Tissue Factor Dependent Par2 Signaling

组织因子依赖性 Par2 信号传导

• 批准号: 7629128
• 负责人: Ruf Wolfram
• 金额: $ 49.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7629128
• 关键词: Arrestin; Arrestins; Binding; Biochemical; Blood Vessels; Cancer Biology; Cardiovascular Diseases; Cellular biology; Coagulation Process; Complex; Cytoplasmic Tail; Data; Development; Endopeptidases; Endothelial Cells; Event; F2R gene; Genetic; Genetic Crosses; In Vitro; Inflammation; Ligand Binding; Link; Maps; Mitogen-Activated Protein Kinase Kinases; Mus; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphorylation; Phosphotransferases; Recruitment Activity; Regulation; Role; Route; Signal Transduction; Specificity; Testing; Thrombin; Thromboplastin; Transmembrane Domain; angiogenesis; base; gain of function; in vivo; in vivo Model; insight; novel; receptor binding; research study; trafficking

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.

凝血级联反应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PAR 1和PAR 2，但目前对这些信号传导事件如何与更好表征的凝血酶信号传导不同知之甚少。我们的初步数据表明，PAR 2的激活特异性地导致TF胞质结构域Ser 258磷酸化，其作为调节衔接子募集到TF胞质结构域的主要开关。此外，TF胞浆结构域缺失小鼠显示胞内结构域在体内血管生成中具有负调节作用。本研究的主要目的是通过体外实验研究TF与PAR 2之间的信号转导，并通过体内遗传学方法验证TF信号转导的重要性。目的1是确定PAR 2在TF胞质结构域磷酸化中的特异性是如何产生的。我们将测试的假设，羧基末端的PAR 2招募一个特定的激酶复合物，磷酸化TF。我们将绘制PAR 2中的相关区域，确定TF跨膜结构域的参与，表征arrestin的作用，并测试MAP激酶途径的Ser/Thr激酶是否磷酸化TF。这些实验将提供新的见解如何特异性的蛋白酶结合受体与PAR的共同信号。目的2是检验PAR 1和PAR 2通过三元起始复合物激活导致不同的运输和内化途径的假设。这些实验将表征PAR信号在血栓形成级联调节中的作用。目的3是确定TF胞质结构域的缺失是否导致血管生成中PAR信号的失调。我们将分析TF胞质结构域缺失小鼠的功能表型的获得是否可以通过与PAR 1或PAR 2缺陷小鼠的遗传杂交来消除。这些实验将产生 将TF胞质结构域与通过特定PAR的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中的机制研究。以协作 通过项目4的努力，我们将阐明与最近鉴定的结合TF胞质结构域的衔接子的配体结合特异性的结构基础。因此，拟议的研究表征TF起始复合物的PAR 2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。这些实验将提供新的洞察内皮细胞生物学TF的发展，心血管疾病和血管生成的相关性。