PLATELET ACTIVATION SIGNALING VIA GPIB-IX 14-3-3

描述：（研究者摘要）血小板膜糖蛋白Ib-IX 复合物（GP Ib-IX）在血小板初始粘附至 损伤血管壁。通过与内皮下血管性血友病因子结合 (vWF)GP Ib-IX不仅介导血小板的物理粘附， 启动激活信号。这会激活血小板整合素 α IIb β 3，其随后介导血小板扩散和聚集。 拟议研究的总体目标是确定GP的机制 Ib-IX介导的信号传导。我们发现GP Ib-IX的胞质结构域 与重要的细胞内信号分子zeta-14 - 3 - 3相互作用 蛋白质（14 - 3 - 3 ζ）。14-3-3 zeta的功能是磷酸化依赖的 连接激酶和其他分子的接头，形成信号复合物。 14-3-3 zeta调节c-Raf和几种蛋白激酶亚型的功能 C.拟议中的研究将检验14 - 3 - 3zeta是一个关键环节的假设， GP Ib-IX和细胞内信号通路之间的联系。在支持 研究，我们已经表征了GP Ib-IX-14 - 3 - 3的结构基础 相互作用，并开发了一系列14 - 3 - 3zeta缺失突变体， GP I α结合位点或raf激酶结合位点。此外，我们还表明， 我们能够在一种新的细胞内重建GP Ib-IX介导的整合素激活。 重组DNA在哺乳动物细胞中的稳定表达模型。这将使我们 应用分子生物学技术（不能用于无核 血小板），以具体检查14 - 3 - 3zeta和其他信号传导的作用 GP Ib-IX介导的信号传导中的分子。我们建议（1）重组和 表征哺乳动物细胞中GPI b-IX介导的信号传导途径 （2）确定14 - 3 - 3zeta和GP Ib-IX介导的 使用哺乳动物细胞表达模型和14 - 3 - 3的突变体和 （3）鉴定14 - 3 - 3介导的GPI b-IX信号传导的下游通路; 和（4）确定GP Ib-IX的磷酸化在调节 在GP Ib-IX介导的细胞凋亡中，GP Ib-IX和14 - 3 - 3zeta之间的相互作用 发信号。

DESCRIPTION: (Investigator's abstract) The platelet membrane glycoprotein Ib-IX complex (GP Ib-IX) plays an important role in the initial platelet adhesion to injured vascular wall. By binding to subendothelial von Willebrand factor (vWF), GP Ib-IX not only mediates the physical adherence of platelets, but also initiates an activation signal. This activates the platelet integrin alphaIIbbeta3 which subsequently mediates platelet spreading and aggregation. Overall objective of the proposed studies is to determine the mechanism of GP Ib-IX-mediated signaling. We have found that the cytoplasmic domain of GP Ib-IX interacts with an important intracellular signaling molecule, zeta-form 14-3-3 protein (14-3-3zeta). 14-3-3zeta functions as a phosphorylation-dependent adapter that links kinases and other molecules, forming signaling complex. 14-3-3zeta regulates functions of c-Raf and several isoforms of protein kinase C. The proposed studies will test the hypothesis that 14-3-3zeta is a key link between GP Ib-IX and the intracellular signaling pathway. In supporting studies, we have characterized the structural basis of GP Ib-IX-14-3-3 interaction, and developed a series of 14-3-3zeta deletion mutants containing GP Ibalpha binding site or raf kinase binding site. Further, we have shown that we are able to reconstitute GP Ib-IX-mediated integrin activation in a recombinant DNA stable expression model in mammalian cells. This will enable us to apply molecular biological techniques (which cannot be used in the anucleate platelet) to specifically examine the roles of 14-3-3zeta and other signaling molecules in GP Ib-IX-mediated signaling. We propose to (1) reconstitute and characterize the GPI b-IX-mediated signaling pathway in a mammalian cell expression model; (2) determine the roles of 14-3-3zeta and GP Ib-IX-mediated signaling using the mammalian cell expression model and mutants of 14-3-3 and GP Ib; (3) identify downstream pathways of 14-3-3 mediated GPI b-IX signaling; and (4) determine the roles of phosphorylation of GP Ib-IX in the regulation of the interaction between GP Ib-IX and 14-3-3zeta in the GP Ib-IX-mediated signaling.