Laminin promotes neurite outgrowth from cultured neuronal cells and promotes the adhesion and motility of glioblastoma cells in vitro. Laminin-containing a cellular fetal membranes also show activity in nerve regeneration in vivo. We have located the neurite-promoting site in human laminin to the end of the long arm using monoclonal antibodies to laminin and proteolytic fragments. We have evidence suggesting that glioblastoma cells interact with the same site and that the receptors on neuronal cells and glioblastoma cells are related. We propose to identify and characterize the neurite-promoting/cell adhesion site of laminin by isolating smaller active proteolytic fragments of laminin and analyzing these fragments by rotary shadowing-electron microscopy, electrophoresis, NH2-terminal sequencing of peptide subunits, and antibody reactivity. Complete structural information on the neurite-promoting site will be obtained from cloning and sequencing of cDNA coding for sequences around the site. We will reconstruct the neurite-promoting site using peptide synthesis and/or synthesis in bacteria and test the constructs for in vitro and in vivo activity. The human cell surface receptor interacting with the neurite-promoting site in laminin will be identified, characterized, and isolated from glioblastoma cells using monoclonal antibodies and affinity-chromatography on natural or synthetic neurite-promoting/cell binding sites of laminin. The relationship of the glioblastoma cell laminin receptor to other laminin receptors on other cell types and to other extracellular matrix receptors will be established. The activity of the laminin receptor will be evaluated in various in vitro assays using specific peptides with neurite-promoting activity and using antibodies specific for this receptor.
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