During embryonic development, regeneration and wound repair, members of the basement membrane laminin family of structural glycoproteins provide important signals for cell adhesion, shape, polarity, migration and differentiation. The long arm of laminin is central to these processes, affecting such functions as kidney tubule development, the migration and differentiation of myoblasts, and the guidance of axonal and dendritic processes to their targets. Some of the cell receptors involved in these functions are highly specific for the long arm of laminin, or its variants, while others are promiscuous in their recognition. For both there is growing evidence that receptor recognition of the long arm is dependent on the trimeric assembly of A, B1 and B2 chain subunits into conformationally- correct rod and globular (G) domains. Furthermore, preliminary studies reveal that specific biological activities can be reconstituted y the intercalation of recombinant with authentic laminin chains. A long term goal is to understand the molecular basis for laminin-cell signaling in which tertiary and quaternary structure play a crucial role and in which different isoform chain substitutions provide different signals. This proposal is based upon an experimental approach which combines genetic engineering, protein biochemistry, immunochemistry, biophysics and cell biology. Recombinant glycoproteins, both normal and containing a variety of sequence modification, will be generated with the eukaryotic baculovirus expression system (with limited comparative studies using mammalian recombinant molecules) and intercalated with authentic or other recombinant chains. The refolded substrate structures will be characterized by biophysical, biochemical and electron microscopy techniques and function will be evaluated in cell interaction and receptor binding studies with the aid of receptor- and substrate-specific neutralizing and immunoprecipitating antibodies. In particular, the focus will be on HT1080 fibrosarcoma cell and C2C12 myoblast adhesion and spreading, PC12 pheochromocytoma cell neurite-outgrowth, olfactory epithelial cell neurite-outgrowth and migration, and the binding interactions of alpha7beta1 and alpha3beta1 integrins to these substrates. The sites of recognition will be mapped and the role of conformation will be determined with the experimental approach divided into an analysis of the contributions of [1] the globular domain, [II] the rod domain, and [III] variant chains of laminin to cell function. Overall, these studies will lead to a better understanding of how basement membranes in general, and laminin in particular, encode specific information for interacting cells.
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