Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors Are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain

doi:10.1074/jbc.270.9.4325

Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors Are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain (*)

From the (1)Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132 and the
(2)Department of Medicine, University of California at San Diego, La Jolla, California 92093

↵^§ Present address: Bristol-Myers Squibb Research Institute, 5 Research Pkwy., Wallingford, CT 06492.

Abstract

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min) and turnover (t 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t 11 h) was similar to that of occupied, kinase-active holo-EGFR (t 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Footnotes

↵* These studies were supported by National Institutes of Health Grants PO1HD28528 (to H. S. W.) and PO1CA 58689 (to G. N. G.) and by National Science Foundation Grant BCS91-11940 (to H. S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGFR

epidermal growth factor receptor

mutant EGFR

are indicated by the carboxyl-terminal amino acid residue (c') and by the amino acids of the segment fused in-frame to the carboxyl terminus (f), i.e. c'688 f945-1186

EGFR corresponds to

EGFR truncated at residue 688 followed in-frame by residues 945-1186

ΔKD-EGFR

EGFR lacking the tyrosine kinase domain (residues 689-944)

k

recycling rate constant

fraction of recycled ligand

bp

base pair(s)

Ab

antibody.
↵²B. H. Will, unpublished observations.
- Received August 23, 1994.
- Revision received November 29, 1994.