The broad thrust of the experiments described in this application is to study the structural and functional aspects of the EGF receptor. The focus is on the receptor itself and these studies do not include, in a major way, analysis of receptor-distal molecules that constitute signal pathways. While more information is available regarding the structure/function aspects of the EGF receptor than any other growth factor receptor, there remains a great deal that is not understood. The EGF receptor plays a central role in the proliferation of various cell types and is implicated at several levels as a biologically significant element in tumor cells. The experimental approaches that are proposed for the experiments in this application include biochemical, cell biological, and molecular biological technologies. Studies of the EGF extracellular or ligand-binding domain will include site-directed mutagenesis to probe the role of oligosaccharide chains in receptor structure and processing. Glycosylation mutants will be tested for their capacity to fold into an active conformation, within the lumen of the ER, to be efficiently precessed to the cell surface, for the presence of mannose phosphate, for ligand binding and signal transducing capacities, and for rapid, EGF-directed internalization and lysosomal sorting. Similar assays will be performed in chimeric receptor molecules in which the EGF binding domain has been replaced by the receptor binding domain for a non- growth factor polypeptide hormone. This study will test the specificity for ligand activation of receptor catalytic domains. The cytoplasmic domain of the receptor will be investigated with studies designed to assess serine/threonine phosphorylation elicited by the two tumor promoters TPA and okadaic acid. Tyrosine phosphorylations in the autophosphorylation domain will be examined in terms of an autoinhibitory domain and in terms of sensitivity to tyrosine phosphatase regulation. Finally, experiments using many of the assays mentioned above are proposed to examine the mechanism of interaction between the E5 papilloma virus transforming protein and the EGF receptor.
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