Signaling mechanisms activated by engagement of NGE-PG

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The NG2 proteoglycan contributes to cell spreading and migration on surfaces coated with type VI collagen. The cytoplasmic domain of the proteoglycan is required for the NG2-dependent component of spreading and migration, suggesting that this domain is involved in transducing signals that influence rearrangements of cytoskeletal machinery. Cell spreading on surface coated with anti-NG2 monoclonal antibodies is now being used to study specific signaling mechanisms activated upon engaged of NG2 by the substratum. This application proposes experiments designed to identify signaling mechanisms mediated by NG2 and to define the impact of these processes on cell behavior. Specific aim 1 focuses on identifying structural elements of the NG2 cytoplasmic domain that are required for cell spreading. These will be defined by using cells transfected with NG2 cDNAs carrying deletions and mutations in key determinants. Specific aim 2 probes the role of NG2 in activating rho family GTPases (rho, rac, and cdc42), molecules that control rearrangements of the actin cytoskeleton. Specific aim 3 examines the SH3 domains. These molecules may provide the link between NG2 and intracellular signaling pathways. Specific aim 4 investigates the interaction of the C-terminal PDZ-binding of NG2 with cytoplasmic molecules that contain PDZ domains. PDZ domain-containing molecules such as MUPPI, FAP-1 and rhophilin may provide additional links to signaling pathways, as well as serving as scaffolds for the physical localization of NG2 to critical cellular microdomains.