The Fc receptor c-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin Oscar Berlanga 1,2 , David Tulasne 1 , Teresa Bori 3 , Daniel C. Snell 1 , Yoshiki Miura 4 , Stephanie Jung 4 , Masaaki Moroi 4 , Jonathan Frampton 2 and Steve P. Watson 1 1 Department of Pharmacology, University of Oxford, UK; 2 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK; 3 Division of Medical Sciences, The Medical School, University of Birmingham, UK; 4 Department of Protein Biochemistry, Institute of Life Sciences, Kurume University, Japan There is extensive evidence that FcR c-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR c-chain into the human erythroleukae- mia cell line K562, which lacks detectable expression of GPVI and the FcR c-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR c-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR c-chain and two mutant versions of GPVI shows that the transmembrane arginine and cyto- plasmic tail of GPVI are necessary for association with the FcR c-chain. These results demonstrate that reconstitution of the GPVI–FcR c-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide. Keywords: collagen; collagen-related peptide (CRP); convulxin; FcR c-chain; glycoprotein VI (GPVI). Glycoprotein VI (GPVI) is the major signalling collagen receptor present in platelets and megakaryocytes. It belongs to the superfamily of immunoblobulin receptors and is closely related to Fca receptor (FcaR) and natural killer receptor [1]. The cloned cDNA of GPVI indicates an ORF encoding a 20 amino-acid signal sequence and a mature protein of 319 amino acids. Its extracellular region has two immunoglobulin-like domains and a mucin-like Ser/Thr region, followed by a transmembrane and short cytoplasmic tail. GPVI forms a complex with the Fc receptor c-chain (FcR c-chain), which is respon- sible for signalling through GPVI [2–4]. Previous reports on two receptors sharing homology with GPVI, namely FcaR and paired immunoglobulin-like receptor A (PIR- A), revealed that they are expressed on the surface of the membrane independently of the FcR c-chain in cell lines [5,6], but coexpression with FcR c-chain increases the level of expression of the receptor at the surface [5,7]. The interaction between FcaRorPIR-AandtheFcR c-chain occurs in the transmembrane region as the result of oppositely charged amino-acid residues [6,8]. The immune receptor tyrosine-based activation motif domain within the cytoplasmic tail of the FcR c-chain is responsible for signalling after engagement of the recep- tor complex [9]. The interaction between platelets and collagen involves adhesion and activation leading to increased strength of adhesion, secretion and ultimately aggregation [10–12]. It is accepted that the integrin a2b1 is the major receptor supporting strong platelet adhesion to collagen, whereas GPVI mediates activation [3,13]. The multimeric nature of collagen means that the development of specific ligands to either receptor is essential for understanding their relative contribution to the overall mechanism of platelet–collagen interaction. Among these, collagen-related peptide (CRP) is thought to signal specifically through GPVI, as demonstra- ted by the lack of response to the peptide in GPVI-deficient platelets [13]. Convulxin, a C-type lectin from the venom of the tropical rattlesnake Crotalus durissus terrificus,also specifically recognizes GPVI. The present results show surface expression of GPVI independently of the FcR c-chain in COS-7 and K562 cells, and that the transmembrane arginine and cytoplasmic domain of GPVI are necessary for association with the FcR c-chain. Moreover, the FcR c-chain is necessary and sufficient to initiate the signalling events after GPVI engagement, as demonstrated for the first time in a reconstituted system in which both GPVI and FcR c-chain have been stably expressed. Correspondence to O. Berlanga, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK. Fax:+ 44 1865 222737, Tel.: + 44 1865 222437, E-mail: oscar.berlanga@pharm.ox.ac.uk Abbreviations: GPVI, glycoprotein VI; FcRc, Fc receptor c-chain; CRP, collagen-related peptide; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; GST, glutathione S-transferase; PIR-A, paired immunoglobulin-like receptor A; PMA, phorbol myristate acetate. (Received 14 November 2001, revised 20 March 2002, accepted 30 April 2002) Eur. J. Biochem. 269, 2951–2960 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02969.x MATERIALS AND METHODS Materials CRP (GCP*[GPP*]10GCP*G, P* ¼ hydroxyproline; the peptide is cross-linked through the C-terminal cysteines) was a gift from Drs Barnes, Knight, and Farndale (Department of Biochemistry, University of Cambridge, UK). Convulxin was supplied by Drs Leduc and Bon (Institute Pasteur, Paris, France). Anti-Syk N-19 was from Santa Cruz (Insight Biotechnology Ltd, Wembley Middlesex, UK). Fluorescein isothiocyanate (FITC)- conjugated anti-(human CD36) was from Serotec (Kidlington, Oxfordshire, UK). FITC-conjugated anti- rabbit IgG (Fab¢) 2 fragments were from Sigma (Poole, Dorset, UK). All other reagents were from previously described sources [3,4,14]. Cell culture and platelet preparation K562 and Jurkat cells were grown in RPMI-1640 medium, and COS-7 cells were grown in Dulbecco’s modified Eagle’s medium. Both cell lines were supple- mented with 1 m M glutamine, 100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin and 10% heat-inactivated fetal bovine serum under 5% CO 2 /95% air in a humidified incubator. Cells were kept at exponential phase of growth. Differentiation of K562 cells was induced with phorbol myristate acetate (PMA) as previously described [14]. Platelets were obtained from drug-free volunteers on the day of the experiment. They were isolated as previously described [3]. Transient and stable transfections Two different methods of transfection were used: electro- poration and the calcium phosphate method. For electro- poration, 5 · 10 6 cells were washed twice in cytomix buffer (120 m M KCl, 0.5 m M CaCl 2 ,10m M K 2 HPO 4 ,10m M KH 2 PO 4 ,25m M Hepes, 2 m M EGTA, 5 m M MgCl 2 , pH 7.6 adjusted with KOH) supplemented on the day of experiment with 5 m M glutathione and 2 m M ATP, and resuspended in 400 lL cytomix buffer, which was added in an electroporation cuvette already containing 5–10 lg plasmid DNA. After electroporation, cells were diluted to a final volume of 5 mL with complete prewarmed medium and placed in the incubator. The calcium phos- phate method was used with COS-7 cells. A mixture containing 500 lL sterile distilled water, 186 lL1 M CaCl 2 and 20 lg plasmid DNA was prepared. After 5 min, 750 lL2· HBS buffer [250 m M NaCl, 10 m M KCl, 1.5 m M Na 2 HPO 4 (anhydrous), 50 m M Hepes, 12 m M dextrose, pH 7.05] was added, and 200 lL of the final mixture per ml of culture medium was added to the cells. For transient expression experiments, cells were used after 2 days of transfection. For stable expression, plasmid DNA was linearized before transfection with a proper restriction enzyme, and the enzyme inactivated by heat. After 24 h, cells were separated into at least 24 wells and exposed to antibiotic (500 lgÆmL )1 G418 or 500 lgÆmL )1 hygromycin, as indicated). After  1 week, individual clones of cells were selected and placed in 96-well plates for expansion. Plasmid constructs cDNAs coding for wild-type human GPVI (F1) or GPVI mutated in the transmembrane arginine residue to alanine (R272A) (FA) or with the cytoplasmic tail deleted (A288STOP) (C1) were cloned into HindIII–XbaIsitesof pRc plasmid (Invitrogen). pMG plasmid (InvivoGen) is a bicistronic plasmid for simultaneous expression of two different proteins. The cDNA coding for human FcR c-chain was subcloned into SmaI–XbaI sites of multicloning site 1. pEGFP plasmid, coding for the green fluorescent protein (GFP), was from Clontech (Basingstoke, Hampshire, UK). A chimeric protein GPVI–GFP was made by inserting the cDNA of GPVI into the EcoRI site of EGFP plasmid. Immunoprecipitation and affinity precipitation Cells [(2–4) · 10 6 ] were lysed in buffer [10 m M Tris 150 m M NaCl, 5 m M EDTA, 1% (v/v) Triton X-100, 0.5 m M phenylmethanesulfonyl fluoride, 1 m M Na 3 VO 4 ,5lgÆmL )1 leupeptin, 5 lgÆmL )1 aprotinin, 0.5 lgÆmL )1 pepstatin A, pH 7.3] and rotated at 4 °C for 30 min; they were then centrifuged for 10 min and the supernatant used to measure protein concentration. Samples were precleared for 1 h with 30 lL protein A–Sepharose (50%, v/v), centrifuged, and supernatant used for immunoprecipitation as previously described [14]. For GPVI affinity precipitation, 5 · 10 8 platelet-protein extract or 500 lg protein extract from the cell lines was incubated with 15 n M convulxin for 2 h. Convulxin antibody (0.4 lgÆmL )1 ) was added, and incuba- tions were carried out overnight. Then 30 lLproteinA– Sepharose was added and the mixture incubated for 1 h. Samples were washed 4–6 times with lysis buffer and resuspended in Laemmli sample buffer. A glutathione S-transferase (GST) fusion protein containing the tandem SH2 domains of Syk (GST-Syk-SH2) was expressed as described previously [15], and bound to glutathione– agarose. Precipitated proteins were separated by SDS/ PAGE and transferred on to poly(vinylidene difluoride) membranes. Immunoblotting and ligand blotting Protein extracts from whole cell lysates or immunoprecip- itated proteins were used for blotting as previously described [14]. GPVI and FcR c-chain blotting was performed under nonreducing conditions. For GPVI detection, membranes were blocked for 1 h using Tris/NaCl-T containing 5% skimmed milk, and then incubated with 10 n M convulxin dissolved in Tris/NaCl/Tween [20 m M Tris, 137 m M NaCl, 0.1% (v/v) Tween 20, pH 7.6] for 1 h at room temperature; they were then washed and incubated with convulxin antibody and secondary antibody, both dissolved in Tris/ NaCl-T. Flow cytometry Cells were resuspended in Tyrodes/Hepes or NaCl/P i buffer containing 1% human serum albumin and 0.02% sodium azide. For some experiments, cells were fixed with 3.7% formaldehyde for 30 min and platelets with 1% formalde- hyde for 1 h, followed by 10 min incubation with 50 m M 2952 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002 NH 4 Cl. All incubation times were 30 min unless otherwise indicated. GPVI was detected as previously described [14]. Briefly, cells were incubated with 20 n M convulxin, washed, andincubatedwith0.4lgÆmL )1 convulxin antibody. After being washed, cells were incubated with FITC-conjugated anti-rabbit IgG for 20 min. Cells were washed again and immediately analyzed by flow cytometry. FITC-conjugated anti-human CD49b (a2 subunit), FITC-conjugated anti- human CD36 and their respective isotype controls were used at a dilution of 1 : 5. All data were analyzed immediately using a FACScalibur (Becton Dickinson). Data were recorded and analyzed using CELLQUEST soft- ware. Calcium fluorimetry A concentration of 2 · 10 6 cells/ml Tyrodes/Hepes buffer (pH 7.3) containing 0.5 m M CaCl 2 was incubated with 1 l M of the calcium reporter dye Fura-2 for 1 h at 37 °C, then washed. Cells were stimulated, with slow stirring, at 37 °Cin a fluorimeter at an excitation wavelength of 530 nm and emission wavelengths of 330 nm and 380 nm. The ratios of Fura-2 emissions were measured and analyzed using phycoerythrin FWINLAB software. Ratiometric analysis was converted into a concentration of Ca 2+ by measuring R max (maximal fluorescence in lysed, labelled cells) and R min (minimum fluorescence in lysed, labelled cells in the presence of 5 m M EGTA). Adhesion assay Ninety-six-well plates were coated with 20 lgÆmL )1 colla- gen, convulxin, CRP, or BSA in NaCl/P i , and incubated overnight at 4 °C, then saturated with 1% BSA for 1 h at room temperature and washed with Tyrodes/Hepes buffer (pH 7.3). Then 2 · 10 5 cells in Tyrodes/Hepes buffer were added per well and allowed to adhere for 1 h at 37 °C. Cells were washed at least three times with Tyrodes/Hepes buffer and fixed with 3% formaldehyde. Assays were performed in triplicate for each condition, and at least 10 different fields of cells per experiment were counted under the microscope. RESULTS GPVI is expressed at the surface independently of FcR c-chain in COS-7 and K562 cells COS-7 cells were transiently transfected with empty plasmid (COS/pRc) or plasmid encoding wild-type human GPVI (COS/F1) or two different mutant constructs of the receptor. Expression of GPVI was detected by flow cytometry using the GPVI-specific ligand convulxin and antibody to convulxin (Fig. 1A). COS/F1 or mutant versions in which the transmembrane arginine of GPVI was mutated to alanine (COS/FA) or the cytoplasmic tail depleted (COS/C1) were expressed on the surface of COS-7 cells with similar efficiency, despite a low percentage of transfection (Fig. 1A). As GPVI is noncovalently and constitutively associated with FcR c-chain in platelets [2], COS-7 cells were also cotransfected with the different constructs of GPVI and FcR c-chain. Cotransfection of the FcR c-chain did not alter the surface expression of the different GPVI constructs, as measured by flow cytometry (Fig. 1A). Relative amounts of the three receptor forms were similar, as detected by ligand blotting in the presence (Fig. 1B) or absence (data not shown) of FcR c-chain. Surface expression of GPVI in the absence of FcR c-chain was further analysed by transient transfection of a chimeric protein (GPVI–GFP) into COS-7 cells. Cellular localization of GPVI–GFP in COS-7 cells was analysed by immuno- histochemistry. Cells transfected with GFP or GPVI–GFP were incubated with convulxin or anti-convulxin and indirectly labelled with an R-phycoerythrin-conjugated anti-rabbit IgG (Fig. 2). GFP localized in the cytoplasm of the cell, whereas GPVI–GFP was detectable at the surface. Moreover, convulxin did not bind to COS-7 cells transfected with GFP, whereas it did bind to cells expressing GPVI–GFP. Magnification of the latter revealed colocali- zation between GPVI–GFP and convulxin, confirming surface expression of GPVI independently of FcR c-chain. These results demonstrate surface expression of GPVI independently of the FcR c-chain, but the possibility remained that FcR c-chain was necessary for stable surface expression of the receptor. To address this, GPVI was stably transfected into the erythroleukaemic cell line K562, which Fig. 1. GPVI is surface-expressed on the membrane independently of the FcR c-chaininCOS-7cells.(A) COS-7 cells were transiently trans- fected with wild-type GPVI (F1), GPVI mutated in the transmembrane arginine to alanine (FA), or depleted of the cytoplasmic tail (C1), together (bottom panels) or not (upper panels) with the FcR c-chain. Control cells were transfected with empty plasmid (pRc). Surface- expressed GPVI was detected by flow cytometry using convulxin or an antibody to convulxin and indirectly labelled with FITC-conjugated anti-rabbit IgG (filled histogram). Background fluorescence was obtained in the absence of convulxin (empty histogram). (B) Whole cell lysates from the above cells were subjected to SDS/PAGE under nonreducing conditions using 10% polyacrylamide gels. GPVI was detected by ligand blotting using convulxin and FcR c-chain by Western blotting using a specific antibody. When 10% polyacrylamide gels were used, a shift in mobility of C1 was observed relative to F1 or FA, according to its lower molecular mass (not shown). Different bands possibly corresponding to differently phosphorylated forms of FcR c-chain are observed in platelets and F1-c cells, but not the others. n ¼ 2. Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2953 lacks detectable expression of FcR c-chain ([16] and Fig. 4). Mock-transfected cells (K562/pRc) did not express GPVI as detected by flow cytometry and ligand blotting (Fig. 3A and Fig. 4, respectively), whereas cells transfected with full- length GPVI (K562/F1) or a cytoplasmic-deleted GPVI (K562/C1) expressed the receptor at the surface (Fig. 3A). When FcR c-chain was cotransfected, there was no major change in surface expression of GPVI (Fig. 3B), showing that the FcR c-chain was not necessary for expression of GPVI in these cells. The level of expression of GPVI and FcR c-chain was increased after treatment of the cells with the phorbol ester PMA (Fig. 3A,B, and Fig. 4). This was an increase in transfected proteins, as endogenous GPVI and FcR c-chain were not detected in mock-transfected cells after PMA treatment, as assessed by flow cytometry (Fig. 3A) and Western or ligand blotting (Fig. 4). It is noteworthy that cells transfected with FcR c-chain only expressed the protein at an undetectable level before treatment with PMA, and to a low level after differentiation with the phorbol ester for 3 days (Fig. 4). However, when GPVI was cotransfected, expression of FcR c-chain was upregulated (Fig. 4), demonstrating that the glycoprotein receptor regulates directly or indirectly the expression of FcR c-chain. It is unclear whether the increase in expression with PMA is an effect of the phorbol ester over the plasmid promoter or whether it is a cellular response to the phorbol ester, although the former seems more likely. Differentiated K562 cells expressing GPVI and the FcR c-chain (K562/F1-c) were analysed for expression of the a2 subunit of the integrin a2b1 and CD36, to determine if collagen receptors other than GPVI were present in these Fig. 3. Surface expression of GPVI in K562 cells. (A) Cells mock- transfected (K562/pRc) or transfected with wild-type GPVI (K562/F1) or a cytoplasmic-tail-deleted mutant (K562/C1) were differentiated with PMA for 1 and 3 days, and expression of GPVI at the surface detected by flow cytometry using convulxin (filled histogram). Back- ground fluorescence was obtained in the absence of convulxin (empty histograms). (B) The above cells were stably cotransfected with the FcR c-chain and GPVI expression at the surface detected as above. Expression of GPVI after 3 days of differentiation is higher than at 1 day. Mock-transfected cells display a low level of GPVI expression, in both the presence (K562/pRc-c) and absence (K562/pRc) of FcR c-chain. Extensive cellular death is detected in the cultures after 3 days of differentiation, and expression of GPVI is not homogeneous within the clonal population of cells (see K562/F1 and K562/F1-c). n ¼ 3. Fig. 2. Convulxin colocalizes at the membrane with the chimeric protein GPVI–GFP in the absence of FcR c-chain. COS-7 cells growing on coverslips were transient transfected with a plasmid encoding either GFP or GPVI–GFP. After 48 h, cells were fixed. GPVI–GFP was visualized by incubation with convulxin or an antibody to convulxin, and indirectly labelled with R-phycoerythin-conjugated anti-rabbit IgG. Fluorescent GFP is directly visualized in the microscope. Upper panel shows cytoplasmic localization of GFP (top left), and absence of binding of convulxin to those cells (top right). The chimeric protein GPVI–GFP localizes to the membrane (bottom left) and is recognized by convulxin (bottom right). Initial magnification · 20. Bottom panels show a magnification of cells transfected with GPVI–GFP and incu- bated with convulxin (right). Arrows indicate spots of colocalization between GPVI–GFP and convulxin. The bright cytoplasmic spot of GFP or GPVI–GFP (upper and low panels, respectively) probably corresponds to the site of synthesis/accumulation of the protein. Bar indicates 30 lm. n ¼ 3. 2954 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002 cells. Flow-cytometry studies showed low levels of expres- sion of both receptors compared with platelets (Fig. 5). FcR c-chain is necessary for GPVI signalling upon convulxin stimulation To investigate whether GPVI was functional, we measured tyrosine phosphorylation of the FcR c-chain and the tyrosine kinase Syk, which is tyrosine phosphorylated and activated early after GPVI engagement. Phosphorylation of FcR c-chain was assessed in K562 cells expressing FcR c-chain along with wild-type GPVI (K562/F1-c) or a mutant receptor lacking the cytoplasmic tail (K562/C1-c). Cells were stimulated with 20 n M convulxin for 90 s, and FcR c-chain precipitated using a fusion protein consisting of GST conjugated with the SH2 domain of Syk, which recognizes the phosphorylated form of FcR c-chain [4]. Tyrosine phosphorylation of the FcR c-chain was observed in cells expressing both wild-type GPVI and FcR c-chain (K562/F1-c), but not in cells expressing only the FcR c-chain (K562/pRc-c) (Fig. 6A). This demonstrates that convulxin phosphorylates the FcR c-chain only when this is coexpressed with GPVI. In contrast, GPVI lacking the cytoplasmic tail was unable to phosphorylate the FcR c-chain under the same conditions (Fig. 6A). Next, we performed a time course analysis of phosphory- lation of Syk in K562 cells expressing GPVI and FcR c-chain (K562/F1-c) after differentiation with PMA. Stimulation of these cells with 2 and 20 n M convulxin led to an increase in tyrosine phosphorylation of Syk (Fig. 6B). The increase was evident after 30 s of stimulation and maximum after 90 s, phosphorylation remaining for up to 270 s. Stimulation with 20 n M convulxin produced slightly stronger phosphorylation than 2 n M after 30 s of stimulation, although longer stimu- lation using both concentrations of convulxin rendered a similar intensity of response (Fig. 6B). Phosphorylation of Syk was also detected in K562/F1-c cells nondifferentiated with PMA, which expressed a lower level of the GPVI–FcR c-chain complex (Fig. 7A). The increase in signal in PMA- treated cells was due to an increase in the number of receptors at the surface of the cells. A similar set of experiments was carried out with K562 cells that had been stably transfected with the cytoplasmic-tail-deleted form of GPVI together with FcR c-chain (K562/C1-c). No increase in phosphorylation of Syk was detected in these cells upon convulxin stimulation (Fig. 7A), demonstrating that deletion of the cytoplasmic tail Fig. 6. Phosphorylation of FcR c-chainandSykinK562cells.(A) K562 cells stably expressing FcR c-chain (pRc/c), or cotransfected with either wild-type GPVI and FcR c-chain (F1/c) or a cytoplasmic-tail- deleted GPVI and FcR c-chain (C1/c) were stimulated for 90 s with 20 n M convulxin. Protein lysates were affinity-precipitated using GST- conjugated SH2 domain of Syk, and precipitated proteins subjected to SDS/PAGE under nonreducing conditions, then blotted to detect tyrosine phosphorylated proteins. Arrows indicate a characteristic doublet representing phosphorylated forms of FcR c-chain. (B) K562 cells stably expressing GPVI and FcR c-chain were stimulated with convulxin at the concentrations and times indicated. Protein lysates were immunoprecipitated using an anti-Syk IgG and subjected to SDS/PAGE, then blotted to detect phosphorylated bands using mAb 4G10. The arrow indicates the position of Syk. The membrane was stripped and reprobed to detect Syk. n ¼ 3. Fig. 5. Expression of a2b1 and CD36 in K562 cells. Cells stably expressing GPVI and the FcR c-chain (K562-F1/c) were differentiated with PMA for 24 h. Expression of the integrin a2b1 and CD36 was detected by flow cytometry using specific antibodies to either receptor (filled histograms). Background fluorescence was obtained with an isotype control antibody (empty histograms). Fig. 4. GPVI and FcR c-chain expression in K562 cells. K562 cells were stably transfected with FcR c-chain together with empty vector (K562/ pRc-c) or coding for wild-type GPVI (K562/F1-c) or a cytoplasmic- tail-depleted GPVI (K562/C1-c). Cells were stimulated with PMA for the times indicated, and protein extracts separated by SDS/PAGE under nonreducing conditions using 12.5% polyacrylamide gels. Membranes were blotted for GPVI or FcR c-chain detection. Arrows indicate the position of GPVI and the FcR c-chain. When 10% polyacrylamide gels were used, a shift in mobility of C1 was observed relative to F1, according to its lower molecular mass (not shown). n ¼ 2. Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2955 renders the receptor unable to signal to the tyrosine kinase. Further, K562 cells stably transfected with only GPVI, and therefore lacking FcR c-chain (K562/F1), did not show a detectable increase in tyrosine phosphorylation of Syk after 20 n M convulxin stimulation for 90 s, even when expressing higher levels of GPVI after PMA treatment (Fig. 7B). Together, our data indicate that, in K562 cells, cotrans- fection of GPVI and the FcR c-chain leads to formation of a receptor complex, which can be activated with the GPVI- specific ligand convulxin, resulting in phosphorylation of the protein kinase Syk, therefore reproducing the proximal events of GPVI signalling in platelets. The FcR c-chain is crucial for the generation of this signal, as cross-linking of GPVI in the absence of FcR c-chain was unable to promote an increase in phosphorylation of Syk. A transmembrane arginine residue and the cytoplasmic tail of GPVI are necessary for its association with FcR c-chain We next performed experiments to determine whether there was a physical association between GPVI and FcR c-chain as described on platelets, and the role of the transmembrane arginine and cytoplasmic tail of GPVI in this association. COS-7 cells were cotransfected with the different GPVI constructs along with FcR c-chain. Affinity precipitation of GPVI, using convulxin and an antibody to convulxin, and subsequent immunoblotting to detect FcR c-chain, showed an association between the FcR c-chain and wild-type GPVI, but not with the other two mutants of GPVI (Fig. 8A). This shows that the transmembrane arginine and the cytoplasmic tail of GPVI are essential for its association with the FcR c-chain. A similar set of experiments was carried out using K562 cells stably expressing either wild-type GPVI or the cyto- plasmic-tail-deleted form, together with FcR c-chain. The cells were stimulated with PMA for 24 h to increase expression of both proteins. As observed in COS-7 cells, only wild-type GPVI was able to bind FcR c-chain (Fig. 8B), confirming a requirement for the cytoplasmic tail in the association with FcR c-chain. The lack of association between the FcR c-chain and a mutant GPVI lacking its cytoplasmic tail explains the failure of the mutant receptor to produce a functional response when stimulated with convulxin, as described above. CRP but not collagen stimulates Syk phosphorylation in GPVI-expressing K562 cells PMA-differentiated K562 cells stably expressing FcR c-chain alone (K562/pRc-c) or together with GPVI (K562/F1-c) were stimulated with collagen and CRP for 90 s and subjected to Syk immunoprecipitation. After transfer, membranes were blotted to detect tyrosine phos- phorylation (Fig. 9). The results were compared with those obtained after convulxin stimulation as above. Convulxin (20 n M ) induced a robust increase in phosphorylation of Syk, whereas 3 lgÆmL )1 CRP induced a weaker increase in phosphorylation. However, collagen was unable to pro- mote an increase in phosphorylation of Syk, even when used at a concentration of 100 lgÆmL )1 , which is 200 times greater than that required for platelet activation. When stimulated with CRP under the same conditions in the absence of GPVI, the cells showed no increase in phos- phorylation of Syk (data not shown). This demonstrates that GPVI expression was sufficient to reproduce the phosphorylation of Syk induced by convulxin and CRP, but not by collagen. GPVI-expressing Jurkat cells bind to collagen, CRP and convulxin, but only convulxin is able to induce calcium release The lack of effect of collagen on GPVI in K562 cells led us to use a second system for expression of GPVI. GPVI was Fig. 7. FcR c-chain and the cytoplasmic tail of GPVI are necessary to initiate the GPVI signalling cascade. (A) K562 cells stably transfected with FcR c-chain and cotransfected with empty vector (pRc/c), wild- type GPVI (F1/c) or a cytoplasmic-tail-depleted GPVI (C1/c) were treated or not with PMA for 24 h, then stimulated with convulxin for 90 s and protein lysates subjected to Syk immunoprecipitation. Immunoprecipitated proteins were separated by SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane. Phosphorylated proteins were detected using mAb 4G10. The arrow indicates the position of Syk. An unidentified phosphorylated band of  100 kDa coprecipitated with phosphorylated Syk. (B) Cells transfected with the different mutant versions of GPVI but lacking expression of FcR c-chain were treated with PMA for 24 h, stimulated with 20 n M con- vulxin for 90 s, and protein lysates immunoprecipitated and subjected to SDS/PAGE as above. Membranes were incubated with mAb 4G10 to detect tyrosine-phosphorylated bands. A sample stimulated with IV.3 antibody to cross-link FccRIIA shows phosphorylation of Syk. However, in the absence of FcR c-chain, cross-linking of GPVI is unable to promote phosphorylation of Syk, although some basal phosphorylation of the tyrosine kinase can be detected. The arrow indicates the position of Syk. n ¼ 2. 2956 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002 stably transfected into the human T-cell line Jurkat. Mock- transfected cells (Jurkat/pRc) do not express GPVI, as detected by flow cytometry, whereas cells transfected with full-length GPVI (Jurkat/F1) express the receptor at the surface (Fig. 10A). Expression of GPVI leads to adhesion of Jurkat cells to a monolayer of collagen, convulxin, or CRP, but not to BSA (Fig. 10B). Convulxin stimulated a similar pattern of tyrosine phosphorylation of proteins in GPVI- expressing Jurkat cells to that induced by cross-linking of the T cell antigen receptor, despite the absence of the c-chain. A possible explanation for this is provided by the observation that the CD3 chain of the T-cell receptor complex is phosphorylated in response to convulxin (data not shown), suggesting that it may associate with GPVI in the absence of the FcR c-chain. Convulxin also induced an increase in calcium in GPVI-expressing Jurkat cells (Fig. 10D). In contrast, however, neither collagen nor CRP stimulated tyrosine phosphorylation or calcium mobilization in the GPVI-transfected Jurkat cells (Fig. 10C,D). These results show that, although expression of GPVI in Jurkat cells supports adhesion to convulxin, collagen and CRP, only the snake venom is able to stimulate the signalling pathway leading to activation. DISCUSSION GPVI–FcR c-chain is unique compared with other FcR c-chain complexes, as it has been reported to act as a collagen receptor with no apparent function in the immune response, despite sharing similar structure and signalling features with other FcRs [1,3,17]. The similarity between GPVI and different FcRs provides a starting point in the study of the structure–function relationship of GPVI and the signalling pathways. Many FcRs and their associated b and c-chains bind to each other through the transmembrane domain resulting in a receptor complex that is able to signal inside the cell. The nature of this binding depends on oppositely charged amino-acid residues within the trans- membrane domain of each subunit. Point mutations of these amino acids have revealed the importance of the arginine residue in the transmembrane domain of the immunoglo- bulin receptor. However, the cytoplasmic domain of these FcRs is not essential for binding to the FcR c-chain. Our results show that GPVI depends on both the transmem- brane arginine and at least part of the cytoplasmic tail for its association with the FcR c-chain, although it is not clear how the cytoplasmic tail supports the association. The molecular basis of this interaction is still being studied. We have shown by transient and stable transfections of GPVI into COS-7 and K562 cells, respectively, that GPVI is expressed at the cell surface independently of the FcR c-chain. This is a property that has previously been described for the GPVI-related receptors PIR-A and FcaR [5,6] and for the FcR c-chain partner FccRI [18] when transfected into a number of cell lines. However, experi- ments using transgenic and knock-out mice showed that in vivo,theFcRc-chain was necessary for stable surface Fig. 8. Transmembrane arginine and cytoplasmic tail of GPVI are necessary for its association with FcR c-chain. (A) COS-7 cells were transiently cotransfected with wild-type or different mutant versions of GPVI. GPVI was affinity-precipitated using convulxin, and samples were subjected to SDS/PAGE under nonreducing conditions. The top part of the membrane was blotted for GPVI detection using convulxin, and the bottom part was blotted for FcR c-chain detection. The positions of GPVI (55 kDa) and other bands of lower molecular mass, probably products of degradation, are indicated by arrows. A platelet sample is included as a positive control. (B) K562 cells stably trans- fected with FcR c-chain alone (pRc/c) or cotransfected with wild-type GPVI (F1/c) or a cytoplasmic-tail-depleted GPVI (C1/c) were stimu- lated or not with PMA for 24 h. GPVI was affinity-precipitated using convulxin and detected by ligand blotting as a band of  55 kDa. Additional bands of lower molecular mass are indicated. The associ- ated FcR c-chain was detected by Western blotting using a specific antibody. A sample of F1/c cells stimulated with PMA was used for precipitation in the absence of convulxin as a control. A platelet sample is included as a positive control. Arrows indicate the position of GPVI and the FcR c-chain. n ¼ 3. Fig. 9. CRP but not collagen induces Syk phosphorylation in K562 cells. Cells stably expressing wild-type GPVI and the FcR c-chain and dif- ferentiated with PMA for 24 h were stimulated with the indicated concentrations of collagen and CRP for 90 s, and Syk immunopre- cipitated. Immunocomplexes were separated by SDS/PAGE and transferred to poly(vinylidene difluoride) membranes, then blotted with mAb 4G10. The arrow indicates the position of phosphorylated Syk. The membrane was stripped and reblotted to detect Syk. Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2957 expression of FcaRandFccRI [18,19] possibly through prevention of their degradation [20]. Consistent with this, mice depleted of the FcR c-chain did not express detectable GPVI [21], raising the possibility that in vivo, unlike in cell lines, GPVI may be degraded in the absence of the FcR c-chain. This may reflect a specific pathway of degradation that prevents expression of functionally uncoupled receptors in certain cell types. The cotransfection of FcR c-chain with GPVI did not increase the level of surface expression of the latter relative to transfection of GPVI alone, either on transient or stable transfections. The same observation has been reported on transient transfections of GPVI into COS-7 cells [22]. This is different from the related PIR-A and Fca-receptor [5,9] and suggests that, in cell lines, the FcR c-chain is acting as a signalling and stabilizing subunit for some receptors, such as FcaR, but only as a signalling subunit for others, such as GPVI. Conversely, GPVI is required for stable expression of the FcR c-chain in the absence of other binding partners. It is well known that the FcR c-chain is essential for signalling by receptors with which it associates [5,9,19]. Mice genetically engineered to lack the FcR c-chain do not express detectable GPVI [21], making it impossible to determine whether GPVI signals in the absence of FcR c-chain in vivo. K562 cells do not express detectable levels of endogenous GPVI or FcR c-chain, even after differentiation with PMA, making them a suitable system for studying the signalling events triggered by transfected GPVI. Convulxin stimulated robust tyrosine phosphorylation of Syk in K562 cells transfected with GPVI/FcR c-chain but not in mock- transfected K562 cells. The increase in phosphorylation of Syk was not accompanied by an increase in phosphoryla- tion of PLCc2 and other downstream proteins, possibly because of the absence of the adapter LAT (O. Berlanga & S.P. Watson, unpublished), which has been shown to play a critical role in GPVI signalling [27]. These studies also show that, although GPVI required the FcR c-chain to generate a signal to convulxin, the latter is not required for recognition of the receptor by the snake venom. This illustrates the dual mechanism of the complex, with one subunit, namely GPVI, responsible for binding to the ligand, and the other subunit, the FcR c-chain, responsible for transmission of the signal within the cell. Studies of GPVI-deficient human [23] and murine platelets [21] have shown that the glycoprotein is essential for platelet activation by collagen. Consistent with this, transient expression of GPVI was demonstrated to confer some calcium mobilization on collagen in the DAMI Fig. 10. GPVI-expressing Jurkat cells bind to collagen, CRP and convulxin, but only convul- xin is able to induce calcium release. (A) In mock-transfected cells (Jurkat/pRc) and GPVI-transfected cells (Jurkat/F1), surface expression of GPVI was detected by flow cytometry using convulxin (filled histogram). Background fluorescence was obtained in the absence of convulxin (empty histograms). (B) Mock-transfected and GPVI-transfected cells (2 · 10 5 ) were incubated for 30 min at 37 °Cina96-wellplatecoatedwithBSA, convulxin, collagen or CRP. After extensive washing and fixation, adherent cells were counted (0.4 mm square). Triplicate samples were counted for each condition and standard deviations are shown. (C) Jurkat cells stably expressing GPVI were stimulated for 90 s with 10 lgÆmL )1 convulxin (Cvx), 50 lgÆmL )1 CRP, or 100 lgÆmL )1 collagen (Coll.). Whole cell protein lysates were subjected to SDS/ PAGE,thenblottedtodetecttyrosine-phos- phorylated proteins using mAb 4G10. (D) Fura-2-loaded mock-transfected cells (pRc) and GPVI-transfected cells (F1) were stimu- lated with 10 lgÆmL )1 convulxin, 50 lgÆmL )1 CRP and 100 lgÆmL )1 collageninaspectro- fluorimeter cuvette. Dual excitation at 340/ 380 nm and emission recorded at 510 nm [Ca 2+ ] were calculated as described in Mate- rials and methods. 2958 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002 megakaryocytic cell line [1]. On the other hand, control cells were unresponsive to collagen despite expression of endo- genous GPVI, the integrin a2b1 [24], and probably other collagen receptors. Interestingly, collagen also has no effect on a number of other megakaryocytic cell lines despite expression of a low level of GPVI sufficient to support activation by convulxin [12]. These observations are similar to those on transfected GPVI in RBL-2H3 cells, a rat basophilic leukaemia cell line that expresses FcR c-chain but not GPVI. Transfected RBL cells show an intracellular signal when stimulated with convulxin and a small response toCRPwhenusedataconcentration500timesinexcessof that required to activate platelets, whereas collagen is inactive [25]. They also correspond to those of the present study in that collagen was inactive with GPVI-transfected K562 and Jurkat cells, with high concentrations of CRP inducing a weak response only in K562 cells. The absence of activation does not appear to be due to the type of collagen used in this study as the same observation was made with bovine collagen types I–V (D. Tulasne & Jarvis, unpub- lished). Further, expression of GPVI in Jurkat cells conferred adhesion of collagen and CRP in the absence of activation. It therefore appears that expression of a low level of GPVI is sufficient to support binding to collagen but not detectable activation. There are several possible explanations for the lack of response to collagen on cell lines transfected with GPVI. One is the absence of a second receptor such as the integrin a2b1. This seems unlikely, however, because collagen is also inactive on a number of megakaryocytic cell lines that express a low level of GPVI together with other receptors for collagen [28]. A second explanation is that GPVI expression may fail to reach the critical level for activation by collagen. A third possibility is that the interaction of collagen with GPVI generates a signal that falls below the detection limits of the assays used in this system. Collagen is a far weaker stimulus than convulxin in platelets, its response being heavily dependent on the liberation of ADP and thromboxanes for activation [29]. However, collagen still stimulates calcium increases in single platelets and induces tyrosine phosphorylation in the presence of ADP and thromboxane receptor antagonists [30] (B. T. Atkinson & S. P. Watson, unpublished). The most likely explanation for these observations is that a certain receptor density is required for functional responses to collagen, whereas adhesion can be supported by lower receptor densities. Direct evidence to support this is provided by a recently published study [31]. In conclusion, we have demonstrated that reconstitution of GPVI with the FcR c-chain restores its responses to convulxin and CRP, but that GPVI is unable to signal in the absence of a protein bearing the immune receptor tyrosine-based activation motif. In addition, we have shown that collagen binds to transfected GPVI but is unable to induce its activation in cells expressing a low level of receptors. ACKNOWLEDGEMENTS We thank Drs Mireille Leduc and Cassian Bon for the gift of convulxin. This work was supported by the British Heart Foundation. S.P.W. is a British Heart Foundation Senior Research Fellow. J. F. is a Wellcome Trust Senior Research Fellow. REFERENCES 1. Clemetson, J.M., Polgar, J., Magnenat, E., Wells, T.N.C. & Clemetson, K.J. (1999) The platelet receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcaR and the natural killer receptor. J. Biol. Chem. 274, 29019– 29024. 2. Tsuji, M., Ezumi, Y., Arai, M. & Takayama, H. (1997) A novel association of Fc receptor c-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J. Biol. Chem. 272, 23528–23531. 3. Gibbins, J.M., Okuma, M., Farndale, R., Barnes, M. & Watson, S.P. (1997) Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor c-chain. FEBS Lett. 413, 255–259. 4. Poole, A., Gibbins, J.M., Turner, M., Van Gut, M.J., Dan de Winkel, J.G.J., Saito, T., Tybulewicz, V.L.J. & Watson, S.P. (1997) The Fc receptor c-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 16, 2333–2341. 5. Craig-Morton, H., van den Herik-Oudijk, I.E., Vossebeld, P., Snijders, A., Verhoeven, A.J., Capel, P.J.A. & van de Winkel, J.G.J. (1995) Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR c-chain. J. Biol. Chem. 270, 29781–29787. 6.Ono,M.,Yuasa,T.,Ra,C.&Takai,T.(1999)Stimulatory function of paired immunoglobulin-like receptor-A in mast cell line by associating with subunits common to Fc receptors. J. Biol. Chem. 274, 30288–30296. 7. Hayami, K., Fukuta, D., Nishikawa, Y., Yamashita, Y., Inui, M., Ohyama, Y., Hikida, M., Ohmori, H. & Takai, M. (1997) Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors. J. Biol. Chem. 272, 7320–7327. 8. Taylor, L.S. & McVicar, D.W. (1999) Functional association of FceRIc with arginine 632 of paired immunoglobulin-like receptor (PIR)-A3 in murine macrophages. Blood 94, 1790–1796. 9. Maeda, A., Kurosaki, M. & Kurosaki, T. (1998) Paired immunoglobulin-like receptor (PIR)-A is involved in activating mast cells through its association with Fc receptor c-chain. J. Exp. Med. 188, 991–995. 10. Morton, L.F., Peachey, A.R. & Barnes, M.J. (1989) Platelet- reactive sites in collagens type I and III. Evidence for separate adhesion and aggregatory sites. Biochem. J. 258, 157–163. 11. Santoro, S.A., Walsh, J.J., Staatz, W.D. & Baranski, K.J. (1991) Distinct determinants on collagen support alpha 2 beta 1 integrin- mediated platelet adhesion and platelet activation. Cell Regul. 2, 905–913. 12. Watson, S.P., Berlanga, O., Best, D. & Frampton, J. (2000) Update on collagen receptor interactions in platelets: is the two- state model still valid? Platelets 11, 252–258. 13. Kehrel, B., Wierwille, S., Clemetson, K.J., Anders, O., Steiner, M., Knight, C.G., Farndale, R.W., Okuma, M. & Barnes, M.J. (1998) Glycoprotein VI is a major collagen receptor for platelet activa- tion: it recognizes the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood 91, 491–499. 14. Berlanga, O., Bobe, R., Becker, M., Murphy, G., Leduc, M., Bon, C., Barry, F.A., Gibbins, J.M., Garcia, P., Frampton, J. & Watson, S.P. (2000) Expression of the collagen receptor gly- coprotein VI during megakaryocyte differentiation. Blood 96, 2740–2745. 15. Yanaga, F., Poole, A., Asselin, J., Blake, R., Schieven, G., Clark, E.A., Law, C L. & Watson, S.P. (1995) Syk interacts with tyrosine phosphorylated proteins in human platelets activated by collagen and cross-linking of the FccRIIA receptor. Biochem. J. 311, 471–478. Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2959 16. Ernst, L.K., Duchemin, A M. & Anderson, C.L. (1993) Asso- ciation of the high-affinity receptor for IgG (FccRI) with the c subunit of the IgE receptor. Proc. Natl. Acad. Sci. USA 90, 6023–6027. 17. Jandrot-Perrus, M., Busfield, S., Lagrue, A.H., Xiong, X., Debili, N., Chickering, T., Le Couedic, J.P., Goodearl, A., Dussault, B., Fraser, C., Vainchenker, W. & Villeval, J.L. (2000) Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily. Blood 96, 1798–1807. 18. Takai, T., Li, M., Sylvestre, D., Clynes, R. & Ravetch, J.V. (1994) FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 76, 519–529. 19. Van Egmond, M., van Vuuren, A.J., Morton, H.C., van Spriel, A.B., Shen, L., Hofhuis, F.M., Saito, T., Mayadas, T.N., Verbeek, J.S. & van de Winkel, J.G. (1999) Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18). Blood 93, 4387–4394. 20. Kurosaki, T., Gander, I. & Ravetch, J.V. (1991) A subunit com- mon to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci. USA 88, 3837–3841. 21. Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner,J.E.&Zirngibl,H.(2000)Expressionandfunctionofthe mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain. J. Biol. Chem. 275, 23998–24002. 22. Ezumi, Y., Uchiyama, T. & Takayama, H. (2000) Molecular cloning, genomic structure, chromosomal localization, and alternative splicing forms of the platelet collagen receptor glycoprotein VI. Biochem. Biochem. Biophys. Res. Commun. 277, 27–36. 23. Moroi, M., Jung, S.M., Okuma, M. & Shinmyozu, K. (1989) A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion. J. Clin. Invest. 84, 1440–1445. 24. Strouse, R.J. & Daniel, J.L. (1996) The Dami cell possesses the platelet collagen receptor VLA-2, but does not mobilise calcium. Thromb. Res. 82, 485–493. 25. Zheng, Y.M., Changdong, L., Chen, H., Locke, D., Ryan, J.C. & Kahn, M.L. (2001) Expression of the platelet receptor GPVI confers signaling via the Fc receptor c-chain in response to the snake venom convulxin but not collagen. J. Biol. Chem. 276, 12999–13006. 26. Lagrue-Lak-Hal, A.H., Debili, N., Kingbury, G., Lecut, C., Le Couedic, P., Villeval, J.L., Jandrot-Perrus, M. & Vainchenker, W. (2001) Expression and function of the collagen receptor GPVI during megakaryocyte differentiation. Blood 276, 15316–15325. 27. Pasquet, J M., Gross, B., Quek, L., Asazuma, N., Zhang, W., Sommers, C.L., Schweighoffer, E., Tybulewicz, V., Judd, B., Lee, J.R., Koretzky, G., Love, P.E., Samelson, L.E. & Watson, S.P. (1999) LAT is required for tyrosine phosphorylation of phos- pholipase cgamma2 and platelet activation by the collagen receptor GPVI. Mol. Cell Biol. 19, 8326–8334. 28. Mountford, J.C., Melford, S.K., Bunce, C.M., Gibbins, J. & Watson, S.P. (1999) Collagen or collagen-related peptide cause (Ca 2+ ) i elevation and increased tyrosine phosphorylation in human megakaryocytes. Thromb. Haemost. 82, 1153–1159. 29. Atkinson, B.T., Stafford, M.J., Pears, C.J. & Watson, S.P. (2001) Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin. Eur. J. Biochem. 268, 5242– 5248. 30. Poole, A.W. & Watson, S.P. (1995) Regulation of cytosolic calcium by collagen in single human platelets. Br.J.Pharmacol. 115, 101–106. 31. Chen, H., Locke, D., Liu, Y., Liu, C. & Kahn, M.L. (2002) The platelet receptor GPVI mediates both adhesion and signaling responses to collagen in a receptor density-dependent fashion. J. Biol. Chem. 277, 3011–3019. 2960 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002 . The Fc receptor c-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin Oscar. reconstitution of the GPVI–FcR c-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and