The erbB-2 oncogene encodes a 185 kDa transmembrane protein which has been suggested to be a growth factor receptor due to its homology with the EGF receptor (EGFR). We have identified two ligands of the erbB-2 oncoprotein, gp30 and p75. gp30 binds to EGFR and pl85erbB2, and the p75 binds exclusively to pl85erbB-2. We have identified and purified the gp30 growth factor that is secreted by MDA-231 human breast cancer cells. This growth factor binds to the EGFR and induces NRK cell colony formation (1). We have reported that gp30 is also a ligand for the pl85erbB-2 protein and induces growth inhibition of cells with erbB-2 amplification and over expression. At very low concentrations however, gp30 stimulated the proliferation of SKBr-3 overexpressing erbB-2 breast cancer cells. We have demonstrated that gp30 interferes directly with the binding of a specific erbB-2 antibody (4D5) which has been shown to induce growth inhibition of overexpressing erbB-2 cells. We can utilize this specific competition binding assay to detect other possible ligands for erbB-2, including the p75 described in this proposal. Preliminary studies, with the competitive erbB-2 receptor binding assay, suggest that SKBr-3 cells secrete a putative erbB-2 ligand. Further-more, purification of this erbB-2 ligand from SKBr-3 cells, using erbB-2 extracellular domain coupled to an affinity column yielded a p75 ligand which competes with 4D5 of binding to erbB-2. We now intend to continue the biological, biochemical and molecular characterization of gp30 and p75 by several approaches which include protein sequencing to generate polyclonal and monoclonal antibodies, as well as cDNA cloning and gene regulation. We also will compare their sequence homology and biological activity. The prognostic, diagnostic and therapeutic implications of this proposed work for breast, ovarian and possibly gastric cancers are of substantial significance and will be explored systematically in this project.
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