchased from R & D Systems Inc. (Minneapolis, MN). Monoclonal α-CD209 (DC-SIGN) was purchased from Pharmingen. Rabbit polyclonal antibodies α-Syk, α-PLCγ2, and α-Btk have been described previously (18Pearce A.C. Senis Y.A. Billadeau D.D. Turner M. Watson S.P. Vigorito E. J. Biol. Chem. 2004; 279: 53955-53962Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Atkinson B.T. Ellmeier W. Watson S.P. Blood. 2003; 102: 3592-3599Crossref PubMed Scopus (129) Google Scholar). Anti-phosphotyrosine monoclonal antibody 4G10, α-SLP-76 polyclonal antibody, and α-LAT polyclonal antibody were purchased from Upstate Biotechnology Inc. (TCS Biologicals Ltd., Bucks, UK). Anti-human Vav3 antibody was a kind gift from Dr. Daniel Billadeau and was raised in rabbits as described previously (20Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). Anti-MYC antibody was purchased from Cell Signaling Technology (New England Biolabs, Herts, UK). F(ab′)2 fragments of the anti-human FcγRIIA antibody IV.3 were generated as described previously (21Inoue O. Suzuki-Inoue K. Dean W.L. Frampton J. Watson S.P. J. Cell Biol. 2003; 160: 769-780Crossref PubMed Scopus (200) Google Scholar). Fluorescein isothiocyanate-conjugated donkey anti-goat IgG secondary antibody was from Jackson ImmunoResearch. Horseradish peroxidase-conjugated sheep anti-mouse secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, and enhanced chemiluminescence reagents (ECL) were purchased from Amersham Biosciences. GST fusion proteins corresponding to single or tandem SH2 domains of Syk were prepared as described previously (22Qi R. Ozaki Y. Asazuma N. Satoh K. Yatomi Y. Law C.L. Hato T. Nomura S. Biochim. Biophys. Acta. 1999; 1451: 353-363Crossref PubMed Scopus (11) Google Scholar). Rhodocytin was purified by Dr. Johannes Eble as described previously (23Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The GPIIbIIIa antagonist lotrafiban was a gift from GlaxoSmithKline (King of Prussia, PA), and the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide was obtained from Peptide Institute (Osaka, Japan). The Src kinase inhibitor PD0173952 was a gift from Pfizer Global Research and Development (Ann Arbor, MI). The Src kinase inhibitor PP2 was purchased from Calbiochem. All other reagents were purchased from Sigma or from previously described sources (18Pearce A.C. Senis Y.A. Billadeau D.D. Turner M. Watson S.P. Vigorito E. J. Biol. Chem. 2004; 279: 53955-53962Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 24Bori-Sanz T. Inoue K.S. Berndt M.C. Watson S.P. Tulasne D. J. Biol. Chem. 2003; 278: 35914-35922Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).Platelet Studies—Blood was drawn on the day of experiment from healthy, drug-free volunteers into 1:10 (v/v) sterile sodium citrate and 1:9 (v/v) acid citrate dextrose (ACD: 120 mm sodium citrate, 110 mm glucose, 80 mm citric acid). Washed platelets were prepared as described previously (18Pearce A.C. Senis Y.A. Billadeau D.D. Turner M. Watson S.P. Vigorito E. J. Biol. Chem. 2004; 279: 53955-53962Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Platelet aggregation studies were carried out using washed platelets at a concentration of 2 × 108/ml in a Born aggregometer (ChronoLog, Havertown, PA) at 37 °C with continuous stirring at 1200 rpm for 5 min. Aggregation of platelets in response to rhodocytin (300 nm) or α-CLEC-2 antibody (10 μg/ml) was recorded by measuring change in optical density. Platelets were preincubated with IV.3 F(ab′)2 (12 μg/ml), PP2 (10 μm), or PD0173952 (25 μm) for 10 min prior to stimulation where indicated. Platelets were used at 1 × 109/ml for protein studies. Lotrafiban (10 μm) or GRGDS peptide (1 mm) was included in the resuspension buffer to block aggregation and signaling through GPIIbIIIa. Stimulations were carried out in a Born aggregometer for the times shown. Following stimulation platelets were lysed with an equal volume of 2× lysis buffer (300 mm NaCl, 20 mm Tris, 2 mm EGTA, 2 mm EDTA, 2% Nonidet P-40, pH 7.4, with 2.5 mm Na3VO4, 100 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 0.5 μg/ml pepstatin).Constructs—Human CLEC-2 cloned into pcDNA3 has been described previously (25Pohlmann S. Zhang J. Baribaud F. Chen Z. Leslie G.J. Lin G. Granelli-Piperno A. Doms R.W. Rice C.M. McKeating J.A. J. Virol. 2003; 77: 4070-4080Crossref PubMed Scopus (341) Google Scholar). For these experiments CLEC-2 was subcloned into pEF6 vector with a C-terminal MYC tag, pEF6/Myc-His A (Invitrogen). A point mutation of the cytoplasmic tyrosine residue of CLEC-2 to a phenylalanine (Y7F) was generated by a two-step PCR method (26Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2087) Google Scholar). The mutating primers CLEC-2-Y7F-REV (5′-GTT TTA ATA TTT AAG GTG ATG AAT CCA TCT TCA TCC TG-3′) and CLEC-2-Y7F-FWD (5′-CAG GAT GAA GAT GGA TTC ATC ACC TTA AAT ATT AAA AC-3′) were used along with vector specific primers T7 and 4150 (5′-AGG CAC AGT CGA GGC TGA TC-3′). D3A, E4A, D5A, and G6A CLEC-2 were generated by a single step PCR approach. The mutating primers were D3A-FWD (5′-TAG TAG GGA TCC ATG CAG GCT GAA GAT GGA TAC-3′), E4A-FWD (5′-TAG TAG GGA TCC ATG CAG GAT GCA GAT GGA TAC-3′), D5A-FWD (5′-TAG TAG GGA TCC ATG CAG GAT GAA GCT GGA TAC ATC-3′), G6A-FWD (5′-TAG TAG GGA TCC ATG CAG GAT GAA GAT GCA TAC ATC ACC-3′), and hCLEC-2-REV (5′-TAG TAG GCG GCC GCA GGT AGT TGG TCC ACC TTG GTC-3′). Porcine Syk cloned into pcDNA3 has been described previously (27Taniguchi T. Kobayashi T. Kondo J. Takahashi K. Nakamura H. Suzuki J. Nagai K. Yamada T. Nakamura S. Yamamura H. J. Biol. Chem. 1991; 266: 15790-15796Abstract Full Text PDF PubMed Google Scholar). Inactivating point mutants of each SH2 domain of Syk were made by mutating Arg-37 or Arg-190 to Ala. In both cases T7 and BGH were used as outside primers. Specific primers for the point mutants were Syk-R37A-FWD (5′-GGG CTC TAC CTG CTT GCC CAG AGC CGC AAC TAC-3′), Syk-R37A-REV (5′-GTA GTT GCG GCT CTG GGC AAG CAG GTA GAG CCC-3′), Syk-R190A-FWD (5′-GGG AAG TTT TTG ATC GCG GCC AGG GAC AAC GGG-3′), Syk-190A-REV (5′-CCC GTT GTC CCT GGC CGC GAT CAA AAA CTT CCC-3′). Human GPVI cloned into pRC plasmid has been described previously (24Bori-Sanz T. Inoue K.S. Berndt M.C. Watson S.P. Tulasne D. J. Biol. Chem. 2003; 278: 35914-35922Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For these experiments hGPVI was subcloned into pcDNA3 with a C-terminal MYC tag (Invitrogen). Human FcR γ-chain DNA was amplified from HEL cell cDNA by PCR (hFcRγ-FWD (5′-TAG TAG GGA TCC CAG CCC AAG ATG ATT CCA GC-3′) and hFcRγ-REV (5′-TAG TAG GCG GCC GCC TAC TGT GGT GGT TTC TCA TG-3′)) and cloned into pEF6 vector with no tag (Invitrogen). Murine Dectin-1 was amplified from cDNA prepared from murine spleen by PCR (mDectin-1-FWD (5′-TAG TAG GGA TCA TGA AAT ATC ACT CTC ATA TAG-3′) and mDectin-1-REV (5′-TAG TAG GCG GCC AGT TCC TTC TCA CAG ATA C-3′)) and cloned into pEF6 vector with a C-terminal MYC tag, pEF6/Myc-His. All sequences were verified by sequencing. Wild type DC-SIGN in pcDNA3 has been described previously (28Pohlmann S. Baribaud F. Lee B. Leslie G.J. Sanchez M.D. Hiebenthal-Millow K. Munch J. Kirchhoff F. Doms R.W. J. Virol. 2001; 75: 4664-4672Crossref PubMed Scopus (200) Google Scholar). A point mutation of the YXXL tyrosine (Tyr-31) to phenylalanine was generated by a two-step PCR method. The mutating primers were hDCSIGNY31F-FWD (5′-CGA CAG ACT CGA GGA TTC AAG AGC TTA GCA GGG-3′) and hDCSIGNY31F-REV (5′-CCC TGC TAA GCT CTT GAA TCC TCG AGT CTG TCG-3′). The NFAT luciferase reporter contains three copies of the distal NFAT site from the IL-2 promoter (29Shapiro V.S. Mollenauer M.N. Greene W.C. Weiss A. J. Exp. Med. 1996; 184: 1663-1669Crossref PubMed Scopus (69) Google Scholar) and was kindly provided by Prof. A. Weiss. The pEF6-lacZ expression construct was obtained from Invitrogen.Cell Culture and Transfection—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 mercaptoethanol, and 20 mm GlutaMAX. DT40 cells rendered deficient for SYK (30Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar), LYN (30Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar), LYN/SYK (31Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar), BLNK (32Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), BTK (31Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (424) Google Scholar), and PLCγ2 (33Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar) were described previously and kindly provided by Dr. T. Kurosaki (Kansai Medical University, Moriguchi, Japan). Jurkat T cells were grown in RPMI supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 20 mm GlutaMAX. Jurkat derivatives JCaM1 and JCaM1/Lck and J14 and J14/SLP-76 (J14-76) were kindly provided by Dr. A. Weiss (University of California, San Francisco) and have been described previously (34Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 35Yablonski D. Kuhne M.R. Kadlecek T. Weiss A. Science. 1998; 281: 413-416Crossref PubMed Scopus (353) Google Scholar). Cells were transfected in a volume of 400 μl of nonsupplemented RPMI by electroporation using a GenePulser II (Bio-Rad) set at 350 V and 500 microfaradays for DT40 and 250 V and 950 microfaradays for Jurkat cells. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 20 mm Glutamax. 293T cells were transfected with 5 μg of DNA of each DNA construct by calcium phosphate precipitation.Cell Line Protein Studies—Cells were transfected as described above with 10 μg of CLEC-2 or 10 μg of Y7F CLEC-2. Twenty hours following transfection cells were washed and resuspended in nonsupplemented RPMI. Cells were stimulated with 500 nm rhodocytin at room temperature for 10 min. Where indicated, inhibitors were preincubated with the cells for 10 min prior to stimulation. Following stimulation cells were lysed with an equal volume of 2× lysis buffer. 293T cells were harvested 48 h after transfection, washed once in phosphate-buffered saline, and lysed in 500 μl of 1× lysis buffer.Luciferase Assay—Cells were transfected as described above with either 10 μg of CLEC-2, 10 μg of Y7F CLEC-2, 10 μg of Dectin-1, or 2 μg of GPVI and 2 μg of FcR γ-chain constructs, in addition to 15 μg of the luciferase reporter construct and 2 μg of pEF6-lacZ to control for transfection efficiency. Where indicated the receptor of interest was cotransfected along with 5 μg of wild type Syk, R37ASyk, or R190ASyk into Syk-deficient DT40 cells. Twenty hours after transfection, live cells were counted by trypan blue exclusion, and samples were divided for luciferase assay, β-galactosidase assay, and flow cytometry. Luciferase assays were as described previously (36Tomlinson M.G. Kane L.P. Su J. Kadlecek T.A. Mollenauer M.N. Weiss A. Mol. Cell. Biol. 2004; 24: 2455-2466Crossref PubMed Scopus (69) Google Scholar). For luciferase assays, rhodocytin was used at 50 nm, α-CLEC-2 antibody at 40 μg/ml, α-DC-SIGN at 10 μg/ml cross-linked with sheep α-mouse F(ab′)2 fragments at 30 μg/ml, zymosan at 250 μg/ml, and convulxin at 10 μg/ml. Luciferase activity was measured with a Centro LB 960 microplate luminometer (Berthold Technologies, Germany). Data are expressed either as luminescence units normalized to β-galactosidase activity or as fold increase in luminescence units over basal as indicated. All luciferase data are averaged from three readings. Data are represented as one experiment representative of three ± S.E. for the three readings of the experiment. β-Galactosidase assays were performed with half a million cells using the Galacto-Light chemiluminescent reporter assay, according to the manufacturer's instructions (Applied Biosystems, Bedford, MA). β-Galactosidase activity was measured in triplicate using a microplate luminometer. All luciferase assay data were normalized to β-galactosidase values.Flow Cytometry—Expression of each receptor was confirmed by flow cytometry. For CLEC-2 or Dectin-1 detection, 5 × 105 cells were stained in 50-μl volume for 20 min with either 10 μg/ml goat α-CLEC-2 or 10 μg/ml goat α-Dectin-1 antibody alongside goat IgG as a negative control. Cells were then washed and incubated for 20 min with 15 μg/ml fluorescein isothiocyanate-conjugated α-goat IgG secondary antibody. Stained cells were analyzed using a FACSCalibur (BD Biosciences). Data were collected and analyzed using Cellquest software.Immunoprecipitation, Pulldowns, and Western Blotting—Cell lysates were precleared, and detergent-insoluble debris was removed as described (37Suzuki-Inoue K. Inoue O. Frampton J. Watson S.P. Blood. 2003; 102: 1367-1373Crossref PubMed Scopus (81) Google Scholar). Following preclearing, 50-μl aliquots of the stimulation were added to an equal volume of 2× Laemmli sample buffer for whole cell phosphorylation studies. For immunoprecipitation and pulldown studies, lysates were incubated with the indicated antibodies and a mixture of Protein A-Sepharose and Protein G-Sepharose or GST fusion proteins corresponding to single or tandem SH2 domains of Syk associated with glutathione-Sepharose. Following immunoprecipitation, the Sepharose beads were washed, and resulting protein complexes were eluted with 2× Laemmli sample buffer. The resulting whole cell lysates and immunoprecipitates were resolved by SDS-PAGE, and Western blotting was carried out as described previously (18Pearce A.C. Senis Y.A. Billadeau D.D. Turner M. Watson S.P. Vigorito E. J. Biol. Chem. 2004; 279: 53955-53962Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Analysis of Data—Experiments were performed on at least three occasions and are shown as representative data from one experiment. Where experiments were carried out in triplicate, results are presented as the mean of the data.RESULTSAnti-CLEC-2 Antibody and Rhodocytin Stimulate Similar Patterns of Tyrosine Phosphorylation in Platelets—The snake toxin rhodocytin binds to multiple receptors on the platelet surface therefore making it unclear which signaling events are mediated through CLEC-2 or by other receptors for the snake toxin. To address this, we used an α-CLEC-2 antibody, which we have shown previously is able to induce platelet aggregation and phosphorylation of CLEC-2 in platelets independent of the low affinity immune receptor, FcγRIIA (4Suzuki-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 (370) Google Scholar). Experiments were carried out in the presence of F(ab′)2 fragments of the antibody IV.3 to block the FcγRIIA receptor on platelets. The antibody to CLEC-2 (10 μg/ml) stimulated platelet shape change and aggregation, whereas a nonspecific goat IgG control antibody had no effect (Fig. 1A, panel i). The onset of aggregation in response to the CLEC-2 antibody occurs after a lag time that is characteristic of platelet aggregation to rhodocytin (Fig. 1A, panel i). Aggregation to the CLEC-2 antibody is completely inhibited by the Src family kinase inhibitor, PP2 (Fig. 1A, panel ii), as is also the case for rhodocytin (4Suzuki-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 (370) Google Scholar). The same result was observed with the structurally distinct Src kinase inhibitor PD0173952 (data not shown). Tyrosine phosphorylation of platelet lysates induced by rhodocytin and the CLEC-2 antibody was compared by Western blotting with the anti-phosphotyrosine antibody 4G10. The two agonists stimulated a similar pattern of tyrosine phosphorylation suggesting that the increase in tyrosine phosphorylation induced by rhodocytin is mediated through CLEC-2 (Fig. 1B). No increase in whole cell tyrosine phosphorylation was observed in platelets incubated with nonspecific goat IgG antibody (Fig. 1B). In addition, immunoprecipitation studies confirmed that the CLEC-2 antibody induced tyrosine phosphorylation of the same set of proteins that are regulated by rhodocytin in platelets, namely Syk, PLCγ2, Vav3, LAT, SLP-76, and Btk (Fig. 1C). The greater level of tyrosine phosphorylation of Syk, LAT, and PLCγ2 induced by the CLEC-2 antibody may reflect slight differences in the kinetics of activation or differences in the level of stimulation. Importantly, none of these proteins became phosphorylated following stimulation with control goat IgG. These results demonstrate that CLEC-2 is sufficient to cause platelet activation and suggest that the major mechanism of platelet activation by rhodocytin is through the lectin receptor.CLEC-2 Expressing DT40 Cells and Jurkat Cells Are Activated by Stimulation of the Receptor—To further investigate the mechanism of CLEC-2 signaling, CLEC-2 was cloned into an expression vector and transiently transfected into cell lines. DT40 B cells and Jurkat T cells were used as model systems for studying CLEC-2 signaling because B cells and T cells express many of the same signaling proteins as platelets, and mutants of both cell lines are available with deficiencies in the key signaling proteins. Transfection of CLEC-2 into DT40 cells and Jurkat cells led to expression at the cell surface as measured by flow cytometry, although it was absent from mock-transfected cells (Fig. 2A).
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