Functional Cooperation of Tissue Factor and Integrins

描述（由申请人提供）：这一竞争性继续申请是基于三个主要发现，证明组织因子（TF），凝血级联的起始蛋白酶受体，调节与血管生成相关的整合素依赖性细胞迁移。(1) TF细胞外区域的一个特异性表位与β -1整合素依赖的上皮细胞迁移有关，TF与β -1整合素异源二聚体以及α -v- β -3相互作用。(2)在细胞内，磷脂酰肌醇-3激酶的p85亚基，一个参与细胞迁移的接头，以Ser 258磷酸化调节的方式与TF细胞质结构域结合。(3) TF细胞质结构域缺失小鼠血管生成和内皮细胞萌发增强，为TF在整合素功能中的生理调控作用提供了证据。我们提出表征内皮细胞中TF和整合素之间的相互作用，具体目的如下。目的1是绘制TF上的整合素结合位点，并表征内皮细胞中TF与整合素相关的特征。这些研究将定义TF的新型分子相互作用，并为解决TF“非止血”相互作用中的整合素特异性提供工具。目的2是表征内皮细胞TF细胞质结构域Ser 258磷酸化与整合素的关系。除了cys245的棕榈酰化调控外，Ser 258的磷酸化还受到细胞质域外相互作用的影响。该实验将确定整合素相互作用是否调节TF细胞质结构域磷酸化，Ser 258磷酸化的TF是否与特异性整合素相关，以及磷酸化如何调节TF和整合素组装到内皮细胞来源的微粒中。目的3是明确TF细胞质结构域在肿瘤生长和血管生成中的作用。本实验的目的是表征TF胞质结构域缺失小鼠的肿瘤发展特征，并在体内和体外实验中确定TF胞质结构域抑制血管生成的机制。总之，这些实验将表征整合素与内皮细胞迁移相关的TF合作的新途径。

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.