Evidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets

Gross, Barbara; Melford, Steven K.; Watson, Steve P.

doi:10.1046/j.1432-1327.1999.00560.x

Cited by 64 publications

(51 citation statements)

References 52 publications

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“…A substrate for Syk is the adaptor LAT (Zhang et al, 1998), which possesses multiple phosphorylation sites that act as docking sites for recruitment of additional proteins to form a signalling complex (Gibbins et al, 1998;Pasquet et al, 1999b;Sarkar, 1998). For example, PLCγ2 and PI3K (p85/p110) are brought to the vicinity of their substrates at the plasma membrane through interaction with tyrosinephosphorylated LAT (Gibbins et al, 1998;Gross et al, 1999b).…”

Section: Signalling Through Gpvimentioning

confidence: 99%

Platelet adhesion signalling and the regulation of thrombus formation

Gibbins

2004

Journal of Cell Science

259

232

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Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin α2β1. These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin αIIbβ3, a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins α2β1 and αIIbβ3 contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.

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Section: Signalling Through Gpvimentioning

confidence: 99%

Platelet adhesion signalling and the regulation of thrombus formation

Gibbins

2004

Journal of Cell Science

259

232

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“…Where indicated, the proteins were dissolved using Laemmli sample buffer without 2-mercaptoethanol (non-reducing condition). Precipitation of proteins with the fusion construct was performed as described previously (19).…”

Section: Binding Of Gst-sh3 Domain Fusion Proteins To Mbp-gpvi Fusionmentioning

confidence: 99%

“…MBP and MBP-GPVI expressed in E. coli were purified on amylose-Sepharose according to the manufacturer's instructions (New England Biolabs). GST-SH3 domains of Fyn, Lyn, Src, Fgr, Btk, or PLC␥2 expressed in E. coli were purified on glutathione-agarose as described previously (19). Where appropriate, GST-Lyn-SH3 and GST-Src-SH3 were radioiodinated with sodium […”

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confidence: 99%

Association of Fyn and Lyn with the Proline-rich Domain of Glycoprotein VI Regulates Intracellular Signaling

Suzuki‐Inoue¹,

Tulasne²,

Shen³

et al. 2002

Journal of Biological Chemistry

146

115

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The glycoprotein VI (GPVI)-Fc receptor (FcR)␥The adhesion and activation of platelets by subendothelial collagen fibers initiates aggregate formation at sites of vessel damage. Glycoprotein (GP) 1 VI plays a critical role in the activatory events induced by collagen as shown by the lack of response to collagen in human and mice platelets deficient in the glycoprotein (1, 2). A collagen-related peptide and a snake venom toxin, convulxin, interact specifically with GPVI and mimic many of the responses to collagen (3-5).Because of the physiological importance of GPVI, the mechanism of the GPVI-mediated signaling system has been extensively investigated (6 -8). GPVI is present as a complex with Fc receptor (FcR) ␥-chain in the platelet membrane (8 -10). The Src family kinases, Fyn and Lyn, are associated with GPVIFcR ␥-chain complex in platelets and initiate activation through phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) in the FcR ␥-chain leading to binding and activation of the tyrosine kinase Syk. A series of adapter molecules including LAT and SLP76 orchestrate a carefully regulated signaling network leading to activation of PLC␥2, phosphoinositol 3-kinase, and small molecular weight G proteins, leading to platelet activation (6, 7).The cloning of GPVI (11-14) has revealed it to be a member of the immunoglobulin (Ig) superfamily, showing close homology to Fc␣RI. GPVI has a charged arginine residue in its transmembrane domain. This arginine, together with elements within the cytoplasmic domain, is crucial for association of GPVI with FcR ␥-chain and GPVI-mediated signal transduction (15,16). In addition, the cytoplasmic tail of GPVI has a cluster of 6 proline residues of unknown function (11)(12)(13)(14). This sequence of GPVI, RPLPPLPPLP, contains a consensus Src family kinase-SH3 recognition motif (RPLPPLP) (17,18), and provides a potential site of interaction with Fyn and Lyn via their SH3 domains.In this study, we demonstrate that depletion of the prolinerich domain in GPVI abolishes the association with Fyn and Lyn and prevents tyrosine phosphorylation of FcR ␥-chain and downstream responses. From these findings, we suggest that Fyn/Lyn directly bind the proline-rich domain of GPVI and that this association is necessary for phosphorylation of the ITAM and downstream signals.

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“…Syk is able to bind to the tyrosine-phosphorylated ITAM of the FcR ␥-chain via its tandem SH2 domains (12) leading to autophosphorylation and activation. Syk plays a critical role in the regulation of PLC␥2 through phosphorylation of a number of key proteins including the adapters LAT and SLP-76 (13)(14)(15). These signaling events occur upon collagen-induced GPVI cross-linking and following GPVI binding to collagen-related peptides (CRPs) (15)(16)(17).…”

mentioning

confidence: 99%

Evidence for Two Distinct Epitopes within Collagen for Activation of Murine Platelets

Schulte¹,

Snell²,

Bergmeier³

et al. 2001

Journal of Biological Chemistry

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It has recently been shown that the monoclonal antibody JAQ1 to murine glycoprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody crosslinking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem. 275, 23998 -24002). In the present study, we have shown that crosslinking of GPVI by JAQ1 results in tyrosine phosphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) ␥-chain, Syk, LAT, SLP-76, and phospholipase C␥2. In contrast, platelet aggregation and tyrosine phosphorylation of these proteins were inhibited when mouse platelets were preincubated with JAQ1 in the absence of cross-linking and were subsequently stimulated with a collagen-related peptide (CRP) that is specific for GPVI and low concentrations of collagen. However, at higher concentrations of collagen, but not CRP, aggregation of platelets and tyrosine phosphorylation of the above proteins (except for the adapter LAT) is re-established despite the presence of JAQ1. These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, is occupied in the presence of high concentrations of collagen. Although this could be a second site on GPVI that is activated by a novel motif within the collagen molecule, the absence of LAT phosphorylation in response to collagen in the presence of JAQ1 suggests that this is more likely to be caused by activation of a second receptor that is also coupled to the FcR ␥-chain. The possibility that this response is mediated by a receptor that is not coupled to FcR ␥-chain is excluded on the grounds that aggregation is absent in platelets from FcR ␥-chain-deficient mice.The platelet collagen receptor GPVI 1 plays a pivotal role in platelet activation following receptor cross-linking by collagen. This is highlighted by the impaired response to collagen in GPVI-deficient patients (2-4) and the bleeding disorders associated with this. GPVI is a 60 -65-kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily (5), which forms a complex with the FcR ␥-chain at the cell surface in human and mouse platelets (1, 6, 7). Signaling through GPVI occurs via a similar pathway to that used by immunoreceptors (8) as revealed by the tyrosine phosphorylation of the FcR ␥-chain immunoreceptor tyrosine-based activation motif (ITAM) by a Src-like kinase (9, 10). The Src-like kinases Fyn and Lyn are believed to be involved in this phosphorylation (9 -11). Syk is able to bind to the tyrosine-phosphorylated ITAM of the FcR ␥-chain via its tandem SH2 domains (12) leading to autophosphorylation and activation. Syk plays a critical role in the regulation of PLC␥2 through phosphorylation of a number of key proteins including the adapters LAT and SLP-76 (13-15). These signaling events occur upon colla...

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Evidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets

Cited by 64 publications

References 52 publications

Platelet adhesion signalling and the regulation of thrombus formation

Platelet adhesion signalling and the regulation of thrombus formation

Association of Fyn and Lyn with the Proline-rich Domain of Glycoprotein VI Regulates Intracellular Signaling

Evidence for Two Distinct Epitopes within Collagen for Activation of Murine Platelets

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