The overall goal of this research is to elucidate the structure-function relationships of laminin. In the next 3-year period, the structural requirements for the biological effects of laminin on cells will be investigated. The interaction of laminin with cells will be monitored primarily by measuring the capacity of laminin to stimulate neurite production from neuronal cells. This activity of laminin will be compared to its ability to promote cell adhesion and spreading and to support cell proliferation. These studies will mainly be carried out using human laminin. Monoclonal antibodies will be made against nearly intact human laminin isolated from peptic digests of placenta. These preparations of laminin have retained the full neurite-promoting activity of intact laminin from rodents and are active at picomolar concentrations in assays with cultured neurons. Monoclonal antibodies which block the neurite-promoting activity of laminin will be selected. The active site(s) in laminin will be identified by analysis of complexes of laminin and blocking antibody by electron microscopy after rotary shadowing. Blocking antibodies will also be used to isolate smaller, active fragments of laminin generated by proteolytic digestion. Active fragments will be used to identify and isolate the laminin """"""""receptor"""""""" on neuronal and other cells. These experiments will characterize the nature of the laminin-cell interaction(s) and show whether laminin has different binding sites for different cell types. The work proposed will contribute to an understanding of the structure and function of basement membranes.
