Hrs Interacts with Sorting Nexin 1 and Regulates Degradation of Epidermal Growth Factor Receptor*
- From the Department of Pharmacology and of Cell and Molecular Physiology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
Abstract
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a ∼550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using deletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.
Footnotes
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↵* This work was supported by a grant from the University of North Carolina Research Council (to L.-S. C.) and by Grant NS37939 from the National Institutes of Health (to L. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, Rm. 1025A Thurston-Bowles, University of North Carolina, Chapel Hill, NC 27599-7178. Tel.: 919-966-0503; Fax: 919-966-5679; E-mail: LianLi@med.unc.edu.
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Published, JBC Papers in Press, December 7, 2000, DOI 10.1074/jbc.M004129200
- Abbreviations:
- EGFR
-
epidermal growth factor receptor
- Hrs
-
hepatocyte growth factor-regulated tyrosine kinase substrate
- SNX1
-
sorting nexin 1
- EGF
-
epidermal growth factor
- PDGF
-
platelet-derived growth factor
- STAM
-
signal transducing adaptor molecule
- Hbp
-
Hrs-binding protein
- EEA1
-
early endosome antigen 1
- GST
-
glutathione S-transferase
- PAGE
-
polyacrylamide gel electrophoresis
- HA
-
hemagglutinin
- GFP
-
green fluorescent protein
- Vps
-
vacuolar protein sorting
- PX
-
Phox homology
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- Received May 15, 2000.
- Revision received December 4, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
