CELL/ECM LINKAGE VIA NG2 PG--TYPE VI COLLAGEN COMPLEX

描述：能够与的特定组件交互 细胞外基质(ECM)对正常发育和 细胞的功能。目前的工作主要集中在综合能力的培养上。 膜蛋白多糖NG2通过与细胞外基质结合介导与细胞外基质的相互作用 VI型胶原蛋白。免疫共沉淀实验，以及 免疫组织化学共定位和共同封顶研究表明， NG2和VI型胶原之间存在物理相互作用。此外, 将NG2基因导入NG2阴性细胞系 能够将VI型胶原锚定在细胞表面，证实 NG2作为一种有效的细胞表面受体的能力 VI胶原蛋白。 此应用程序包含旨在增加对 NG2-VI型胶原在分子水平上的相互作用并提供 洞察这种相互作用在细胞生理学中扮演的角色。 具体目标1侧重于确定NG2核心的结构域 负责与VI型胶原结合的蛋白质。三、分析了 NG2缺失突变体对VI型胶原的锚定能力 细胞表面将作为识别NG2关键片段的一种手段 与VI型胶原蛋白结合所需。为了证实这些发现， 重组NG2片段代表由 删除分析将在固相结合分析中进行测试，以确定 它们与VI型胶原蛋白相互作用的能力。在特定的目标中2电子 显微镜也将被用来检查NG2之间的空间关系 和VI型胶原在原位以及在纯化的复合体之间 分子。 在特定的目标3中，NG2-VI型胶原相互作用的能力 将通过测试细胞黏附来检查影响生物过程的因素， 在涂有类型VI的衬底上的扩散、迁移和扩散 胶原蛋白和其他细胞外基质成分，如纤维连接蛋白。为了评估 NG2在这些过程中的重要性，配对的NG2阳性和 NG2阴性细胞将在每一次检测中进行比较。比较将会 在NG2-VI型胶原介导的作用和 整合素-纤维连接蛋白介导的作用。

DESCRIPTION: The ability to interact with specific components of the extracellular matrix (ECM) is critical to the normal development and function of cells. The present work focuses on the ability of the integral membrane proteoglycan NG2 to mediate interaction with the ECM by binding to type VI collagen. Co-immunoprecipitation experiments, as well as immunohistochemical co-localization and co-capping studies, suggest that a physical interaction exists between NG2 and type VI collagen. In addition, transfection of NG2-negative cell lines with NG2 cDNA endows these cells with the ability to anchor type VI collagen at the cell surface, confirming the ability of NG2 to serve as an effective cell surface receptor for type VI collagen. This application contains experiments designed to increase understanding of the NG2-type VI collagen interaction at the molecular level and to provide insight into the role that this interaction plays in cell physiology. Specific Aim 1 focuses on identification of the domain of the NG2 core protein responsible for binding to type VI collagen. Analysis of the ability of transfected NG2 deletion mutants to anchor type VI collagen at the cell surface will serve as one means of identifying key segments of NG2 required for binding to type VI collagen. To confirm these findings, recombinant NG2 fragments representing key domains identified by the deletion analysis will be tested in solid phase binding assays to determine their ability to interact with type VI collagen. In Specific Aim 2 electron microscopy will also by used to examine the spatial relationship between NG2 and type VI collagen in situ as well as in complexes between purified molecules. In Specific Aim 3 the ability of the NG2-type VI collagen interaction to affect biological processes will be examined by testing cell adhesion, spreading, migration, and proliferation on substrates coated with type VI collagen and other ECM components such as fibronectin. In order to assess the importance of NG2 in these processes, matched pairs of NG2-positive and NG2-negative cells will be compared in each of the assays. Comparisons will be made between NG2-type VI collagen mediated effects and integrin-fibronectin mediated effects.