ACTIVATION OF EPIDERMAL GROWTH FACTOR RECEPTOR

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The long term objective of this project is to elucidate all of the steps in the molecular mechanism by which epidermal growth factor (EGF) activates the tyrosine kinase of epidermal growth factor receptor (EGF receptor). The phosphorylation of various proteins within the cell by this tyrosine kinase initiates a cascade of events leading to cell division and growth. It has been shown that cells containing certain mutant forms of EGF receptor grow uncontrollably in a transformed state and certain viral oncogenes are altered viral forms of this cellular receptor. These observations suggest that this protein could be a point of initiation for abnormal oncogenic growth. The mechanism of activation of EGF receptor involves binding of EGF, dimerization of the EGF receptor, activation of the tyrosine kinase, and self-phosphorylation of the receptor. Each of these steps will be examined in detail. the affinity of monomeric and dimeric forms of EGF receptor for EGF will be measured to determine whether or not dimerization of monomeric EGF receptor and activation of the tyrosine kinase is linked to this binding. The second-order rate constant for the dimerization of the extracellular domain will be measured so that it can be compared directly to that for dimerization of the intact protein. We will look for conformational changes in the cytoplasmic domain of intact EGF receptor that are initiated by binding EGF but that precede dimerization. The free cytoplasmic domain of EGF receptor will be dimerized with bivalent immunoglobulins to see if dimerization is sufficient to activate the tyrosine kinase. the activation of a truncated EGF receptor lacking all of the self-phosphorylation sites will be studied to verify the requirement for dimerization in the process of activation even in the absence of self-phosphorylation. The ability of a promoter of EGF receptor to phosphorylate itself in the activated dimer will be assessed. The progress of self-phosphorylation will be correlated temporally with dimerization and activation of the tyrosine kinase. The reversibility of the dimerization of EGF receptor and the activation of the tyrosine kinase will be examined. The results from these experiments will increase our understanding of the various steps in the mechanism of activation of this growth factor receptor.