The family of receptors involved with cellular adhesion has been termed integrins because of the predicted function of these proteins in integrating the extracellular matrix with the cytoskeletal framework. The prototypic integrin, the fibronectin receptor, mediates the interaction of cells with fibronectin. The purpose of this study is to elucidate the cytoplasmic interactions of the fibronectin receptor and ultimately to characterize the molecular basis for the fibronectin receptor-cytoskeleton association. The work proposed herein will concentrate on identifying and characterizing intracellular proteins that interact with cytoplasmic domains of each subunit of the fibronectin receptor. Each of the two cytoplasmic domains of the receptor possesses the potential to interact with intracellular proteins. Synthetic polypeptides representing the individual cytoplasmic domains will be coupled to Sepharose and used as affinity matrices for isolation of binding proteins from detergent extracts of cells or tissue. Preliminary chromatography experiments using an affinity matrix of a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit coupled to Sepharose has been used to identify a novel protein. This protein, which we have named fibulin, may mediate cytoplasmic connections of the fibronectin receptor as well as the other adhesion receptors that share the beta subunit. Such connections potentially include an interaction with the cytoskeleton. In addition, this molecule may be involved with receptor signal transduction and modulation of receptor function. A significant portion of the work proposed in this study will concentrate on characterization of the structure and biological function of fibulin. A complete amino acid sequence of fibulin will be determined from a combination of protein and cDNA sequencing. Particular emphasis will be placed on characterizing the interaction of fibulin with the fibronectin receptor, with itself, and with other cytoplasmic proteins. Using cDNA and antibody probes the expression of fibulin will be examined in both normal and tumor cells. Additional affinity chromatography experiments are proposed to identify and protein(s) that might interact with the fibronectin receptor alpha subunit cytoplasmic domain.
