Protein transport and sorting are essential processes for many cellular functions. Understanding molecular interactions that regulate intracellular protein transport is therefore a fundamental question in cell biology. The present proposal, in its fifth year of funding, seeks continued support for investigating EGF receptor (EGFR) degradation induced by a novel human group C adenovirus (Ad) protein called E3- 13.7/11.3 kDa. This application has been revised since it was originally reviewed in October, 1993, in response to the comments of the study section and our additional preliminary data. Our major accomplishments in the last funding period have been: a) The discovery that E3-13.7/11.3 kDa is translated in Ad-infected cells, and is an integral membrane protein with two potential membrane-spanning alpha-helices, an extracellular loop domain connecting the helices, and a cytosolic tail region essential for function. b) The discovery that the EGFR cytosolic juxtamembrane domain is required for E3-13.7/11.3 kDa-induced degradation. c) The discovery that the insulin receptor, IGF1 receptor and pp185(c-neu) are also down- regulated by E3-13.7/11.3 kDa, but the PDGF receptor and aFGF receptor are not. d) The discovery that E3-13.7/11.3 kDa increases trafficking of constitutively internalized EGFRs to multivesicular bodies (MVBs) where they are sorted to lysosomes, but does not increase the rate of EGFR internalization or induce intrinsic EGFR tyrosine kinase activity. These findings have led us to hypothesize that E3-13.7/11.3 kDa regulates EGFR plasma membrane (PM) to lysosome (Ly) sorting. If this supposition is correct, this is the first viral protein identified that uses this mechanism, and as such will be a valuable model system for characterizing host cell proteins that regulate membrane protein traffick. Towards that end, we have recently identified cellular proteins that bind specifically to a bacterial fusion protein expressing the E3-13.7/11.3 kDa cytosolic tail in vitro. In order to understand the interaction between EGFR and E3- 13.7/11.3 kDa in proper physiological context, we have also demonstrated the feasibility of studying this process in filter-grown polarized epithelial cells where EGFRs are located predominantly in the basolateral (BL) PM. We have recently shown that E3-13.7/11.3 kDa selectively sorts to the BL PM and also induces EGFR degradation in polarized cells. Surprisingly, a significant fraction of EGFRs missort to the apical (Ap) PM in cells infected with an Ad E3-13.7/11.3 kDa deletion mutant. This suggests that E3-13.7 kDa mediated EGFR degradation may counteract EGFR missorting induced by a separate event during early Ad infection. The new specific aims are: 1. To learn whether E3-13.7/11.3 kDa affects recycling of endocytosed EGFRs to PM, or transport of newly synthesized EGFRs from trans Golgi network to PM. 2. To learn whether E3-13.7/11.3 kDa is an organelle-associated protein that regulates EGFR protein transport. This will be achieved by determining the steady-state subcellular distribution of E3-13.7/11.3 kDa; which amino acids in the cytosolic tail of 13.7/11.3 kDa are important for proper expression and function; and whether E3- 13.7/11.3 kDa interacts with host cell proteins. 3. To precisely define the minimum receptor tyrosine kinase sequence required for E3-13.7/11.3 kDa-induced degradation in EGFR, the insulin receptor, and pp185(c-neu). 4. To establish a physiological model for studying Ad-induced effects on EGFR trafficking, by studying this effect in polarized epithelial cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA049540-10
Application #
2608053
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Wong, May
Project Start
1989-05-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1999-11-30
Support Year
10
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Physiology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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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
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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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