FIBRONECTIN AND INTEGRIN INTERACTIONS

细胞外基质的组成和结构在心血管系统的模式形成中起着核心作用。除了发育作用外，细胞外基质还经常引发血栓的形成。项目2的重点是控制细胞外基质的一种成分--不溶性纤维连接蛋白纤维--组装的基本机制。这些研究将解决控制整合素功能的细胞内机制，整合素功能是基质组装过程中必不可少的。特别是，这个项目将分析整合素α5β1的高亲和力形式“激活”的机制反应及其与细胞骨架的物理联系。这些目标将通过分离整合素α5β1激活所需的细胞通路缺陷的细胞系来实现。然后，表达cdna克隆将识别补充这些缺陷的基因。另一种策略是利用互补辐射杂交体的富集化来确定互补基因的染色体位置。然后，通过使用序列标记的位点进一步定位这些基因，通过用P1克隆转染来挽救，最后通过转染候选cDNA来挽救。整合素-细胞骨架连接的分析将使用新的重组结构模拟物的β细胞质结构域，并分析它们与两个肌动蛋白结合蛋白，丝氨酸和Talin的相互作用。整合素和细胞骨架蛋白中的相互作用部位将被绘制出来。这些信息将被用来检验这样的假设，即这些蛋白质与不同整合素的不同结合导致了整合素特有的功能。这些研究将为血管细胞细胞外基质的关键成分的组装提供基本的见解。

The composition and structure of the extracellular matrix plays a central role in the patterning of the cardiovascular system. In addition to its developmental role, the extracellular matrix often initiates thrombus formation. Project 2 is focused on the fundamental mechanisms controlling the assembly of one component of the extracellular matrix, insoluble fibronectin fibrils. The studies will address intracellular mechanisms that control integrin functions essential for the matrix assembly process. In particular, this project will analyze the mechanisms response for "activation" of the high affinity form of integrin alpha5beta1 and for its physical linkage to the cytoskeleton. These aims will be accomplished by isolating cell lines with defects in cellular pathways required for activation of integrin alpha5beta1. Expression cDNA cloning will then identify Genes that complement those defects. An alternative strategy will employ enrichment of complemented radiation hybrids to identify chromosomal positions of complementing genes. The genes will then be further localized through use of sequence tagged sites, rescue by transfection with P1 clones and finally rescue by transfection of candidate cDNAs. Analysis of integrin-cytoskeleton linkage will employ novel recombinant structural mimics of the beta cytoplasmic domains and analyze their interactions with two actin binding proteins, filamin and talin. Interactive sites in the integrin and the cytoskeletal proteins will be mapped. This information will be used to test the hypothesis that the differential binding of these proteins to different integrins leads to integrin-specific functions. The studies will provide fundamental insight into the assembly of a critical component of the extracellular matrix of vascular cells.