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

描述：与特定组件交互的能力。 细胞外基质（ECM）对正常发育至关重要， 细胞的功能。 目前的工作重点是能力的积分 膜蛋白聚糖NG 2，通过结合 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加帽研究表明， NG 2与VI型胶原之间存在物理相互作用。 此外，本发明还提供了一种方法， 用NG 2 cDNA转染NG 2阴性细胞系， 具有将VI型胶原锚在细胞表面的能力，证实了 NG 2作为有效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以下内容的理解的实验 NG 2-VI型胶原蛋白在分子水平上的相互作用， 深入了解这种相互作用在细胞生理学中的作用。 具体目标1侧重于确定NG 2核心的结构域 负责与VI型胶原结合的蛋白质。 分析 转染的NG 2缺失突变体在20 ℃下锚VI型胶原的能力 细胞表面将作为识别NG 2关键片段的一种手段 需要结合VI型胶原蛋白。 为了证实这些发现， 重组NG 2片段，代表由免疫组织化学鉴定的关键结构域。 缺失分析将在固相结合试验中进行测试，以确定 它们与VI型胶原蛋白相互作用的能力。 在特定目标2电子 显微镜也将被用来检查NG 2之间的空间关系 和VI型胶原蛋白原位以及在纯化的 分子。 在具体目标3中，NG 2-VI型胶原相互作用的能力， 影响生物过程将通过测试细胞粘附来检查， 在涂有VI型的基质上的扩散、迁移和增殖 胶原蛋白和其它ECM成分如纤连蛋白。 为了评估 NG 2在这些过程中的重要性，NG 2阳性和 将在每项试验中比较NG 2阴性细胞。 比较将 在NG 2-VI型胶原蛋白介导的作用和 整合素-纤连蛋白介导的作用。

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.