Integrin Associated Protein in a Thrombospondin Receptor

描述（由申请人提供）：整合素相关蛋白（IAP，CD 47）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。CD 47可以与β 1、β 2和β 3整合素结合并增强其功能。因此，CD 47信号传导的生物学后果取决于在给定细胞类型中表达的特定整联蛋白，并且可以导致增强的细胞运动性、扩散、血小板活化、吞噬作用、氧化爆发和甚至凋亡。CD 47的许多作用被细胞的百日咳毒素处理阻断，表明异源三聚体Gi家族蛋白在将CD 47连接至不同整联蛋白及其信号传导途径中的共同作用。我们已经分离出含有整联蛋白、CD 47和Gi的去污剂可溶性复合物，并发现该复合物的稳定性需要CD 47和胆固醇，这为该复合物在富含胆固醇的脂质结构域中的定位提供了理论基础。我们现在已经建立了实验系统，以确定该信号复合物的组分的相互作用的位点和机制。本提案的主要目标是阐明CD 47、与其相关的整合素和信号传导组分（包括异源三聚体Gi和下游信号传导效应物）之间的分子相互作用。具体目标是：（1）使用分子生物学和化学交联方法来定义负责CD 47-整联蛋白-Gi复合物的组装和信号传导的分子相互作用。(2)采用一组单独和共同表达目标整合素和CD 47的细胞，鉴定在mAb或配体包被磁珠上快速分离的CD 47/整合素信号传导复合物的组分。(3)使用共定位研究、抗体、肽和化学抑制剂（如可用）、显性阴性和其他突变形式的蛋白质表达以及反义和遗传缺失策略，检测Aim 2中鉴定的候选蛋白质在CD 47 α-d整联蛋白信号传导中的作用。

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.