NG2 is a chondroitin sulfate proteoglycan that is expressed on immature precursor cells of a number of different lineages, including 02A glial progenitors in the central nervous system. NG2 expression in these cells is down-regulated when the precursors stop dividing and undergo terminal differentiation. NG2 is upregulated in some pathological conditions characterized by an increase in cell proliferation. These findings have led to speculation that NG2 may be involved in regulation of cell proliferation and/or differentiation. The applicant s overall goal is to understand the role of NG2 in the development of 02A progenitor cells. The 300 kDa core glycoprotein of NG2 is a membrane-spanning molecule that interacts with both extracellular and intracellular ligands, and thus could be involved in transmembrane signaling. Extracellular matrix-associated ligands for NG2 include type VI collagen, thrombospondin, tenascin, and laminin, while membrane associated ligands include the PDGF alpha receptor. Intracellularly, NG2 appears to be anchored to the actin cytoskeleton via its cytoplasmic domain. The ability of NG2 to associate with extracellular matrix and cell surface components will be further explored using both cellular and cell-free (i.e. purified proteins) systems. For each of the putative binding partners the applicant will assess the specificity and affinity of binding, and attempt to define the portion of the NG2 core protein responsible for mediating the interaction. The latter goal will be accomplished by comparing the properties of wild type NG2 with those of mutant molecules lacking specific regions of the core protein. The biological importance of NG2 interaction with these ligands will be studied by examining the role of the proteoglycan in assays of cell adhesion and spreading, cell migration, and behavior of matched pairs of cells differing only in their expression of NG2. Whenever possible the applicant will utilize NG2-positive and NG2-negative 02A cells derived from normal and NG2 gene knockout mice to compare with the NG2-negative parental cells. When biological functions for NG2 are established in these assays, the applicant will identify NG2 domains responsible for function by repeating the assays with cells expressing mutant NG2 molecules lacking defined portions of the core protein.
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