Integrin Associated Protein in a Thrombospondin Receptor

描述（由申请人提供）：整合素相关蛋白（IAP, CD47）是血小板反应蛋白（tsp）和信号抑制调节蛋白a （SlRPa）的普遍表达受体。CD47可以与81、32和03个整合蛋白结合并增强其功能。因此，CD47信号传导的生物学后果取决于特定细胞类型中表达的特定整合素，并可导致细胞运动性增强、扩散、血小板活化、吞噬、氧化破裂甚至凋亡。CD47的许多作用被百日咳毒素处理细胞阻断，这表明异源三聚体Gi家族蛋白在将CD47连接到不同整合素及其信号通路中的共同作用。我们已经分离出一种含有整合素、CD47和Gi的洗涤剂可溶性复合物，并发现该复合物的稳定性需要CD47和胆固醇，这为该复合物定位于富含胆固醇的脂质结构域提供了理论依据。我们现在已经建立了实验系统来确定这个信号复合体的组成部分的相互作用的位置和机制。本研究的主要目标是阐明CD47、与其相关的整合素和包括异三聚体Gi和下游信号效应物在内的信号成分之间的分子相互作用。具体目标是：(1)利用分子生物学和化学交联方法确定cd47 -整合素- gi复合物组装和信号传导的分子相互作用。(2)鉴定CD47/整合素信号复合物的组成部分，快速分离在单抗或配体包被的磁珠上，使用一组细胞单独或一起表达感兴趣的整合素和CD47。(3)通过共定位研究、抗体、多肽和化学抑制剂、显性阴性和其他突变形式的蛋白表达、反义和基因缺失策略，测试Aim 2中鉴定的候选蛋白在CD47 ar~d整合素信号传导中的作用。

DESCRIPTION (provided by applicant): Integrin-associated protein (IAP, CD47) is a ubiquitously expressed receptor for thrombospondins (TSPs) and for Signal Inhibitory Regulatory Protein a (SlRPa). CD47 can associate with, 81, 32 and 03 integrins and augment their functions. Thus the biological consequence of CD47 signaling depends on the particular integrin expressed in a given cell type and can lead to enhanced cell motility, spreading, platelet activation, phagocytosis, oxidiative burst and even apoptosis. Many of the effects of CD47 are blocked by pertussis toxin treatment of cells indicating a common role for heterotrimeric Gi family proteins in connecting CD47 to different integrins and their signaling pathways. We have isolated a detergent soluble complex containing integrin, CD47 and Gi and find that stability of this complex requires CD47 and cholesterol, providing a rationale for localization of the complex in cholesterol-rich lipid domains. We have now established experimental systems with which to determine the sites and mechanisms of interaction of the components of this signaling complex. The primary goals of the current proposal are to elucidate the molecular interactions among CD47, the integrins with which it associates and signaling components including heterotrimeric Gi and downstream signaling effectors. The specif~c aims are: (1) To define the molecular interactions responsible for assembly of and signaling by the CD47-integrin-Gi complex using molecular biological and chemical crosslinking approaches. (2) To identify the components of the CD47/integrin signaling complexes, rapidly isolated on mAb- or ligand-coated magnetic beads, employing a panel of cells expressing the integrin of interest and CD47 singly and together. (3) To test the roles of candidate proteins identified in Aim 2 in CD47 ar~d integrin signaling using co-localization studies, antibody, peptide and chemical inhibitors as available, expression of dominant negative and other mutant forms of the proteins, and antisense and genetic deletion strategies.