Expression of the Platelet Receptor GPVI Confers Signaling via the Fc Receptor gamma -Chain in Response to the Snake Venom Convulxin but Not to Collagen*

Yun-Min ZhengDagger §, Changdong LiuDagger §, Hong ChenDagger , Darren LockeDagger , James C. Ryan, and Mark L. KahnDagger ||

From the Dagger  Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the  Department of Medicine, University of California at San Francisco and the Veterans Administration Medical Center, San Francisco, California 94121

Received for publication, October 13, 2000, and in revised form, December 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fcepsilon receptor gamma -chain (Fc Rgamma ), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagen-related peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc Rgamma partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc Rgamma and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the alpha 2beta 1 integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Platelet activation is essential for both normal hemostasis and arterial thrombosis that occurs in the setting of vascular diseases such as stroke and myocardial infarction. One of the earliest steps in arterial thrombosis is the adhesion of circulating platelets to areas of injured vessel wall and the activation of adherent platelets, which recruits additional platelets to form a hemostatic plug. Activation of platelets at sites of vascular injury occurs in response to exposed subendothelial matrix proteins, the most important of which is collagen.

Exposed collagen initiates two essential platelet functions: the adhesion of circulating platelets to the site of injury and the activation of platelet signaling, which stimulates thrombus growth. Platelet adhesion to collagen has been shown to occur both indirectly, via interaction of platelet GPIb with plasma von Willebrand's factor bound to exposed collagen (1), and directly, via collagen interaction with the platelet integrin alpha 2beta 1 (2). In contrast, although the activation of platelets by collagen has been observed for over 30 years (3), the receptors and signaling pathways that mediate platelet activation by collagen are only beginning to be fully understood. Indirect evidence suggests that both alpha 2beta 1 and GPIb can initiate signaling when bound to collagen (1, 4, 5). However, this signaling does not appear to be sufficient to account for the magnitude of the platelet response to collagen.

GPVI is a recently cloned 62-kDa surface protein (6, 7) first identified by iodination of platelet surface glycoproteins (8). GPVI was proposed as a signaling receptor for collagen following the description of individuals with bleeding disorders whose platelets could not be activated by collagen and lacked GPVI despite having normal levels of the platelet integrin alpha 2beta 1 (9, 10). Significant evidence suggests that GPVI signaling is sufficient to activate platelets and that GPVI may mediate collagen signaling in platelets. Platelets are activated by cross-linked anti-GPVI antibodies (11) and by convulxin (CVX),1 a multimeric snake venom protein isolated from a South American rattlesnake which is capable of desensitizing platelets specifically to collagen and which binds specifically to GPVI (12, 13). In addition, collagen signaling and GPVI signaling in platelets both employ the immunoreceptor signaling pathway (14) and require Fc Rgamma (15). These data have led to a model of collagen activation of platelets in which adhesive roles are played by the integrin alpha 2beta 1 as well as GPIb and signaling roles are played by GPVI-Fc Rgamma , alpha 2beta 1, as well as perhaps GPIb (14). This model has not been adequately tested, however, because of the absence of systems in which the contribution of each receptor can be studied in isolation.

The recent cloning of human and mouse GPVI (6, 7) reveals that GPVI is a type I transmembrane protein whose deduced amino acid sequence identifies it as an Ig domain-containing receptor homologous to the Fc and killer Ig-like receptors, some of which are known to signal via Fc Rgamma (16). Consistent with its putative role as an Fc Rgamma partner, GPVI has a charged arginine residue in its transmembrane domain that may mediate interaction with the Fc Rgamma transmembrane domain in a manner analogous to that of the known Fc Rgamma partners Fcalpha RI and PIR-A (16-18). Direct functional evidence demonstrating that GPVI is a collagen receptor and that GPVI signaling is mediated by Fc Rgamma , however, are lacking. Transient expression of GPVI has been demonstrated to confer a slight calcium signal to collagen in the DAMI megakaryocytic cell line (6), but these cells express endogenous GPVI (data not shown and Ref. 6), alpha 2beta 1 (19), and perhaps other collagen receptors; it is therefore not clear if that response is mediated directly by GPVI or if GPVI expression is sufficient to enhance signaling by alpha 2beta 1 or other identified (e.g. p65 (20)) or unidentified collagen receptors.

To address the functional role of GPVI, we have stably expressed the receptor in RBL-2H3 cells, a rat basophilic leukemia cell line that expresses Fc Rgamma and reproduces the platelet collagen responses of intracellular calcium mobilization and degranulation but does not express endogenous GPVI or alpha 2beta 1. Our studies reveal that GPVI cross-linking by the GPVI-specific ligand convulxin initiates intracellular signaling but that GPVI alone is incapable of mediating a signaling response to collagen. A small signaling response is elicited by collagen-related peptides (CRPs), however, and static adhesion studies support interaction with convulxin and CRP but not collagen. Finally, site-directed mutagenesis of the GPVI transmembrane domain and intracellular C-tail demonstrates that both the GPVI transmembrane arginine and the receptor C-tail are necessary for Fc Rgamma interaction and intracellular signaling. These results provide insight into GPVI signal transduction and suggest that GPVI-signaling in response to collagen requires coreceptors for both ligand binding and intracellular signal transduction.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Type I collagen derived from equine tendons was obtained from Chronolog (Havertown, PA) and used for all studies shown. Studies were confirmed with type I collagen derived from bovine tendon and type III collagen derived from calf skin (Sigma). Convulxin was obtained from Sigma and purified from the venom of the Crotalus durissus rattlesnake using gel filtration as previously described (13). CRPs were synthesized as previously described using cross-linked cysteine residues (21). All other reagents were obtained from Sigma.

Cloning and Epitope Tagging of GPVI-- A GPVI cDNA was generated by PCR from human platelet cDNA using primers based on published 5'- and 3'-untranslated sequences (sense strand primer: 5'- TCAGGACAGGGCTGAGGAACC-3'; antisense strand primer: 5'-TTGGATACGACCGTGCCTGGG-3'). Three distinct amplified 1.1-kilobase pair products were sequenced to obtain a consensus sequence that exhibited several differences from the published cDNA (6) but agreed with a cDNA sequence deposited directly in GenBankTM (accession number AB035073). All GPVI receptor amino acids reported here correspond to the protein predicted by the open reading frame of this cDNA starting at nucleotide number 13. FLAG-tagged GPVI was generated by replacing the endogenous signal peptide with that of interleukin-1 and placing the FLAG epitope (DYKDDDDK) in frame with GPVI at amino acid number 21, the predicted site of signal peptide cleavage (SignalP VI.I). HA-tagged GPVI was generated in an identical manner. Wild-type and epitope-tagged GPVI were expressed using the mammalian expression vector pcDNA3.0 (Invitrogen).

Site-directed Mutagenesis of GPVI-- Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). The oligonucleotide used for the R272L mutation was 5'- GCAACCTGGTCCGGATATGCCTCGGGGCTGTG-3'. The oligonucleotide used for the R295STOP mutation was 5'-GGCAGAGGACTGGCACAGCTAGAGGAAGCGCCTGC-3'. All mutants were made as epitope-tagged receptors as described above.

Platelet Aggregation Studies-- Blood was collected into citrate buffer and platelet-rich plasma obtained as previously described (22). All studies were performed using platelet-rich plasma at a platelet density of 2 × 108 platelets/ml.

Creation and Screening of RBL-2H3 Cells Stably Expressing GPVI-- RBL-2H3 cells (ATCC, Manassas, VA) were electroporated with linearized expression plasmids and selected in G418 (1.0 mg/ml active concentration; Life Technologies, Inc.) as previously described (23). Wild-type GPVI-expressing clones were directly tested for signaling in response to convulxin (below) and epitope-tagged clones tested for receptor expression by flow cytometry using M2 anti-FLAG antibody (Sigma) or anti-HA antibody (Sigma) as primary antibodies and fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody.

Intracellular Calcium Studies-- Increases in cytoplasmic calcium in response to convulxin, CRP, collagen, and thrombin were measured using the calcium-sensitive dye Fura-2 as previously described at a final cell concentration of 2 × 106 cells/ml (24). The buffer used for these studies was RPMI 1640 (Life Technologies) with HEPES 25 mM and 1 mg/ml BSA.

Functional Rescue of Surface FLAG-Fc Rgamma in HEK-293T Cells to Measure GPVI-Fc Rgamma Interaction-- A stable HEK-293T cell line that stably expressed an N-terminally FLAG-tagged Fc Rgamma in a manner identical to that previously described for DAP-12 (25) was a kind gift of Steve Spusta (University of California at San Francisco). These cells expressed FLAG-Fc Rgamma at a level such that no cell surface FLAG-Fc Rgamma was detectable by fluorescence-activated cell sorting despite readily detectable intracellular FLAG-Fc Rgamma by Western blot analysis (data not shown). HA-tagged wild-type and mutated GPVI receptors were transiently expressed in FLAG-Fc Rgamma -expressing HEK-293T cells (Fugene6 transfection reagent; Roche Molecular Biochemicals). Surface expression of GPVI was followed with anti-HA antibody, and surface expression of Fc Rgamma was followed with anti-FLAG antibody using flow cytometry as previously described for DAP-12 (25). Rescue of surface expression of FLAG-Fc Rgamma was quantitated by comparing surface FLAG expression in GPVI-transfected versus mock-transfected 293T-FLAG-Fc Rgamma cells as previously described (25).

Biochemical Precipitation of GPVI and GPVI Mutants-- CVX was coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech) at a concentration of 200 nM per ml of swollen gel. RBL-2H3 cells (total of ~1 × 107) were lysed for 2 h at 4 °C in ice-cold lysis buffer (1% (w/v) digitonin (Calbiochem), 0.12% (v/v) Triton X-100, 150 mM NaCl, 0.01% (w/v) NaN3, 20 mM triethanolamine, pH 7.8, and containing a 1:100 (v/v) dilution of a mammalian protease inhibitor mixture (Sigma)). Detergent-insoluble cellular debris was pelleted at 10,000 × gav for 15 min, and 50 µl of CVX-Sepharose beads were used to immunoprecipitate GPVI from the supernatant in a 2-h incubation period. Sepharose beads that had been activated by 1 mM HCl and blocked in 0.1 M Tris-HCl, pH 8.0, containing 0.5 M sodium chloride were used as controls for nonspecific binding. Beads were pelleted by centrifugation and washed three times in ice-cold washing buffer (50 mM Tris, 150 mM NaCl, pH 8.0, 5 mM CHAPS, containing 1:100 (v/v) protease inhibitors). Finally, beads were heated to 100 °C in an equal volume of 2× Laemmli sample buffer (1 M Tris-HCl, pH 6.8, 0.2 M DTT, 4% (w/v) SDS, 0.004% bromphenol blue, 20% glycerol), and an aliquot was run on 5-20% (v/v) gradient SDS-polyacrylamide gels using a standard electrophoresis buffer (25 mM Tris-HCl, 0.25 M glycine, and 0.1% (w/v) SDS). Gels were transferred (50 mM Tris-HCl, 40 mM glycine, 0.037% (w/v) SDS, 20% (v/v) methanol) to Hybond-P nitrocellulose membrane (Amersham Pharmacia Biotech), blocked overnight in blocking buffer (0.1 M Tris-buffered saline, pH 7.4, containing 5% (w/v) nonfat milk, 5% (w/v) BSA, and 0.02% (v/v) Tween 20), and probed with antibodies to the FLAG epitope (Bio-M2 (Sigma)) and the Fc Rgamma (Upstate Biotechnology, Inc., Lake Placid, NY) diluted 1:3000 (v/v) and 1:1500 (v/v), respectively, in blocking buffer. These antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Sigma) diluted 1:10,000 (v/v) in 0.1 M Tris-buffered saline, pH 7.4, and membranes were developed using the ECL method (ECL-Plus; Amersham Pharmacia Biotech).

Measurement of RBL Cell Adhesion-- 96-Well polystyrene high binding plates (Costar 3590; Corning Glass) were coated with 50 µl/well of 20 µg/ml BSA, convulxin, collagen, and fibronectin or 15 µg/ml CRP in PBS with 0.9 mM calcium and 0.4 mM magnesium overnight at 4 °C. Prior to application, the pH of the diluted protein solution was adjusted to pH 7-8. The plates were blocked with 150 µl of 10 mg/ml BSA for 2 h at room temperature and washed with PBS with 0.9 mM calcium and 0.4 mM magnesium twice. RBL-2H3 cells were detached from plates with 5 mM EDTA, washed with PBS or saline solution containing 3 mM Ca2+ and 1.5 mM Mg2+, and suspended in the same solution at a cell concentration of 2 × 106/ml. 2 × 105 cells were applied to each well. After a 1-h incubation at room temperature, the plates were washed with the same solution seven times. To read the number of cells bound to each well, the beta -hexosaminidase activity assay was used as previously described (26). Briefly, 20 µl of 0.5% Triton X-100 in PBS were added to each well to lyse the bound cells. 80 µl of 1 mM substrate (p-nitrophenol N-acetyl-beta -D-glucosaminide; Sigma catalog no. N9376) in 0.05 M citrate buffer, pH 4.5, were subsequently added. After 1 h of incubation at 37 °C, 100 µl of 0.1 M sodium carbonate, 0.1 M sodium bicarbonate were added per well. The A405 was measured with an Emax precision microplate reader (Molecular Devices, Inc., Sunnyvale, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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GPVI-expressing RBL-2H3 Cells Signal in Response to Convulxin and CRP but Not to Collagen-- RBL-2H3 cells stably expressing wild-type GPVI (GPVI-RBL) and FLAG-tagged GPVI (FLAG GPVI-RBL) were identified using adhesion to convulxin and flow cytometry to detect surface FLAG epitope, respectively. The ability of the putative GPVI ligands collagen, CRP, and convulxin to initiate calcium signaling in RBL, GPVI-RBL, and FLAG GPVI-RBL was tested in parallel with aggregation studies of human platelets performed using the same reagents on the same day (Fig. 1, Table I, and data not shown). GPVI-RBL and FLAG GPVI-RBL, but not untransfected RBL cells, responded to convulxin with a threshold concentration only 1.6-fold greater than that found necessary to aggregate human platelets (0.3 nM). These results demonstrate functional GPVI signaling and close concordance between the two assays for GPVI dose response, suggesting similar GPVI receptor density in the two cell types. In contrast, collagen elicited no response even at concentrations 500 times greater than that necessary to aggregate human platelets (100 µg/ml). Similar results were obtained using three distinct GPVI-RBL clones and three distinct FLAG-GPVI RBL clones. The data shown are representative of experiments performed in RPMI with Chronolog Type I collagen, which elicits the most robust platelet responses in our hands (data not shown). In addition, no signaling responses were observed using a second source of type I collagen, type III collagen, or in the presence of higher cation concentrations (1 mM calcium and 0.5 mM magnesium; data not shown). Interestingly, CRP was capable of eliciting a small calcium response but required a concentration 500 times greater than that necessary to induce platelet aggregation (50 µg/ml). CRP signaling responses were also only seen with the two GPVI-expressing RBL-2H3 cell clones that had the greatest sensitivity to convulxin. These data demonstrate that GPVI is a functional receptor but that GPVI expression alone is insufficient to reproduce the collagen and CRP signaling responses observed in platelets.


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Fig. 1.   GPVI signaling in RBL-2H3 cells. Wild type (a) and RBL cells stably expressing GPVI (b-e) were exposed to collagen (100 µg/ml), convulxin (1 nM), CRP (50 µg/ml), or thrombin (10 nM), and calcium signaling was measured using the calcium-sensitive dye Fura-2. a, wild-type RBL cells have no response to convulxin or CRP but signal in response to thrombin. b, RBL cells expressing GPVI signal in response to the GPVI ligand CVX. c, GPVI-expressing RBL cells do not respond to collagen. d and e, GPVI-expressing RBL cells signal weakly in response to CRP. Results are representative of eight experiments performed with three distinct GPVI-expressing lines of RBL cells.

                              
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Table I
Comparison of signaling by putative GPVI ligands in GPVI-expressing RBL cells and in platelets
The threshold concentrations of the platelet agonists convulxin, CRP, and collagen necessary to induce platelet aggregation were determined using platelet aggregometry and compared with those necessary for detection of calcium signaling in GPVI-expressing RBL cells using fluorimetry. Note the close concordance between the two assays for the GPVI-specific ligand convulxin and the significant discrepancy for the putative GPVI ligands CRP and collagen.

Adhesion of RBL and GPVI-RBL to the Putative GPVI Ligands Convulxin, Collagen, and CRP-- To determine whether expression of GPVI is sufficient to mediate adhesion but not signaling to collagen, static adhesion assays were performed with wild-type and GPVI-expressing RBL cells (GPVI RBL and FLAG-GPVI RBL generated identical results; data not shown). Expression of GPVI conferred strong adhesion to convulxin and moderate adhesion to CRP, but no adhesion to collagen was detected (Fig. 2). GPVI R272L- and GPVI R295STOP-expressing RBL clones, which express ~5-fold and 10-50-fold more surface GPVI than FLAG-GPVI, respectively (Fig. 3), also failed to bind collagen despite binding both CRP and CVX. Performing the assay using normal saline with 1 mM calcium and 0.5 mM magnesium yielded identical results (data not shown). In addition, no adhesion of GPVI-expressing or wild-type RBL cells was observed to bovine type I collagen or to bovine type III collagen (data not shown). Wild-type RBL cells adhered only to fibronectin (Fig. 2). Unlike fibronectin binding, GPVI-mediated adhesion to CRP and convulxin was not disrupted by 5 mM EDTA (Fig. 2B), consistent with a nonintegrin-mediated mechanism of adhesion. Thus, adhesion assays of GPVI-expressing RBL cells are consistent with signaling assays and show strong GPVI interaction with convulxin, weaker GPVI interaction with CRP, and no GPVI interaction with collagen.


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Fig. 2.   Adhesion of GPVI-expressing RBL cells to putative GPVI ligands. Microtiter plates were coated with type I collagen, CRP, CVX, fibronectin, or BSA, and adhesion of wild-type and GPVI-expressing RBL cells was measured at A405 using a colorimetric substrate of the endogenous RBL enzyme hexosaminidase. a, adhesion of wild type RBL cells (open bars) and GPVI-expressing RBL cells (black bars) in the PBS with 0.9 mM calcium and 0.4 mM magnesium. b, adhesion of wild type RBL cells (open bars) and GPVI-expressing RBL cells (black bars) in the presence of EDTA (5 mM). The results shown are the mean and S.D. of quadruplicate samples from a single experiment. Each experiment is representative of 3-5 similar experiments performed with distinct GPVI-expressing clones. Note the strong adhesion of GPVI-expressing cells to CVX, intermediate adhesion to CRP, and lack of detectable adhesion to collagen.


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Fig. 3.   Expression and function of GPVI transmembrane and C-tail mutants in RBL-2H3 cells. FLAG-tagged wild-type (WT), R272L, and R295STOP GPVI receptors were stably expressed in RBL cells and tested for their signaling responses to the GPVI ligand CVX using the calcium-sensitive dye Fura-2. a, schematic diagrams of the wild-type and mutant receptors expressed are shown to highlight the sites of mutation and the proposed point of interaction with the signaling adaptor Fc Rgamma . EC, receptor extracellular domain; TM, receptor transmembrane domain; IC, receptor intracellular domain; R, the GPVI transmembrane domain arginine; L, the leucine substituted for R272 in the R272L mutant; D, the Fc Rgamma transmembrane domain aspartate; Y, Fc Rgamma ITAM tyrosine residues. b, receptor surface expression measured with anti-FLAG antibody in control RBL cells (thin lines) and GPVI-expressing RBL cells (thick lines). c, calcium signaling of GPVI-expressing RBL cells in response to CVX (10 nM) and thrombin (10 nM). These results are representative of identical experiments performed with 3-5 distinct clones for each receptor type.

The GPVI Transmembrane Arginine and Intracellular C-tail Are Both Necessary for GPVI Signaling-- The signaling roles of the GPVI transmembrane (TM) domain arginine (R272) and intracellular C-tail were tested by generating RBL cell lines expressing FLAG-tagged receptors in which the TM arginine is replaced by leucine (R272L-RBL) and the C-tail is truncated shortly following the TM domain (R295STOP-RBL). Both mutant GPVI receptors were expressed on the surface of RBL cells at levels equal to or greater than clones expressing wild-type GPVI (Fig. 3B). Consistent with the results of analogous mutations in related receptors, R272L-RBL did not signal in response to convulxin, confirming a necessary role for the GPVI TM domain arginine for signal transduction. Surprisingly, unlike similar C-tail truncation mutants of the Fc Rgamma partners Fcepsilon RI and Fcgamma RIII (see Fig. 6), RBL cells expressing the GPVI C-tail truncation mutant R295STOP also failed to signal to convulxin, demonstrating an unexpected necessary role for the GPVI C-tail (Fig. 3C).

Loss of the GPVI Transmembrane Domain Arginine and C-tail Results in Loss of Coupling to Fc Rgamma -- To determine why the R272L and R295STOP mutants of GPVI no longer supported signaling by CVX, we compared the ability of wild-type and mutant GPVI receptors to interact with Fc Rgamma with a functional assay in HEK-293T cells stably expressing FLAG Fc Rgamma and by direct biochemical means using coprecipitation. As for HEK-293T cells engineered to express low levels of the homologous immunoreceptor signaling adaptor DAP-12(25), FLAG-Fc Rgamma is not expressed on the cell surface of these cells in the absence of a coexpressed Fc Rgamma partner (Fig. 4A). The ability of an expressed receptor to rescue surface expression of FLAG-Fc Rgamma therefore measures functional association with Fc Rgamma . Wild-type GPVI expression rescued 10 times more surface FLAG-Fc Rgamma than mock-transfected cells (Fig. 4A). In contrast, GPVI R272L expression failed to rescue any FLAG-Fc Rgamma , consistent with a complete loss of association with Fc Rgamma (Fig. 4A). GPVI R295STOP expression also failed to rescue surface FLAG-Fc Rgamma , indicating a lack of Fc Rgamma interaction (Fig. 4A). Surface staining for the HA epitope confirmed that wild-type and mutant GPVI receptors were expressed at equivalent levels (Fig. 4B) and demonstrates that, as for the related Fc Rgamma partner PIRalpha (18), GPVI expression in HEK-293T cells does not require Fc Rgamma interaction. Loss of Fc Rgamma interaction in GPVIR272L and GPVIR295 STOP was confirmed biochemically using convulxin to precipitate GPVI and subsequently assaying for associated Fc Rgamma by immunoblotting (Fig. 5). Convulxin precipitation of wild-type GPVI, but neither mutant receptor resulted in coprecipitation of Fc Rgamma (Fig. 5). Interestingly, immunoblotting of GPVI-R295STOP protein with anti-FLAG antibody reveals the presence of mature protein at the predicted molecular mass of ~57 kDa (5 kDa smaller than the wild-type and R272L receptors) and the presence of a large amount of protein at 36-38 kDa, the predicted size for unglycosylated, incompletely processed protein. It is possible that this lower molecular mass species represents protein that cannot reach the cell surface because it cannot couple to Fc Rgamma partners and is therefore targeted for degradation. The fact that the abundantly expressed R272L mutant escapes this fate supports the role of the transmembrane arginine in targeting unpartnered receptors for degradation. These results show that the GPVI transmembrane arginine is necessary but not sufficient for functional association with the receptor's signaling coreceptor Fc Rgamma and that loss of signaling following truncation of the GPVI C-tail is due to loss of Fc Rgamma interaction rather than loss of an unidentified, distinct signaling function.


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Fig. 4.   Wild-type and mutant GPVI receptor interaction with Fc Rgamma . The interaction between wild type and mutant GPVI receptors and the signaling coreceptor Fc Rgamma was measured using surface rescue of FLAG-Fc Rgamma in a HEK-293T cell line that stably expresses a low level of FLAG-tagged Fc Rgamma as described under "Experimental Procedures" a, surface FLAG-Fc Rgamma expression in cells transfected with HA-tagged wild-type GPVI (WT), HA-tagged GPVI R272L (R272L), and HA-tagged GPVI R295STOP (R295STOP) is shown relative to mock-transfected cells (control). These results are the mean and S.D. of three experiments. b, expression of wild-type GPVI (blue line), GPVI R272L (red line), and GPVI R295STOP (green line) receptors was measured using anti-HA antibody and is shown relative to non-GPVI-transfected cells (black line).


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Fig. 5.   Coprecipitation of Fc Rgamma with wild-type and mutant GPVI receptors expressed in RBL cells. The interaction of Fc Rgamma with wild-type and mutant GPVI receptors in RBL cells was determined biochemically by precipitation of GPVI receptors with convulxin-coated beads. CVX-precipitated protein was probed for GPVI using anti-FLAG antibody (upper panel) and for Fc Rgamma using anti-Fc Rgamma antibody (lower panel). RBL, untransfected RBL cells; RBL-GPVI, RBL cells stably expressing wild-type GPVI; RBL-GPVIR272L, RBL cells stably expressing GPVIR272L; RBL-GPVIR295STOP, RBL cells stably expressing GPVI R295STOP; +, precipitation with convulxin-coated beads; -, control precipitations with BSA-coated beads. The GPVI-expressing RBL cell lines used were the same as those analyzed for surface expression and signaling in Fig. 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent studies of human platelets that are unresponsive to collagen (10), mouse knockouts (27), and platelet signaling (reviewed in Ref. 14) have generated the hypothesis that the platelet surface protein GPVI mediates collagen signaling and does so through its interactions with the immunoreceptor signaling adaptor Fc Rgamma . We have expressed GPVI in RBL-2H3 cells and studied GPVI signaling in a heterologous system to directly and formally address this hypothesis. RBL-2H3 cells express endogenous Fc Rgamma and are a model cell line for studying Fcepsilon RI receptor signaling (28). Like platelets, the signaling end points achieved by Fcepsilon RI receptor cross-linking and Fc Rgamma signaling in RBL-2H3 cells include mobilization of intracellular calcium and degranulation. Unlike the megakaryocytic cell lines DAMI, HEL, and MEG-01, however, RBL-2H3 cells express neither endogenous GPVI nor the integrin receptor for collagen alpha 2beta 1 (data not shown). Thus, RBL-2H3 cells express the appropriate signaling machinery to study GPVI signaling without the ambiguity of endogenous collagen receptor expression.

Expression of wild-type and FLAG-GPVI conferred robust calcium signaling to the snake venom protein convulxin at a threshold concentration equivalent to that necessary to activate human platelets but no detectable response to collagen at a concentration more than 500 times greater than that necessary to activate human platelets (Fig. 1). Unlike collagen, convulxin has been demonstrated to directly bind GPVI (13, 29) and was used by Clemetson et al. (6) to purify the GPVI protein from platelets. Thus, GPVI is a functional receptor in RBL-2H3 cells and signaling in RBL-2H3 cells closely resembles that in human platelets, but GPVI alone is not sufficient for collagen signaling.

To address GPVI-ligand interaction independent of signal transduction, we tested the adhesion of GPVI-RBL to putative GPVI ligands. GPVI expression conferred strong binding to convulxin and weaker binding to CRP but no detectable binding to collagen. Thus, the adhesion to immobilized proteins conferred by GPVI expression parallels the signaling responses observed to soluble agonists. These results are in contrast to those recently reported by Jandrot-Perrus et al. (7), who detected a small amount of collagen binding in a monocytic cell line (U937) stably expressing human or mouse GPVI. This discrepancy could reflect a difference in GPVI receptor density between the stable cell lines used or differences in methodology. In our hands, however, even clones expressing very high levels of GPVI such as the R272L clones (whose extracellular domains are wild type) confer adhesion to both CRP and convulxin but not collagen (data not shown and Fig. 3).

Is GPVI a bona fide collagen receptor, and, if so, why is GPVI expression insufficient to confer collagen signaling? Inadequate receptor density on RBL-2H3 cells is not a likely explanation, since the dose-response to CVX is similar in platelets and in GPVI-expressing RBL cells (Table I). One potential explanation for these results is that GPVI does mediate collagen signaling but that another coreceptor is required. This coreceptor might facilitate direct GPVI-collagen binding, or GPVI might mediate collagen signaling indirectly by linking a ligand-binding coreceptor to the signaling adaptor Fc Rgamma . Of the reported platelet collagen receptors, including the integrin alpha 2beta 1 (2), glycoprotein IV (30), and p65 (20), the most likely candidate is the alpha 2beta 1 integrin, whose high affinity for collagen may bring collagen to the platelet surface in an apparent concentration and/or configuration necessary for GPVI binding and signal transduction, although the precise role of alpha 2beta 1 remains controversial (31). Alternatively, GPVI may not be involved in collagen signaling, and an as yet unrecognized Fc Rgamma partner may be the true collagen receptor.

Several lines of evidence support the model that collagen is a GPVI ligand but that GPVI absolutely requires a coreceptor such as alpha 2beta 1 for productive collagen interaction. CRPs, which structurally closely resemble collagen but are more potent activators of platelets (21), initiate a small amount of intracellular signaling in GPVI-expressing but not wild-type RBL-2H3 cells (Fig. 1). The CRP signaling response in GPVI-expressing RBL-2H3 cells, however, requires 500 times the concentration necessary to activate platelets, suggesting that the lack of observed signaling to the related ligand collagen may reflect an extremely low affinity rather than a complete lack of direct interaction. A necessary role for alpha 2beta 1 as a coreceptor for collagen signal transduction through GPVI is also supported by the description of an individual with reduced levels of alpha 2beta 1 whose platelets failed to aggregate in response to collagen (32), but the lack of genetic and molecular characterization of this individual precludes exclusion of associated defects in the platelet expression of GPVI, Fc Rgamma , or other unidentified platelet receptors. Finally, platelets from an individual lacking GPVI also demonstrated a loss of Fc Rgamma expression despite normal Fc Rgamma expression in other cell types (33), suggesting that GPVI may be the only Fc Rgamma partner expressed in platelets and therefore must play a role in collagen signaling. Lack of genetic and molecular characterization of this individual, however, limits interpretation of this observation, and a megakaryocyte-specific block in Fc Rgamma expression cannot be excluded. Thus, the preponderance of data support a complex model of collagen signal transduction at the platelet surface with necessary roles played by no fewer than four transmembrane proteins, GPVI, alpha 2beta 1, and Fc Rgamma .

Studies of Fc Rgamma -deficient mouse platelets have revealed that, like Fc Rgamma partners expressed in immune cells, GPVI is not expressed in the absence of Fc Rgamma (27). Amino acid analysis of human and mouse GPVI reveals the presence of an arginine in the receptor transmembrane domain in a position identical to that of related Fc Rgamma partners such as the Fcalpha receptor and the NK receptors PIRalpha and NKp46 (6). As found for the Fcalpha and PIRalpha receptors (16, 18), mutation of this arginine does not interfere with receptor expression but results in a complete loss of receptor signaling (Fig. 3) and loss of interaction with the Fc Rgamma (Figs. 4 and 5). Thus, the GPVI transmembrane arginine is required for Fc Rgamma interaction, and Fc Rgamma interaction is required for GPVI signaling.

The human GPVI C-tail is ~50 amino acids long and can be divided into basic, proline-rich, and serine/threonine-rich domains (Fig. 6). The mouse GPVI C-tail is shorter and lacks the serine/threonine-rich region (data not shown, and see Ref. 7). Truncation of the GPVI C-tail does not interfere with receptor expression but results in complete loss of signaling in RBL cells and loss of Fc Rgamma interaction despite the presence of the transmembrane arginine (Figs. 3-5). Thus, both the transmembrane arginine and the receptor C-tail are necessary, but neither alone is sufficient for intracellular signaling. Interestingly, similar truncation mutants (within 5-10 amino acids of the TM domain) have been studied with two other Fc Rgamma partners, the Fcepsilon RI (34) and the Fcgamma RIII (35) receptors, with no loss of Fc Rgamma interaction or signaling. It is intriguing to note that while all of these receptors couple functionally to the Fc Rgamma , the transmembrane domains of Fcepsilon RI and Fcgamma RIII are homologous to each other but demonstrate little homology to those of GPVI or Fcalpha receptor and lack the signature arginine residue of that subfamily of Fc Rgamma partners. In addition, Fcepsilon RI and Fcgamma RIII share a chromosomal locus on human chromosome 1 (36), while GPVI and the related receptors discussed share a locus on chromosome 19, consistent with the existence of distinct ancestral receptors from which two receptor families may have evolved. Our results suggest that GPVI couples to the signaling adaptor Fc Rgamma in a manner distinct from that of the previously studied Fc Rgamma partners Fcepsilon RI and Fcgamma RIII. Precisely how the GPVI C-tail facilitates Fc Rgamma interaction and whether GPVI-related receptors also require their C-tails for Fc Rgamma coupling remains uncertain and awaits further mutational analysis.


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Fig. 6.   Amino acid alignment of the transmembrane and intracellular domains of GPVI and other Fc Rgamma receptor partners. Alignment of the deduced amino acid sequences of human GPVI (hGPVI), mouse GPVI (mGPVI), Fcalpha , PIRalpha , Fcepsilon RI, and Fcgamma RIII receptors was performed using the ClustalW program (Macvector). Amino acid identities shared among the GPVI, Fcalpha , and PIRalpha receptors are shaded. Amino acid identities shared between the Fcepsilon RI and Fcgamma RIII receptors are boxed. hGPVI R272 is in boldface type. *, the site of C-tail truncation for GPVI R295STOP; **, the site of C-tail truncation for an Fcepsilon RI receptor mutant; ***, the site of C-tail truncation for an Fcgamma RIII receptor mutant; TM, transmembrane domain; basic, portion of GPVI C-tail containing a significant number of basic amino acids; proline, portion of GPVI C-tail containing a cluster of proline residues; S/T, portion of human GPVI C-tail with a significant number of serine and threonine residues.

These studies provide the first functional analysis of GPVI as a signaling receptor and raise several important questions regarding the role of GPVI in vivo. The inability of GPVI to respond directly to collagen may suggest the evolution of a receptor adapted to operate in a highly specialized cellular environment in cooperation with other collagen receptors such as the integrin alpha 2beta 1 (a hypothesis supported by the megakaryocytic-specific pattern of expression of the receptor's mRNA (data not shown, and see Ref. 27), but the possibility that GPVI does not mediate collagen signaling cannot yet be definitively excluded. Our results extend the proposed model of platelet collagen signaling to one requiring no fewer than four receptor subunits and establish a heterologous system in which to further dissect this signaling pathway. Identification of the receptors involved in collagen activation of platelets and the molecular basis for this response may provide novel targets for anti-platelet therapies, which act at a critical point in thrombogenesis, the activation of newly adherent platelets at sites of atherosclerotic rupture.

    ACKNOWLEDGEMENTS

We acknowledge the helpful suggestions of Dr. Reuben Siraganian for generation of RBL-2H3 cell clones and the thoughtful comments of Drs. Skip Brass and Gary Koretzky regarding the manuscript.

    FOOTNOTES

* This work was supported by grants from the American Heart Association and the W. W. Smith Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: University of Pennsylvania, 421 Curie Blvd., BRB II/III Rm. 952, Philadelphia, PA 19104-6100; Tel.: 215-898-9007; Fax: 215-573-2094; E-mail: markkahn@mail.med.upenn.edu.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M009344200

    ABBREVIATIONS

The abbreviations used are: CVX, convulxin; Fc Rgamma , Fc gamma -chain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; TM, transmembrane; HA, hemagglutinin; BSA, bovine serum albumin; CRP, collagen-related peptide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Savage, B., Almus-Jacobs, F., and Ruggeri, Z. M. (1998) Cell 94, 657-666[Medline] [Order article via Infotrieve]
2. Santoro, S. A. (1986) Cell 46, 913-920[Medline] [Order article via Infotrieve]
3. Wilner, G. D., Nossel, H. L., and LeRoy, E. C. (1968) J. Clin. Invest. 47, 2616-2621[Medline] [Order article via Infotrieve]
4. Du, X., Harris, S. J., Tetaz, T. J., Ginsberg, M. H., and Berndt, M. C. (1994) J. Biol. Chem. 269, 18287-18290[Abstract/Free Full Text]
5. Keely, P. J., and Parise, L. V. (1996) J. Biol. Chem. 271, 26668-26676[Abstract/Free Full Text]
6. Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019-29024[Abstract/Free Full Text]
7. Jandrot-Perrus, M., Busfield, S., Lagrue, A. H., Xiong, X., Debili, N., Chickering, T., Couedic, J. P., Goodearl, A., Dussault, B., Fraser, C., Vainchenker, W., and Villeval, J. L. (2000) Blood 96, 1798-1807[Abstract/Free Full Text]
8. Clemetson, K. J., McGregor, J. L., James, E., Dechavanne, M., and Luscher, E. F. (1982) J. Clin. Invest. 70, 304-311[Medline] [Order article via Infotrieve]
9. Sugiyama, T., Okuma, M., Ushikubi, F., Sensaki, S., Kanaji, K., and Uchino, H. (1987) Blood 69, 1712-1720[Abstract]
10. Moroi, M., Jung, S. M., Okuma, M., and Shinmyozu, K. (1989) J. Clin. Invest. 84, 1440-1445[Medline] [Order article via Infotrieve]
11. Moroi, M., Okuma, M., and Jung, S. M. (1992) Biochim. Biophys. Acta 1137, 1-9[Medline] [Order article via Infotrieve]
12. Jandrot-Perrus, M., Lagrue, A. H., Okuma, M., and Bon, C. (1997) J. Biol. Chem. 272, 27035-27041[Abstract/Free Full Text]
13. Polgar, J., Clemetson, J. M., Kehrel, B. E., Wiedemann, M., Magnenat, E. M., Wells, T. N. C., and Clemetson, K. J. (1997) J. Biol. Chem. 272, 13576-13583[Abstract/Free Full Text]
14. Watson, S. P. (1999) Thromb. Haemostasis 82, 365-376[Medline] [Order article via Infotrieve]
15. Poole, A., Gibbins, J. M., Turner, M., van Vugt, M. J., van de Winkel, J. G., Saito, T., Tybulewicz, V. L., and Watson, S. P. (1997) EMBO J. 16, 2333-2341[Abstract/Free Full Text]
16. Morton, H. C., van den Herik-Oudijk, I. E., Vossebeld, P., Snijders, A., Verhoeven, A. J., Capel, P. J., and van de Winkel, J. G. (1995) J. Biol. Chem. 270, 29781-29787[Abstract/Free Full Text]
17. Taylor, L. S., and McVicar, D. W. (1999) Blood 94, 1790-1796[Abstract/Free Full Text]
18. Ono, M., Yuasa, T., Ra, C., and Takai, T. (1999) J. Biol. Chem. 274, 30288-30296[Abstract/Free Full Text]
19. Strouse, R. J., and Daniel, J. L. (1996) Thromb. Res. 82, 485-493[CrossRef][Medline] [Order article via Infotrieve]
20. Chiang, T. M., Rinaldy, A., and Kang, A. H. (1997) J. Clin. Invest. 100, 514-521[Abstract/Free Full Text]
21. Morton, L. F., Hargreaves, P. G., Farndale, R. W., Young, R. D., and Barnes, M. J. (1995) Biochem. J. 306, 337-344[Medline] [Order article via Infotrieve]
22. Kahn, M. L., Nakanishi-Matsui, M., Shapiro, M. J., Ishihara, H., and Coughlin, S. R. (1999) J. Clin. Invest. 103, 879-887[Abstract/Free Full Text]
23. Hamawy, M. M., Swieter, M., Mergenhagen, S. E., and Siraganian, R. P. (1997) J. Biol. Chem. 272, 30498-30503[Abstract/Free Full Text]
24. Connolly, A. J., Ishihara, H., Kahn, M. L., Farese, R. V., and Coughlin, S. R. (1996) Nature 381, 516-519[CrossRef][Medline] [Order article via Infotrieve]
25. Bakker, A. B., Baker, E., Sutherland, G. R., Phillips, J. H., and Lanier, L. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9792-9796[Abstract/Free Full Text]
26. Posner, R. G., Subramanian, K., Goldstein, B., Thomas, J., Feder, T., Holowka, D., and Baird, B. (1995) J. Immunol. 155, 3601-3609[Abstract]
27. Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem. 275, 23998-24002[Abstract/Free Full Text]
28. Barsumian, E. L., Isersky, C., Petrino, M. G., and Siraganian, R. P. (1981) Eur. J. Immunol. 11, 317-323[Medline] [Order article via Infotrieve]
29. Francischetti, I. M., Saliou, B., Leduc, M., Carlini, C. R., Hatmi, M., Randon, J., Faili, A., and Bon, C. (1997) Toxicon 35, 1217-1228[CrossRef][Medline] [Order article via Infotrieve]
30. Nakamura, T., Jamieson, G. A., Okuma, M., Kambayashi, J., and Tandon, N. N. (1998) J. Biol. Chem. 273, 4338-4344[Abstract/Free Full Text]
31. Monnet, E., Sizaret, P., Arbeille, B., and Fauvel-Lafeve, F. (2000) Thromb. Res. 98, 423-433[CrossRef][Medline] [Order article via Infotrieve]
32. Nieuwenhuis, H. K., Akkerman, J. W., Houdijk, W. P., and Sixma, J. J. (1985) Nature 318, 470-472[Medline] [Order article via Infotrieve]
33. Tsuji, M., Ezumi, Y., Arai, M., and Takayama, H. (1997) J. Biol. Chem. 272, 23528-23531[Abstract/Free Full Text]
34. Alber, G., Miller, L., Jelsema, C. L., Varin-Blank, N., and Metzger, H. (1991) J. Biol. Chem. 266, 22613-22620[Abstract/Free Full Text]
35. Lanier, L. L., Yu, G., and Phillips, J. H. (1991) J. Immunol. 146, 1571-1576[Abstract/Free Full Text]
36. Le Coniat, M., Kinet, J. P., and Berger, R. (1990) Immunogenetics 32, 183-186[Medline] [Order article via Infotrieve]


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