Information on the molecular mechanisms involved in cell growth control is important for understanding the events surrounding normal development and those underlying carcinogenesis. The epidermal growth factor (EGF) is a ligand-stimulated protein-tyrosine kinase that undergoes rapid EGF-induced self-phosphorylation in its extreme carboxy- (C-) terminus. The EGF receptor is homologous to the retroviral transforming protein v-erbB, and it is overexpressed in certain human tumors. Of interest are two general mutations in erbB that increase its oncogenic capacity: an amino-terminal deletion that removes the EGF binding site, and various C-terminal truncations. In this regard, our initial studies have suggested that removal of the C-terminal self-phosphorylation domain decreases high affinity binding, increases tyrosine kinase activity in vivo and disrupts receptor association with the cell cytoskeleton. Moreover, these and other investigations strongly suggest that the receptor C-terminal domain and receptor interaction with the cell cytoskeleton can both exert control over EGF receptor binding and kinase activities. Because EGF receptor kinase activity is essential for proper biological function, the following specific aims are proposed: 1) Examine the mechanisms by which the EGF receptor C-terminus regulates protein-tyrosine kinase activity. These studies will involve an analysis of the substrate specificity, kinetic mechanism and sensitivity to protein activators of both normal and C- terminally mutated EGF receptors. 2) Characterize the role of the EGF receptor C-terminus in promoting cytoskeletal attachment, and ascertain the processes by which cytoskeletal association affects receptor high affinity binding and protein-tyrosine kinase activity. This work will focus on the influence of C-terminal domains and protein kinase C activation on receptor cytoskeletal distribution, and will also asses the effects of cytoskeletal association on receptor high affinity binding, tyrosine kinase activity and substrate specificity. 3) Identify and analyze specific proteins involved in the EGF receptor-cytoskeletal association using direct binding studies, as well as co-immunoprecipitation and crosslinking analyses. These experiences are expected to more rigorously establish the mechanisms by which specific domains, and their interaction with the cellular cytoskeleton, can play a key role in the regulation of EGF receptor function in both normal and abnormal cell growth.
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