Tissue Factor Dependent Par2 Signaling

组织因子依赖性 Par2 信号传导

基本信息

批准号：
7432439
负责人：
Ruf Wolfram
金额：
$ 50.5万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-01 至 2009-05-31
项目状态：
已结题

项目摘要

The activation of the coagulation cascade is intimately linked to vascular cell signaling in development, inflammation and cancer biology. The TF initiation complex in primary endothelial cells activates PAR1 and PAR2, but it is currently poorly understood how these signaling events are distinct from the better characterized thrombin signaling. Our preliminary data show that activation of PAR2 specifically results in TF cytoplasmic domain Ser258 phosphorylation which serves as the major switch to regulate adaptor recruitment to the TF cytoplasmic domain. Furthermore, TF cytoplasmic domain deleted mice show that the intracellular domain has negative regulatory roles in angiogenesis in vivo. The primary objective of this project is to characterize the signaling cross-talk of TF with PAR2 by in vitro experiments and to validate significance of TF signaling by genetic approaches in vivo. Aim 1 is to define how the PAR2 specificity in phosphorylation of the TF cytoplasmic domain is generated. We will test the hypothesis that the carboxyl-terminus of PAR2 recruits a specific kinase complex that phosphorylates TF. We will map the relevant region in PAR2, establish the involvement of the transmembrane domain of TF, characterize the role of arrestin and test whether Ser/Thr kinases of the MAP kinase pathway phosphorylate TF. These experiments will provide novel insight into how specificity is created in the co-signaling of protease-binding receptors with PARs. Aim 2 is to test the hypothesis that PAR1 and PAR2 activation by the ternary initiation complex results in distinct trafficking and internalization routes. These experiments will characterize the role of PAR signaling in the regulation of the thrombogenic cascade. Aim 3 is to determine whether deletion of the TF cytoplasmic domain results in deregulated PAR signaling in angiogenesis. We will analyze whether the gain of function phenotype of TF cytoplasmic domain deleted mice can be ablated by genetic crosses with PAR1 or PAR2 deficient mice. These experiments will generate genetic evidence that links the TF cytoplasmic domain to signaling through a specific PAR and validate the mechanistic studies in the previous aims in a relevant in vivo model. In a collaborative effort with Project 4, we will elucidate the structural basis of ligand binding specificity with recently identified adaptors that bind the TF cytoplasmic domain. The proposed studies thus characterize the biochemical basis, cell biology and in vivo relevance of the PAR2-dependent cell signaling specificity of the TF initiation complex. These experiments will provide novel insight into the endothelial cell biology of TF of relevance for development, cardiovascular disease and angiogenesis.

凝血级联的激活与血管细胞信号传导密切相关发育、炎症和癌症生物学。原代内皮细胞中的 TF 起始复合物激活 PAR1 和 PAR2，但目前人们对这些信号转导事件如何与更好表征的凝血酶信号转导不同还知之甚少。我们的初步数据表明，PAR2 的激活特异性导致 TF 细胞质结构域 Ser258 磷酸化，这是调节接头招募至 TF 细胞质结构域的主要开关。此外，TF胞质结构域缺失的小鼠表明胞内结构域在体内血管生成中具有负调节作用。该项目的主要目标是通过体外实验表征 TF 与 PAR2 的信号传导串扰，并通过体内遗传方法验证 TF 信号传导的重要性。目标 1 是确定 TF 胞质结构域磷酸化中的 PAR2 特异性是如何产生的。我们将检验以下假设：PAR2 的羧基末端招募一种磷酸化 TF 的特定激酶复合物。我们将绘制 PAR2 中的相关区域图，确定 TF 跨膜结构域的参与，表征抑制蛋白的作用，并测试 MAP 激酶途径的 Ser/Thr 激酶是否磷酸化 TF。这些实验将为了解如何在蛋白酶结合受体与 PAR 的共同信号传导中产生特异性提供新的见解。目标 2 是检验三元起始复合物激活 PAR1 和 PAR2 导致不同运输和内化途径的假设。这些实验将表征 PAR 信号传导在血栓形成级联调节中的作用。目标 3 是确定 TF 胞质结构域的缺失是否会导致血管生成中 PAR 信号传导失调。我们将分析 TF 胞质结构域缺失小鼠的功能表型增益是否可以通过与 PAR1 或 PAR2 缺陷小鼠的遗传杂交而消除。这些实验将产生通过特定 PAR 将 TF 胞质结构域与信号传导联系起来的遗传证据在相关的体内模型中验证先前目标的机制研究。在协作中通过项目 4，我们将通过最近鉴定的结合 TF 胞质结构域的接头来阐明配体结合特异性的结构基础。因此，拟议的研究描述了 TF 起始复合物的 PAR2 依赖性细胞信号传导特异性的生化基础、细胞生物学和体内相关性。这些实验将为与发育、心血管疾病和血管生成相关的 TF 内皮细胞生物学提供新的见解。