THROMBOSPONDIN 1 OPENS ENDOTHELIAL PARACELLULAR PATHWAY

血小板反应蛋白（TSP）-1是一种约420 kDa的三聚体糖蛋白，由包括内皮细胞（EC）在内的许多宿主组织分泌。 这种多结构域分子识别几种EC受体，并诱导多种有时相互冲突的信号传导事件和生物反应。 EC既能产生TSP-1，又能对TSP-1产生应答，这表明EC具有自分泌/旁分泌调节作用。 TSP-1包括EC附着和铺展在基质上、肌动蛋白组织、细胞运动和血管生成。 最近，我们已经证明TSP-1通过蛋白酪氨酸磷酸化打开肺血管内皮细胞旁通路。 粘附小带（ZA）是与肌动蛋白细胞骨架物理耦合并通过蛋白酪氨酸磷酸化调节的细胞间粘附连接。 在EC中，一组细胞质蛋白，统称为连环蛋白，形成多蛋白复合物，将肌动蛋白拴系到血管内皮（VE）-钙粘蛋白的细胞质结构域。钙粘蛋白是介导嗜同性细胞间粘附的表面受体。 我们现在已经证明，TSP-1诱导两个ZA组件，γ-连环蛋白和p120 Cas的酪氨酸磷酸化。 为了扩展我们的研究结果，我们提出了以下具体目标：1。 确定TSP-1中2个特异性序列的相互作用是否与EC受体相互作用，该受体与酪氨酸磷酸化事件和内皮细胞旁通路的开放偶联。 2. 确定TSP-1是否通过酪氨酸磷酸化依赖性ZA分解打开内皮细胞旁途径。 3. 确定TSP-1是否通过酪氨酸磷酸化依赖性肌动蛋白重组和/或破坏ZA-肌动蛋白细胞骨架连接打开内皮细胞旁途径。所提出的研究旨在鉴定TSP-1序列、EC受体和酪氨酸磷酸化事件，TSP-1通过这些事件干扰蛋白质-蛋白质相互作用，促进ZA分解、肌动蛋白重组和/或ZA-肌动蛋白细胞骨架连接的破坏，并最终打开内皮细胞旁途径。

Thrombospondin (TSP)-1 is an approximately 420kDa trimeric glycoprotein that is secreted by numerous host tissues including the endothelial cell (EC). This multidomain molecule recognizes several EC receptors and induces multiple and sometimes conflicting signaling events and biological responses. That EC can both produce and respond to TSP-1 suggests autocrine/ paracrine regulation. TSP-1 includes EC attachment to and spreading on substrates, actin organization, cell motility, and angiogenesis. Recently we have demonstrated that TSP-1 opens the pulmonary vascular endothelial paracellular pathway through protein tyrosine phosphorylation. The zonula adherens (ZA) is an intercellular adherens junction that is physically coupled to the actin cytoskeleton and regulated through protein tyrosine phosphorylation. In EC, a group of cytoplasmic proteins, collectively termed catenins, form a multiprotein complex that tethers actin to the cytoplasmic domain of vascular endothelial (VE)-cadherin. the Cadherins are surface receptors that mediate homophilic intercellular adhesion. We now have demonstrated that TSP-1 induces tyrosine phosphorylation of two ZA components, gamma-catenin and p120Cas. To extend our findings, we propose the following Specific Aims: 1. To determine whether the interaction(s) Of 2 specific sequence(s) within the TSP-1 interacts with an EC receptor(s) that is coupled to tyrosine phosphorylation events and opening of the endothelial paracellular pathway. 2. To determine whether TSP-1 opens the endothelial paracellular pathway through tyrosine phosphorylation-dependent ZA disassembly. 3. To determine whether TSP-1 opens the endothelial paracellular pathway through tyrosine phosphorylation-dependent actin reorganization and/or disruption of the ZA-actin cytoskeletal linkage. The proposed studies are designed to identify the TSP-1 sequence(s), the EC receptor, and the tyrosine phosphorylation events through which TSP-1 perturbs protein-protein interactions promoting ZA disassembly, actin reorganization, and/or disruption of ZA-actin cytoskeletal linkage, and finally, opening of the endothelial paracellular pathway.