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• W2105341630 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. 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. epidermal growth factor receptor hepatocyte growth factor-regulated tyrosine kinase substrate sorting nexin 1 epidermal growth factor platelet-derived growth factor signal transducing adaptor molecule Hrs-binding protein early endosome antigen 1 glutathione S-transferase polyacrylamide gel electrophoresis hemagglutinin green fluorescent protein vacuolar protein sorting Phox homology Vesicular trafficking, the process by which a transport vesicle buds from a donor membrane and fuses with its target, is fundamental to the function of eukaryotic cells. For example, it is becoming increasingly clear that vesicular trafficking of ligand-activated receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)1 plays a critical role in controlling diversity, intensity, and duration of tyrosine kinase signaling (1Di Fiore P.P. Gill G.N. Curr. Opin. Cell Biol. 1999; 11: 483-488Crossref PubMed Scopus (117) Google Scholar, 2Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). Binding of EGF triggers the dimerization of EGFR and the activation of the tyrosine kinase at the cytoplasmic domain of the receptor, which then activates downstream signal transduction pathways (3Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar). After ligand binding, the ligand-receptor complexes are recruited to clathrin-coated pits and internalized. Following endocytosis, the ligand-receptor complexes are transported to early endosomes, where a sorting decision must be made between recycling back to the cell surface or delivery to lysosomes for degradation. The internalized EGF·EGFR complexes are primarily transported to lysosomes, and their degradation represents a major mechanism for attenuating EGF signaling (4Mellman I. Curr. Opin. Cell Biol. 1996; 8: 497-498Crossref PubMed Scopus (50) Google Scholar). Moreover, accumulating evidence indicates that the internalized EGF·EGFR continues to bind and phosphorylate downstream signaling proteins in pre-degradative intracellular compartments, leading to activation of signaling pathways that are distinct from those originated at the cell surface (2Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar, 5Bergeron J.J. Di Guglielmo G.M. Baass P.C. Authier F. Posner B.I. Biosci. Rep. 1995; 15: 411-418Crossref PubMed Scopus (47) Google Scholar). To ensure proper temporal and spatial signaling, the endocytic and lysosomal trafficking of EGF receptors is tightly regulated. Whereas the major events in endocytosis are fairly well understood, the molecular mechanisms underlying lysosomal trafficking of these receptors remain poorly characterized.Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was identified originally as a phosphoprotein whose tyrosine phosphorylation is induced upon stimulation by hepatocyte growth factor (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar). Subsequent studies demonstrate that the tyrosine phosphorylation of Hrs is also induced by a variety of other growth factors and cytokines, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin 2, and granulocyte-macrophage colony-stimulating factor (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Hrs exists in cytosolic and membrane-associated forms, and appears to function in both signaling and vesicular trafficking (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar). Hrs is thought to function in cell growth signaling by cytokines via its interaction with signal transducing adaptor molecule (STAM) (7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, it was reported that Hrs also interacts with Hbp/STAM2, an STAM isoform involved in cytokine signaling and degradation of PDGF receptors (10Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (69) Google Scholar, 11Endo K. Takeshita T. Kasai H. Sasaki Y. Tanaka N. Asao H. Kikuchi K. Yamada M. Chenb M. O'Shea J.J. Sugamura K. FEBS Lett. 2000; 477: 55-61Crossref PubMed Scopus (62) Google Scholar). In addition, our previous work has demonstrated that Hrs regulates the exocytotic fusion process via its interaction with SNAP-25, an essential component of the membrane fusion machinery (9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar). Hrs shares 20% sequence identity and similar domain structure with Vps27p, a yeast protein that is required for trafficking of proteins from a prevacuolar/endosomal compartment to Golgi and vacuole, the yeast equivalent of the lysosome (8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar, 12Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (340) Google Scholar). Targeted disruption of Hrs gene in mice leads to abnormally enlarged early endosomes that are reminiscent of exaggerated “class E” compartment in yeast vps27 mutant, suggesting that Hrs may have an analogous function in vesicular trafficking through mammalian endosomes (8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 13Komada M. Soriano P. Genes Dev. 1999; 13: 1475-1485Crossref PubMed Scopus (193) Google Scholar). However, it has yet to be demonstrated whether Hrs actually acts in endosome-to-Golgi and endosome-to-lysosome trafficking in mammalian cells.To understand the action of Hrs in vesicular trafficking and signaling, we performed a search for proteins that interact with Hrs using a yeast two-hybrid screen. We report here the isolation and characterization of a Hrs-interacting protein that is the rat counterpart of the human sorting nexin 1 (SNX1) (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). SNX1 was first identified as a protein that interacts with the lysosomal targeting signal-containing cytoplasmic region of EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). Overexpression of SNX1 accelerates degradation of EGFR, suggesting a role for SNX1 in endosome-to-lysosome trafficking (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). It remains controversial as to whether SNX1 interacts only with EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar) or additionally with multiple types of other cell surface receptors, including the receptors for PDGF, insulin, leptin, and transferrin (15Haft C.R. de la Luz Sierra M. Barr V.A. Haft D.H. Taylor S.I. Mol. Cell. Biol. 1998; 18: 7278-7287Crossref PubMed Scopus (212) Google Scholar). Interestingly, SNX1 is homologous to Vps5p, a yeast protein that is required for endosome-to-Golgi trafficking (16Horazdovsky B.F. Davies B.A. Seaman M.N. McLaughlin S.A. Yoon S. Emr S.D. Mol. Biol. Cell. 1997; 8: 1529-1541Crossref PubMed Scopus (184) Google Scholar, 17Nothwehr S.F. Hindes A.E. J. Cell Sci. 1997; 110: 1063-1072Crossref PubMed Google Scholar, 18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). Recent evidence indicates that Vps5p is a molecular component of a multimeric membrane-associated protein complex termed the retromer complex, which serves as a novel membrane coat acting in the formation of vesicles for endosome-to-Golgi trafficking (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). It thus likely that SNX1 may function in a similar manner in mammalian cells, acting as a key component of the lysosomal sorting machinery by incorporating cargo proteins into a retromer-like membrane coat.In the present study, we demonstrate that Hrs interacts with SNX1 bothin vitro and in vivo. We define the structural requirement for this novel interaction and show that the Hrs-binding site of SNX1 overlaps with its EGFR-binding site. In addition, gel filtration analysis and coimmunoprecipitation studies reveal that SNX1 and Hrs form a ∼550-kDa complex that excludes EGFR. We characterize the expression pattern and subcellular localization of SNX1 and show that it colocalizes with Hrs on early endosomes. Furthermore, we show that Hrs and SNX1 are involved in the regulation of the ligand-induced degradation of EGF receptors, but not in the internalization of these receptors. Our data suggest that Hrs may regulate lysosomal sorting and trafficking pathways via its interaction with SNX1.DISCUSSIONTrafficking of EGFR from endosome to lysosome plays a key role in attenuating EGF signaling. However, little is known at the molecular level about the mechanisms that regulate the lysosomal trafficking pathway. Previous studies have defined the lysosome-targeting signals within the cytoplasmic domain of EGFR that are responsible for the EGF-induced lysosomal degradation (42Opresko L.K. Chang C.P. Will B.H. Burke P.M. Gill G.N. Wiley H.S. J. Biol. Chem. 1995; 270: 4325-4333Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The first sorting nexin, SNX1, which recognizes the EGFR lysosome-targeting signals, has recently been identified and shown to function in lysosomal trafficking of EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). In this paper, we describe a novel interaction between SNX1 and Hrs, a protein that is implicated in both vesicular trafficking and cell growth signaling. The interaction of SNX1 with Hrs was demonstrated in the yeast two-hybrid system and confirmed byin vitro binding studies and coimmunoprecipitation experiments. Deletion analysis reveals that the Hrs-SNX1 interaction involves multiple coiled-coil domains and complex folded structures of the Hrs central region (residues 225–541) and of the SNX1 COOH-terminal region (residues 300–522). Several lines of evidence support a physiological significance of the observed interaction between SNX1 and Hrs. 1) Hrs and SNX1 are ubiquitously expressed proteins that exist in both cytosolic and membrane-associated pools. 2) Coimmunoprecipitation experiments demonstrate that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. 3) Gel filtration analysis reveals the presence of an endogenous ∼550-kDa protein complex containing SNX1 and Hrs. 4) Subcellular fractionation studies show that SNX1 cofractionates with Hrs and early endosomal markers on an Optiprep density gradient. 5) Double immunofluorescence analysis demonstrates that Hrs and SNX1 colocalize on early endosomes. 6) Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but has no effect on EGFR internalization. Together, these data suggest that the interaction between SNX1 and Hrs may be involved in the regulation of endosome-to-lysosome trafficking of EGFR.Our results indicate that SNX1 and Hrs share similar properties with their yeast homologues, Vps5p and Vps27p. In yeast, both Vps5p and Vps27p are localized to the prevacuolar/endosomal compartment, although it is not known whether they colocalize with each other (17Nothwehr S.F. Hindes A.E. J. Cell Sci. 1997; 110: 1063-1072Crossref PubMed Google Scholar, 18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar, 44Piiper A. Stryjek-Kaminska D. Jahn R. Zeuzem S. Biochem. J. 1995; 309: 621-627Crossref PubMed Scopus (10) Google Scholar). In mammalian cells, we found that Hrs and SNX1 colocalize on the early endosome, a sorting compartment where membrane proteins destined for degradation are sorted away from proteins that are recycled back to cell surface. The involvement of SNX1 and Hrs in lysosomal trafficking of EGFR is consistent with the role of Vps5p and Vps27p in yeast vesicular trafficking. Recently, it was reported that Vps5p assembles with Vps17p, Vps26p, Vps29p, and Vps35p to form a novel coat complex called the retromer complex (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). The presence of mammalian homologues of other retromer components Vps26p, Vps29p and Vps35p suggest that SNX1 may function in a manner that is analogous to Vps5p by forming a retromer-like coat complex in mammalian cells (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). Since SNX1 directly interacts with lysosome-targeting signals on cargo proteins such as EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar), it is likely that SNX1 performs its sorting function by selectively recruiting specific cargo proteins into the retromer-like coat complex.The mutually exclusive interaction of Hrs and of EGFR with SNX1 indicates that the association of Hrs with SNX1 is likely to interfere with the ability of SNX1 to bind and recruit EGFR into a functional coat complex for delivery to lysosomes. Supporting this view, overexpression of Hrs or its SNX1-binding domain in HeLa cells leads to an inhibition of lysosomal trafficking of EGFR for degradation. Based upon these data, a model for the role of Hrs in lysosomal trafficking can be envisaged. Hrs, by interacting with SNX1, might serve as a regulator for the assembly of functional sorting machinery. The association of Hrs with SNX1 keeps SNX1 in an inactive state, unavailable to interact with cargo proteins and/or with other components of the retromer-like coat complex. Disruption of this association by protein phosphorylation (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) or interaction with signaling proteins such as STAM and STAM2/Hbp (7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (69) Google Scholar, 11Endo K. Takeshita T. Kasai H. Sasaki Y. Tanaka N. Asao H. Kikuchi K. Yamada M. Chenb M. O'Shea J.J. Sugamura K. FEBS Lett. 2000; 477: 55-61Crossref PubMed Scopus (62) Google Scholar) would then increase the availability of SNX1 and promote cargo recruitment and assembly of functional coat complexes, and hence facilitate lysosomal sorting and trafficking. Future studies will test this model and determine the molecular mechanisms by which Hrs and SNX1 regulate vesicular trafficking. Vesicular trafficking, the process by which a transport vesicle buds from a donor membrane and fuses with its target, is fundamental to the function of eukaryotic cells. For example, it is becoming increasingly clear that vesicular trafficking of ligand-activated receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)1 plays a critical role in controlling diversity, intensity, and duration of tyrosine kinase signaling (1Di Fiore P.P. Gill G.N. Curr. Opin. Cell Biol. 1999; 11: 483-488Crossref PubMed Scopus (117) Google Scholar, 2Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar). Binding of EGF triggers the dimerization of EGFR and the activation of the tyrosine kinase at the cytoplasmic domain of the receptor, which then activates downstream signal transduction pathways (3Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1287) Google Scholar). After ligand binding, the ligand-receptor complexes are recruited to clathrin-coated pits and internalized. Following endocytosis, the ligand-receptor complexes are transported to early endosomes, where a sorting decision must be made between recycling back to the cell surface or delivery to lysosomes for degradation. The internalized EGF·EGFR complexes are primarily transported to lysosomes, and their degradation represents a major mechanism for attenuating EGF signaling (4Mellman I. Curr. Opin. Cell Biol. 1996; 8: 497-498Crossref PubMed Scopus (50) Google Scholar). Moreover, accumulating evidence indicates that the internalized EGF·EGFR continues to bind and phosphorylate downstream signaling proteins in pre-degradative intracellular compartments, leading to activation of signaling pathways that are distinct from those originated at the cell surface (2Ceresa B.P. Schmid S.L. Curr. Opin. Cell Biol. 2000; 12: 204-210Crossref PubMed Scopus (256) Google Scholar, 5Bergeron J.J. Di Guglielmo G.M. Baass P.C. Authier F. Posner B.I. Biosci. Rep. 1995; 15: 411-418Crossref PubMed Scopus (47) Google Scholar). To ensure proper temporal and spatial signaling, the endocytic and lysosomal trafficking of EGF receptors is tightly regulated. Whereas the major events in endocytosis are fairly well understood, the molecular mechanisms underlying lysosomal trafficking of these receptors remain poorly characterized. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was identified originally as a phosphoprotein whose tyrosine phosphorylation is induced upon stimulation by hepatocyte growth factor (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar). Subsequent studies demonstrate that the tyrosine phosphorylation of Hrs is also induced by a variety of other growth factors and cytokines, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin 2, and granulocyte-macrophage colony-stimulating factor (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Hrs exists in cytosolic and membrane-associated forms, and appears to function in both signaling and vesicular trafficking (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar). Hrs is thought to function in cell growth signaling by cytokines via its interaction with signal transducing adaptor molecule (STAM) (7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, it was reported that Hrs also interacts with Hbp/STAM2, an STAM isoform involved in cytokine signaling and degradation of PDGF receptors (10Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (69) Google Scholar, 11Endo K. Takeshita T. Kasai H. Sasaki Y. Tanaka N. Asao H. Kikuchi K. Yamada M. Chenb M. O'Shea J.J. Sugamura K. FEBS Lett. 2000; 477: 55-61Crossref PubMed Scopus (62) Google Scholar). In addition, our previous work has demonstrated that Hrs regulates the exocytotic fusion process via its interaction with SNAP-25, an essential component of the membrane fusion machinery (9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar). Hrs shares 20% sequence identity and similar domain structure with Vps27p, a yeast protein that is required for trafficking of proteins from a prevacuolar/endosomal compartment to Golgi and vacuole, the yeast equivalent of the lysosome (8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Kwong J. Roundabush F.L. Moore P.H. Montague M. Oldham W. Li Y. Chin L. Li L. J. Cell Sci. 2000; 113: 2273-2284PubMed Google Scholar, 12Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (340) Google Scholar). Targeted disruption of Hrs gene in mice leads to abnormally enlarged early endosomes that are reminiscent of exaggerated “class E” compartment in yeast vps27 mutant, suggesting that Hrs may have an analogous function in vesicular trafficking through mammalian endosomes (8Komada M. Masaki R. Yamamoto A. Kitamura N. J. Biol. Chem. 1997; 272: 20538-20544Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 13Komada M. Soriano P. Genes Dev. 1999; 13: 1475-1485Crossref PubMed Scopus (193) Google Scholar). However, it has yet to be demonstrated whether Hrs actually acts in endosome-to-Golgi and endosome-to-lysosome trafficking in mammalian cells. To understand the action of Hrs in vesicular trafficking and signaling, we performed a search for proteins that interact with Hrs using a yeast two-hybrid screen. We report here the isolation and characterization of a Hrs-interacting protein that is the rat counterpart of the human sorting nexin 1 (SNX1) (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). SNX1 was first identified as a protein that interacts with the lysosomal targeting signal-containing cytoplasmic region of EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). Overexpression of SNX1 accelerates degradation of EGFR, suggesting a role for SNX1 in endosome-to-lysosome trafficking (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). It remains controversial as to whether SNX1 interacts only with EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar) or additionally with multiple types of other cell surface receptors, including the receptors for PDGF, insulin, leptin, and transferrin (15Haft C.R. de la Luz Sierra M. Barr V.A. Haft D.H. Taylor S.I. Mol. Cell. Biol. 1998; 18: 7278-7287Crossref PubMed Scopus (212) Google Scholar). Interestingly, SNX1 is homologous to Vps5p, a yeast protein that is required for endosome-to-Golgi trafficking (16Horazdovsky B.F. Davies B.A. Seaman M.N. McLaughlin S.A. Yoon S. Emr S.D. Mol. Biol. Cell. 1997; 8: 1529-1541Crossref PubMed Scopus (184) Google Scholar, 17Nothwehr S.F. Hindes A.E. J. Cell Sci. 1997; 110: 1063-1072Crossref PubMed Google Scholar, 18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). Recent evidence indicates that Vps5p is a molecular component of a multimeric membrane-associated protein complex termed the retromer complex, which serves as a novel membrane coat acting in the formation of vesicles for endosome-to-Golgi trafficking (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). It thus likely that SNX1 may function in a similar manner in mammalian cells, acting as a key component of the lysosomal sorting machinery by incorporating cargo proteins into a retromer-like membrane coat. In the present study, we demonstrate that Hrs interacts with SNX1 bothin vitro and in vivo. We define the structural requirement for this novel interaction and show that the Hrs-binding site of SNX1 overlaps with its EGFR-binding site. In addition, gel filtration analysis and coimmunoprecipitation studies reveal that SNX1 and Hrs form a ∼550-kDa complex that excludes EGFR. We characterize the expression pattern and subcellular localization of SNX1 and show that it colocalizes with Hrs on early endosomes. Furthermore, we show that Hrs and SNX1 are involved in the regulation of the ligand-induced degradation of EGF receptors, but not in the internalization of these receptors. Our data suggest that Hrs may regulate lysosomal sorting and trafficking pathways via its interaction with SNX1. DISCUSSIONTrafficking of EGFR from endosome to lysosome plays a key role in attenuating EGF signaling. However, little is known at the molecular level about the mechanisms that regulate the lysosomal trafficking pathway. Previous studies have defined the lysosome-targeting signals within the cytoplasmic domain of EGFR that are responsible for the EGF-induced lysosomal degradation (42Opresko L.K. Chang C.P. Will B.H. Burke P.M. Gill G.N. Wiley H.S. J. Biol. Chem. 1995; 270: 4325-4333Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The first sorting nexin, SNX1, which recognizes the EGFR lysosome-targeting signals, has recently been identified and shown to function in lysosomal trafficking of EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). In this paper, we describe a novel interaction between SNX1 and Hrs, a protein that is implicated in both vesicular trafficking and cell growth signaling. The interaction of SNX1 with Hrs was demonstrated in the yeast two-hybrid system and confirmed byin vitro binding studies and coimmunoprecipitation experiments. Deletion analysis reveals that the Hrs-SNX1 interaction involves multiple coiled-coil domains and complex folded structures of the Hrs central region (residues 225–541) and of the SNX1 COOH-terminal region (residues 300–522). Several lines of evidence support a physiological significance of the observed interaction between SNX1 and Hrs. 1) Hrs and SNX1 are ubiquitously expressed proteins that exist in both cytosolic and membrane-associated pools. 2) Coimmunoprecipitation experiments demonstrate that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. 3) Gel filtration analysis reveals the presence of an endogenous ∼550-kDa protein complex containing SNX1 and Hrs. 4) Subcellular fractionation studies show that SNX1 cofractionates with Hrs and early endosomal markers on an Optiprep density gradient. 5) Double immunofluorescence analysis demonstrates that Hrs and SNX1 colocalize on early endosomes. 6) Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but has no effect on EGFR internalization. Together, these data suggest that the interaction between SNX1 and Hrs may be involved in the regulation of endosome-to-lysosome trafficking of EGFR.Our results indicate that SNX1 and Hrs share similar properties with their yeast homologues, Vps5p and Vps27p. In yeast, both Vps5p and Vps27p are localized to the prevacuolar/endosomal compartment, although it is not known whether they colocalize with each other (17Nothwehr S.F. Hindes A.E. J. Cell Sci. 1997; 110: 1063-1072Crossref PubMed Google Scholar, 18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar, 44Piiper A. Stryjek-Kaminska D. Jahn R. Zeuzem S. Biochem. J. 1995; 309: 621-627Crossref PubMed Scopus (10) Google Scholar). In mammalian cells, we found that Hrs and SNX1 colocalize on the early endosome, a sorting compartment where membrane proteins destined for degradation are sorted away from proteins that are recycled back to cell surface. The involvement of SNX1 and Hrs in lysosomal trafficking of EGFR is consistent with the role of Vps5p and Vps27p in yeast vesicular trafficking. Recently, it was reported that Vps5p assembles with Vps17p, Vps26p, Vps29p, and Vps35p to form a novel coat complex called the retromer complex (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). The presence of mammalian homologues of other retromer components Vps26p, Vps29p and Vps35p suggest that SNX1 may function in a manner that is analogous to Vps5p by forming a retromer-like coat complex in mammalian cells (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). Since SNX1 directly interacts with lysosome-targeting signals on cargo proteins such as EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar), it is likely that SNX1 performs its sorting function by selectively recruiting specific cargo proteins into the retromer-like coat complex.The mutually exclusive interaction of Hrs and of EGFR with SNX1 indicates that the association of Hrs with SNX1 is likely to interfere with the ability of SNX1 to bind and recruit EGFR into a functional coat complex for delivery to lysosomes. Supporting this view, overexpression of Hrs or its SNX1-binding domain in HeLa cells leads to an inhibition of lysosomal trafficking of EGFR for degradation. Based upon these data, a model for the role of Hrs in lysosomal trafficking can be envisaged. Hrs, by interacting with SNX1, might serve as a regulator for the assembly of functional sorting machinery. The association of Hrs with SNX1 keeps SNX1 in an inactive state, unavailable to interact with cargo proteins and/or with other components of the retromer-like coat complex. Disruption of this association by protein phosphorylation (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) or interaction with signaling proteins such as STAM and STAM2/Hbp (7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (69) Google Scholar, 11Endo K. Takeshita T. Kasai H. Sasaki Y. Tanaka N. Asao H. Kikuchi K. Yamada M. Chenb M. O'Shea J.J. Sugamura K. FEBS Lett. 2000; 477: 55-61Crossref PubMed Scopus (62) Google Scholar) would then increase the availability of SNX1 and promote cargo recruitment and assembly of functional coat complexes, and hence facilitate lysosomal sorting and trafficking. Future studies will test this model and determine the molecular mechanisms by which Hrs and SNX1 regulate vesicular trafficking. Trafficking of EGFR from endosome to lysosome plays a key role in attenuating EGF signaling. However, little is known at the molecular level about the mechanisms that regulate the lysosomal trafficking pathway. Previous studies have defined the lysosome-targeting signals within the cytoplasmic domain of EGFR that are responsible for the EGF-induced lysosomal degradation (42Opresko L.K. Chang C.P. Will B.H. Burke P.M. Gill G.N. Wiley H.S. J. Biol. Chem. 1995; 270: 4325-4333Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 43Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The first sorting nexin, SNX1, which recognizes the EGFR lysosome-targeting signals, has recently been identified and shown to function in lysosomal trafficking of EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar). In this paper, we describe a novel interaction between SNX1 and Hrs, a protein that is implicated in both vesicular trafficking and cell growth signaling. The interaction of SNX1 with Hrs was demonstrated in the yeast two-hybrid system and confirmed byin vitro binding studies and coimmunoprecipitation experiments. Deletion analysis reveals that the Hrs-SNX1 interaction involves multiple coiled-coil domains and complex folded structures of the Hrs central region (residues 225–541) and of the SNX1 COOH-terminal region (residues 300–522). Several lines of evidence support a physiological significance of the observed interaction between SNX1 and Hrs. 1) Hrs and SNX1 are ubiquitously expressed proteins that exist in both cytosolic and membrane-associated pools. 2) Coimmunoprecipitation experiments demonstrate that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. 3) Gel filtration analysis reveals the presence of an endogenous ∼550-kDa protein complex containing SNX1 and Hrs. 4) Subcellular fractionation studies show that SNX1 cofractionates with Hrs and early endosomal markers on an Optiprep density gradient. 5) Double immunofluorescence analysis demonstrates that Hrs and SNX1 colocalize on early endosomes. 6) Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but has no effect on EGFR internalization. Together, these data suggest that the interaction between SNX1 and Hrs may be involved in the regulation of endosome-to-lysosome trafficking of EGFR. Our results indicate that SNX1 and Hrs share similar properties with their yeast homologues, Vps5p and Vps27p. In yeast, both Vps5p and Vps27p are localized to the prevacuolar/endosomal compartment, although it is not known whether they colocalize with each other (17Nothwehr S.F. Hindes A.E. J. Cell Sci. 1997; 110: 1063-1072Crossref PubMed Google Scholar, 18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar, 44Piiper A. Stryjek-Kaminska D. Jahn R. Zeuzem S. Biochem. J. 1995; 309: 621-627Crossref PubMed Scopus (10) Google Scholar). In mammalian cells, we found that Hrs and SNX1 colocalize on the early endosome, a sorting compartment where membrane proteins destined for degradation are sorted away from proteins that are recycled back to cell surface. The involvement of SNX1 and Hrs in lysosomal trafficking of EGFR is consistent with the role of Vps5p and Vps27p in yeast vesicular trafficking. Recently, it was reported that Vps5p assembles with Vps17p, Vps26p, Vps29p, and Vps35p to form a novel coat complex called the retromer complex (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). The presence of mammalian homologues of other retromer components Vps26p, Vps29p and Vps35p suggest that SNX1 may function in a manner that is analogous to Vps5p by forming a retromer-like coat complex in mammalian cells (18Seaman M.N. McCaffery J.M. Emr S.D. J. Cell Biol. 1998; 142: 665-681Crossref PubMed Scopus (542) Google Scholar). Since SNX1 directly interacts with lysosome-targeting signals on cargo proteins such as EGFR (14Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (314) Google Scholar), it is likely that SNX1 performs its sorting function by selectively recruiting specific cargo proteins into the retromer-like coat complex. The mutually exclusive interaction of Hrs and of EGFR with SNX1 indicates that the association of Hrs with SNX1 is likely to interfere with the ability of SNX1 to bind and recruit EGFR into a functional coat complex for delivery to lysosomes. Supporting this view, overexpression of Hrs or its SNX1-binding domain in HeLa cells leads to an inhibition of lysosomal trafficking of EGFR for degradation. Based upon these data, a model for the role of Hrs in lysosomal trafficking can be envisaged. Hrs, by interacting with SNX1, might serve as a regulator for the assembly of functional sorting machinery. The association of Hrs with SNX1 keeps SNX1 in an inactive state, unavailable to interact with cargo proteins and/or with other components of the retromer-like coat complex. Disruption of this association by protein phosphorylation (6Komada M. Kitamura N. Mol. Cell. Biol. 1995; 15: 6213-6221Crossref PubMed Scopus (144) Google Scholar, 7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) or interaction with signaling proteins such as STAM and STAM2/Hbp (7Asao H. Sasaki Y. Arita T. Tanaka N. Endo K. Kasai H. Takeshita T. Endo Y. Fujita T. Sugamura K. J. Biol. Chem. 1997; 272: 32785-32791Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Takata H. Kato M. Denda K. Kitamura N. Genes Cells. 2000; 5: 57-69Crossref PubMed Scopus (69) Google Scholar, 11Endo K. Takeshita T. Kasai H. Sasaki Y. Tanaka N. Asao H. Kikuchi K. Yamada M. Chenb M. O'Shea J.J. Sugamura K. FEBS Lett. 2000; 477: 55-61Crossref PubMed Scopus (62) Google Scholar) would then increase the availability of SNX1 and promote cargo recruitment and assembly of functional coat complexes, and hence facilitate lysosomal sorting and trafficking. Future studies will test this model and determine the molecular mechanisms by which Hrs and SNX1 regulate vesicular trafficking. We are grateful to Drs. Paul Worley (The Johns Hopkins University, Baltimore, MD) and Hamid Band (Harvard Medical School, Cambridge, MA) for providing the rat hippocampal/cortical cDNA library and the pAlterMAX-EGFR construct, respectively. We thank Drs. Yi Zhang and Hengbing Wang for advice and help in the analysis of protein complexes using size-exclusion chromatography." @default.
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• W2105341630 title "Hrs Interacts with Sorting Nexin 1 and Regulates Degradation of Epidermal Growth Factor Receptor" @default.
• W2105341630 cites W1595436183 @default.
• W2105341630 cites W1967759145 @default.
• W2105341630 cites W1972049807 @default.
• W2105341630 cites W1978104052 @default.
• W2105341630 cites W1979153707 @default.
• W2105341630 cites W1979171720 @default.
• W2105341630 cites W1992628345 @default.
• W2105341630 cites W2003554177 @default.
• W2105341630 cites W2007797957 @default.
• W2105341630 cites W2017886031 @default.
• W2105341630 cites W2018435413 @default.
• W2105341630 cites W2024645073 @default.
• W2105341630 cites W2029001718 @default.
• W2105341630 cites W2030013085 @default.
• W2105341630 cites W2030854565 @default.
• W2105341630 cites W2032504743 @default.
• W2105341630 cites W2032516003 @default.
• W2105341630 cites W2036673687 @default.
• W2105341630 cites W2055267308 @default.
• W2105341630 cites W2060854650 @default.
• W2105341630 cites W2065033398 @default.
• W2105341630 cites W2066017487 @default.
• W2105341630 cites W2067000579 @default.
• W2105341630 cites W2079911954 @default.
• W2105341630 cites W2088013881 @default.
• W2105341630 cites W2090286778 @default.
• W2105341630 cites W2102026555 @default.
• W2105341630 cites W2102677721 @default.
• W2105341630 cites W2106223472 @default.
• W2105341630 cites W2107502575 @default.
• W2105341630 cites W2118893166 @default.
• W2105341630 cites W2123363603 @default.
• W2105341630 cites W2131424376 @default.
• W2105341630 cites W2140364885 @default.
• W2105341630 cites W2145206503 @default.
• W2105341630 cites W2154534551 @default.
• W2105341630 cites W2155274044 @default.
• W2105341630 cites W2156458318 @default.
• W2105341630 cites W2166961904 @default.
• W2105341630 cites W2169290377 @default.
• W2105341630 cites W2188339793 @default.
• W2105341630 cites W2188988257 @default.
• W2105341630 cites W2418320998 @default.
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