Cell type-specific alternative splicing of at least three regions of the rat fibronectin (FN) gene transcript generates multiple mRNAs encoding different FN subunits. This structural diversity may reflect functional differences required for FN's involvement in wound healing, blood clotting, cell migration, and oncogenic transformation. In the initial stages of the proposed work, we plan to complete the analysis of the known variations at the RNA and protein levels along with mapping any other regions of alternative splicing, should they exist. The major portion of this proposal uses cDNA-encoded FN polypeptides expressed by cultured mammalian cells to study the intermolecular interactions of FN in normal and transformed cells. We have designed a retroviral-based vector system to express parts of the FN cDNAs in mouse 3T3 cells. The truncated FN polypeptides (containing the C-terminal 40% of FN) are secreted but are only functional in the form of heterodimers with the endogenous 3T3 FN monomers. With the addition of 5' segments of cDNA we should be able to generate truncated molecules that are completely autonomous, i.e., that function independently of 3T3 FN. We will then introduce these cDNAs recombinant retroviruses into oncogenically transformed cells and analyze the potential of cDNA-encoded FN polypeptides for reverting the transformed phenotype. This approach will enable us to analyze the involvement of FN in transformation and in normal cellular behavior. With this combination of molecular and cell biology, we intend to locate precisely the segments of FN involved in FN self association and in binding to cells and collagen by using site-specific and deletion mutagenesis of FN cDNAs. Furthermore, by varying the inserts within the polypeptides, we hope to assign functional and/or structural roles to the segments that are included or omitted by alternative splicing. The outlined experiments will extend our understanding of the structure-function relationships within specific domains, how these domains mediate the intermolecular interactions required for FN activity, and why FN subunits vary in a cell type-specific fashion. In addition, they will provide a basis for determining the role of different forms of FN in multiple processes in vivo.
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