THROMBOSPONDIN 1 OPENS ENDOTHELIAL PARACELLULAR PATHWAY

凝血酶原蛋白(TSP)-1是一种大小约为420 kDa的三聚体糖蛋白，由包括内皮细胞在内的多种宿主组织分泌。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事件和生物反应。EC既能产生TSP-1，又能对TSP-1作出反应，这提示了自分泌/旁分泌调节。TSP-1包括EC在底物上的附着和扩散、肌动蛋白的组织、细胞运动和血管生成。最近，我们发现TSP-1通过蛋白酪氨酸磷酸化打开肺血管内皮细胞旁细胞通路。ZA是一种细胞间黏附连接，物理上与肌动蛋白细胞骨架偶联，并通过蛋白质酪氨酸磷酸化来调节。在EC中，一组细胞质蛋白，统称为连接素，形成一个多蛋白复合体，将肌动蛋白连接到血管内皮细胞(VE)-钙粘附素的细胞质区域。钙粘附素是一种表面受体，可以介导细胞间的同嗜性黏附。我们现在已经证明，TSP-1诱导两个ZA组分--γ-连环素和p120Cas的酪氨酸磷酸化。为了扩大我们的发现，我们提出了以下具体目标：1.确定TSP-1中2个特定序列(S)的相互作用(S)是否与EC受体(S)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有关。2.确定TSP-1是否通过酪氨酸磷酸化依赖的ZA分解开放内皮细胞旁通路。3.确定TSP-1是否通过酪氨酸磷酸化依赖的肌动蛋白重组和/或破坏ZA-肌动蛋白细胞骨架连接而开放内皮细胞旁通路。这些研究旨在确定TSP-1序列(S)、EC受体和酪氨酸磷酸化事件，TSP-1通过这些事件扰乱促进ZA分解、肌动蛋白重组和/或破坏ZA-肌动蛋白细胞骨架连接的蛋白质-蛋白质相互作用，最终开放内皮细胞旁细胞途径。

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.