INTEGRIN ASSOCIATED PROTEIN/CD47 IS A THROMBOSPONDIN RECEPTOR

描述（由申请人提供）：血栓反应蛋白-1 （TSP1）及其受体长期以来被认为在调节血管细胞中具有重要作用，包括循环细胞和壁细胞。在之前的资助期间，我们已经发现TSP1和CD47（整合素相关蛋白）调节血管细胞中一氧化氮（NO）信号的动态范围。因此，TSP1-CD47相互作用不仅在血管生成调节中很重要，而且在组织灌注的快速调节和许多其他NO维持心血管系统健康的作用中也很重要。我们已经确定了TSP1的c端结构域上与CD47相互作用的结合表面。我们还发现了一种新的通过CD47激活整合素的细胞外机制。本文主要研究TSP1、CD47和23整合素之间的分子相互作用。这些相互作用的分子细节将通过所有三个相互作用伙伴的突变来推断。突变蛋白将在体外结合试验和CD47和整合素功能试验中进行测试。我们在1v23结构中发现的整合素激活的细胞外扣机制将在培养细胞和用表达活化的23整合素构建体的骨髓重新填充的23-null小鼠中进行测试。一个类似的方法将测试CD47通过与培养细胞和表达突变CD47转基因的小鼠中的环相互作用激活23个整合素的能力。TSP1-CD47和cd47 -整合素复合物形成的条件将使用物理方法和几种EM方法进行评估。这些方法将用于评估突变蛋白的相互作用，以确定每个突变蛋白的结构元素，这些结构元素对它们的分子相互作用很重要。基于这些结果，CD47与TSP1 c端结构域和1v23整合素配合物的结晶和x射线衍射研究将开始。这些研究将首次提供tsp1 - cd47 -整合素相互作用的物理模型。鉴于我们的血管系统中TSP1-CD47调控NO信号的新范式，这一信息将对设计调节内源性NO信号的新策略至关重要，这是改善心血管疾病的新方法。

DESCRIPTION (provided by applicant): Thrombospondin-1 (TSP1) and its receptors have long been thought to have important roles in regulating vascular cells, both circulating and mural. During the preceding grant period we have discovered that TSP1 and CD47 (integrin-associated protein) regulate the dynamic range of nitric oxide (NO) signaling in vascular cells. Thus TSP1-CD47 interactions are important not only in angiogenic regulation but in rapid regulation of tissue perfusion and many other roles where NO maintains the health of the cardiovascular system. We have identified the binding surface on the C-terminal domain of TSP1 that interacts with CD47. We have also discovered a new extracellular mechanism for integrin activation via CD47. This proposal focuses on the molecular interactions among TSP1, CD47 and 23 integrins. The molecular details of these interactions will be deduced by mutagenesis of all three interacting partners. Mutant proteins will be tested in vitro in binding assays and assays of CD47 and integrin function. The extracellular clasp mechanism for integrin activation that we identified within the 1v23 structure will be tested in cultured cells and in 23-null mice repopulated with bone marrow expressing activated 23 integrin constructs. A similar approach will test the ability of CD47 to activate 23 integrins via interaction with the clasp in cultured cells and in mice expressing mutant CD47 transgenes. Conditions for formation of TSP1-CD47 and CD47-integrin complexes will be evaluated using physical methods and several EM approaches. These methods will then be used to evaluate the interactions of mutant proteins in order to identify structural elements of each that are important for their molecular interactions. Based on these results, crystallization and X-ray diffraction studies will be initiated of CD47 in complexes with the TSP1 C-terminal domain and with 1v23 integrin. The sum of these studies will provide for the first time a physical model of TSP1-CD47-integrin interactions. Given our new paradigm for TSP1-CD47 regulation of NO signaling in the vascular system, this information will be vital in designing new strategies to modulate endogenous NO signaling, a new approach to ameliorating cardiovascular disease.