The overall goal of the proposal is to determine the role of the newly described ganglioside binding sites of both fibroblasts and its 140 kD cell surface receptor protein in the mechanism of cell adhesion. The PI recently demonstrated a ganglioside binding site in the amino terminal domain of fibronectin and proposes to further define the role of the ganglioside binding site in cell adhesion. Using a quantitative binding assay, termed fluoresence polarization, he will study the structure and and function of the ganglioside binding sites of both fibronectin and its receptor. The fluorescence polarization assay will be used for studying the binding of gangliosides to the 140 kD fibronectin receptor and platelet membrane glycoprotein IIb/IIIa. He will also determine the specificity of the receptors for both various ganglioside species and divalent cations. Another objective is to determine the precise site on fibronectin which binds gangliosides by (a) limited proteolytic digestion of fibronectin and (b) expression of the region of the fibronectin gene that codes for the ganglioside binding site. Fluorescence polarization will also be used to quantitatively measure cooperative interactions between the ganglioside binding site and other fibronectin binding sites. Cell adhesion mutants will be used to examine the role of gangliosides in cell adhesion, and the molecular basis of the phenotype will be analyzed. The molecular basis of specific and non-specific interactions between cells, and between cells and the substrate, is a important area of research in cell biology. The proposed work promises to provide important new information concerning one type of interaction, adhesion, and combines both biochemical and molecular genetic approaches in a clever and productive way.
