A Leucine-based Determinant in the Epidermal Growth Factor Receptor Juxtamembrane Domain Is Required for the Efficient Transport of Ligand-Receptor Complexes to Lysosomes*
- From the ‡Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University and the ¶Case Western Reserve University Cancer Center, Cleveland, Ohio 44106-4970
Abstract
Ligand binding causes the epidermal growth factor (EGF) receptor to undergo accelerated internalization with eventual degradation in lysosomes. The goal of this study was to investigate the molecular basis of endocytic sorting, focussing on post-internalization events. We have identified a sequence located between amino acid residues 675 and 697, encompassing a dileucine motif at residues 679 and 680, that enhances endosome-to-lysosome transport when conformational restraints in the EGF receptor carboxyl terminus are removed by truncation. The same dileucine motif is also necessary for efficient lysosomal transport of ligand-occupied full-length EGF receptors. A L679A,L680A substitution diminished the degradation of occupied full-length EGF receptors without affecting internalization but had a significant effect on recycling. Rapid recycling of mutant receptors resulted in reduced intracellular retention of occupied EGF receptors and delayed down-regulation of cell surface receptors. We propose that the L679A,L680A substitution acts primarily to impair transport of ligand-receptor complexes through an early endosomal compartment, diverting occupied receptors to a recycling compartment at the expense of incorporation into lysosome transport vesicles. We also found that mutant receptors with truncations at the distal half of tyrosine kinase domain (residues 809–957) were not efficiently delivered to the cell surface but were destroyed in an endoplasmic reticulum-associated degradative pathway.
Footnotes
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↵* This work was supported by National Institutes of Health Grants CA49540 and DK45669 (to C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported in part by National Institutes of Health Training Grant T32-HL07717.
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↵‖ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, School of Medicine, 10900 Euclid Ave., Cleveland, OH, 44106-4970. Tel.: 216-368-8939; Fax: 216-368-5586; E-mail: cxc39{at}po.cwru.edu.
- Abbreviations:
- EGF
-
epidermal growth factor
- EGFR
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EGF receptor
- ECV/MVB
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endosomal carrier vesicle/multivesicular body
- mAb
-
monoclonal antibody
- PAGE
-
polyacrylamide gel electrophoresis
- BFA
-
brefeldin A
- endo H
-
endoglycosidase H
- ER
-
endoplasmic reticulum
- PCR
-
polymerase chain reaction
- DMEM
-
Dulbecco’s modified Eagle’s medium
- BSA
-
bovine serum albumin.
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- Received April 1, 1998.
- Revision received October 20, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
