INTEGRIN ASSOCIATED PROTEIN/CD47 IS A THROMBOSPONDIN RECEPTOR

描述（由申请人提供）：血栓反应蛋白-1（TSP 1）及其受体长期以来被认为在调节循环和壁血管细胞中具有重要作用。在之前的研究期间，我们发现TSP 1和CD 47（整合素相关蛋白）调节血管细胞中一氧化氮（NO）信号传导的动态范围。因此，TSP 1-CD 47相互作用不仅在血管生成调节中重要，而且在组织灌注的快速调节和NO维持心血管系统健康的许多其他作用中也很重要。我们已经确定了与CD 47相互作用的TSP 1的C-末端结构域上的结合表面。我们还发现了一种新的细胞外机制，通过CD 47激活整合素。本研究的重点是TSP 1、CD 47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伴侣的诱变来推导。将在体外结合试验以及CD 47和整联蛋白功能试验中检测突变蛋白。我们在1v 23结构中鉴定的整合素活化的细胞外卡环机制将在培养的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缺失小鼠中进行测试。类似的方法将测试CD 47通过与培养细胞和表达突变体CD 47转基因的小鼠中的卡环相互作用来激活23种整联蛋白的能力。将使用物理方法和几种EM方法评价TSP 1-CD 47和CD 47-整联蛋白复合物形成的条件。然后，这些方法将用于评估突变蛋白质的相互作用，以确定对它们的分子相互作用重要的每个结构元件。基于这些结果，将启动CD 47与TSP 1 C末端结构域和1v 23整联蛋白复合物的结晶和X射线衍射研究。这些研究的总和将首次提供TSP 1-CD 47-整联蛋白相互作用的物理模型。鉴于我们的新范式TSP 1-CD 47调节NO信号在血管系统中，这一信息将是至关重要的，在设计新的策略来调节内源性NO信号，一种新的方法来改善心血管疾病。

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.