EGF RECEPTOR FUNCTION AND CONTROL BY PHOSPHORYLATION

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A detailed analysis of the biochemical mechanisms involved in cellular growth and differentiation is crucial to our understanding of the molecular events surrounding normal growth and development, as well as those underlying carcinogenesis. The epidermal growth factor (EGF) receptor is an EGF-responsive protein-tyrosine kinase that undergoes rapid ligand-induced self- phosphorylation on three tyrosine residues (Tyrs 1173, 1148, 1068) located in the extreme carboxy-terminal region of the molecule. The EGF receptor exhibits extensive homology with certain retroviral transforming proteins (also self-phosphorylating tyrosine kinases, e.g. erb B and src) and its overexpression in various human tumors suggests that it plays a critical role in cell proliferation and transformation. This proposal is designed to examine EGF receptor regulation by phosphorylation, with an emphasis on self-phosphorylation. Specifically, it is anticipated that the proposed research plan will elucidate the following: 1) The biochemical properties and regulation by self-phosphorylation of EGF receptor protein-tyrosine kinase and ligand binding activities, 2) the effect of ligand-induced EGF receptor self- phosphorylation on biological events including intracellular tyrosine phosphorylation, DNA synthesis, Ca++ uptake and cell division, and 3) identification and characterization of additional EGF receptor sites of tyrosine and serine/threonine phosphorylation and an initial analysis of their effect on receptor function. This work will use both normal human EGF receptors and available site-directed mutant receptors wherein specific sites of tyrosine self-phosphorylation have been altered to phenylalanine residues (i.e. Tyr 1173 alone, Tyrs 1173 plus 1068, Tyrs 1173, 1068 and 1148, and a mutant wherein these tyrosine residues have been deleted entirely). These studies involve the use and development of methods for kinetic analyses, protein isolation and structural characterization, cell culture and the hormonal control of cellular processes. The knowledge gained from these studies is expected to more rigorously establish the mechanisms by which self-phosphorylation can modulate EGF receptor function and should also prove valuable in understanding possible control mechanisms for other hormone receptors/enzymes that appear regulated by self-phosphorylation and which are important in normal and abnormal cellular metabolism and development.