Functional Cooperation of Tissue Factor and Integrins

描述（由申请人提供）：本竞争性继续申请基于三项主要发现，这些发现证明组织因子（TF）（凝血级联反应的起始蛋白酶受体）调节与血管生成相关的整合素依赖性细胞迁移。(1)TF细胞外结构域中的特异性表位涉及β-1-整联蛋白依赖性上皮细胞迁移，TF与β-1整联蛋白异二聚体亚群以及α-v-β-3相互作用。(2)在细胞内侧，磷脂酰肌醇-3激酶的p85亚基（参与细胞迁移的衔接子）以Ser 258磷酸化调节的方式结合TF胞质结构域。(3)TF胞质结构域缺失的小鼠表现出增强的血管生成和内皮细胞发芽，为TF在整合素功能中的生理相关调节作用提供了证据。我们提出的特点TF和整合素之间的相互作用在内皮细胞与以下具体目标。目的1是定位TF上的整合素结合位点，并表征TF在内皮细胞中与哪些整合素结合。这些研究将定义TF的新分子相互作用，并提供工具来解决TF的“非止血”相互作用中的整合素特异性。目的二是研究内皮细胞中整合素与TF胞浆区Ser 258磷酸化的关系。除了通过Cys 245的棕榈酰化调节外，Ser 258磷酸化还受到胞质结构域外的相互作用的影响。拟议的实验将建立是否整合素相互作用调节TF胞质结构域磷酸化，是否Ser 258磷酸化TF与特定的整合素，以及如何TF和整合素组装成内皮细胞衍生的微粒是由磷酸化调节。目的3是明确TF胞浆区在肿瘤生长和血管生成中的作用。所提出的实验的目标是表征TF胞质结构域缺失小鼠中的肿瘤发展，并定义TF胞质结构域在离体和体外测定中抑制血管生成的机制。总之，这些实验将表征整合素与TF合作与内皮细胞迁移相关的新途径。

DESCRIPTION (provided by applicant): This competing continuation application is based on three major findings that demonstrate that tissue factor (TF), the initiating protease receptor of the coagulation cascade, regulates integrin-dependent cell migration of relevance for angiogenesis. (1) A specific epitope in the TF extracellular domain is implicated in beta-1-integrin-dependent epithelial cell migration and TF interacts with a subset beta-1 integrin heterodimers as well as alpha-v-beta-3. (2) On the intracellular side, the p85 subunit of phosphatidylinositol-3 kinase, an adaptor involved in cell migration, binds to the TF cytoplasmic domain in a Ser 258 phosphorylation regulated manner. (3) TF cytoplasmic domain-deleted mice show enhanced angiogenesis and endothelial cell sprouting, providing evidence for a physiologically relevant regulatory role of TF in integrin function. We propose to characterize the interaction between TF and integrins in endothelial cells with the following specific aims. Aim 1 is to map the integrin binding site on TF and to characterize with which integrins TF associates in endothelial cells. These studies will define novel molecular interactions of TF and provide tools to address integrin-specificity in the "non-hemostatic" interactions of TF. Aim 2 is to characterize the relationship of integrins and Ser 258 phosphorylation of the TF cytoplasmic domain in endothelial cells. In addition to regulation by palmitoylation of Cys 245, Ser 258 phosphorylation is influenced by interactions outside the cytoplasmic domain. The proposed experiments will establish whether integrin interactions regulate TF cytoplasmic domain phosphorylation, whether Ser 258 phosphorylated TF is associated with specific integrins, and how TF and integrin assembly into endothelial cell-derived microparticles is regulated by phosphorylation. Aim 3 is to define the role of the TF cytoplasmic domain in tumor growth and angiogenesis. The goals of the proposed experiments are to characterize tumor development in TF cytoplasmic domain deleted mice and to define the mechanism by which the TF cytoplasmic domain suppresses angiogenesis in ex vivo and in vitro assays. Together, these experiments will characterize a novel pathway of integrin cooperation with TF of relevance for endothelial cell migration.