A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor

Experimental Cell Research 297 (2004) 380 – 391 www.elsevier.com/locate/yexcr

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor Chitose Morino, a Masaki Kato, a Akitsugu Yamamoto, b Emi Mizuno, a Akira Hayakawa, a Masayuki Komada, a and Naomi Kitamura a,* a

Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori, Yokohama 226-8501, Japan b Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, Tamura-cho, Nagahama 526-0829, Japan Received 10 August 2003, revised version received 14 March 2004 Available online 17 April 2004

Abstract Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs. D 2004 Elsevier Inc. All rights reserved. Keywords: EGF receptor; Early endosome; Endosomal sorting; Hrs; FYVE domain

Introduction The binding of growth factors to their receptors on the cell surface triggers the activation of the tyrosine kinase in the cytoplasmic domain of the receptors, which then activates downstream signaling pathways [1]. After growth factor binding, growth factor –receptor complexes are rapidly internalized and transported to early endosomes. Growth factor-unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to early endosomes. At early endosomes, a sorting decision is made between recycling back to the cell surface and delivery to lysosomes for degradation. The growth factor-stimulated receptors are mainly sorted to the lysosomal degradation * Corresponding author. Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8501, Japan. Fax: +81-45-9245771. E-mail address: [email protected] (N. Kitamura). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.03.038

pathway, which leads to attenuation of growth factor-receptor signaling, whereas unstimulated receptors are mostly recycled back to the cell surface [2]. Little is known about the molecular mechanism of the endosomal sorting of ligand-stimulated and unstimulated growth factor receptors. Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) is an early endosomal protein [3], and is suggested to be involved in vesicular trafficking during endocytosis and exocytosis [4]. Several lines of experimental evidence suggest that Hrs may regulate trafficking of growth factor– receptor complexes on early endosomes. First, Hrs is tyrosine-phosphorylated in cells treated with growth factors [5]. Second, overexpression of Hrs inhibits degradation of epidermal growth factor (EGF) and ligand-induced degradation of EGF receptor (EGFR) [6– 8]. Third, Hrs interacts with signal transducing adaptor molecule 2 (STAM2), sorting nexin 1 (SNX1) and Eps15 [6,9,10]. STAM2, also known as Hbp (Hrs binding protein), plays an essential role in the degradation of platelet derived growth factor– receptor complexes, and the association with Hrs is critical for STAM2 to

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exert its function [9]. SNX1 recognizes the lysosomal targeting code of EGFR and participates in lysosomal trafficking of the receptor [11]. Eps15 plays an essential role in receptor-mediated endocytosis through interaction with aadaptin, a subunit of the AP2 complex of the clathrin-coated pits [12]. Fourth, elimination of Drosophila Hrs expression impairs endosomal membrane invagination and the formation of multivesicular bodies (MVBs) and causes enhanced tyrosine kinase signaling as a result of failure to degrade activated receptor tyrosine kinases, including EGFR and Torso receptor [13]. Finally, Hrs knockdown by treatment with siRNA inhibits degradation of EGFR and the hepatocyte growth factor receptor [14,15]. Thus, Hrs may regulate the sorting of growth factor receptors on early endosomes. Hrs consists of several characteristic structural and functional domains including a FYVE domain [5]. FYVE domains were identified in proteins implicated in vesicular transport such as EEA1 [16], Fab1p [17], Vps27p [18], and Vac1p/Pep7p [19,20]. The FYVE domains of these proteins bind specifically to phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro [21 – 23], and are implicated in the endosomal localization of these proteins. The FYVE domain

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of Hrs also binds specifically to PtdIns(3)P in vitro and can replace the FYVE domain of EEA1 with respect to the endosomal targeting of EEA1 [22]. Furthermore, a point mutation in the FYVE domain of Hrs, which reduces the binding affinity with PtdIns(3)P induces an accumulation of the mutant Hrs in proteinaceous aggregates devoid of membranes [24]. Thus, the early endosomal localization of Hrs appears to be mediated through its FYVE domain by binding to PtdIns(3)P. In addition to the FYVE domain, a sequence within the coiled-coil domain of Hrs is also required for targeting of Hrs to early endosomes [24]. In this study, we examined whether Hrs regulates sorting of growth factor receptors on early endosomes. We found that overexpression of Hrs inhibited trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. We also found that overexpression of mutant Hrs with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These findings suggest that Hrs regulates sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to

Fig. 1. EGFR trafficking in HeLa cells overexpressing Hrs. HeLa cells expressing DsRed-tagged Hrs were untreated (EGF ( ), A – C) or treated with EGF for 45 min (EGF (45 min), D – F) or 60 min (EGF (60 min), G – I). After treatment, cells were fixed, permeabilized, and incubated with a mouse anti-EGFR monoclonal antibody followed by an Alexa 488-conjugated anti-mouse immunoglobulin antibody (A, D, and G). The expression of DsRed-tagged Hrs analyzed by confocal microscopy is shown in B, E, and H. The yellow stain indicates colocalization (C, F, and I). Scale bars, 20 Am.

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reside in an early endosomal microdomain, plays an essential role in the function of Hrs.

Materials and methods Expression vectors Expression vectors encoding fusion proteins of Hrs and DsRed were constructed as follows. Expression vectors, pmiw-FLAG-Hrs, pmiw-FLAG-HrsDCC, and pmiwFLAG-HrsC215S, were described previously [25]. Inserts of these vectors were excised with NotI and inserted into the NotI site of pBluescript II SK vector. The SphI – BamHI fragment of Hrs, which encodes a part of Hrs without its termination codon was obtained by PCR using oligonucleotide primers, 5V-GGTGTACTCTACCAGGCC-3V and 5VGGGGGATCCCCGTCAAAGGAGATGAGCTGGG-3V, and pBluescript-FLAG-Hrs as a template. The SacII – SphI fragments were excised from pBluescript-FLAG-Hrs, pBluescript-FLAG-HrsDCC, and pBluescript-FLAG-HrsC215S, and inserted together with the SphI –BamHI fragment into the SacII and BamHI sites of pDsRed-N1 vector (Clontech). pBluescript-FLAG-Hrs mutants encoding HrsR183A, HrsDPro-rich (lacking amino acids 290 – 390), Hrs 219 – 775, and HrsDVHS (lacking amino acids 1 – 154) were obtained with QuikChange site-directed mutagenesis Kit

(Stratagene). The SacII –SphI fragments were excised from the plasmids and inserted together with the SphI– BamHI fragment into the SacII and BamHI sites of pDsRed-N1 vector. Human EGFR cDNA described previously [26] was inserted into an expression vector, pRc/CMV. An expression vector (pME18S) encoding FLAG-STAM2 was described previously [27]. Antibodies and reagents Antibodies and growth factors were purchased as follows. An anti-EGFR monoclonal antibody was from MBL, an antiFLAG monoclonal antibody (M2) from Sigma, Horseradish peroxidase (HRP)-conjugated anti-mouse Ig and anti-rabbit Ig from Amersham Pharmacia Biotech., Alexa Fluork 488 conjugated goat anti-mouse IgG from Molecular Probes, recombinant human EGF from Pepro Tech, and EGF biotinylated, complexed to Alexak 488 streptavidin from Molecular Probes. Cell culture and transfection HeLa cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. CHO cells were cultured in Ham’s F12 medium with 10% fetal bovine serum. Plasmid DNAs were transfected using FuGENE6 transfection reagent (Roche Molecular Biochemicals).

Fig. 2. Association of EGFR with Hrs. CHO cells were transfected with a vector encoding EGFR alone (MOCK) or cotransfected with vectors encoding EGFR and FLAG-tagged Hrs (FLAG-Hrs), FLAG-tagged HrsDCC (FLAG-HrsDCC) (A) or FLAG-tagged STAM2 (FLAG-STAM2) (B), and untreated ( ) or treated with EGF (+). Lysates of the cells were immunoprecipitated (IP) with an anti-EGFR antibody and the immunoprecipitates were immunoblotted (IB) with an anti-FLAG antibody (top panel). Equivalent levels of protein expression were verified by immunoblotting using an anti-FLAG antibody (middle panel) or antiEGFR antibody (bottom panel).

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Immunoprecipitation and immunoblotting CHO cells were cotransfected with expression vectors encoding EGFR and FLAG-tagged Hrs, HrsDCC, or STAM2. At 24 h after transfection, the medium was replaced with serum-free medium. Then, cells were cultured for 24 h, and were incubated with or without 100 ng/ml EGF for 2 h at 4jC. Immunoprecipitation and immunoblotting were performed as described previously [5]. For immunoprecipitation, 0.1 Ag of an anti-EGFR antibody was used. For immunoblotting, 0.5 Ag/ml of an anti-EGFR or 5 Ag/ml of an anti-FLAG antibody was used. Indirect immunofluorescence staining HeLa cells were transfected with expression vectors encoding DsRed-tagged FLAG-Hrs or its mutants. At 24 h after transfection, the medium was replaced with serumfree medium. Then, cells were cultured for 24 h and were incubated with or without 100 ng/ml EGF for 1 h at 4jC then for 45 or 60 min at 37jC. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde in PBS for 30 min at 4jC and permeabilized with 0.1%

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Triton X-100 in PBS for 5 min at 4jC. The primary antibody was a mouse anti-EGFR monoclonal antibody. The secondary antibody was an Alexa 488-conjugated anti-mouse immunoglobulin antibody. Cells were examined by confocal immunofluorescence microscopy. Immunoelectron microscopy HeLa cells were transfected with expression vectors encoding FLAG-tagged Hrs-DsRed or its mutants on collagen-coated plastic sheets (Sumitomo Bakelite), fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 30 min. For immunoelectron microscopy, the preembedding immunogold method followed by silver enhancement procedure was used as described previously [28]. The cells were frozen in 14% glycerol and 35% sucrose in liquid nitrogen and then thawed. They were incubated with the rabbit anti-FLAG epitope antibody (10 Ag/ml) overnight and then with colloidal gold (1.4-nm diameter) conjugated secondary antibody for 2 h. The gold labeling was intensified using a silver enhancement kit (HQ silver; Nanoprobes). The cells were postfixed with 0.5% OsO4 for 90 min on ice, incubated with 50% ethanol for 10 min, and

Fig. 3. Structures and expression of wild-type Hrs and Hrs mutant constructs transfected into HeLa cells. (A) The domain organization of Hrs is shown on top. Below it, transfected constructs are shown. (B) Expression of the proteins was verified by immunoblotting using an anti-FLAG antibody.

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block-stained with 2% uranyl acetate for 2 h. The cells were dehydrated with a graded series of ethanol and embedded in epoxy resin. Ultrathin sections were observed under an electron microscope.

Results Overexpression of Hrs causes accumulation of EGFR on early endosomes In ligand-unstimulated cells, EGFR is constitutively internalized from the cell surface, transported to early endosomes, and recycled back to the cell surface. In ligandstimulated cells, EGFR forms complexes with EGF and EGF – EGFR complexes are internalized from the cell surface, and then transported to early endosomes. Most of the EGF – EGFR complexes escape recycling back to the cell surface and are transported to late endosomes, and then to lysosomes for degradation [2]. To investigate the role of Hrs in growth factor receptor trafficking through early endosomes, we overexpressed DsRed-tagged Hrs in HeLa cells and analyzed the subcellular distribution of EGFR by immunofluorescence staining using an anti-EGFR antibody and

confocal microscopy. As was previously observed [3], overexpression of Hrs induced aggregation of early endosomes, and Hrs localized to the early endosomes (Fig. 1B). In ligand-unstimulated cells, a large amount of EGFR accumulated on the Hrs-localized endosomal compartments in Hrstransfected cells, whereas no such accumulation was observed in Hrs-untransfected cells (Figs. 1A –C). At 45 min after ligand stimulation, almost all EGFR was internalized and accumulated on the Hrs-localized compartments in Hrstransfected cells (Figs. 1D – F). Quantitative analysis showed that 95% of internalized EGFR was localized to the Hrspositive compartments. At 60 min, EGFR remained accumulated on the same compartments (Figs. 1G – I). These results suggest that EGFR recycling from early endosomes in ligand-unstimulated cells and EGFR trafficking from early endosomes to late endosomes in ligand-stimulated cells are inhibited in the presence of overexpressed Hrs. Association of EGFR with Hrs The accumulation of EGFR on the Hrs-localized compartments suggests that these proteins may be found in the same molecular complexes. The association of EGFR with Hrs was examined by coimmunoprecipitation. EGFR and

Fig. 4. EGFR trafficking in HeLa cells overexpressing deletion mutants of Hrs lacking each of the structural domains. HeLa cells expressing DsRed-tagged HrsDVHS, HrsDPro-rich, HrsDCC or Hrs219-775 were untreated (A – C, G – I, M – O and S – U) or treated with EGF for 60 min (D – F, J – L, P – R, and V – X), and analyzed as described in the legend to Fig. 1. Scale bars, 20 Am.

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FLAG-tagged Hrs were coexpressed in CHO cells, and the cells were untreated or treated with EGF. Lysates of the cells were immunoprecipitated with an anti-EGFR antibody, and the immunoprecipitates were immunoblotted with an antiFLAG antibody. FLAG-tagged Hrs was detected in the immunoprecipitates with the anti-EGFR antibody both in untreated cells and in EGF-treated cells (Fig. 2A). The level of the coimmunoprecipitated Hrs was higher in the immunoprecipitates from the EGF-treated cells than those from untreated cells. Hrs possesses a coiled-coil motif, which mediates interaction with binding proteins such as STAM1 and STAM2 [9,29]. EAST, a chicken homolog of STAM2, has been shown to associate with EGFR [30]. Thus, the association of EGFR with Hrs might be mediated through these proteins. To test this, a FLAG-tagged mutant Hrs (HrsDCC) lacking the coiled-coil motif was expressed with EGFR in CHO cells, and their association was examined by

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coimmunoprecipitation. The deletion mutant of Hrs was not detected in the immunoprecipitates with the anti-EGFR antibody, suggesting that EGFR does not directly associate with Hrs, but associates with some other protein, which in turn interacts with Hrs through the coiled-coil motif. To examine whether STAM proteins associate with EGFR, EGFR and FLAG-tagged STAM2 were coexpressed in CHO cells, and cells were untreated or treated with EGF. Lysates of the cells were immunoprecipitated with an antiEGFR antibody, and the immunoprecipitates were immunoblotted with an anti-FLAG antibody. FLAG-tagged STAM2 was detected in the immunoprecipitates with the anti-EGFR antibody both in untreated cells and in EGF-treated cells (Fig. 2B). The level of the coimmunoprecipitated STAM2 was higher in the immunoprecipitates from the EGF-treated cells than those from untreated cells. These results suggest that EGFR associates with Hrs through STAM proteins.

Fig. 5. EGFR trafficking in HeLa cells overexpressing Hrs mutants with a point mutation within the FYVE domain. HeLa cells expressing DsRed-tagged HrsC215S or HrsR183A were untreated (A – C and G – I) or treated with EGF for 60 min (D – F and J – L), and analyzed as described in the legend to Fig. 1. Scale bars, 20 Am.

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FYVE domain mutants does not cause accumulation of EGFR on early endosomes Hrs possesses several characteristic structural domains [5]. To examine the structural domains involved in EGFR trafficking on early endosomes, we constructed four deletion mutants which lacked the structural domains of Hrs (Fig. 3A) and transiently transfected them into HeLa cells. Immunoblotting analysis showed that these deletion mutants were stably expressed in HeLa cells (Fig. 3B). The subcellular distribution of EGFR in these cells was analyzed by immunofluorescence staining using an anti-EGFR antibody and confocal microscopy. As was observed with the wildtype Hrs, the Hrs mutants lacking the VHS domain (DVHS), proline-rich domain (DPro-rich), or coiled-coil motif (DCC) induced aggregation of early endosomes, and internalized EGFR accumulated on the Hrs-localized compartments both in unstimulated cells and in cells stimulated with EGF for 60 min (Figs. 4A –F, G –L, and M –R). On the other hand, internalized EGFR was not colocalized with the deletion mutant of Hrs lacking both VHS and FYVE domains (219 – 775) both in unstimulated cells and in cells stimulated with EGF for 60 min (Figs. 4S – X). Similar result was obtained on cells expressing the deletion mutant of Hrs lacking the FYVE domain (data not shown). These results suggest that the FYVE domain is required for the accumulation of EGFR on early endosomes in the cells overexpressing Hrs. To further examine the role of the FYVE domain, we constructed two Hrs mutants with a point mutation in the FYVE domain. A mutant was constructed by replacing the cysteine residue at 215 with a serine (C215S) and transiently transfected into HeLa cells. This mutation has been shown

to reduce the binding affinity with PtdIns(3)P [22]. Immunoblotting analysis showed that this mutant Hrs was expressed in HeLa cells, although its expression level was much lower than that of other mutants (Fig. 3B). Internalized EGFR was not colocalized with the mutant Hrs both in unstimulated cells and in cells stimulated with EGF for 60 min (Figs. 5A –F). It has been shown that this mutation causes structural distortion of the FYVE domain, and this distortion induces accumulation of the mutant Hrs in proteinaceous aggregates devoid of membranes [24]. Thus, another mutation was introduced into the FYVE domain by replacing the arginine residue at 183 with alanine (R183A). It has been shown that this mutation does not cause structural distortion of the FYVE domain, but causes a loss of affinity for PtdIns(3)P [24]. The mutant construct was transiently transfected into HeLa cells. Immunoblotting analysis showed that this mutant Hrs was well expressed in HeLa cells (Fig. 3B). Internalized EGFR was not colocalized with the mutant Hrs both in unstimulated cells and in cells stimulated with EGF for 60 min (Figs. 5G –L). These results confirmed that the FYVE domain of Hrs is required for the accumulation of EGFR on early endosomes in the cells overexpressing Hrs. The subcellular localization of HrsR183A was examined in HeLa cells stimulated with EGF for 15 min. As was previously described, at 15 min after the stimulation, EGFR was localized to early endosomes. Similar to the wild-type Hrs, most of the mutant HrsR183A was colocalized with EGFR (Figs. 6A – C and D – F), suggesting that HrsR183A is localized on early endosomes. This result is not consistent with the result in the previous report showing that HrsR183A accumulates in the cytosol in the majority of the transfected cells [24]. A more detailed analysis of

Fig. 6. EGFR trafficking in HeLa cells overexpressing wild-type Hrs or HrsR183A treated with EGF for 15 min. HeLa cells expressing DsRed-tagged Hrs (A – C) or HrsR183A (D – F) were treated with EGF for 15 min, and analyzed as described in the legend to Fig. 1. Scale bars, 20 Am.

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Fig. 7. EGF trafficking in HeLa cells overexpressing Hrs. HeLa cells expressing DsRed-tagged Hrs were treated with 20 ng/ml Alexa 488-conjugated EGF. Upon the treatment (A – C) or at 60 min after the treatment (D – F), cells were fixed, permeabilized, and analyzed by confocal microscopy. Scale bars, 20 Am.

HrsR183A localization by immunoelectron microscopy will be described below. The accumulation of EGFR on early endosomes in cells overexpressing Hrs may result in extension of the half-life of EGFR. To examine whether overexpression of Hrs, but not overexpression of Hrs with mutations within the FYVE domain, leads to extension of the half-life of EGFR, we transfected the vector encoding DsRed-tagged Hrs or

its mutants into HeLa cells, and analyzed the level of EGFR by immunoblotting. We were not able to obtain convincing results showing extension of the half-life of EGFR in cells expressing Hrs. This may be because transfection efficiency of the vector encoding DsRedtagged Hrs into HeLa cells was low and EGFR from untransfected cells disturbed detection of the level of EGFR from transfected cells.

Fig. 8. EGF trafficking in HeLa cells overexpressing Hrs mutants. HeLa cells expressing various mutant forms of DsRed-tagged Hrs were treated with 20 ng/ml Alexa 488-conjugated EGF. At 60 min after the treatment, cells were fixed, permeabilized, and analyzed by confocal microscopy. Scale bars, 20 Am.

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Accumulation of EGF on early endosomes in Hrs-overexpressing cells After binding to its receptor, EGF together with EGFR is internalized and transported to early endosomes. Thus, it is assumed that overexpression of Hrs also causes accumulation of EGF on early endosomes. Intracellular trafficking of EGF was examined in HeLa cells overexpressing Hrs. HeLa cells transfected with DsRed-tagged Hrs were stimulated with Alexa-488-labeled EGF, and the subcellular distribution of EGF was analyzed by confocal microscopy. On treatment with EGF, EGF was distributed to the cell surface (Fig. 7A). At 60 min after EGF treatment, EGF was internalized and accumulated on the Hrs-localized endosomal compartments in cells overexpressing wild-type Hrs (Figs. 7D –F). Next, to examine the structural domains of Hrs involved in EGF trafficking on early endosomes, HeLa cells were transfected with DsRed-tagged Hrs mutants, and the subcellular distribution of EGF was analyzed. As was observed with EGFR,

EGF also accumulated on the Hrs-localized compartments in cells overexpressing the mutant Hrs lacking the VHS domain (DVHS), proline-rich domain (DPro-rich) or coiled-coil motif (DCC) (Figs. 8A –C). On the other hand, EGF was not colocalized with the mutant Hrs lacking both VHS and FYVE domains (219 – 775), or with a point mutation at cysteine 215 (C215S) or arginine 183 (R183A) (Figs. 8D – F). These results suggest that EGF trafficking from early endosomes is inhibited in the presence of overexpressed Hrs, and that the FYVE domain is required for the inhibition. Immunoelectron microscopic detection of intracellular localization of Hrs mutants To investigate in more detail the localization of Hrs mutants, we detected FLAG-tagged Hrs mutants in transfected cells with immunoelectron microscopy. HeLa cells transfected with FLAG-Hrs mutants were fixed, permeabilized, and incubated with a rabbit anti-FLAG epitope anti-

Fig. 9. Immunoelectron microscopic detection of subcellular localization of Hrs mutants. HeLa cells expressing FLAG-tagged wild-type Hrs and Hrs mutants were fixed, permeabilized, and incubated with a rabbit anti-FLAG epitope antibody. The cells were then incubated with an antibody against rabbit immunoglobulin G conjugated with 1.4 nm gold particles, and the localization of wild-type Hrs (A) and Hrs mutants (B – G) was visualized using a silver enhancement procedure. Arrows in E and F show early endosomes. Scale bar, 500 nm.

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body. The cells were then incubated with an antibody against rabbit immunoglobulin G conjugated with 1.4 nm gold particles, and the localization of Hrs mutants was visualized using a silver enhancement procedure. The deletion mutants of Hrs lacking the VHS domain (DVHS), proline-rich domain (DPro-rich), or coiled-coil motif (DCC) were detected on the cytoplasmic surface of aggregating early endosomes, similar to the wild-type Hrs (Figs. 9A –D). On the other hand, the deletion mutant lacking both VHS and FYVE domains (219 –775) was not detected on early endosomes, but detected in proteinaceous aggregates (Fig. 9E). As was previously reported [24], the mutant with a point mutation (C215S) was also detected in proteinaceous aggregates (Fig. 9F). Consistent with the result from the confocal microscopy, the mutant with a point mutation (R183A) was detected on the cytoplasmic surface of aggregating early endosomes (Fig. 9G). Thus, in our transfection system, HrsR183A localized to early endosomes in HeLa cells overexpressing the Hrs mutant.

Discussion In this study, we examined the effect of overexpression of Hrs on the intracellular trafficking of EGF and EGFR. Overexpression of Hrs induced the aggregation of early endosomes and caused the accumulation of EGFR on Hrslocalized endosomal compartments not only in ligand-stimulated cells but also in unstimulated cells. The accumulation of EGFR on overexpression of Hrs in unstimulated cells has been shown in CHO cells expressing EGFR fused to the green fluorescence protein [31]. Moreover, overexpression of Hrs caused the accumulation of EGF on Hrs-localized early endosomes. In unstimulated cells, EGFR is constitutively internalized to early endosomes and recycled back to the cell surface [2]. This default recycling back from early endosomes may be inhibited in the cells overexpressing Hrs, and as a result, EGFR accumulates on Hrs-localized endosomal compartments. In ligand-stimulated cells, the EGF – EGFR complexes are internalized and transported to early endosomes, then sorted to the lysosomal degradation pathway [2]. This sorting may be inhibited in cells overexpressing Hrs, leading to the accumulation of the EGF – EGFR complexes in early endosomal compartments. Hrs resides in an early endosomal microdomain that contains clathrin, dynamin, and Eps15 [7]. Because EGFR accumulated on Hrs-localized endosomal compartments in both unstimulated and ligand-stimulated cells overexpressing Hrs, it appears that EGFR both with and without EGF binding interacts with the Hrs-containing early endosomal microdomain, and that EGFR without EGF binding is transferred to the default recycling pathway, whereas EGFR with EGF binding is transferred to the lysosomal degradation pathway. Thus, Hrs may regulate the sorting of growth factor receptors on early endosomes between recycling back to the cell surface and delivery to lysosomes for degradation.

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Binding of growth factors to their receptors induces tyrosine phosphorylation of the receptors, and in addition it induces ubiquitination of the receptors through the action of Cbl ubiquitin ligases, which bind to the activated receptors [32]. The ubiquitination of the receptors has become recognized as an important sorting signal for the receptors to be incorporated into the luminal vesicles of the late endosomes/ MVBs [33]. Hrs binds to ubiquitinated proteins via its ubiquitin-interacting motif (UIM) [34]. STAM proteins that tightly bind to Hrs also bind to ubiquitinated proteins, and this binding is mediated through their VHS domain and UIM [27]. Overexpression of STAM2, but not its mutants lacking the ubiquitin-binding activity, causes the accumulation of ligand-activated EGFR on early endosomes [27]. Furthermore, eps15, which associates with Hrs, binds to ubiquitinated proteins via its UIM [35]. Hrs, STAM1/STAM2, and eps15 form a ternary complex [14]. Thus, Hrs-STAM-eps15 complexes appear to interact with the ubiquitinated EGFR on early endosomes. In yeast, ESCRT-I (endosomal sorting complex required for transport-I) composed of class E vacuolar protein sorting (Vps) proteins, Vps23p, Vps28p, and Vps37p, has been identified as a 350-kDa complex involved in the endosomal sorting of ubiquitinated cargo into internal MVB vesicles [36]. The human ortholog of Vps23p, tumor susceptibility gene 101 (Tsg101), is part of a protein complex that contains human Vps28p, and binds to ubiquitin [8,37]. Depletion of Tsg101 or human Vps28p causes an alteration in EGF trafficking [8,37]. Thus, HrsSTAM-eps15 complexes may transfer the ubiquitinated EGFR to protein complexes including Tsg101 and Vps28p in the MVB pathway. On the other hand, EGFR that is not ubiquitinated may not be able to be transferred to this pathway and be recycled back to the cell surface. Coimmunoprecipitation analysis showed that Hrs was coimmunoprecipitated with EGFR and a Hrs mutant lacking the coiled-coil motif was not. It also showed that STAM2 was coimmunoprecipitated with EGFR. These results suggest that EGFR associates with Hrs through STAM proteins because Hrs forms a tight complex with STAM proteins through the coiled-coil motif [9,29]. However, EGFR might associate with Hrs through other protein such as SNX1 and Huntingtin-associated protein 1 (HAP1) because the coiledcoil motif of Hrs is required to mediate the interaction of Hrs with these proteins [6,38]. Furthermore, the possibility cannot be ruled out that EGFR directly associates with Hrs through the coiled-coil motif. Two mechanisms are considered for the accumulation of EGF and EGFR on early endosomes in the cells overexpressing Hrs. One is that overexpression of Hrs may induce loss of function of early endosomes. Overexpression of Hrs induces the aggregation of early endosomes ([3]; this study). This aggregation may result in loss of normal function of early endosomes, leading to accumulation of EGF and EGFR. The other mechanism is that overexpression of Hrs may form unbalanced protein complexes. Hrs has been shown to interact with various molecules implicated in

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protein trafficking through endosomes such as STAM, eps15, and SNX1 [6,9,10,29]. Overexpression of Hrs may trap these proteins, and inhibit their function, resulting in accumulation of EGF and EGFR. A Hrs mutant (R183A) with a point mutation within the FYVE domain induced aggregation of early endosomes, but did not accumulate EGF and EGFR on the early endosomes, suggesting that the aggregation of early endosomes may not result in the accumulation of EGF and EGFR. Thus, the latter mechanism is more likely. Immunoelectron microscopic analysis revealed that the deletion mutant (Hrs219-775) lacking the FYVE domain and the point mutant (HrsC215S) with a replacement of the cysteine at 215 with a serine did not localize to early endosomes, and accumulated in proteinaceous aggregates devoid of membranes ([24]; this study). Our previous confocal microscopic analysis showed that HrsC215S and the deletion mutant (HrsDFYVE) lacking the FYVE domain colocalized with transferrin receptors. Based on these results, we concluded that these Hrs mutants localized to early endosomes [3,25]. However, this analysis could not distinguish localization of these Hrs mutants on early endosomes from that in proteinaceous aggregates. EGF and EGFR did not accumulate on early endosomes in cells expressing Hrs219-775 or HrsC215S. Thus, trafficking of EGF –EGFR complexes through early endosomes is not affected in these cells, and the complexes are normally transported to lysosomes for degradation. Confocal microscopic and immunoelectron microscopic analysis showed that the point mutant Hrs (HrsR183A) with a replacement of the arginine residue at 183 with an alanine localized to early endosomes. However, EGF and EGFR did not accumulate on early endosomes in cells expressing this mutant. The FYVE domain of Hrs binds to PtdIns(3)P [22] and is required for early endosomal localization of Hrs [24]. In addition to the FYVE domain, a sequence within the coiled-coil domain of Hrs is also required for targeting of Hrs to early endosomes [24]. The mutant HrsC215S has reduced binding affinity with PtdIns(3)P [22] and has a distorted structure of the FYVE domain [24]. On the other hand, the mutation R183A does not cause structural distortion of the FYVE domain, but causes a loss of affinity for PtdIns(3)P [24]. Thus, a lack of the FYVE domain or reduced binding affinity with PtdIns(3)P and a distorted structure of the FYVE domain probably inhibits early endosomal targeting of Hrs. A loss of binding affinity with PtdIns(3)P without structural distortion of the FYVE domain may inhibit targeting of Hrs to a microdomain in early endosomes. Size variation of endosomes among cells expressing deletion mutants of Hrs was observed on immunoelectron microscopic analysis. The size of endosomes in cells expressing HrsDVHS was much smaller than that in cells expressing HrsDPro-rich or HrsDCC. These observation suggests that the VHS domain of Hrs may regulate morphology of endosomes. However, the molecular mechanism of the regulation is not clear. We need further characteriza-

tion of the function of the VHS domain in morphology of endosomes. EGF and EGFR accumulated on early endosomes in the cells overexpressing Hrs mutants lacking the VHS domain, the proline-rich domain or the coiled-coil motif. The VHS domain is present in proteins involved in vesicular trafficking in eukaryotic cells [39]. The VHS domains in GGA proteins bind to sortilin and mannose 6-phosphate receptors at the TGN [40 –43]. The VHS domains in STAM proteins bind to ubiquitin [27]. Thus, the VHS domain in Hrs is suggested to participate in the interaction of Hrs with one of the proteins involved in the trafficking of growth factor– receptor complexes, although it has not been identified yet. The proline-rich domain in Hrs mediates the interaction of Hrs with eps15 [10], and together with the coiled-coil motif, mediates the interaction of Hrs with SNX1 and HAP1 [6,38]. HAP1 colocalizes with Hrs on early endosomes, and overexpression of HAP1 inhibits the degradation of internalized EGFR, suggesting the involvement of HAP1 in trafficking of EGFR on early endosomes [38]. The coiled-coil motif in Hrs mediates the interaction of Hrs with STAM proteins [9,29]. The deletion mutants of Hrs lacking each of these domains are probably able to associate with some of these binding partners, and thus overexpression of these mutants inhibits the trafficking of EGF and EGFR, leading to their accumulation on early endosomes. Thus, further characterization of the role of these binding partners of Hrs is required to understand the molecular mechanisms of endosomal trafficking of growth factor –receptor complexes.

Acknowledgments This work was supported by a grant-in-aid from the Ministry of Education, Science, sports and Culture of Japan (No. 15370053).

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