This research proposal is aimed at understanding the mechanism of action of human epidermal growth factor (EGF) by studying its structure-function relationships. A goal of these studies is the possible development of EGF analogs that might act as inhibitors of cell proliferation and possess anti-tumor activity. Utilizing the human EGF gene, recently cloned in the applicant's laboratory as a bacterial secretory protein, structure- function analysis will be performed by alteration of targeted amino acid residues by oligonucleotide-directed mutagenesis. The amino acid residues to be substituted will be chosen on the basis of their species invariance, the solution structure of EGF, reported studies of chemically synthesized peptide fragments of EGF, and preliminary structure-function analysis of EGF in this laboratory. The mutant EGF's will be purified and tested for their 10 binding to the EGF receptor of human cells, 20 stimulation of the protein-tyrosine kinase activity of the egf receptor, and 30 stimulation of DNA synthesis and growth of cells in culture. In designing the mutant EGFs, special attention will be placed on the possibility of uncoupling the primary binding to the receptor from the stimulation of the receptor;s tyrosine kinase activity and subsequent stimulation of DNA synthesis and cell growth. Such EGF analogs could then be potential candidates to be tested for possible anti-tumor activity. At the very least, these studies should help identify the amino acid residues in EGF that are crucial for its biological activity.
