Integrin Associated Protein in a Thrombospondin Receptor

血小板反应蛋白受体中的整合素相关蛋白

• 批准号: 6752865
• 负责人: WILLIAM A FRAZIER
• 金额: $ 38.25万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752865
• 关键词: CD47 molecule; antisense nucleic acid; biological signal transduction; chemical association; clone cells; crosslink; fluorescence microscopy; guanine nucleotide binding protein; immunomagnetic separation; integrins; monoclonal antibody; protein engineering; protein kinase; protein localization; protein protein interaction; protein tyrosine phosphatase; proteomics; thrombospondins; tissue /cell culture

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.

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）的普遍表达的受体。 CD47 可以与 81、32 和 03 整合素结合并增强其功能。因此，CD47信号传导的生物学结果取决于给定细胞类型中表达的特定整合素，并且可以导致细胞运动、扩散、血小板活化、吞噬作用、氧化爆发甚至细胞凋亡增强。 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 家族蛋白在将 CD47 与不同整合素及其信号传导途径连接方面具有共同作用。我们分离出了一种含有整合素、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，这为将该复合物定位在富含胆固醇的脂质结构域中提供了理论依据。我们现在已经建立了实验系统，用于确定该信号复合物成分相互作用的位点和机制。当前提案的主要目标是阐明 CD47、与其关联的整合素以及包括异三聚体 Gi 和下游信号传导效应器在内的信号传导成分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 CD47-整合素-Gi 复合物的组装和信号传导的分子相互作用。 (2) 使用一组单独和一起表达目的整合素和 CD47 的细胞，在 mAb 或配体包被的磁珠上快速分离 CD47/整合素信号传导复合物的成分。 (3) 使用共定位研究、可用的抗体、肽和化学抑制剂、显性失活和其他蛋白质突变形式的表达以及反义和基因删除策略来测试目标2中鉴定的候选蛋白质在CD47和整联蛋白信号传导中的作用。