Lysosomal Targeting of Epidermal Growth Factor Receptors via a Kinase-dependent Pathway Is Mediated by the Receptor Carboxyl-terminal Residues 1022-1123

doi:10.1074/jbc.271.48.30340

Lysosomal Targeting of Epidermal Growth Factor Receptors via a Kinase-dependent Pathway Is Mediated by the Receptor Carboxyl-terminal Residues 1022-1123*

From the ^‡ Department of Pharmacology and
^‡‡ Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262,
the ^§ Institute of Cytology, Russian Academy of Science, Tikhoretsky 4, St. Petersburg, 194064 Russia, and
^∥ Laboratorio di Oncologia Molecolare, DIBIT and Instituto di Neuroscienze e Biommagini del Consiglio Nazionale delle Ricerche, H. S. Raffaele, Milano, 20132 Italy

^§§ Supported by National Institutes of Health Grant DK46817 and Grant SA039 from the Council for the Tobacco Research. To whom correspondence should be addressed:
Dept. of Pharmacology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262.
Tel.: 303-315-7252; Fax: 303-315-7097; E-mail: alexander. sorkin{at}uchsc.edu.

Abstract

Binding of epidermal growth factor (EGF) to its receptor induces rapid internalization and degradation of both ligand and receptor via the lysosomal pathway. To study the mechanism of intracellular sorting of EGF-EGF receptor complexes to lysosomes, NIH 3T3 cells transfected with wild-type and mutant EGF receptors were employed. The kinetics of ¹²⁵I-EGF trafficking was analyzed using low concentrations of the ligand to avoid saturation of the specific sorting system. The relative size of the pool of internalized ¹²⁵I-EGF-receptor complexes that were capable of recycling decreased as receptors traversed the endosomal system. The rate of ¹²⁵I-EGF sequestration from the recycling pathway correlated with the rate of ¹²⁵I-EGF transition from early to late endosomes as measured by Percoll gradient fractionation. Deletion of the last 63 amino acids of the EGF receptor cytoplasmic tail did not inhibit the process of sequestration and targeting to the late endosomes and lysosomes. Truncation of the 123 residues, however, resulted in impaired lysosomal targeting and increased recycling of EGF. Receptor mutant in which 165 residues were deleted displayed maximal ability to recycle and a minimal extent of sorting to the late endosomes. The data suggest that two regions of the EGF receptor molecule, residues 1022-1063 and to a lesser extent residues 1063-1123, contribute in the regulation of routing of EGF receptors to the degradation pathway. The kinase-negative receptor mutant recycled EGF more intensively compared with the wild-type receptor, and the transport of this mutant to late endosomes was inhibited. These results support the view that the receptor kinase activity is important for ligand-induced sorting of EGF receptors to the pathway of lysosomal degradation.

Footnotes

↵^∥ Supported by Grant SA039 from the Council for Tobacco Research.
↵** Supported by grants from Associazione Italiana Ricerche sul Cancro (AIRC) and from Consiglio Nazionale delle Ricerche (CNR; PF-ACRO).
↵* 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.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

DMEM

Dulbecco's modified Eagle's medium

PNS

postnuclear supernatant

TES

triethanolamine buffer

MVE

multivesicular endosomes

WT

wild-type

KN

kinase-negative mutant.
↵² A. Sorkin and L. Beguinot, manuscript in preparation.
- Received July 18, 1996.
- Revision received September 19, 1996.