Epidermal growth factor acts via a receptor with intrinsic protein tyrosine kinase activity to signal not only cell growth but also differentiation and cell-specific responses. Mutations that impair EGFR function cause defects in development while mutations that result in excessive EGFR function cause cell transformation. Goals are to understand how EGFR functions: how ligand binding activates PTK, how PTK functions, how receptor expression is controlled and how specificity in signaling is achieved. Comparison with erbB-2 will be used to define specificity. 1. Structure-function analysis of EGFR. We propose to use site-specific mutations, functional assays and structural analyses to study the subdomain of holo EGFR. a. The importance of coiled.coil interactions in the ecto domain for ligand-induced dimerization and transmembrane signal transduction will be investigated. b. The structure of the intracellular domain and features essential for ATP and substrate binding and for catalysis will b investigated using mutagenesis and x-ray crystallography. The structure of the C' terminal regulatory domain will also be studied. 2. Signal transduction by EGFR. To investigate mechanisms involved in specific responses to EGF we will determine the function of genes cloned based on specific binding to the EGFR core kinase domain using a yeast 2-hybrid screen. We will evaluate their function biochemically as substrates, activators and inhibitors of EGFR and biologically as components of specific response and general proliferative pathways. Interactions with EGFR and functions will be compared with those involved in erbB-2 signaling. Initial focus will be on 4 novel proteins identified: core kinase domain interactor 1 and 2, a nuclear protein that undergoes tyr phosphorylation and a PTK that is activated by EGF-dependent tyr phosphorylation.3. Regulation of transcription; A transcription factor that acts via two palindromes in the erbB-2 promoter will be isolated, characterized and its role in erbB-2 expression in breast cancer studied.
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