During abnormal processes such as metastasis in cancer and also normal biological processes such as wound healing and embryonic development, cells respond to components of the extracellular matrix (ECM). As one of the best-characterized components of the ECM, the adhesive glycoprotein fibronectin is a useful model system for the investigation of cell-ECM interactions. Cells bind fibronectin via specific surface receptors. One class of fibronectin receptors consists of two or three glycoprotein components of approximately 140 kilodaltons and are termed 140K complexes. This proposal describes studies that will examine the biosynthesis, structure, and function of the 140K complexes using cells in culture. Mechanisms of biosynthesis and regulation of the 140K complex will be examined by metabolic labeling methods, immunoaffinity purification of 140K from cell lysates, and detection by polyacrylamide gel electrophoresis and autoradiography. The role of carbonhydrates in the biosynthesis, intracellular transport, and acquisition of biological activity of 140k will be examined in detail. Modifications in the biosynthesis of 140k associated with metabolic changes occurring with cellular transformation, or treatment with exogenous agents such as growth factors and tumor promoters will also be investigated. Of particular interest is the possibility that phosphorylation of 140k occurs after treatment of cells in culture with growth factors or tumor promoters. The structure and function of the 140k complexes from different sources will be investigated using biochemical and immunological techniques, and also functional biological assays for cell-substrate interactions. Biochemical techniques include affinity chromatography, binding studies, and receptor reconstitution. Monoclonal antibodies that inhibit specific functions of 140k will be characterized. Fibronectin receptors on B16 melanoma cells, which recognize a different adhesion signal and may be unrelated to those of the 140k class, will be purified and characterized. The melanoma cell fibronectin receptors will be compared to those of the 140k class by immunological methods using monoclonal and polyclonal antibodies. A physical description of 140k will be obtained by chemical crosslinking studies, and quantitiative protein analysis. Results of these studies are expected to help explain cellular adhesion and migration in molecular detail.
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