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.
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