Adhesion of nerve cells to extracellular matrices (ECM) is essential to the formation of proper connections between nerves and their targets (such as muscles). The long-term objectives of this project are to identify and characterize the cell-surface molecules that mediate neuronal adhesion to two ECM components, collagen and laminin, and to understand the mechanism of this adhesion and its function in development. PC12 cells, an established line of cells derived from a rat pheochromocytoma, resemble sympathetic neurons in many respects, including extension of neurites in response to nerve growth factor. Active attachment of PC12 cells to collagen and laminin requires Mg2+ and is inhibited at low temperature or in the presence of azide, whereas passive attachment to substrata coated with polylysine or wheat germ agglutinin occurs in the absence of divalent cations, at low temperature, and in the presence of azide. Also, neurite extension occurs on collagen or laminin (provided Mg2+ is present), but not on polylysine or wheat germ agglutinin, indicating that the active adhesion mechanism functions at the growth cone. A recently isolated monoclonal antibody that inhibits active, Mg -dependent, attachment of PC12 cells to both collagen and laminin (but not passive adhesion), and that reacts with an antigen on embryonic rat neurons (blocking their adhesion to laminin), has been used to identify (by immunoprecipitation) a putative collagen/laminin receptor, an apparently novel protein of about 200 kD. Preliminary findings suggest that this protein can be purified, from both PC12 cells and rat brain, by affinity chromatography on antibody-coupled beads. It is proposed to purify sufficient putative receptor for production of polyclonal rabbit antiserum, for N-terminal sequence analysis, and for incorporation into liposomes (to confirm that it binds to collagen and laminin). Mono- and polyclonal antibodies will be employed to inhibit adhesion in studies of the mechanism, to localize the antigen on nerve and other cells at different developmental stages, and to assess relationship(s) to other known matrix adhesion molecules. These studies promise to elucidate the molecular basis of an important class of cell-ECM adhesion mechanisms. The results should ultimately contribute to a better understanding of neural development and neuromuscular diseases, several of which are thought to involve altered nerve-muscle interactions.
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