Tissue Factor Dependent Par2 Signaling

凝血级联反应的激活与血管细胞信号转导密切相关 发展、炎症和癌症生物学。原代内皮细胞中的TF起始复合体激活PAR1和PAR2，但目前尚不清楚这些信号事件与特征更好的凝血酶信号有何不同。我们的初步数据表明，PAR2的激活特异性地导致了TF胞浆区域Ser258的磷酸化，Ser258是调节接头募集到TF胞浆区域的主要开关。此外，Tf胞浆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控作用。本项目的主要目的是通过体外实验来表征Tf和PAR2的信号串扰，并通过体内遗传学方法来验证Tf信号的意义。目的1是确定PAR2在转录因子胞浆结构域磷酸化中的特异性是如何产生的。我们将检验这一假设，即PAR2的羧基末端招募一种特定的激酶复合体，使TF磷酸化。我们将定位PAR2中的相关区域，建立Tf跨膜区的参与，表征arrestin的作用，并测试MAP激酶途径的Ser/Thr激酶是否使Tf磷酸化。这些实验将为如何在蛋白酶结合受体与PAR的共同信号中产生特异性提供新的见解。目的2是验证三元引发复合体激活PAR1和PAR2导致不同的运输和内化途径的假设。这些实验将表征PAR信号在调节血栓形成级联反应中的作用。目的3是确定缺失Tf胞浆结构域是否导致血管生成中PAR信号的失控。我们将分析Tf胞浆结构域缺失小鼠与PAR1或PAR2缺陷小鼠的遗传杂交是否可以消除其功能表型的获得。这些实验将产生 将Tf细胞质结构域与通过特定的PAR和TRAR的信号联系起来的遗传证据 在相关的活体模型中验证先前目标中的机制研究。在协作环境中 在项目4的努力下，我们将用最近发现的结合Tf细胞质结构域的适配器来阐明配体结合特异性的结构基础。因此，拟议的研究表征了Tf起始复合体的PAR2依赖细胞信号特异性的生化基础、细胞生物学和体内相关性。这些实验将为TF与发育、心血管疾病和血管生成相关的内皮细胞生物学提供新的见解。

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.