FIBRONECTIN AND INTEGRIN INTERACTIONS

细胞外基质的组成和结构在心血管系统的形成中起着核心作用。除了它的发育作用，细胞外基质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个组成部分，不溶性纤维连接蛋白原纤维组装的基本机制。该研究将解决细胞内机制，控制整合素的功能至关重要的基质组装过程。特别是，本项目将分析高亲和形式的整合素alpha5beta1的“激活”及其与细胞骨架的物理联系的机制。这些目标将通过分离具有整合素α 5β 1激活所需细胞通路缺陷的细胞系来实现。cDNA克隆将鉴定出弥补这些缺陷的基因。另一种策略将采用富集互补辐射杂交体来鉴定互补基因的染色体位置。然后，基因将通过使用序列标记位点进一步定位，通过转染P1克隆进行修复，最后通过转染候选cdna进行修复。整合素-细胞骨架连接的分析将采用新的重组结构模拟β细胞质结构域，并分析它们与两种肌动蛋白结合蛋白，丝蛋白和talin的相互作用。整合素和细胞骨架蛋白的相互作用位点将被绘制出来。这一信息将用于验证这样一个假设，即这些蛋白质与不同整合素的差异结合导致整合素特异性功能。这些研究将为血管细胞细胞外基质的关键组成部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.