Interactions between extracellular matrix proteins such as tenascin and fibronectin and their integrin receptors are recognized as important in tumor cell motility and adhesion and are key players in the process of tumor invasion, metastatis and angiogenesis. The overall goal of this application is to gain an understanding of the structures of fibronectin and tenascin binding sites recognized by integrin receptors. The focus of this proposal is to engineer short peptide ligands into a context protein and examine the biological activity, receptor binding characteristics and structure of the recombinant protein. The approach is comparable to peptide mimetic studies but is innovative in providing a means of: 1) rapidly generating peptide ligands in the context of a protein; 2) efficiently generating structure-function mutants and 3) providing a protein host suitable for functional and structural study. The context protein chosen is tryptophan repressor, TrpR, a small gene regulatory protein which has been characterized in terms of its structure- function mutants, and its NMR and X-ray crystal structure. Our preliminary studies have resulted in the successful application of this approach. We have made a novel recombinant integrin receptor binding protein. The protein is the result of cloning the cell binding site SRRGDMS of human tenascin within TrpR. The recombinant protein is a cell attachment protein that is recognized by the RGD-dependent integrin alphav beta3. We have also succeeded in obtaining preliminary NMR structural data and crystals for X-ray crystallography on the recombinant protein. The results demonstrate the potential to examine a variety of ligand site mutants in terms of their biological function and conformations. Such information is important in providing the basic data for understanding the mechanisms of receptor recognition and specificity as well as for the design of biologically active compounds to inhibit or potentiate the effects of ligand-receptor interactions in a variety of diseases including cancer.
