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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National Cancer Institute (NCI)
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Saint Louis University
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Crooks, D; Kil, S J; McCaffery, J M et al. (2000) E3-13.7 integral membrane proteins encoded by human adenoviruses alter epidermal growth factor receptor trafficking by interacting directly with receptors in early endosomes. Mol Biol Cell 11:3559-72
Kil, S J; Carlin, C (2000) EGF receptor residues leu(679), leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments. J Cell Physiol 185:47-60
Kil, S J; Hobert, M; Carlin, C (1999) A leucine-based determinant in the epidermal growth factor receptor juxtamembrane domain is required for the efficient transport of ligand-receptor complexes to lysosomes. J Biol Chem 274:3141-50
Hobert, M E; Kil, S J; Medof, M E et al. (1997) The cytoplasmic juxtamembrane domain of the epidermal growth factor receptor contains a novel autonomous basolateral sorting determinant. J Biol Chem 272:32901-9
Stewart, A R; Tollefson, A E; Krajcsi, P et al. (1995) The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane. J Virol 69:172-81
Wold, W S; Tollefson, A E; Hermiston, T W (1995) E3 transcription unit of adenovirus. Curr Top Microbiol Immunol 199 ( Pt 1):237-74
Wold, W S; Hermiston, T W; Tollefson, A E (1994) Adenovirus proteins that subvert host defenses. Trends Microbiol 2:437-43
Hoffman, P; Carlin, C (1994) Adenovirus E3 protein causes constitutively internalized epidermal growth factor receptors to accumulate in a prelysosomal compartment, resulting in enhanced degradation. Mol Cell Biol 14:3695-706
Kuivinen, E; Hoffman, B L; Hoffman, P A et al. (1993) Structurally related class I and class II receptor protein tyrosine kinases are down-regulated by the same E3 protein coded for by human group C adenoviruses. J Cell Biol 120:1271-9
Hoffman, B L; Takishima, K; Rosner, M R et al. (1993) Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking. J Cell Physiol 157:535-43

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