Huntingtin-associated Protein 1 Interacts with Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate and Functions in Endosomal Trafficking*

Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein huntingtin. Here we report that HAP1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking. This novel interaction was identified in a yeast two-hybrid screen using full-length Hrs as bait, and confirmed byin vitro binding assays and co-immunoprecipitation experiments. Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins. Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes. Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors. Whereas overexpression of HAP1 does not affect either constitutive or ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes. These findings implicate, for the first time, the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments.

Huntington's disease (HD) 1 is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch (Ն36 glutamines) at the N terminus of huntingtin, a ubiquitously expressed protein of unknown function (1,2). A search for huntingtin-interacting proteins has led to the identification of a novel protein called huntingtin-associated protein 1 (HAP1) (3). HAP1 interacts with the N-terminal domain of both wild type and mutant huntingtin. The binding affinity of HAP1 to huntingtin is enhanced by the increasing length of the polyglu-tamine stretch, implying a possible role for HAP1 in the pathogenesis of HD. The amino acid sequence of HAP1 exhibits no homology to any known protein in the data base, and virtually nothing is known about the cellular function of HAP1. Biochemically, HAP1 has been shown to interact with p150 glued , a dynactin subunit that participates in microtubule-based transport (4,5), and with Duo/Kalirin, a Rac1 guanine nucleotide exchange factor that regulates actin cytoskeleton dynamics (6,7), although the functional role of these interactions has not yet been examined. Electron microscopic studies reveal that HAP1 is associated with many types of membranous organelles, including endosomes, tubulovesicular structures, and synaptic vesicles (8,9). Based on these observations, it has been speculated that HAP1 may have a role in vesicular trafficking and/or organelle transport (4,6,8). However, it remains to be determined experimentally whether HAP1 indeed plays such a role(s).
In the present study, we identify a novel interaction between HAP1 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), an early endosome-associated phosphoprotein involved in the regulation of vesicular trafficking and signal transduction (10 -12). Hrs is tyrosine phosphorylated upon stimulation by a variety of growth factors and cytokines (13,14), and is thought to participate in signaling downstream of the cytokine and of the transforming growth factor-␤ receptor via interaction with signal transducing adaptor molecules and Smad2, respectively (14 -17). Hrs appears to be a mammalian homologue of Vps27p, a yeast protein required for vesicular trafficking from a prevacuolar/endosomal compartment to Golgi and vacuole (18). Gene targeting studies reveal that mutant mice with Hrs null mutation or a Hrs C-terminal deletion are embryonic lethal, perhaps as a result of defective vesicular trafficking and/or signaling (17,19). Interestingly, Hrs null mutant mice contain abnormally enlarged early endosomes that are reminiscent of the exaggerated "class E" compartment in the yeast vps27 mutant, suggesting an analogous role for Hrs in vesicular trafficking through mammalian endosomes. In support of this role, our previous work demonstrates that Hrs regulates the trafficking of EGF receptors from early endosomes to lysosomes via its interaction with sorting nexin 1, a mammalian homologue of yeast Vps5p that binds to the lysosomal targeting region of the EGF receptor (20). Very recently, it was reported that elimination of Drosophila Hrs expression impairs the ability of endosomal membranes to invaginate and form internal vesicles of the multivesicular body, and causes enhanced tyrosine kinase signaling as a result of failure to degrade activated receptor tyrosine kinases, including EGFR (21).
In the present study, we demonstrate that HAP1 associates with Hrs both in vitro and in vivo, and define the structural requirement underlying this novel interaction. Moreover, we show that HAP1 co-localizes with Hrs on early endosomes, and provide evidence supporting a role for HAP1 in the regulation of vesicular trafficking from early endosomes to late endocytic compartments. Our findings, together with the observation that mutant huntingtin binds differently than wild-type huntingtin to HAP1, suggest that the aberrant interaction between mutant huntingtin and HAP1 may cause abnormalities in the endocytic trafficking pathway, leading to neurodegeneration in HD.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Screens-A bait plasmid, pPC97-Hrs, was constructed by subcloning the entire open reading frame of rat Hrs (23) into the pPC97 vector (3,22). The yeast strain CG-1945 (CLONTECH) was transformed sequentially with pPC97-Hrs and a rat hippocampal/cortical two-hybrid cDNA library (3). Positive clones were selected on 3-aminotriazole-containing medium lacking leucine, tryptophan, and histidine, and confirmed by a filter assay for ␤-galactosidase activity. Prey plasmids from positive clones were rescued and re-transformed into fresh yeast cells with the Hrs bait or various control baits to confirm the specificity of the interaction.
Expression Constructs-An HAP1-A cDNA in pBluescript was obtained as a generous gift from Dr. Gillian Bates (24), in which the first four nucleotides of the open reading frame were missing. The fulllength HAP1-A was then constructed by PCR and the sequence was confirmed by DNA sequencing. Conventional molecular biological techniques (25) were used to subclone the DNA fragments encoding fulllength or truncated forms of Hrs and HAP1 into the following vectors: the prokaryotic expression vector pGEX-5x-2 (Amersham Pharmacia Biotech) and pET28c (Novagen) for the production of GST-and His 6tagged fusion proteins, and the mammalian expression vector pEGFP and pCHA for transfection into HeLa cells. The deletion mutants encoded by the truncated forms of Hrs and HAP1 are indicated in Fig. 2. HAP1⌬H, an HAP1-A fragment lacking the Hrs-binding domain, was generated by deletion of amino acid residues 261-368.
Fusion Protein Expression and Purification-GST-or His 6 -tagged fusion proteins were expressed in Escherichia coli BL21 cells as previously described (26). GST fusion proteins were affinity purified by using the glutathione-agarose beads (Sigma). His 6 -tagged proteins were purified using the His⅐Bind Resin and Buffer kit (Novagen). Protein concentrations were estimated by Coomassie Blue staining of protein bands following SDS-PAGE, using bovine serum albumin as standard.
Rat Brain GST Pull-down Assays-Rat brain extracts were prepared by homogenizing the brains in a homogenization buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 300 mM sucrose, plus protease inhibitors including 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml benzamidine, 1 M aprotinin, 10 M leupeptin, 1 M pepstatin A, and 1 M bestatin). Triton X-100 was added to the homogenates to a final concentration of 1% and incubated at 4°C for 30 min. Insoluble material was removed by centrifugation at 100,000 ϫ g for 1 h at 4°C, and the supernatant was used as the Triton X-100 extracts of rat brain. For binding experiments, brain extracts (100 l) were incubated with various GST-Hrs or GST-HAP1 fusion proteins immobilized on the glutathione-agarose beads at 4°C for 1 h. After extensive washes, bound proteins were eluted by boiling in the Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting.
In Vitro Binding Assays-Equal amounts of GST or various GST-HAP1 fusion proteins immobilized on the glutathione-agarose beads were incubated with soluble His 6 -tagged Hrs in the phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4, plus protease inhibitors) at 4°C for 1 h under gentle rocking. After extensive washes, bound proteins were analyzed by SDS-PAGE and immunoblotting.
Co-immunoprecipitation-To detect endogenous HAP1⅐Hrs com-plexes, rat brain extracts (1 ml) were subjected to immunoprecipitation by mouse anti-HAP1 or control mouse IgG, as described previously (27). The immunocomplexes were recovered by incubation with protein Gagarose beads (Sigma) for 1 h at 4°C. After extensive washes, the immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. Immunofluorescence Microscopy-PC12 cells were grown on poly-Llysine-coated glass coverslips and differentiated with nerve growth factor (50 ng/ml) for 48 h. HeLa cells were transfected with the indicated Hrs and HAP1 expression constructs using LipofectAMINE (Invitrogen). Cells were fixed with 4% paraformaldehyde, stained with appropriate primary and secondary antibodies, and processed for immunofluorescence microscopy as previously described (26) using a Zeiss LSM 510 confocal microscope.
Subcellular Fractionation-Confluent PC12 cells were collected by centrifugation, and the pellet was homogenized in 1 ml of ICT buffer (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 10 mM EGTA, 50 mM HEPES/ KOH, pH 7.0) plus 250 mM sucrose (20,28). After a 5-min centrifugation at 1,000 ϫ g, the supernatant was placed on a 5-20% linear Opti-prep (Nycomed) gradient formed in ICT buffer containing 42 mM sucrose, and centrifuged at 4°C for 20 h at 125,000 ϫ g in a SW41 rotor (Beckman). Following centrifugation, the gradient was harvested into 300-l fractions using an Auto Densi-Flow gradient harvester (Labconco). Equal volumes of each fraction were analyzed by SDS-PAGE and by sequential immunoblotting for HAP1, Hrs, and organelle markers.
Endocytic Trafficking Assays-For measurement of transferrin or EGF endocytosis, pEGFP-HAP1-transfected HeLa cells were incubated in serum-free medium for 1 h, then treated with 100 g/ml Texas Red-conjugated transferrin at 37°C for 30 min, or with 3 g/ml Texas Red-conjugated EGF in the presence of 0.1% bovine serum albumin at 37°C for 10 min. The cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for direct fluorescence visualization. For measurement of EGF trafficking after internalization, pEGFP-HAP1-transfected HeLa cells were incubated in serum-free medium for 1 h and then treated with 3 g/ml Texas Red-conjugated EGF in the presence of 0.1% bovine serum albumin at 37°C for 10 min. The cells were washed three times with HeLa medium to remove extracellular Texas Red-conjugated EGF and incubated at 37°C for 3 h, then fixed with 4% paraformaldehyde and processed for immunofluorescence microscopy.
EGFR Degradation Assays-70% confluent HeLa cells on 150-mm plates were transiently transfected with 1.0 g of pAlterMAX-EGFR (29) in combination with 15 g of pCHA-HAP1, pCHA-HAP1⌬1, pCHA-HAP1⌬H, or pCHA vector. Sixteen hours after transfection, cells were split onto two 150-mm plates and incubated for another 20 h. Cells were incubated in serum-free medium for 1 h and then incubated in the absence or presence of 100 ng/ml EGF for 45 min at 37°C. Cells were lysed for 30 min at 4°C in a lysis buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1% Triton X-100, 1% Nonidet P-40 plus protease inhibitors). Equal amounts (500 g) of protein from each lysate were immunoprecipitated using monoclonal anti-EGFR antibody 528 or mouse IgG. The immunoprecipitates were subjected to immunoblotting with anti-EGFR antibody 1005 and detection by enhanced chemiluminescence (ECL). The intensity of the EGFR band (180 kDa) was quantified using MCID M4 software (Imaging Research Inc.).

Yeast Two-hybrid Screens Reveal an Interaction between Hrs
and HAP1-To identify novel binding partners of Hrs, we used the full-length rat Hrs as bait to screen a two-hybrid rat hippocampal/cortical cDNA library. One of the positive clones, clone C24, encodes the middle portion of HAP1 (Fig. 1A). HAP1 was first identified as a novel huntingtin-associated protein with two alternatively spliced isoforms, HAP1-A and HAP1-B (3). HAP1 contains three putative coiled-coil domains, H1, H2, and H3, followed by a huntingtin-binding region (4). Although HAP1 was initially reported as a brain-specific protein (3), EST data base searches reveal the presence of HAP1 mRNAs in many non-neuronal tissues and cells, including mammary organ, melanocyte, retina, kidney, and lymph node. The broad distribution of HAP1 suggests that HAP1 may have a more general function than previously proposed. The specificity of the Hrs-HAP1 interaction was confirmed by yeast re-transformation experiments, in which the HAP1 clone interacts only with Hrs but not with irrelevant baits such as SNAP-25 or synaptophysin (data not shown).
Hrs and HAP1 Associate in Vitro and in Vivo-To verify the interaction between Hrs and HAP1 detected in the yeast twohybrid screens, we performed in vitro binding assays using recombinant Hrs and HAP1 proteins. GST or GST-Hrs immobilized on glutathione-agarose beads was incubated with a His 6 -tagged HAP1 fusion protein generated from the HAP1 two-hybrid clone C24. Bound proteins were analyzed by immunoblotting with an antibody against the His tag. The result shows that HAP1 binds selectively to GST-Hrs but not to the GST control, indicating a direct and specific interaction between recombinant Hrs and HAP1 (Fig. 1B). To further determine whether Hrs was able to bind endogenous HAP1, pulldown assays were performed by incubation of rat brain Triton X-100 extracts with GST or GST-Hrs immobilized on glutathione-agarose beads. As shown in Fig. 1C, the GST-Hrs fusion protein, but not the GST control, was able to pull down endogenous HAP1-A and HAP1-B from the brain extracts.
To determine whether Hrs associates with HAP1 in vivo, we performed co-immunoprecipitation experiments, using an anti-HAP1 antibody to precipitate HAP1 and its associated proteins from rat brain Triton X-100 extracts. Hrs was detected in the immunocomplexes precipitated by the anti-HAP1 antibody, demonstrating an in vivo association of Hrs with HAP1-A and HAP1-B (Fig. 1D). In contrast, neither HAP1 nor Hrs was precipitated by control IgG, confirming the specificity of Hrs and HAP1 co-immunoprecipitation detected by the anti-HAP1 antibody.
Identification of the Binding Domains Mediating the Association between Hrs and HAP1-The Hrs-interacting clone (C24) isolated from the yeast two-hybrid screen encodes residues 246 -425 of HAP1 (Fig. 1A), indicating that the N-(residues 1-245) and C-terminal (residues 426 -599 of HAP1-A or 426 -629 of HAP1-B) regions are dispensable for the association of HAP1 with Hrs. Because the HAP1-coding region of clone C24 contains two predicted coiled-coil domains, H2 (residues 262-305) and H3 (residues 328 -370), and the huntingtin-binding domain HB (residues 371-420), we sought to further map the Hrs-binding domain of HAP1 by deletion analysis. A series of HAP1 deletion mutants were generated as GST fusion proteins and their interactions with recombinant Hrs were analyzed in the in vitro binding assays ( Fig. 2A). Only the fusion proteins (HAP1⌬1 and HAP1⌬2) containing both coiled-coil domains H2 and H3 of HAP1 were capable of binding Hrs, whereas neither of these two domains alone (HAP1⌬4 or HAP1⌬5) was sufficient for interacting with Hrs. Furthermore, the huntingtinbinding domain HB (HAP1⌬6) was not involved in binding Hrs. The same results were obtained when we used pull-down assays to analyze the ability of these GST-HAP1 deletion mutants to bind endogenous Hrs from brain extracts (data not shown). These data indicate that Hrs and huntingtin associate with different regions of HAP1, however, the simultaneous binding of Hrs and huntingtin to HAP1 was not yet examined.
To delineate the minimal structural requirement for Hrs to bind HAP1, we performed similar deletion analysis by examining the interaction of various Hrs deletion mutants with endogenous HAP1 in brain extracts (Fig. 2B). A Hrs central region (residues 225-541) consisting of both H1 and H2 coiledcoil domains and the proline-rich linker was found to be responsible for binding HAP1, whereas the N-terminal VHS and FYVE domains and the C-terminal proline-rich domain of Hrs were not involved. Furthermore, neither the H2 domain nor the H1 domain with the proline-rich linker were able to bind HAP1, suggesting that multiple sites and/or a complex folded structure of the Hrs central region (residues 225-541) were involved in the interaction with HAP1.
Co-localization of Hrs and HAP1 by Immunofluorescence Microscopy-To provide additional evidence for an in vivo association of Hrs with HAP1, we first compared the intracellular localization of endogenous Hrs and HAP1 in nerve growth factor-differentiated PC12 cells by immunofluorescence confo-

FIG. 1. Specific and direct interaction between Hrs and HAP1.
A, domain structure of HAP1. HAP1 has two alternatively spliced isoforms, HAP1-A and HAP1-B, which differ only at the C terminus. Three putative coiled-coil domains (H1, H2, and H3) and the HB are indicated. The location of the Hrs-interacting clone (C24) isolated from the yeast two-hybrid screens is shown below the domain structure of HAP1. B, in vitro association of Hrs and HAP1. Soluble His-tagged HAP1 (C24) was incubated with equal amounts of immobilized GST or GST-Hrs fusion proteins. Bound HAP1 proteins were detected by immunoblotting. C, binding of endogenous HAP1 to immobilized GST-Hrs fusion proteins. Rat brain extracts were incubated with immobilized GST or GST-Hrs fusion proteins. Endogenous HAP1 proteins "pulled-down" by Hrs were detected by immunoblotting. Input, 30% of the brain extracts used in the incubation. D, co-immunoprecipitation of Hrs with HAP1. Rat brain extracts were subjected to immunoprecipitation with anti-HAP1 antibody or control mouse IgG. The immunocomplexes were analyzed by immunoblotting for HAP1 and Hrs. Input, 30% of the brain extracts used for the immunoprecipitation. cal microscopy. As shown in Fig. 3, A and B, Hrs and HAP1 both exhibit a punctate staining pattern, which was consistent with previous reports (4,23). Simultaneous staining of the same cells with anti-Hrs and anti-HAP1 antibodies revealed a substantial overlap between Hrs and HAP1 immunoreactivity, particularly in the neuritic processes, including the tip of neurites (compare Fig. 3, A and B). These staining data indicate that at least a subpopulation of HAP1 co-localizes with Hrs in PC12 cells.
We then examined the intracellular distribution of HAP1 and Hrs in HeLa cells transfected with the N-terminal HA-or GFP-tagged full-length Hrs and HAP1-A. The N-terminal HA or GFP tags did not affect the protein localization, because the staining pattern of the tagged HAP1 or Hrs was identical to that of the untagged protein (data not shown). In agreement with previous studies (20,30), we observed that recombinant Hrs, when expressed at high levels in cells, causes the formation of enlarged vesicular structures (data not shown). Interestingly, when transfected alone at high expression levels, recombinant HAP1 also led to formation of enlarged vesicular structures (Fig. 4, A and D). In cells co-expressing Hrs and HAP1, we found that a significant population of vesicular structures, both small and large in size, contains both Hrs and HAP1 proteins (Fig. 3, C-F). These results indicate that HAP1 and Hrs also co-localize in transfected HeLa cells, further supporting an in vivo association between these two proteins.
HAP1 Is Localized to Early Endosomes and Overexpression of HAP1 Causes the Formation of Enlarged Early Endosomes-It is well established that Hrs is primarily localized to early endosomes and overexpression of Hrs results in the formation of exaggerated early endosomes (20,30,31). The co-localization of HAP1 with Hrs (Fig. 3) thus gives the first hint that the HAP1-positive vesicular structures may be early endosomes. Formation of enlarged structures in the cytoplasm upon trans-fection of recombinant HAP1 has been observed previously (8,32). However, these HAP1-positive structures were interpreted as the cytoplasmic inclusions based on the observation that these structures were not labeled by markers for various organelles, namely, endoplasmic reticulum, Golgi, late endosomes, and lysosomes. Because these studies did not use any markers for early endosomes, one could not rule out the possibility that these HAP1-positive structures may represent early endosomes. Interestingly, these HAP1-positive structures were remarkably similar in appearance to the enlarged early endosomes observed in cells transfected with Hrs or a GTPasedeficient mutant form of Rab5, Rab5 Q79L (31,33,34), again suggesting an early endosomal localization for HAP1.
To test whether the HAP1-positive vesicular structures were indeed early endosomes, we performed double immunofluorescence experiments to compare the distribution of HAP1 with early endosome antigen 1 (EEA1). EEA1 is a core component of early endosome fusion machinery and has been widely used as a marker for early endosomes (35,36). The result reveals that the majority of HAP1-positive vesicular structures were labeled by EEA1, most notably for the enlarged vesicular structures caused by high-level HAP1 expression (Fig. 4 A-F). In contrast, no co-localization was observed between the distribution of HAP1 and that of LAMP1 and LAMP2, markers for late endosomes and lysosomes (Fig. 4, D-I). To further confirm the early endosomal localization of HAP1, HeLa cells were treated with Texas Red-conjugated EGF at 37°C for 10 min, under which conditions the internalized EGF proteins were almost exclusively localized to early endosomes (37,38). We observed that most of the HAP1-positive structures contained internalized EGF (Fig. 7, A-C), providing additional evidence for the association of HAP1 with early endosomes. To determine whether HAP1 associates with recycling endosomes, HeLa cells were treated with Texas Red-conjugated transferrin at 37°C

FIG. 2. Identification of interaction domains of HAP1 and Hrs.
A, mapping of the Hrs-binding domain of HAP1. Schematic illustration of HAP1-A and its deletion mutants encoded by the GST fusion cDNA constructs is shown on the left. These GST fusion proteins and GST control were immobilized on glutathione-agarose beads and incubated with His-Hrs. Bound Hrs was detected by immunoblotting. B, mapping of the HAP1-binding domain of Hrs. Rat brain extracts were incubated with immobilized GST or GST fusion proteins containing full-length or truncated forms of Hrs as indicated. Bound HAP1 proteins were detected by immunoblotting. for 30 min, under which conditions the internalized transferrin-receptor complexes were known to accumulate at the recycling endosomes, which were often concentrated at the microtubule organizing center (39). Little co-localization was observed between HAP1 and transferrin label organelles, indicating that HAP1 was not associated with recycling endosomes (Fig. 7, D-F). Together, these data clearly demonstrate that HAP1 mainly associates with early endosomes and, when expressed at high levels, causes the formation of enlarged early endosomes.
Co-fractionation of Endogenous HAP1 with Hrs and Early Endosomal Markers on a Density Gradient-To further confirm the co-localization of HAP1 and Hrs within the cell, we performed subcellular fractionation experiments to determine whether endogenous HAP1 and Hrs associate with the same membrane compartment (Fig. 5). Upon fractionation of PC12 postnuclear supernatants on a 5-20% linear Opti-prep gradient, a portion of endogenous HAP1 and Hrs was detected as soluble proteins in very low-density regions (fractions 2-8), consistent with previous reports that a significant percentage of HAP1 and Hrs exists in a cytosolic pool (3,23). In addition, a clear co-fractionation between HAP1 and Hrs was observed in the regions of higher density (fractions 21-23), demonstrating that the membrane-bound pools of these two proteins associate with the same membrane compartment. This membrane compartment was likely to be early endosomes as it overlaps with the membranes (fractions 21-24) labeled by the early endosome marker Rab5 (40), but not with the membranes (fractions 28 -32) labeled by the late endosome/lysosome marker LAMP2 (41). As reported previously (20, 28), a large portion of Rab5 was present in the soluble fractions (fractions 2-9), which may either reflect a cytosolic pool or because of the dissociation of Rab5 from membranes upon homogenization. We also compared the fractionation profiles of HAP1 and Hrs with the distribution of synaptophysin, a marker protein for synapticlike microvesicles and early endosomes in PC12 cells (42)(43)(44)(45)(46). Synaptophysin was detected in fractions 19 -24, which partially overlaps with the Rab5-positive early endosomal membranes (fractions 21-24). These results suggest that HAP1 and Hrs primarily associate with early endosomes, although we cannot exclude the possibility that a small percentage of these proteins were also localized to synaptic-like microvesicles.
Overexpression of HAP1 Inhibits the Degradation but Not the Internalization of EGFR-The ligand-induced down-regulation of EGFR has been widely used as a model to study endocytic trafficking. It is well established that binding of EGF to its surface receptors triggers the rapid internalization of the receptors followed by efficient sorting in the early endosome and eventual degradation of the receptors in the lysosome (47). Because we have shown previously that Hrs regulates the degradation of EGFR (20), the association and co-localization of HAP1 with Hrs raise the possibility that HAP1 may also be involved in the degradation of EGFR. To test this possibility, we evaluated the effect of overexpressing full-length or deletion mutants of HAP1 on the down-regulation of EGFR by using a well established assay (20,48,49). Consistent with previous studies (20,49), in vector-transfected control cells stimulation with EGF for 45 min led to a large decrease in the amount of mature EGFR (Fig. 6). In comparison, the amount of EGFR remaining after 45 min of EGF treatment was significantly increased in cells overexpressing full-length HAP1-A (Fig. 6). The observed effect of HAP1-A overexpression on the EGFR

FIG. 3. Comparison of the intracellular distribution of HAP1 with Hrs by confocal fluorescence microscopy.
A and B, nerve growth factor-differentiated PC12 cells were simultaneously stained with a primary antibody against Hrs, which was detected by a secondary antibody conjugated to fluorescein (A), and with an anti-HAP1 antibody followed by detection with a Texas Red-conjugated secondary antibody (B). C-F, HeLa cells were transiently co-transfected with pEGFP-Hrs and pCHA-HAP1-A (C and D) or with pCHA-Hrs and pEGFP-HAP1-A (E and F). The distribution of GFP-Hrs (C) and GFP-HAP1-A (F) were directly visualized by green fluorescence emitted by the GFP. The same cells were stained with a primary antibody against the HA tag (D and E) that were detected by a secondary antibody conjugated to Texas Red. The arrows indicate vesicular structures clearly visible in both panels (co-localized), whereas the arrowheads mark the vesicular structure visible in one panel but not in the other (not colocalized). Scale bar ϭ 10 m.
down-regulation seems to depend specifically on the HAP1-Hrs interaction as the same effect was also observed in cells overexpressing HAP1⌬1, a HAP1 fragment containing the Hrsbinding domain. In contrast, overexpression of HAP1⌬H, a HAP1 fragment lacking the Hrs-binding domain, had little effect (Fig. 6). These results suggest that HAP1 may be involved in the ligand-induced down-regulation of EGFR via its interaction with Hrs.
To determine whether the effect of overexpressing HAP1 on the down-regulation of EGFR was because of altered internalization of EGFR, HeLa cells were transiently transfected with the GFP-HAP1 construct, and transfected cells were tested for their capacity to internalize Texas Red-conjugated EGF. We observed that cells expressing GFP-HAP1 internalized a similar amount of Texas Red-conjugated EGF compared with untransfected cells (Fig. 7, A-C), indicating that overexpression of HAP1 does not alter the ligand-induced internalization of EGF⅐EGFR complexes. To examine whether overexpression of HAP1 affects constitutive clathrin-mediated internalization, we assessed the effect of overexpression of HAP1 on endocytosis of transferrin, a widely used marker for constitutive receptor-mediated endocytosis (50). The results reveal that cells  GFP (A, D, G, and J). Cells were stained with primary antibodies against EEA1 (B and E), LAMP1 (H), or LAMP2 (K), which were detected by secondary antibodies conjugated to Texas Red. Merged images (C, F, I, and L) demonstrate that the distribution of HAP1 partially overlaps with that of EEA1, but not with that of LAMP1 or LAMP2. Scale bar ϭ 10 m.

FIG. 5. Co-fractionation of endogenous HAP1 with Hrs on an Opti-prep gradient.
Postnuclear supernatants from PC12 cells were fractionated on a 5-20% linear Opti-prep gradient. The gradient was divided into 36 fractions, with fraction 1 corresponding to the top of the gradient. Equal volumes of each fraction were analyzed by SDS-PAGE and sequential immunoblotting for HAP1, Hrs, Rab5, synaptophysin, and LAMP2.
expressing GFP-HAP1 internalized Texas Red-conjugated transferrin as well as untransfected cells (Fig. 7, D-F), demonstrating that overexpression of HAP1 has little effect on the constitutive receptor-mediated internalization.
Overexpression of HAP1 Inhibits Vesicular Trafficking from Early to Late Endosomes-The apparent lack of effect on EGFR internalization (Fig. 7) suggests that the inhibition of EGFR degradation by overexpression of HAP1 (Fig. 6) was likely because of an altered trafficking of internalized EGFR from early endosomes to lysosomes. To address this issue, we assessed the effect of HAP1 overexpression on intracellular trafficking of EGFR using a "pulse-chase" assay. In this assay, transfected HeLa cells were allowed to internalize Texas Redconjugated EGF for 10 min. After extensive washes to remove extracellular Texas Red-conjugated EGF, the internalized Texas Red-conjugated EGF were chased for 3 h. Untransfected cells did not exhibit any detectable red fluorescence after the 3-h chase (Fig. 8), indicating that the internalized Texas Redconjugated EGF had been completely degraded. In contrast, cells overexpressing HAP1 still retained a significant amount of the internalized Texas Red-conjugated EGF after the same chase period (Fig. 8, A, B, and D). These results were consistent with the biochemical data (Fig. 6) showing that overexpression of HAP1 results in a strong reduction of EGF degradation. Simultaneous staining of HAP1 expressing cells with EEA1 demonstrates that a majority of vesicular structures that retained Texas Red-conjugated EGF after the 3-h chase contained EEA1 (Fig. 8, C, E, and F). These data indicate that overexpression of HAP1 potently blocks the trafficking from the early endosome to the late endosome, leading to accumulation of internalized EGF on early endosomes. DISCUSSION The expansion of the N-terminal polyglutamine stretch in huntingtin was the causative mutation for Huntington's disease. To understand the physiological function of huntingtin and the pathogenic mechanism by which mutant huntingtin causes neurodegeneration, a major research effort in the field has been directed in the search for binding partners of huntingtin. This effort has led to the isolation of several huntingtin interactors, including two novel proteins named HAP1 (3) and huntingtin-interacting protein 1 (HIP1) (51,52). HIP1 is the mammalian orthologue of Sla2p/End4p, a yeast protein that regulates endocytosis and cytoskeleton function (53). Furthermore, HIP1 contains an epsin N-terminal homology domain and canonical clathrin-and adaptor protein 2-binding motifs that were present in several endocytosis accessory proteins, including AP180, amphiphysin, and epsin (54). These similarities have led to recent a demonstration of a role for HIP1 in the regulation of clathrin-mediated endocytosis (55,56). Unlike HIP1, analysis of the HAP1 protein sequence has not provided any clues to the function of HAP1, as HAP1 bears no close similarity to any other protein and contains no recognizable domain except putative coiled-coil regions (Fig. 1A). Intriguingly, the present study reveals that HAP1 binds and co-localizes with Hrs, and provides the first evidence supporting a role for HAP1 as a functional partner of Hrs in the regulation of vesicular trafficking from early endosomes to late endosomes.
Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain, and the association of Hrs with early endosomes was well established (20,30,31,33). By comparison, the subcellular localization of HAP1 has not yet been fully characterized. Previous electron microscopic studies demonstrate that HAP1 is associated with endosomes and synaptic vesicles as well as other types of tubulovesicular structures and membranous organelles (8,9). Consistent with these observations, our results of immunofluorescence and subcellular fractionation studies reveal that in PC12 cells, endogenous HAP1 was primarily associated with early endosomes and a significant percentage of HAP1 co-localizes with Hrs. Furthermore, when expressed in HeLa cells, exogenous HAP1 protein was targeted to an early endosomal compartment that contains Hrs. These results, together with the biochemical data of the HAP1-Hrs interaction, suggest that HAP1 was well positioned to regulate endocytic trafficking through early endosomes.
Overexpression of HAP1 has previously been shown to cause the formation of exaggerated structures in the cytoplasm (8,32), however, the identity of these exaggerated structures is unclear. Our studies indicate that these structures were most likely to be enlarged early endosomes because they were labeled by early endosomal marker EEA1 and contain internalized EGF after a short (10 min) stimulation. The formation of FIG. 6. Effect of overexpression of Hrs and HAP1 on the degradation of EGFR. HeLa cells were transiently transfected with pAl-terMAX-EGFR in combination with pCHA-HAP1-A, pCHA-HAP1⌬1, pCHA-HAP1⌬H, or pCHA vector control. 24 h after transfection, cells were subjected to serum starvation, followed by incubation in the absence or presence of 100 ng/ml EGF for 45 min at 37°C. Cells were lysed, and equal amounts (500 g) of the protein from each lysate were immunoprecipitated using anti-EGFR antibody 528. The immunoprecipitates were analyzed by immunoblotting with anti-EGFR antibody 1005 (A). The corresponding lysates (50 g of protein/lane) were immunoblotted with anti-HA antibody (B) or anti-actin antibody (B). The remaining EGFR level after EGF stimulation was quantified and expressed as a percentage of the EGFR level of the unstimulated control cells (C). Data are mean Ϯ S.E. (error bar) of the results from four independent experiments. exaggerated early endosomes induced by HAP1 overexpression was remarkably similar to the effect of Hrs overexpression, which has been reported by several groups to cause the formation of abnormally enlarged early endosomes (20,30,31,33). Interestingly, null Hrs mutation studies in mice and Drosophila reveal that loss of Hrs expression also results in the formation of enlarged early endosomes (19,21). The fact that both elimination of expression and overexpression of Hrs lead to the same phenotype of enlarged early endosomes suggests that the Hrs protein itself and its interaction with other proteins play a crucial role in endosomal trafficking. The finding that overexpression of HAP1 results in a similar phenotype of enlarged early endosomes was consistent with the hypothesis that HAP1 was a functional partner of Hrs in the regulation of endosomal trafficking.
In addition to causing the formation of enlarged early endosomes, overexpression of HAP1 inhibits the degradation but not the internalization of EGFR, and blocks trafficking of en-docytosed EGFR from early endosomes to late endosomes. These phenotypes could result from specific effects of HAP1 overexpression, reflecting a functional role of endogenous HAP1 in regulation of endosomal trafficking via interaction with Hrs. Alternatively, these phenotypes could be nonspecific, "gain-of-function" artifacts because of accumulation of exogenous HAP1 in early endosomes. We favor the first interpretation because these phenotypes were very similar to the phenotypes associated with overexpression of Hrs as well as with elimination of Hrs overexpression (20,21,31). Furthermore, the inhibition of EGFR degradation was observed only with overexpression of full-length HAP1 or of a Hrs-binding domaincontaining HAP1 fragment, but not with overexpression of a mutant form of HAP1 bearing the internal deletion of the Hrs-binding domain. However, we cannot rule out the second possibility. A direct demonstration of the endogenous HAP1 function requires the use of "loss-of-function" approaches, such as examining the functional consequences of elimination or FIG. 7. Overexpression of HAP1 does not alter the internalization of EGF and transferrin. HeLa cells were transiently transfected with pEGFP-HAP1-A, and transfected cells were identified by the green fluorescence emitted by the GFP (A and D). Cells were treated at 37°C with Texas Red-conjugated EGF for 10 min (A-C), or with Texas Red-conjugated transferrin for 30 min (D-F). Internalized EGF or transferrin were visualized by the red fluorescence emitted by Texas Red (B and E). Superimposed images are shown in C and F. Scale bar ϭ 10 m.
FIG. 8. Overexpression of HAP1 inhibits trafficking from early to late endosomes. HeLa cells transfected with pEGFP-HAP1-A were treated with Texas Red-conjugated EGF at 37°C for 10 min, then washed with medium and incubated at 37°C for 3 h. Transfected cells were identified by the green fluorescence emitted by the GFP (A), and internalized EGF was visualized by the red fluorescence emitted by Texas Red (B). The same cells were stained with an anti-EEA1 primary antibody and a secondary antibody conjugated to CY5 (C). Superimposed images (D-F) show the co-localization between HAP1 and EGF (yellow), HAP1 and EEA1 (turquoise), and EGF and EEA1 (purple). Scale bar ϭ 10 m. reduction of HAP1 expression using antisense or knockout strategies. Despite repeated efforts, we are currently unable to reduce the levels of endogenous HAP1 using the antisense approach. It will be important in the future to determine whether targeted disruption of HAP1 gene expression impairs endocytic trafficking.
Our previous work (20,57) has shown that Hrs interacts with sorting nexin 1, a mammalian homologue of yeast Vps5p that is thought to be a key component of the endosomal sorting machinery. We speculate that HAP1, by interacting with Hrs, may serve as a regulator for the assembly of a functional endosomal sorting machinery and thus regulates the trafficking of cargo proteins from early endosomal to the late endocytic compartments. Alternatively, because it was recently reported that Hrs is able to recruit clathrin onto early endosomes (58), HAP1 may function together with Hrs as an endosomal clathrin adaptor to sort cargo destined for the late endosome/lysosome. Further studies are required to distinguish these possibilities and clarify the action of HAP1 and Hrs in endocytic trafficking.
By deletion analysis, we have mapped the Hrs-binding domain to the central region (residues 246 -372) of HAP1 containing coiled-coil domains H2 and H3 ( Fig. 2A). Because the Hrsbinding domain does not overlap with the previously reported huntingtin-binding domain HB (residues 371-420), it is possible that Hrs and huntingtin are capable of simultaneous interactions with HAP1 and forming a ternary Hrs⅐ HAP1⅐huntingtin complex. Alternatively, because the Hrsbinding domain and the huntingtin-binding domain HB are localized next to each other, binding of huntingtin to HAP1 may cause changes in the conformation of HAP1, thus preventing the interaction of Hrs with HAP1, and vice versa. Additional studies will be needed to determine whether Hrs and huntingtin interact competitively or simultaneously with HAP1, and how the mutation of huntingtin affects these protein-protein interactions. In addition, it will be important to determine whether huntingtin is a component of the trafficking machinery, cooperating with HAP1 and Hrs to regulate endocytic trafficking, or a cargo protein, whose trafficking is regulated by HAP1 and Hrs.
Given that the polyglutamine expansion alters the binding affinity of huntingtin for HAP1 (3), it is conceivable that the mutation in huntingtin may cause abnormal interactions of HAP1 with Hrs and perhaps with other components of the endocytic trafficking machinery, resulting in aberrant endocytic trafficking. The aberrant endocytic trafficking would alter protein degradation in lysosomes, which may include the degradation of mutant huntingtin itself, leading to neurodegeneration. Consistent with this notion, examination of biopsy tissues from HD patients performed in the 1970s reveals the presence of numerous multivesicular bodies, endosomes, and lysosomes, indicating a disturbed endocytic trafficking pathway in HD (59,60). Furthermore, huntingtin has been found to accumulate abnormally in the endosomal-lysosomal organelles of the HD brain compared with the control brain (61). Further investigation of the role of huntingtin and its interacting protein HAP1 in endocytic trafficking should lead to a better understanding of the pathogenic mechanisms underlying HD.
In addition to a role in endocytic trafficking, we have previously shown that Hrs also participates in neuronal exocytosis via its interaction with SNAP-25, an essential component of synaptic vesicle fusion machinery (23). Because both HAP1 and huntingtin are present at nerve terminals where they are associated with synaptic vesicles (8), it is possible that HAP1 and huntingtin may act in conjunction with Hrs and SNAP-25 in the regulation of synaptic vesicle exocytosis. Elucidation of the functional role of HAP1 and huntingtin at various stages of vesicular trafficking will not only advance our understanding of the mechanisms underlying vesicular trafficking, but may also provide insights into the physiological function of huntingtin and the pathogenesis of HD.