We have found that group c human adenoviruses (Ad) down-regulate the receptor for epidermal growth factor (EGF-R); i.e. EGF-K is no longer on the cell surface. Using virus mutants, we mapped the effect to a putative 10,400 MW (10.4K) protein encoded in the E3 transcription unit. This represents an important discovery because the function of E3 is largely a mystery. Moreover, EGF-K is a key growth regulatory molecule, and inappropriate EGF signal transduction is often associated with malignancy. Using EGF-R antibodies, we are unable to detect mature EGF-K in 10.4K- expressing cells when EGF-R is metabolically labeled with (35S)Cys, labeled on the cell surface with 125I, or autophosphorylated in vitro. Also, 10.4K prevents binding of EGF to EGF-R. EGF induces a variety of effects in cells with specific receptors. EGF stimulates the intrinsic protein tyrosine kinase of EGF-R; the EGF/EGF-R complex then clusters in clathrin-coated pits, internalizes via endosomes, is transported to lysosomes, and degraded. This process transduces EGF signals which activate cellular metabolism, DNA synthesis, and mitosis. Although 10.4K differs in sequence from EGF and is presumably a membrane rather than a secreted protein, 10.4K may mimic some of the EGF effects on EGF-R. This hypothesis is based on our data above, as well as immunofluorescence, which suggest that 10.4K does not inhibit synthesis of EGF-R, but it does induce internalization and degradation of EGF-R. Activation of EGF-R should benefit Ad in vivo, since Ad multiplication is probably more efficient in stimulated versus quiescent cells. Our major goals are to determine the mechanism of action of 10.4K, and whether it stimulates or abrogates the cellular responses to EGF signal transduction. We will develop antibodies against 10.4K, characterize 10.4K biochemically, generate 10.4K mutants for structure/function studies, and express wild type and mutant forms of 10.4K in eukaryotic and prokaryotic expression vectors. We will determine {i) the sub-cellular localization of 10.4K and whether it binds to EGF-R, (ii) which domains in EGF-R and 10.4K are important for the 10.4K effect, (iii) whether 10.4K alone can stimulate the EGF-R kinase, nutrient uptake, transcription of specific genes, DNA synthesis, and/or mitosis, (iv) whether 10.4K acts on other receptors with protein tyrosine kinase activity, and (v) whether all six groups of human Ads and mouse Ad have a 10.4K- equivalent protein. This project is an ideal merging of the expertise of C. Carlin in EGF-R and cell biology, and of W. Wold in the molecular biology of Ad region E3.
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