A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets:: Involvement of G6f, a novel platelet Grb2-binding membrane adapter
A global proteomics approach identifies novel phosphorylated signaling proteins in GPVI-activated platelets:: Involvement of G6f, a novel platelet Grb2-binding membrane adapter
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DOI：
10.1002/pmic.200600299
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发表时间：
2006-10-01
期刊：
影响因子：
3.4
通讯作者：
Zitzmann, Nicole
中科院分区：
文献类型：
--
作者：
Garcia, Angel;Senis, Yotis A.;Zitzmann, Nicole
胶原相关肽 (CRP) 通过糖蛋白 VI (GPVI)-FcR 伽玛链复合物刺激血小板的强力活化。我们结合蛋白质组学和传统生物化学方法来研究 CRP 激活血小板的蛋白质组，重点关注酪氨酸磷酸化。在两种不同的方法中，使用磷酸酪氨酸免疫沉淀，然后进行 1-D-PAGE 和 2-DE 来分离蛋白质。通过 MS 鉴定蛋白质。通过采用这些方法，发现 96 种蛋白质会响应人血小板中的 CRP 进行 PTM，其中包括 11 种新型血小板蛋白，例如 Dok-1、SPIN90、破骨细胞刺激因子 1 和 beta-Pix。有趣的是，I 型跨膜蛋白 G6f 被发现在 Tyr-281 上特异性磷酸化，以响应 CRP 激活血小板，为接头 Grb2 提供对接位点。还发现 G6f 酪氨酸磷酸化会响应胶原蛋白而发生，但不会响应 G 蛋白偶联受体激动剂、凝血酶和 ADP。此外，我们还首次证明 Grb2 及其同源 Gad 在 CRP 刺激的血小板中被酪氨酸磷酸化。该研究为通过GPVI胶原蛋白受体激活血小板的机制提供了新的见解，有助于为血栓性疾病新药物靶点的开发奠定基础。
Collagen-related-peptide (CRP) stimulates powerful activation of platelets through the glycoprotein VI (GPVI)-FcR gamma-chain complex. We have combined proteomics and traditional biochemistry approaches to study the proteome of CRP-activated platelets, focusing in detail on tyrosine phosphorylation. In two separate approaches, phosphotyrosine immunoprecipitations followed by 1-D-PAGE, and 2-DE, were used for protein separation. Proteins were identified by MS. By following these approaches, 96 proteins were found to undergo PTM in response to CRP in human platelets, including 11 novel platelet proteins such as Dok-1, SPIN90, osteoclast stimulating factor 1, and beta-Pix. Interestingly, the type I transmembrane protein G6f was found to be specifically phosphorylated on Tyr-281 in response to platelet activation by CRP, providing a docking site for the adapter Grb2. G6f tyrosine phoshporylation was also found to take place in response to collagen, although not in response to the G protein-coupled receptor agonists, thrombin and ADP. Further, we also demonstrate for the first time that Grb2 and its homolog Gads are tyrosine-phosphorylated in CRP-stimulated platelets. This study provides new insights into the mechanism of platelet activation through the GPVI collagen receptor, helping to build the basis for the development of new drug targets for thrombotic disease.