Integrin Associated Protein in a Thrombospondin Receptor

描述(申请人提供)：整合素相关蛋白(IAP，CD47)是一种普遍表达的凝血酶敏感蛋白(TSPs)和信号抑制调节蛋白a(SlRPA)的受体。CD47可与81、32和03整合素结合并增强其功能。因此，CD47信号的生物学效应依赖于特定细胞类型中表达的特定整合素，并可导致细胞运动、扩散、血小板激活、吞噬、氧化猝发甚至细胞凋亡。CD47的许多作用被百日咳毒素处理细胞所阻断，这表明异源三聚体GI家族蛋白在连接CD47与不同的整合素及其信号通路中具有共同的作用。我们分离了一个含有整合素、CD47和GI的洗涤剂可溶性复合体，发现该复合体的稳定性需要CD47和胆固醇，这为将该复合体定位于高胆固醇脂类区域提供了理论基础。我们现在已经建立了实验系统，用来确定这个信号复合体组件的相互作用位置和机制。目前建议的主要目标是阐明CD47、与其相关的整合素以及包括异三聚体GI和下游信号效应器在内的信号成分之间的分子相互作用。其具体目的是：(1)利用分子生物学和化学交联法确定负责CD47-整合素-GI复合体的组装和信号传递的分子相互作用。(2)利用一组单独和共同表达目的整合素和CD47的细胞，鉴定CD47/整合素信号复合体的组成，该复合体快速分离在mAb或配体包被的磁珠上。(3)利用共定位研究、抗体、多肽和化学抑制剂、显性负性和其他突变型蛋白的表达以及反义和基因缺失策略，测试AIM 2中确定的候选蛋白在CD47Ad~d整合素信号转导中的作用。

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.