Studies demonstrating the structural and functional specificities of various cell-ECM attachment sites will be continued, with particular emphasis on altered molecular mechanisms of cell adhesion, cell de-adhesion, and invasion in transformed cells. Our working hypotheses are: 1) distinct, specific cell surface receptors are involved in organizing cell attachments to fibronectin and collagen during dynamic processes of cell adhesion and invasion, 2) ligands such as fibronectin can help to regulate the distribution of their receptors and the cytoskeleton, and 3) localized cell surface proteases are responsible for cell contact-related dissolution of the ECM, which permits invasion. Recent studies have demonstrated the existence of transformation-induced cell surface proteases that degrade fibronectin and collagen at cell contact sites. Thus, the immediate goals of the present proposal are to use monoclonal antibody probes to localize these transformation-sensitive, invasion-associated proteases, to identify protease structural domains, and to perturb molecular interactions involving proteases. By this we plan to determine the possible role of these proteases in transformed cell invasion or as indicators of the metastatic potential of tumor cells. The proposed studies will also define possible collagen attachment sites at the electron microscopic level, and determine whether such attachments function via collagen receptors. To do this, we will use in situ localization and antibody inhibition approaches similar to those used for the characterization of the fibronectin-receptor association. Possible regulatory roles of fibronectin and collagen in the organization of attachment sites and in the invasion of the tumor cells will then be studied by allowing these molecules to bind to the cell surface and inhibiting their binding using specific monoclonal antibody probes and synthetic peptides. More specifically, we will seek to determine: 1) the molecular structure and biochemical properties of cell surface proteases by the use of hybridoma technology, 2) the roles of cell surface proteases in transformation and invasion into fibronectin and collagen substrata, 3) the expression and localization of cell surface receptors for collagen and fibronectin at distinct attachment sites, 4) the regulatory roles of fibronectin and collagen in the organization of adhesion sites in transformed cells, and 5) the in vivo biological significance of cell surface proteases or receptors in migration and invasion of tumor cells.
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