Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cγ2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor γ-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor γ-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor γ-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin α2β1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcγRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor γ-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin α2β1 and involves phosphorylation of the Fc receptor γ-chain, its association with Syk and subsequent phosphorylation of phospholipase Cγ2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

The adhesive and stimulatory properties of the extracellular matrix protein collagen on platelets are vital for the maintenance of hemeostasis. Upon vascular damage, platelets adhere to subendothelial collagen which stimulates a tyrosine kinase dependent pathway leading to platelet degranulation, aggregation and development of a hemeostatic plug. The mechanism of collagen stimulation of platelets is poorly understood, and the distinction between adhesion and stimulation ill defined. Several platelet glycoproteins have been implicated as potential collagen receptors, including the integrin ␣ 2 ␤ 1 (1), glycoprotein IV (GPIIIb, CD36) (2), glycoprotein VI (3), and uncharacterized 65-kDa (4) and 85-90-kDa glycoproteins (5). Patients whose platelets express abnormally low numbers of these proteins, or who possess autoantibodies to them, have limited bleeding defects (3,(5)(6)(7)(8)(9).
Collagen stimulation of platelets activates tyrosine kinasedependent mechanisms which involve tyrosine phosphorylation of Syk and phospholipase C ␥ 2 (PLC ␥ 2) 1 (10 -12). Syk is a nonreceptor tyrosine kinase which is assembled into signaling complexes via interaction between its tandem Src homology 2 (SH2) domains and a tyrosine phosphorylated activation motif found in receptors of the immune system or their associated chains. The motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), has the amino acid sequence YXXL/ IX 6 -8 YXXL/I (13), and is phosphorylated on the conserved tyrosine residues by a member of the Src kinase family upon receptor activation.
We recently proposed (12) that a collagen receptor or an associated protein may contain an ITAM motif to allow interaction with Syk. In this report we show that stimulation of platelets with collagen induces tyrosine phosphorylation of the Fc receptor ␥-chain (FcR ␥-chain), which contains an ITAM motif, and that this promotes an association between the FcR ␥-chain and Syk. This allows the construction of a model linking collagen receptor ligation to activation of Syk and a tyrosine kinase signaling cascade.

EXPERIMENTAL PROCEDURES
Materials-Anti-FcR ␥-chain antiserum (14) was kindly provided by Dr. J.-P. Kinet (Beth Israel Hospital, Boston, MA), the monoclonal antibody (mAb) 6F1 was a gift from Dr. B. Coller (Mount Sinai School of Medicine, New York, NY), and the rabbit polyclonal antibody to the T cell -chain was kindly donated by Dr. D. Cantrell (ICRF, London). Collagen fibers, as Horm collagen, a suspension of type I fibers from equine tendon, were obtained from Nycomed (Munich, Germany). A collagen-related peptide (CRP: GCP*(GPP*) 10 GCP*G; single amino acid code P* ϭ hydroxyproline) was synthesized and cross-linked as described previously (15). Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology (TCS Biologicals Ltd., Botolph Claydon, Bucks, UK), anti-Syk antibodies: Syk(LR) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and mAb 101 from Wako GmbH (Neuss, Germany), and anti-Fc␥RII mAb IV.3 was purchased from Medarex Inc. (Annandale, NJ). Other reagents were from previously described * This work was supported by the Wellcome Trust and the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To who correspondence should be addressed. sources (12). Preparation and Stimulation of Platelets-Platelets were prepared as described previously (12) and suspended in modified Tyrodes-Hepes buffer (134 mM NaCl, 0.34 mM Na 2 HPO 4 , 2.9 mM KCl, 12 mM NaHCO 3 , 20 mM Hepes, 5 mM glucose, 1 mM MgCl 2 , pH 7.3) containing 1 mM EGTA and 10 M indomethacin to a density of 8 ϫ 10 8 cells/ml. Mg 2ϩ was omitted, and 1 mM EDTA was added to the buffer where stimulation in the absence of Mg 2ϩ was required. Stimulation of platelets (675 l) with collagen (100 g/ml, 90 s), collagen-related peptide (CRP, 3 g/ml, 90 s), and wheat germ agglutinin (WGA, 100 g/ml, 60 s) was performed at 37°C in an aggregometer with continuous stirring at 800 rpm. Stimulation by cross-linking of the low affinity receptor for immunoglobulin G, Fc␥RIIA, was performed by preincubation of platelets for 60 s with monoclonal antibody IV.3 (1 g/ml) followed by stimulation for 60 s by addition of anti-mouse IgG F(abЈ) 2 (30 g/ml).
Immunoblotting Studies-Platelet stimulation was terminated by the addition of an equal volume of Laemmli sample-treatment buffer. Proteins were separated by SDS-PAGE on 10 -18% gradient slab gels and transferred to polyvinylidene difluoride membranes which were then blocked by incubation in 10% (w/v) bovine serum albumin dissolved in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6). Primary and secondary antibodies were diluted in TBS-T containing 2% (w/v) bovine serum albumin and incubated with Western blots for 1 h at room temperature. Blots were washed for 2 h in TBS-T following each incubation with antibodies, and then developed using an enhanced chemiluminescence (ECL) detection system. Anti-phosphotyrosine and anti-Syk immunoblots were performed using mAb 4G10 and mAb 101, respectively, at 1 g/ml; anti-FcR ␥-chain and anti-T cell -chain immunoblots were made with rabbit antiserum diluted 1:10000. Horseradish peroxidase-conjugated secondary antibodies were diluted 1:10000.
Immunoprecipitation Studies-Platelet stimulation was terminated by the addition of an equal volume of ice-cold lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM EDTA, 2% (v/v) Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 g/ml pepstatin A, pH 7.3). Detergent-insoluble debris was removed, and the lysates were precleared by mixing with protein A-Sepharose for 1 h at 4°C (20 l of a 50% (w/v) suspension of protein A-Sepharose in TBS-T). Preclearing was omitted in samples stimulated by cross-linking of Fc␥RIIA, since this would result in removal of Fc␥RIIA from the lysate. FcR ␥-chain was immunoprecipitated from 1.2 ml of lysate using 4 l of rabbit antiserum, and Syk was immunoprecipitated from 100 l of lysate using 4 g of a polyclonal antibody Syk(LR). Following rotation at 4°C for 1 h, 25 l of a 50% (w/v) suspension of protein A-Sepharose was added to each sample, and mixing was continued for 1 h. The Sepharose pellet was washed in lysis buffer and then TBS-T, before addition of Laemmli sample-treatment buffer. Proteins were separated by SDS-PAGE using 10 -18% gradient slab gels and transferred to polyvinylidene difluoride membranes.
Fusion Protein Precipitation Studies-A GST fusion protein containing the tandem SH2 domains of Syk (GST-Syk SH2) was constructed and expressed as described previously (12) and bound to glutathioneagarose. Precipitation of platelet proteins with the fusion construct was performed as described previously (12). Precipitated proteins were separated by SDS-PAGE using 10 -18% gradient slab gels under either reducing or nonreducing conditions and transferred to polyvinylidene difluoride membranes.
In Vitro Kinase Assay-The FcR ␥-chain or Syk was immunoprecipitated as described above and assayed for kinase activity while immobilized on protein A-Sepharose following the final washing step. The supernatant was removed and replaced with assay buffer (105 mM NaCl, 20 mM Hepes, 5 mM MnCl 2 , 5 mM MgCl 2 , 10 M ATP, and 5 Ci of [␥-32 P]ATP). Samples were incubated at 22°C for 10 min, whereupon reactions were stopped by addition of 500 l of 50 mM EDTA at 4°C. Sepharose was pelleted by brief centrifugation, the supernatant was removed, and Laemmli sample buffer was added to the pellets. Proteins were separated by SDS-PAGE on 10 -18% gradient slab gels and transferred to polyvinylidene difluoride membranes. Kinase activity was recorded by autoradiography.

RESULTS
Tyrosine Phosphorylation of the FcR ␥-Chain-In a strategy designed to identify ITAM-containing proteins which undergo phosphorylation following collagen stimulation, platelet lysates were incubated with the tandem SH2 domains of Syk expressed as a GST fusion construct (GST-Syk SH2). Precipitation and immunoblotting for tyrosine phosphorylation re-vealed a reproducible association of two bands of 11 and 13 kDa along with several higher molecular mass bands. Candidates for the tyrosine-phosphorylated doublet which are known to contain an ITAM include the T cell receptor-associated -chain and the FcR ␥-chain. The -chain was not detected in whole platelet lysates or in GST-Syk SH2 precipitates following immunoblotting with a specific antibody. Immunoblotting for the FcR ␥-chain detected its presence in whole cell lysates and in GST-Syk SH2 precipitates from cells stimulated with collagen.
Proteins precipitated by GST-Syk SH2 and separated by SDS-PAGE under reducing conditions were immunoblotted using anti-FcR ␥-chain antiserum (Fig. 1a(i)). Four proteins of 13, 11, 8.5, and 6.5 kDa were detected in samples precipitated from platelets stimulated with collagen, CRP, and WGA, but were not present in basal cells or cells stimulated by cross-linking Fc␥RIIA (Fig. 1a) or by thrombin (not shown). Reprobing with an anti-phosphotyrosine antibody demonstrated that only the upper two bands were tyrosine-phosphorylated (Fig. 1a(ii)), indicating that tyrosine phosphorylation of the FcR ␥-chain is associated with a mobility shift on SDS-PAGE. Two uncharacterized proteins of 65 and 70 kDa were also tyrosine-phosphorylated and precipitated from collagen-, CRP-, and WGA-stimulated platelets (Fig. 1a(ii)). Fig. 1a(ii) also demonstrates association of the tyrosine phosphorylated Fc␥RIIA with GST-Syk SH2 in cells stimulated by cross-linking of the receptor, as previously reported (12), and also by WGA. The absence of tyrosine-phosphorylated proteins other than those mentioned above in WGA-stimulated platelets suggests that platelets do not express other ITAM-containing proteins which undergo tyrosine phosphorylation on cross-linking of cell surface glycosylated proteins.
FcR ␥-chain is expressed in cells as a homodimer linked by a disulfide bridge. Precipitation of non-tyrosine-phosphorylated FcR ␥-chain with the SH2 domains of Syk may therefore be due to covalent association with a tyrosine-phosphorylated FcR ␥-chain. To examine this, GST-Syk SH2 precipitates were subjected to SDS-PAGE under nonreducing conditions and immunoblotted for FcR ␥-chain and for phosphotyrosine residues. Under nonreducing conditions, the FcR ␥-chain protein remains dimerized and migrates a distance consistent with an approximate doubling of its molecular mass (Fig. 1b(i)). In accordance with this, there was an approximate doubling in molecular mass of the tyrosine phosphorylated bands under nonreducing conditions (Fig. 1b(ii)).
The SH2 domains of Syk precipitated similar amounts of the tyrosine-phosphorylated and non-tyrosine-phosphorylated forms of the FcR ␥-chain, suggesting that only 1 molecule in each pair becomes phosphorylated. Phosphorylation of one chain in each dimer may prevent phosphorylation of the other chain or, alternatively, one chain may not be accessible to the kinase. The existence of four bands under reduced conditions may reflect the fact that the FcR ␥-chain has additional tyrosine residues to those of the ITAM motif and can also undergo phosphorylation on threonine and serine residues (16 -18). The appearance of non-tyrosine-phosphorylated FcR ␥-chain as a doublet on SDS-PAGE has been observed in other cells (16).
Time Course of Phosphorylation of FcR ␥-Chain-FcR ␥-chain was immunoprecipitated from lysates of basal and stimulated cells and the level of tyrosine phosphorylation examined by immunoblot analysis. A time course of FcR ␥-chain phosphorylation in response to collagen revealed that tyrosine phosphorylation occurred within 20 s and reached a maximum by 90 s (Fig. 2(i)). The FcR ␥-chain, along with the tyrosine kinase Syk (12), is one of the earliest proteins to undergo tyrosine phosphorylation in collagen-stimulated platelets.
Association of the FcR ␥-Chain with Syk-While the above demonstrates that tyrosine-phosphorylated FcR ␥-chain from collagen-activated platelets can bind to the SH2 domains of Syk, it is important to establish whether the FcR ␥-chain associates with Syk in vivo. Immunoprecipitation of the FcR ␥-chain combined with immunoblotting and in vitro kinase assays confirmed this interaction in collagen-and CRP-stimulated platelets. Minimal kinase activity was present in FcR ␥-chain immunoprecipitates from resting cells, whereas marked phosphorylation of the FcR ␥-chain was observed in immunoprecipitates from collagen-and CRP-stimulated cells (Fig. 3a(i)). Phosphorylation of a protein which co-migrates with Syk was also observed, and the presence of Syk was confirmed by immunoblot analysis (Fig. 3a(ii)). Immunoblotting with mAb 4G10 revealed that Syk was already tyrosinephosphorylated following co-immunoprecipitation with FcR ␥-chain from collagen-and CRP-stimulated platelets (Fig.  3b(i)). Confirmation of the association between Syk and FcR ␥-chain in collagen-stimulated cells was obtained through an in vitro kinase assay on Syk immunoprecipitates. Phosphorylation of a protein which co-migrates with the larger of the FcR ␥-chain bands (13 kDa) was observed in stimulated cells (Fig. 3c). Equal amounts of the FcR ␥-chain were immunoprecipitated in control and stimulated cells Fig. 3b(ii). This blot, however, shows only the lower molecular mass doublet of FcR ␥-chain, although in longer exposures the upper doublet is visible (not shown). It appears that on collagen stimulation only a small proportion of the total cellular FcR ␥-chain is tyrosine-phosphorylated. Tyrosine phosphorylation of the FcR ␥-chain and the resultant gel shift are most easily observed where the FcR ␥-chain is concentrated by either immunoprecipitation or by GST-Syk SH2 precipitation.
Tyrosine Phosphorylation of FcR ␥-Chain in Response to Collagen Is Not Dependent on the Integrin ␣ 2 ␤ 1 -Although platelets adhere to collagen via the integrin ␣ 2 ␤ 1 through a Mg 2ϩ -dependent mechanism (1), the role of this integrin in collagen signaling is uncertain. Significant phosphorylation of the FcR ␥-chain in collagen-stimulated platelets is observed in the absence of Mg 2ϩ (Fig. 2(ii)), demonstrating independence from ␣ 2 ␤ 1 . Consistent with this, tyrosine phosphorylation of the FcR ␥-chain by CRP, which does not bind to the ␣ 2 ␤ 1 integrin (15), is also maintained in the absence of Mg 2ϩ . Tyrosine phosphorylation of the FcR ␥-chain is also observed in response to both collagen and CRP in the presence of an ␣ 2 ␤ 1 integrin blocking mAb 6F1 (Fig. 2(iii)). The different levels of basal FcR ␥-chain phosphorylation in 2(ii) and 2(iii) are due to the use of different platelet preparations. DISCUSSION The present study identifies a signaling protein which is involved in collagen-stimulated activation of platelets. The FcR ␥-chain becomes tyrosine phosphorylated when platelets are stimulated with collagen enabling binding of Syk and the assembly of a signaling complex. Phosphorylation of the FcR ␥-chain is rapid and detectable within 20 s of stimulation and reaches maximal levels by 90 s. This time course is similar to that observed for the onset of tyrosine phosphorylation in collagen-stimulated platelets and suggests an early involvement FIG. 1. Association of tyrosine-phosphorylated FcR ␥-chain with GST-Syk SH2 domains. Platelets were stimulated with collagen (100 g/ml, 90 s), CRP (3 g/ml, 90 s), WGA (100 g/ml, 60 s), and by cross-linking Fc␥RIIA (60 s), and proteins were precipitated from cell lysates using 10 g of GST-Syk SH2 or a molar equivalent of GST as described under "Experimental Procedures." Proteins were separated by SDS-PAGE under a, reducing, or b, nonreducing, conditions and immunoblotted for FcR ␥-chain and phosphotyrosine residues. a(i) shows FcR ␥-chain precipitated from collagen-, CRP-, and WGA-stimulated cells; a(ii) is the same blot reprobed for phosphotyrosine residues. b(i) shows the FcR ␥-chain precipitated from collagen-, CRP-, and WGA-stimulated cells run under nonreducing conditions, which was subsequently reprobed for phosphotyrosine residues (b(ii)). In a(ii) and b(ii), the position of tyrosine-phosphorylated Fc␥RIIA from Fc␥RIIA-and WGA-stimulated cells is indicated. of the FcR ␥-chain in collagen signaling.
The FcR ␥-chain is recognized for its role in signaling by the high affinity receptors for IgE (Fc⑀RI) (19 -21) and IgG (Fc␥RI) (16,22) and by the low affinity IgG receptor (Fc␥RIII) (22). Clustering and activation of Fc⑀RI, Fc␥RI, and Fc␥RIII stimulates tyrosine phosphorylation of the ITAM on the FcR ␥-chain enabling recruitment of Syk which becomes tyrosine-phosphorylated and activated (16,19,23,24). The promiscuous nature of the FcR ␥-chain is further supported by the recent finding that it forms a functional association with the IgA receptor (Fc␣RI) in cells transfected to express both proteins (25). The FcR ␥-chain is also vital for the assembly and surface expression of Fc⑀RI and Fc␥RIII, by preventing inappropriate degradation in the endoplasmic reticulum (26). The expression of Fc⑀RI, Fc␥RI, Fc␥RIII, and the FcR ␥-chain in platelets has not been reported.
The kinase which is responsible for FcR ␥-chain tyrosine phosphorylation in platelets requires identification. There is a precedent for the involvement of Src-like kinases in the phosphorylation of the ITAM (17,(27)(28)(29). The kinase assays shown in Fig. 3, which were performed on FcR ␥-chain and Syk immunoprecipitates, indicate that a kinase activity capable of phosphorylating the FcR ␥-chain is co-immunoprecipitated from collagen-and CRP-stimulated cells. It is not known whether Syk or a co-precipitated Src-family kinase is the source of this activity.
The identity of the collagen receptor which leads to tyrosine phosphorylation of the FcR ␥-chain is not known. Several lines of evidence suggest that tyrosine phosphorylation of the FcR ␥-chain is not dependent on the integrin ␣ 2 ␤ 1 . Tyrosine phosphorylation of FcR ␥-chain is maintained in the absence of Mg 2ϩ or presence of mAb 6F1, conditions which prevent the association of collagen with the integrin. In addition, CRP also stimulates tyrosine phosphorylation of FcR ␥-chain, yet is not capable of binding to the integrin (15). This suggests that collagen stimulation of platelets involves an uncharacterized receptor which is linked to the FcR ␥-chain. Candidates for this receptor include those proteins described in the Introduction.
Collagen is the only physiological platelet agonist that has been identified to induce phosphorylation of the FcR ␥-chain, suggesting that this event plays a unique role in collagen signaling. Platelets express a low affinity IgG receptor, Fc␥RIIA, which also contains an ITAM sequence, but its activation does not induce phosphorylation of the FcR ␥-chain. Clustering of the platelet Fc␥-IIA receptors results in tyrosine phosphorylation of its integral ITAM and tyrosine phosphorylation of Syk and PLC ␥ 2 (12,30). The lectin WGA, which is recognized as a powerful stimulus of platelet activation (31)(32)(33)(34), elicits tyrosine phosphorylation of Syk (35) and PLC ␥ 2 (10), and also induces phosphorylation of FcR ␥-chain and Fc␥RIIA. WGA therefore manifests its effects on platelets, at least in part, by activating components of both collagen and Fc␥RIIA pathways. Thrombin also stimulates tyrosine phosphorylation of Syk (36) but induces very weak or no phosphorylation of PLC ␥ 2 (10) and does not induce tyrosine phosphorylation of the FcR ␥-chain. This indicates that other pathways lead to phosphorylation of Syk in the platelet and it can be speculated that phosphorylation of an ITAM containing protein may be essential in leading to tyrosine phosphorylation of PLC ␥ 2.
A model for collagen stimulation of platelets can be proposed in light of the results of this study. Under the physiological condition of flow, platelets adhere to extracellular matrix collagen via the integrin ␣ 2 ␤ 1 . This brings collagen into association with a separate cell surface receptor which is coupled to the FcR ␥-chain. Receptor clustering leads to tyrosine phosphorylation of the FcR ␥-chain, possibly by a Src-family kinase, enabling binding of Syk which becomes tyrosine phosphoryl-FIG. 2. Time course of FcR ␥-chain tyrosine phosphorylation in collagen-stimulated platelets and role of the integrin ␣ 2 ␤ 1 . i, the FcR ␥-chain was immunoprecipitated from lysates of platelets stimulated by collagen (100 g/ml) at the indicated times. Proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted for phosphotyrosine residues as described under "Experimental Procedures." ii, platelets were stimulated with collagen (100 g/ml, 90 s) and CRP (3 g/ml, 90 s) in the absence of extracellular Mg 2ϩ and the presence of EDTA (1 mM). FcR ␥-chain immunoprecipitates were immunoblotted for phosphotyrosine residues as described above. iii, the procedure in part ii was repeated using platelets which had been preincubated for 5 min prior to stimulation with monoclonal antibody 6F1 (10 g/ml) which blocks the interaction of collagen with the integrin ␣ 2 ␤ 1 .

FIG. 3.
In vitro kinase activity in FcR ␥-chain and Syk immunoprecipitates from collagen-and CRP-stimulated platelets. a(i), an in vitro kinase assay was performed on immunoprecipitated FcR ␥-chain as described under "Experimental Procedures." Proteins were separated by SDS-PAGE, and phosphorylation was detected by autoradiography. ii, the same membrane was immunoblotted for Syk as described under "Experimental Procedures." b(i), a duplicate set of samples were immunoblotted for tyrosine phosphorylation and ii, reprobed for FcR ␥-chain to ensure equal loading of samples. c, an in vitro kinase assay was performed on Syk immunoprecipitates from platelets as described under "Experimental Procedures." The arrow indicates a low molecular mass protein which becomes phosphorylated in the assay. ated and activated. This initiates a series of events which may involve other kinases and adaptor proteins leading to phosphorylation and activation of PLC ␥ 2. A defect in collagen signaling may contribute to the hemorrhaging in utero which has recently been reported in mice engineered to lack the tyrosine kinase Syk (37,38).