Hsc/Hsp70 Interacting Protein (Hip) Associates with CXCR2 and Regulates the Receptor Signaling and Trafficking*

The ligand-induced trafficking of chemokine receptors plays a significant role in the regulation of inflammatory processes and human immunodeficiency infec-tion. Although many chemokine receptors have been demonstrated to internalize through clathrin-coated vesicles, a process that involves the binding of arrestins to the receptors, accumulating evidence has suggested the possible existence of other regulators. In a yeast two-hybrid screening using the C-terminal domain of CXCR2 as a bait, the Hsc70-interacting protein (Hip) was identified to interact with CXCR2. Hip binds CXCR2 through its C-terminal domain binding to the C-terminal leucine-rich domain (KILAIHGLI) of CXCR2. Hip associates with CXCR2 or CXCR4 in intact cells, and agonist stimulation increases the association. Mutation of the Ile-Leu motif in the C-terminal domain of CXCR2 blocks the agonist-dependent association of the mutant receptor with Hip. Overexpression of a tetratricopeptide repeat (TPR) deletion mutant form of Hip ( (cid:1) TPR), which is unable to bind Hsc70 (Prapapanich, V., Chen, S., Nair, S. C., Rimerman, R. A., and Smith, D. F. (1996) Mol. En-docrinol. 10, 420–431), but retains the ability to bind CXCR2, does not affect CXCR2-mediated rhodamine-conjugated anti-rabbit antibody for 30 Co-localization studies of FITC-labeled CXCR2 rhodamine-labeled His-tagged Hip were performed using dual excitation (488, and emission (515–540 590–610 rhodamine) of established by examination of single-labeled samples.

chemokine receptors are also involved in AIDS pathogenesis (6). Like other members of the GPCR superfamily, the ability of chemokine receptors to transmit signals may be rapidly attenuated. This attenuation of signaling is tightly regulated by several mechanisms, including phosphorylation-dependent desensitization and internalization (7)(8)(9). After receptor internalization, the receptor may be dephosphorylated in the internal vesicles, recycled back to the cell surface, and resensitized to subsequent exposure to the ligand (10,11).
Chemokine receptor internalization and recycling have been shown to play an important role in the regulation of inflammatory processes and HIV-1 infection. Studies demonstrated that the responses of neutrophils, which consist of the major infiltrate in the course of acute inflammation, could be regulated and desensitized in the processes involving chemokine receptor internalization (12)(13)(14). The CCR5 antagonist aminooxypentane-RANTES (regulated on activation normal T cell expressed and secreted) has been shown to induce a potent inhibition of HIV-1 entry into target cells. This inhibition proved to be a direct result of its ability to promote CCR5 internalization and to facilitate its recycling back to the plasma membrane without dissociation of the ligand, thereby preventing HIV binding to CCR5 (11,15).
Despite being extensively studied, the mechanisms underlying the trafficking of chemokine receptors are still not fully understood. Recent studies demonstrated that many chemokine receptors internalize through clathrin-coated vesicle (CCV), a process that involves the binding of arrestins to the phosphorylated receptors (9, 16 -20). However, as revealed by the studies of other GPCRs, even though arrestins bind clathrin in vitro with high affinity (21), arrestins are not constitutively associated with clathrin-coated vesicles (22). Our previous studies demonstrated that truncation of the C-terminal serine-rich domain in CXCR2, which blocked arrestin binding to the mutant receptor, did not prevent the receptor internalization in certain cell types (23). Similar phenomena have been observed with other chemokine receptors, e.g. CXCR4 (8), as well as other GPCRs, e.g. type A cholecystokinin receptor (24). Although arrestins and dynamin appear to play an important role in phosphorylation-dependent sequestration of GPCRs, the phosphorylation-independent sequestration suggests that other potential regulators may exist in the internalization processes. Because the C-terminal domain of chemokine receptors has been demonstrated to play a key role in receptor phosphorylation, desensitization, and internalization (7,8,18,23), we established a yeast-two hybrid system to screen the intracellular proteins that interact with the C-terminal domain of CXCR2, a chemokine receptor that mediates the migration of neutrophils to inflammatory sites in response to Glu-Leu-Arg (ELR)-expressing CXC chemokines (1,25). A number of proteins have been identified to be potentially involved in the receptor signaling and trafficking. In the present study, we demonstrate that Hsc70-interacting protein (Hip) associates with the chemokine receptors CXCR2 and CXCR4, and a tetratricopeptide repeat (TPR) deletion mutant form of Hip, which is unable to bind Hsc70 but continues to bind CXCR2, attenuates the receptor internalization and the receptor-mediated chemotaxis.

EXPERIMENTAL PROCEDURES
Plasmid Construction-Wild-type CXCR2 in pRC/CMV, and the Cterminal domain of CXCR2 in PAS2 or in pGEX-KG were constructed previously (10). The wild-type and the mutant forms of Hip in pcDNA3.1 were constructed by amplifying the fragments using PCR and inserted into the BamHI and XhoI sites.
Yeast-two Hybrid Assay-Yeast two-hybrid techniques were performed as described previously (10). For screening cDNA libraries, the bait plasmid PAS2/CXCR2 tail was transformed into yeast strain Y190 (CLONTECH) using a lithium acetate protocol (CLONTECH manual). After confirming expression of the bait protein, a human B lymphocyte library in PACT2 (26) was transformed into the strain harboring the bait plasmid. The transformants expressing both the bait and the prey proteins were selected on SD/-Leu/-Try/-His medium. Colonies capable of growing on the SD/-Leu/-Try/-His medium were then tested for ␤-galactosidase activity (LacZ ϩ ) using the filter lift assay. Clones that were consistently phenotypically His ϩ and LacZ ϩ were further characterized. Approximately 2.6 ϫ 10 6 transformants were screened, and several of them were His ϩ and LacZ ϩ . One of the clones chosen for sequencing and characterization, based on its strong His ϩ /LacZ ϩ phenotype, displayed sequence identity with Hip.
Filter Lift Assay-A dry nitrocellulose filter was placed on the yeast colonies. The filter was then carefully lifted, transferred (colonies facing up) into liquid nitrogen, completely submerged for 10 s, and then allowed to thaw at room temperature. The filter was then placed on a Whatman filter paper soaked in Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM Mg 2 SO 4 , pH 7.0) containing 50 mg/ml 5-bromo-4-chloro-3-indolyl-galactopyranoside, with colonies face up until blue color appeared.
In Vitro Binding Assay-Bacteria transformed with plasmids encoding GST or GST fusion proteins were cultured overnight at 37°C, then isopropyl ␤-D-thiogalactopyranoside was added and incubation was continued for another 3 h to induce protein expression. The bacteria were lysed in RIPA buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 10 g each of leupeptin and aprotinin) and then sonicated on ice for 10 s. The supernatant of the bacterial lysate was incubated with glutathione-Sepharose at 4°C for 30 min. After washing three times with RIPA buffer, the purified GST-or GST fusion protein-bound beads were resuspended in RIPA buffer. Aliquots of the purified GST or GST fusion proteins were incubated with HEK293 cell lysate at 4°C for 2 h with rotation. Beads were pelleted by centrifugation (15,000 ϫ g, 2 min), and washed four times with RIPA buffer. Bound proteins were released by boiling in SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol for 5 min and detected by SDS-PAGE and Western blot.
Co-immunoprecipitation and Western Blot-The following cell types were used in these experiments: 1) Human neutrophils, which endogenously express CXCR2, were isolated from fresh heparinized peripheral blood from single human donors as described previously (10); 2) RBL-2H3 cells stably expressing CXCR2 were transiently transfected with plasmids encoding His-tagged wild-type Hip or ⌬TPR; 3) HEK293 cells stably expressing wild-type or the internalization-deficient mutant (I323A/L324A) of CXCR2 were transiently transfected with plasmids encoding His-tagged wild-type Hip or ⌬TPR; 4) HEK293 cells transiently expressing myc-CXCR4 and wild-type Hip or ⌬TPR. Cells were treated with carrier buffer (control) or agonists (100 ng/ml CXCL8 for CXCR2 and 100 nM CXCL12 for CXCR4) for 5 min unless indicated, washed three times with ice-cold phosphate-buffered saline, and lysed in 1 ml of RIPA buffer. The cell debris was removed by centrifugation (15,000 ϫ g, 15 min). The supernatant was pre-cleared by incubation with 40 l of protein A/G-agarose (Pierce) for 1 h at 4°C to reduce nonspecific binding. After removal of the protein A/G-agarose by centrifugation (15,000 ϫ g, 1 min), the cleared supernatant was collected and 10 l of affinity-purified anti-CXCR2 antibody (26) or anti-myc antibody (9E10, Santa Cruz Biotechnology) was added for overnight precipitation at 4°C. 40 l of protein A/G was then added and incubated at 4°C for 2 h. The protein A/G-antibody-antigen complex was then collected by washing three times with ice-cold RIPA buffer. The final pellet was re-suspended in 50 l of SDS sample buffer containing 5% ␤-mercaptoethanol and heated to 50°C for 10 min. 20 l of this preparation was electrophoresed on a 10% SDS-polyacrylamide gel, and the proteins on the gel were transferred to nitrocellulose membranes (Bio-Rad). Co-precipitated proteins were detected by Western blotting using specific antibodies.
Confocal Microscopy-Confocal microscopy was performed on a Zeiss LSM-510 laser scanning microscope using a Zeiss 63 ϫ 1.3 numerical aperture (NA) oil immersion lens. HEK293 cells stably expressing CXCR2 and transiently expressing His-tagged Hip were grown on coverslips. Cells were treated with carrier buffer or CXCL8 (100 ng/ml) for 5 min and fixed with methanol. Cells were washed with phosphate-buffered saline and incubated with a mixture of a mouse monoclonal anti-CXCR2 antibody and a rabbit polyclonal anti-His antibody for 30 min. Cells were washed and incubated with a mixture of a fluorescein isothiocyanate (FITC)-conjugated anti-mouse and a rhodamine-conjugated anti-rabbit antibody for 30 min. Co-localization studies of FITC-labeled CXCR2 and rhodamine-labeled His-tagged Hip were performed using dual excitation (488, 568 nm) and emission (515-540 nm, FITC; 590 -610 nm rhodamine) filter sets. Specificity of labeling and absence of signal crossover were established by examination of single-labeled samples.
MAPK Assay-HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids encoding His-tagged wild-type Hip or ⌬TPR. Cells were treated with CXCL8 at 37°C for 10 min then lysed in RIPA buffer. Lysates containing equal amounts of protein were subjected to SDS-PAGE (10%). Proteins were transferred to nitrocellulose membrane, and phosphorylated MAPK (ERK1/ERK2) was detected by Western blotting using a phosphospecific MAPK antibody (SC-7383, Santa Cruz Biotechnology).
Chemotaxis Assay-A 96-well chemotaxis chamber (Neuroprobe Inc.) was used for chemotaxis assays, and the lower compartment of the chamber was loaded with 400-l aliquots of 1 mg/ml ovalbumin/DMEM (chemotaxis buffer) or CXCL1 diluted in the chemotaxis buffer (1-200 ng/ml). Polycarbonate membranes (10-m pore size) were coated on both sides with 20 g/l human collagen type IV, incubated for 2 h at 37°C, then stored at 4°C overnight. To prepare HEK293 cells for chemotaxis assay, cells were removed from the culture dish by trypsinization, washed with Hanks' solution, and incubated in 10% fetal bovine serum/DMEM for 2 h at 37°C to allow time for restoration of receptors. The cells were washed with chemotaxis buffer and then loaded into the upper chamber in the chemotaxis buffer. The chamber was incubated for 4 h at 37°C in humidified air with 5% CO 2 , then the membrane was removed, washed, fixed, and stained with a Diff-Quik kit. Cell chemotaxis was quantified by counting the number of migrating cells present in 10 microscope fields (20 ϫ objective).
Ligand-Receptor Complex Internalization Assay-The acid-wash technique (23) was used to determine the kinetics of CXCL8-induced internalization of CXCR2. HEK293 cells expressing CXCR2 were grown to confluence on 24-well plates, which were pre-coated with 0.1 mg/ml poly-L-lysine (Sigma Chemical Co., M r 30,000 -70,000) for 1 h and washed once with distilled water before use. Cells were incubated at 4°C in 0.5 ml of serum-free DMEM containing 75 nCi/ml 125 I-CXCL8 for 1 h. The medium was subsequently removed, 1 ml of ice-cold serumfree DMEM was added carefully into each well and aspirated, and then another 1-ml aliquot of ice-cold serum-free DMEM was added prior to incubation at 37°C for the indicated time. The medium was removed, and the cells were incubated with 1 ml of ice-cold 0.2 M acetic acid with 0.5 M NaCl for 6 min. After the incubation, the cells were washed once with 1 ml of ice-cold serum-free DMEM, and lysed with 1 ml of 1% SDS with 0.1 N NaOH (lysis solution). The radioactive cell lysate was then counted on a ␥-counter (Gamma 5500, Beckman). Total cell surface receptor binding was measured after incubation with 125 I-CXCL8 medium followed by washing the cells with ice-cold serum-free DMEM. Nonspecific binding was measured by adding ice-cold 0.2 M acetic acid with 0.5 M NaCl after incubation with 125 I-CXCL8 in binding medium. Calculation of the percentage of internalized receptor was performed as described before (23).
FACS Analysis-RBL-2H3 cells were transiently co-transfected with CXCR4 and pcDNA3.1, Hip, or ⌬TPR. Cells (5 ϫ 10 5 /sample) were incubated in HEPES (20 mM)-buffered DMEM at 37°C for 30 min in the presence or absence of CXCL12 (100 nM). Cells were then washed in ice-cold medium and incubated with a monoclonal c-Myc antibody at 4°C for 1 h. Cells were washed with ice-cold medium followed by incubation with fluorescein isothiocyanate-labeled goat anti-mouse IgG at 4°C for 30 min. Cells were washed and fixed in 2% formaldehyde in phosphate-buffered saline and analyzed in FACScan equipped with CellQuest software (Becton Dickinson).

RESULTS
In an attempt to isolate chemokine receptor-associated proteins, we used the yeast two-hybrid system to identify proteins that interact with the C terminus (amino acids 311-355) of CXCR2 (Fig. 1A). Screening of a human B lymphocyte library fused to the GAL4 transactivation domain (27) yielded several potential candidate genes that were both His ϩ and LacZ ϩ . The prey cDNAs were recovered from yeast and transformed into bacteria. The cDNAs were then sequenced using primers complimentary to 5Ј-or 3Ј-ends of the inserts. Among them, one encoding Hip was chosen for further study based on its moderately strong LacZ ϩ . The specificity of the interaction in yeast was tested by re-transforming PACT2/Hip along with the original bait PAS2/CXCR2 C terminus or PAS2 alone back into yeast strain Y190. The interaction between the receptor C terminus and Hip specifically allowed growth of the yeast on SD medium lacking leucine, tryptophan, and histidine (SD/-Leu/-Try/-His) (Fig. 1A, left panel). Deletion of the serine-rich domain (amino acids 331-355) in the receptor C terminus did not affect the interaction, suggesting that the binding domain resides in the region of amino acids 311-330. Neither the bait, PAS2/CXCR2 C terminus, nor the prey was able to activate transcription of the reporter genes in the presence of only empty prey or bait vectors, respectively (Fig. 1A, left panel).
To confirm the specific biochemical interaction between the C terminus of CXCR2 and Hip, we used an in vitro binding assay to test for the direct interaction. GST or GST fusion proteins of the C terminus of wild-type or mutant forms of CXCR2 were incubated with the cell lysate of HEK293 cells, and associated Hip was detected by Western blotting. The results demonstrated that the GST fusion protein of the CXCR2 C terminus, but not the GST alone, associated with Hip. The binding of Hip to the mutant forms of CXCR2 C terminus, K322A/I333A/L324A, I326A/H327A, and G328A/ L329A/I330A, was significantly reduced compared with the binding of Hip to wild-type CXCR2 C terminus, whereas other mutant forms bound a similar amount of Hip as the wild-type CXCR2 C terminus (Fig. 1B). These data suggest that the Hip binding site resides in the KILAIHGLI motif in the C terminus of CXCR2.
To identify the CXCR2 binding site in the Hip protein, PCRdirected mutagenesis was used to prepare mutant Hip cDNAs for the production of truncated or internally deleted Hip proteins. Fig. 2A presents diagrams of the wild-type and mutant forms of Hip prepared. In addition to the N and C termini, three structurally distinctive regions were targeted for mutagenesis. These regions are (i) the TPR at approximately amino acids 100 -200, which is required for Hsc/Hsp70 binding, (ii) a 50-amino acid region enriched in charged residues (ϩϪ ϩϪ), a second binding domain of Hsc/Hsp70, and (iii) a 33amino acid stretch containing degenerate tandem repeats of the sequence glycine-glycine-methionine-proline (G). The Histagged wild-type and mutant Hip cDNAs in pcDNA3.1 were transiently transfected in HEK293 cells, and the protein expression was determined by Western blotting. Fig. 2B shows the expression of the His-tagged wild-type or mutant forms of Hip in HEK293 cells. To identify the CXCR2 binding domain in the sequence of Hip, the GST fusion protein of the CXCR2 C terminus was incubated with the cell lysate of HEK293 cells overexpressing the His-tagged wild-type or the mutant forms of Hip proteins. Co-precipitated wild-type or mutant Hip proteins were detected by Western blotting using a specific anti-His antibody. As shown in Fig. 2C, only the C-terminal parts of Hip were bound to the GST fusion protein of CXCR2 C terminus. Truncation of the C-terminal 66 amino acids completely impaired the interaction, suggesting that the CXCR2 binding site resides in the extreme C-terminal domain of Hip. As expected, the TPR deletion mutant of Hip (⌬TPR) bound an almost equal amount of the GST fusion protein of CXCR2 C terminus (Fig.  2C).
We next examined whether a functional complex consisting of CXCR2 and Hip could be detected in RBL-2H3 cells stably expressing CXCR2 (generous gift from Dr. Ricardo M. Richardson). Immunoprecipitation of CXCR2 from RBL-2H3 cells revealed a basal association of the receptors with Hip, and CXCL8 (100 ng/ml) treatment resulted in a time-dependent increase in the association of Hip with CXCR2, which peaked 5 min after agonist stimulation (Fig. 3A). Similar time-dependent association of CXCR2 with Hip was also observed in HEK293 cells stably expressing CXCR2 (data not shown). In addition, the agonist-induced increase in the association of CXCR2 with Hip was also observed in human neutrophils in response to CXCL8 treatment for 5 min (Fig. 3B). Although in the same time frame, about 50% of the receptors have been internalized (15,23), suggesting that agonist-induced association of CXCR2 with Hip occurs in the internal vesicles, whereas A ␤-galactosidase assay was carried out using a YPD plate replicate (right panel) as described under "Experimental Procedures." B, the indicated GST or GST-CXCR2 C terminus fusion proteins were incubated with the cell lysate of HEK293 cells then absorbed onto glutathione-Sepharose beads. After washing, the beads were resuspended in loading buffer. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Co-precipitated Hip proteins were analyzed by immunoblotting with a polyclonal Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal GST antibody to confirm the protein expression and equal loading. the basal association suggests that a small proportion of CXCR2 associates with Hip on the cell membrane. To test this hypothesis, an internalization-deficient mutant of CXCR2 (I323A/L324A) (23) was used in the coimmunoprecipitation assay. As expected, in HEK293 cells stably expressing I323A/ L324A, agonist stimulation did not induce an increase in the receptor/Hip association (Fig. 3C). To test whether other chemokine receptors associate with Hip, HEK293 cells transiently expressing myc-tagged CXCR4 were treated with carrier buffer or CXCL12 for 5 min, and myc-CXCR4 was immunoprecipitated. As shown in Fig. 3D, Hip was also co-immunoprecipitated with CXCR4 in an agonist-dependent manner.
To visualize the co-localization of CXCR2 with Hip in intact cells, confocal microscopy was performed. CXCR2 and Histagged Hip were stained with specific antibodies in HEK293 cells stably expressing CXCR2 and transiently expressing Histagged Hip. As shown in Fig. 4, CXCR2 was expressed almost exclusively on the cell surface (Fig. 4A), whereas Hip was located largely in the cytoplasm (Fig. 4B). Interestingly, a small proportion of Hip was expressed on the cell surface (Fig.  4B), and co-localized with CXCR2 (Fig. 4C). This may explain the reason for the basal association of CXCR2 with Hip observed in the coimmunoprecipitation assay (Fig. 3). In response to agonist stimulation, a proportion of CXCR2 was internalized in the internal vesicles (Fig. 4D), and the internalized CXCR2 was partially co-localized with Hip (Fig. 4F).
To investigate the potential role of the interaction of CXCR2 with Hip in the receptor signaling and trafficking, a dominant interfering mutant of Hip is needed to block the functions of Hip. Because the major function of Hip characterized so far is to bind Hsc/Hsp70 and regulate their chaperone activities, we chose to use a mutant Hip that is unable to bind Hsc/Hsp70. Because Hip regulates the chaperone activities of Hsc/Hsp70 through its central TPR domain binding to the ATPase domain of Hsc/Hsp70 (28), and ⌬TPR, a dominant interfering mutant form of Hip, has been shown to be unable to bind Hsc/Hsp70 (28), we used ⌬TPR as a probe to test the physiological significance of the interaction between CXCR2 and Hip, and to understand the potential role of Hsc/Hsp70 in the signaling and trafficking of CXCR2. HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids encoding wild-type Hip or ⌬TPR, and the receptor expression, signaling, and trafficking were assessed. Overexpression of wild-type Hip or ⌬TPR did not affect the receptor expression on the cell surface as determined by FACS analysis (data not shown), nor did it significantly affect the receptor-ligand binding as assessed by 125 I-CXCL8 binding assay (data not shown). In addition, agonist-dependent MAPK activation in the CXCR2-expressing cells was not affected by the overexpression of wildtype Hip or ⌬TPR (Fig. 5).
To assess whether Hip is involved in the endocytosis of CXCR2, HEK293 cells stably expressing CXCR2 were transiently transfected with plasmids encoding His-tagged wildtype Hip or ⌬TPR. Cells were incubated with 125 I-CXCL8 at 4°C for 1 h, then warmed to 37°C for different times. After acid washing to eliminate the cell surface 125 I-CXCL8, the internalized 125 I-CXCL8 was detected in a ␥-counter. Fig. 6A shows a time-dependent internalization of 125 I-CXCL8. Overexpression of wild-type Hip did not affect the internalization of 125 I-CXCL8. However, in the cells overexpressing ⌬TPR, the internalization of 125 I-CXCL8 was attenuated about 50% compared with the internalization of 125 I-CXCL8 in the control cells (p Ͻ 0.05). In addition, the internalization of CXCR4 was also tested in RBL-2H3 cells co-transfected with plasmids encoding CXCR4 and His-tagged wild-type Hip or ⌬TPR. Cells were treated in suspension without or with CXCL12 for 30 min, and cell surface expression of CXCR4 was detected by immunofluorescence with anti-c-Myc using FACS analysis. As shown in Fig. 6B, CXCL12 treatment reduced the cell surface expression of CXCR4 in both the control cells and the cells overexpressing Hip proteins, indicating receptor internalization in these cells. CXCR4 was also internalized in the cells expressing ⌬TPR in response to CXCL12 treatment, but the amount of internalization was consistently lower than that observed in the control cells or cells expressing His-tagged Hip. The percentages of CXCR4 internalization from four independent experiments are 75.99 Ϯ 2.45, 81.42 Ϯ 3.54, and 38.29 Ϯ 2.38 for the control cells expressing CXCR4, cells expressing CXCR4 and His-tagged Hip, and cells expressing CXCR4 and Histagged ⌬TPR, respectively. Statistical analysis demonstrated that the internalization of CXCR4 was significantly reduced in the cells expressing His-tagged ⌬TPR (p Ͻ 0.05).
Because one of the most important functions for chemokine receptors is to mediate chemotaxis, and previous studies have demonstrated the important role of CXCR2 internalization in the receptor-mediated chemotaxis (16,23), we examined the potential role of Hip in the chemotaxis of CXCR2-expressing cells. HEK293 cells stably expressing CXCR2 exhibited a typical bell-shape chemotactic response upon treatment by pro-

FIG. 2. Identification of the CXCR2 binding domain in Hip.
A, diagrams of wild-type and mutant Hip forms, highlighting structurally distinct regions. WT, wild-type. B, the wild-type and mutant Hip forms in pcDNA3.1 were transiently transfected in HEK293 cells. Cells were lysed, and proteins were separated by 10% SDS-PAGE. The expression of His-tagged Hip forms was determined by Western blotting using a specific anti-His antibody. C, the GST-CXCR2 C terminus fusion proteins were incubated with the cell lysate of HEK293 cells transiently expressing His-tagged Hip forms. Co-precipitated Hip forms were analyzed by Western blotting using anti-His antibody as described in the Fig. 1B legend. gressive concentrations of CXCL1. Overexpression of wild-type Hip did not affect the cell chemotaxis, whereas overexpression of ⌬TPR significantly reduced the receptor-mediated chemotaxis (Fig. 7). DISCUSSION Chemokine receptor trafficking may play a significant role in the fine-tuning of chemokine-induced inflammatory responses and necessitate a better understanding of the mechanisms responsible for the control of the intracellular trafficking of these receptors. Our present study is the first to provide evidence for novel findings regarding the regulation of chemokine receptor trafficking by Hip, as indicated by the following observations: 1) Hip interacted with CXCR2 through its C-terminal domain binding to the C-terminal leucine-rich domain of CXCR2. CXCR2 and CXCR4 associated with Hip in an agonistdependent manner in intact cells, and an internalization-deficient mutant of CXCR2, I323A/L324A, failed to associate with Hip; 2) overexpression of a dominant interfering mutant of Hip (⌬TPR) inhibited the internalization of CXCR2 and CXCR4. These observations have important physiological implications, because expression of ⌬TPR inhibited the migratory responses of the chemokine receptor-expressing cells as assessed by agonist-induced chemotaxis.
Hip was first noted as a transient component during the cell-free assembly of progesterone receptor complex (29) and was subsequently found to be associated with Hsp70 and Hsc70   FIG. 3. Association of Hip with CXCR2 or CXCR4 in intact cells. A, RBL-2H3 cells stably expressing CXCR2 were exposed to CXCL8 (100 ng/ml) for the indicated times, and CXCR2 was immunoprecipitated from the cell lysate using a rabbit polyclonal anti-CXCR2 antibody. A preimmune rabbit serum (Mock) was used in a parallel experiment to confirm the specificity of the immunoprecipitation. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Co-precipitated Hip proteins were detected using a polyclonal Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal anti-CXCR2 antibody to confirm equal loading. The results represent one of three independent experiments. B, human neutrophils prepared from a single donor were treated without (lane 1) or with (lane 2) CXCL8 (100 ng/ml) for 5 min. The cells were lysed, and CXCR2 was immunoprecipitated described above. Co-precipitated Hip was blotted using specific anti-Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal anti-CXCR2 antibody (E2, Santa Cruz Biotechnologies) to confirm equal loading. C, HEK293 cells stably expressing wild-type (lanes 1 and 2) or I323A/L324A mutant form of CXCR2 (lanes 3 and 4) were treated with carrier buffer (lanes 1 and 3) or CXCL8 (100 ng/ml) (lanes 2 and 4) for 5 min. CXCR2 was immunoprecipitated and co-precipitated Hip proteins were analyzed as described in the Fig. 3A legend. D, HEK293 cells transiently transfected with myc-tagged CXCR4 were treated with carrier buffer (lane 1) or CXCL12 (100 nM) (lane 2) for 5 min. CXCR4 was immunoprecipitated using a monoclonal anti-myc antibody, and co-precipitated Hip proteins were analyzed as described in the Fig. 3A legend. IP, immunoprecipitation; IB, immunoblotting.  1 and 2), His-tagged wild-type Hip constructs (lanes 3 and 4), or His-tagged ⌬TPR constructs (lanes 5 and 6). Cells were treated with carrier buffer (lanes 1, 3, and 5) or CXCL8 (100 ng/ml) (lanes 2, 4, and 6) for 10 min. 30 l of protein from clarified cell lysate was loaded on SDS-polyacrylamide gel per lane and electrophoresed, and Western blot analysis was performed with a phosphospecific MAPK antibody. The blots were striped and re-probed with an antibody against ERK-2 and with an antibody against His, respectively, to ensure equal loading and protein expression. (28,30), members of a molecular chaperone family that participates in the regulation of protein folding and transport (31). Hip binds the ATPase domain of Hsc/Hsp70 in an ADP-dependent manner (32, 33), through a central TPR motif and a downstream highly charged domain (28,30). Besides affecting the Hsc/Hsp70 chaperone activities in vitro and in vivo (34, 35), Hip alone can also bind to unfolded proteins and prevent their aggregation. Yet refolding of proteins to their active state requires cooperation with other chaperones (28,32).
The mechanisms underlying the involvement of Hip in the internalization of chemokine receptors CXCR2 and CXCR4 may be interpreted in two different ways. One interpretation is that, through its own chaperone activity, Hip may facilitate the conformational change of the receptors that is required for the receptors to undergo internalization. Thus overexpression of the Hip mutant, ⌬TPR, which blocks the normal functions of Hip, attenuated the internalization of the chemokine receptors. The second interpretation, which favors an indirect involvement of Hip in the regulation of the chemokine receptor internalization, is that Hip participates in the internalization process of the chemokine receptors by cooperating with Hsc/Hsp70. This is supported by the data that the internalization of CXCR2 and CXCR4 was attenuated by the Hip mutant ⌬TPR, which fails to bind the ATPase domain of Hsc/Hsp70 as reported previously (28,35). Many chemokine receptors, including CXCR2 and CXCR4, internalize through CCV (16,19,36). This process comprises ligand-receptor formation, aggregation of these complexes in coated pits, and formation of CCV (36). Fusion of CCV with endosomes can only occur after the clath- rin-containing coat is released from CCV (38). Hsc/Hsp70 have been suggested to broadly modulate clathrin dynamics throughout the CCV cycle by releasing coat proteins from CCV (39 -41). Studies on the transferrin receptor indicate that the receptor endocytosis is sensitive to antibodies against Hsc70 (42), and the receptor internalization and recycling are blocked by overexpression of ATPase-deficient Hsc70 mutants (43). Although there is no report regarding the direct involvement of Hsc/Hsp70 in the internalization of CXCR2 and CXCR4, a complex formed by CXCR2 and Hsc70 has been observed in intact cells (ongoing study in our laboratory). Moreover, a signal complex containing Hsc70 and CXCR4 (44) and partial co-localization of Hsc73 with the G protein-coupled A1 adenosine receptor in internal vesicles (45) have been demonstrated. These data suggest the potential role of Hsc/Hsp70 in the signaling and trafficking of GPCRs. Further studies regarding the role of Hsc/Hsp70 in the signaling and trafficking of CXCR2 and other chemokine receptors are underway.
Based on the data that overexpression of wild-type Hip or ⌬TPR did not affect CXCL8-induced MAPK activation in CXCR2-expressing cells, we postulate that the immediate downstream signaling of CXCR2 is not regulated by Hip. Although studies on ␤2-adrenergic receptor have demonstrated that in HEK293 cells, the ␤2-adrenergic receptor-mediated MAPK activation is inhibited by blocking receptor internalization (46), CXCR2-mediated MAPK activation appears to be independent of receptor endocytosis. This is supported by the present study showing that ⌬TPR-attenuated CXCR2 internalization, but did not inhibit CXCL8-induced MAPK activation, and by the previous observation that overexpression of a dominant negative mutant of dynamin I (dynamin I K44A) blocked CXCR2 internalization without affecting the receptor-mediated MAPK activation (16). Studies of CXCR2 mutants provide further evidence for separation between CXCR2 internalization and immediate downstream signaling events. A mutant of CXCR2 with deletion of the last 31 amino acids at the Cterminal domain exhibited impaired agonist-induced receptor endocytosis but not inhibition of adenylyl cyclase or MAPK activation (47). Richardson et al. (48) demonstrated that a CXCR2 mutant (331T) exhibiting a deletion of the last 25 amino acid residues of the C-terminal domain lost agonistinduced phosphorylation and sequestration when expressed in RBL-2H3 cells. However, the immediate downstream signaling events such as GTPase activity, phosphatidylinositol hydrolysis, and calcium mobilization were not impaired (48). The reasons for the discrepancy in ligand-induced receptor sequestration and MAPK activation between CXCR2 and ␤2-adrenergic receptor are not clear.
The chemotaxis assay is believed to be the most useful assay for evaluating the signal transduction capacity of chemokine receptors, because it measures the ultimate result of a cascade of intracellular events that are activated by ligand-receptor interaction. More importantly, this assay also provides a functional readout for a process composed of sequential desensitization and resensitization events with respect to receptor activation. Our results clearly demonstrate that overexpression of ⌬TPR attenuates the ligand-induced chemotaxis of CXCR2expressing cells. The equal or similar cell surface expression of receptors was confirmed by immunofluorescence assay to exclude the possibility that the reduced chemotaxis in cells expressing ⌬TPR is due to reduced receptor expression or cell surface targeting. The mechanism underlying the involvement of Hip in CXCR2-mediated chemotaxis remains elusive. We postulate that the effect of Hip in CXCR2-mediated chemotaxis may result from its regulation of the receptor internalization, although we cannot exclude the possibility that Hip directly regulates other proteins involved in the mediation of chemotactic signals. Increasing evidence shows the importance of receptor internalization in receptor-mediated chemotaxis. Studies on CXCR2 have demonstrated that the membrane proximal part of the receptor C-terminal domain consisting of amino acids 317-324 (RHGLLKIL) constituted a minimal requirement for both the receptor sequestration and the receptormediated chemotaxis (25,49). Combined mutations of the LLKIL motif in the C terminus of CXCR2 not only impaired the receptor internalization but also blocked agonist-induced chemotaxis (23). Moreover, overexpression of a dominant negative mutant form of dynamin I, dynamin I K44A, blocked CXCR2 internalization as well as the receptor-mediated chemotaxis (16). An antagonist of CCR1 and CCR3 blocked receptor internalization and the receptor-mediated chemotaxis (50). Because receptor endocytosis permits CXCR2 dephosphorylation and resensitization (10), we therefore postulate that endocytosis, dephosphorylation, and recycling of chemokine receptors are required for gradient response to ligand stimulation. Because ⌬TPR attenuated ligand-induced CXCR2 internalization, it is conceivable that this is the mechanism by which ⌬TPR inhibited the receptor-mediated chemotaxis. However, it is worthy to note that some other chemokine receptor-mediated chemotaxis is independent of receptor internalization (51). These observations suggest that different chemokine receptors are divergently regulated in this respect. Further studies are required to elucidate whether the regulation of CXCR2-mediated chemotaxis by Hip is a generalized mechanism regarding the chemotactic responses of other chemokine receptors.
In conclusion, the present data demonstrate for the first time that Hip interacts with CXCR2 through its C-terminal domain binding to the receptor C-terminal KILAIHGLI motif. Hip forms a complex with CXCR2 and CXCR4 in intact cells in an agonist-dependent manner. Overexpression of a TPR deletion mutant form of Hip (⌬TPR) significantly attenuates the internalization of CXCR2 and CXCR4. Moreover, overexpression of ⌬TPR blocks CXCR2-mediated chemotaxis. Our studies open a possibility that Hip may regulate the signaling and trafficking of chemokine receptors as well as other GPCRs.