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Divisions of
* Viral Products and
Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Correspondence: Hana Golding, Division of Viral Products, Center for Biologics and Evaluation and Research, Building 29B, Room 4NN04, HFM 454, Bethesda, MD 20892. E-mail:
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: monocytes • lymphocytes • chemokine receptor HIV-1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Post-translational modifications, some of which may be associated with constitutive protein turnover and agonist-induced trafficking of CXCR4, could affect the surface density and conformational forms of CXCR4 and have an impact on the overall susceptibility of a given cell for HIV-1 infection [4 , 6 7 8 ]. Some previously reported modifications of CXCR4 include N-linked glycosylation and sulfation [4 , 6 , 9 , 10 ]. Sulfation of CCR5 was also reported and was shown to enhance its function as an HIV coreceptor [6 ]. It was also reported that CXCR4 in the CEM T-lymphocyte line undergoes monoubiquitination in response to SDF-1 binding. This ubiquitination was shown to be responsible for CXCR4 degradation subsequent to internalization from the cell surface [11 ].
In addition to the predicted species of 45 kDa for glycosylated CXCR4 monomers, additional species have been detected in whole cell extracts and immunoprecipitated material from various cells expressing human CXCR4 [6 , 12 , 13 ]. Previously, we reported that CXCR4 in primary monocytes (MO) appeared in Western blots primarily as a 62-kDa species [12 ]. This isoform of CXCR4 constitutively associated with CD4 as determined by coimmunoprecipitation. A direct correlation was seen between the amount of CXCR4 that was coprecipitated with CD4 and the level of fusion with HIV-1 envelope-expressing cells [12 ]. In addition to the 62-kDa species, other isoforms of CXCR4 have been detected in cell extracts from primary cells and a CXCR4-transfected cell line [12 , 13 ], and this heterogeneity could not be explained by modifications that have already been described.
The formation of multiple isoforms is not unique to CXCR4. Similar observations were made for the µ and 
opioid receptors, which are also 7-TM GPCR [14
].
In the current study, we investigated the heterogeneity of CXCR4 isoforms in lymphocytes, MO, macrophages, and the Jurkat T cell line. We provide evidence that this heterogeneity can be partially accounted for by constitutive ubiquitination of CXCR4, which may have an effect on intracellular trafficking and turnover of CXCR4 molecules in human cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the generation of MO-derived macrophages (M
), 2 ml 1.5 x 106 MO/ml in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1000 units/ml granulocyte macrophage-colony stimulating factor (Immunex Corp., Seattle, WA) and 10% pooled, heat-inactivated human serum were added to each well of six-well plates. After 5–6 days, floating cells were removed, and adherent cells were harvested by scraping them from the surface of the wells with rubber policemen.
Lymphocytes were cultured overnight in the presence of 10 ng/ml recombinant human interleukin-4 (IL-4; R & D Systems, Minneapolis, MN) and 10-7 M dexamethasone (Dex) in DMEM medium supplemented with 10% pooled, heat-inactivated human serum to enhance CXCR4 transcription [15 ]. Where indicated, the IL-4 and Dex were omitted.
In some experiments, cells were treated overnight with the proteasome inhibitor lactacystin (Calbiochem, La Jolla, CA) at a final concentration of 10 µM or with the lysosomal inhibitors ammonium chloride (0.5 µg/ml) or monensin (10 µM).
To prepare lymphocytes for immunoprecipitation with anti-CXCR4 and blotting with antiubiquitin, cells were incubated overnight with lactacystin and infected with recombinant vaccinia expressing CXCR4 in the presence of lactacystin for 8 h before lysing in Nonidet P-40 (NP-40) lysis buffer.
The Jurkat cell line is a human T-lymphocyte cell line [16 , 17 ]. It was cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 2 mM glutamine.
Fresh thymus fragments were obtained from Fairfax Hospital (VA) during cardiac surgery from children (ages 1 month to 3 years) with congenital valvular malformations. The tissue was minced, large aggregates were removed by passing through a nylon mesh, and thymocytes were separated by centrifugation on a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, Sweden). Total cell lysate from thymocytes was prepared following the protocol for human lymphocytes.
Vaccinia viruses
In some experiments, lymphocytes and MO were infected for 8 h with recombinant vaccinia virus expressing human CXCR4 (vCBFY1, a gift from Christopher Broder, Uniformed Services University of the Health Sciences, Bethesda, MD, and Edward Berger, National Institute of Allergy and Infectious Diseases, NIH, and referred to as vCXCR4 in figures) or with a control recombinant vaccinia virus (vSC8) encoding the bacterial enzyme ß-galactosidase (a gift from Bernard Moss, NIAID, NIH) at 10 plaque-forming units/cell.
Antibodies and preparation of sepharose beads for immunoprecipitation
The generation of polyclonal rabbit anti-CXCR4 has been described previously [2
, 12
]. The anti-CXCR4 monoclonal antibody (mAb) 4G10 was a kind gift of Edward Berger and Christopher Broder and has been described previously [1
, 18
]. The second monoclonal anti-CXCR4 antibody (mAb 173) was obtained from R & D Systems. All three anti-CXCR4 antibodies recognize sites in the N terminus. The OKT4 hybridoma (anti-CD4) was obtained from American Type Culture Collection (Manassas, VA). Acsites containing 4G10 or OKT4 were generated by Science Applications International Corp. (Frederick, MD).
Murine antiubiquitin mAb were obtained from BD Biosciences (Palo Alto, CA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal rabbit antiubiquitin antibodies were obtained from Sigma Chemical Co. (St. Louis, MO).
Antibodies used for immunoprecipitation were cross-linked to protein G-conjugated sepharose beads using the procedure for conjugating antibodies to protein A-conjugated beads [19 ]. Briefly, for anti-CD4, 5 ml packed protein-G beads (Pierce, Rockford, IL) were added to 1.25 ml OKT4 acsites; for anti-CXCR4, 4 ml packed beads were added to 2 ml 4G10 acsites and 1 mg mAb 173. Beads were mixed overnight at 4°C and washed with 10 vol 0.2 M sodium borate (pH 9.0). The beads were resuspended in 10 vol 0.2 M sodium borate (pH 9.0) with 20 mM dimethylpimelimidate. After rocking for 30 min at room temperature, the reaction was stopped by first washing the beads and then gently mixing for 2 h in 0.2 M ethanolamine (pH 8.0). The beads were finally washed with and suspended in phosphate-buffered saline and stored at 4°C.
Immunoprecipitations
For coprecipitations with CD4, cells were lysed at 1 x 107 cells/ml in BRIJ lysis buffer [0.1% BRIJ 97, 150 mM NaCl, 20 mM Tris (pH 8.2), 5 mM iodoacetamide, 2 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin A]. For direct immunoprecipitations with anti-CXCR4, cells were lysed in NP-40 lysis buffer [1% NP-40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1 mM AEBSF, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin A]. For immunoprecipitation, 8 ml lysed cells and 100 µl packed beads conjugated to mAb were used for each sample. Immunoprecipitation (anti-CXCR4, coprecipitation with anti-CD4, or control antibody), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting procedures have been described previously in detail [2
, 12
].
Mixing experiments
MO, macrophages, and Jurkat cells were lysed at 1 x 107 cells/ml in NP-40 lysis buffer and frozen. Lysates were thawed and kept on ice. MO lysates were mixed with equal volumes of Jurkat or macrophage lysates and incubated at 4°C or 37°C for the designated times. In addition, lysates were incubated with an equal volume of lysis buffer alone. Mixtures were immediately boiled with an equal volume of 2x SDS sample buffer with dithiothreitol (DTT) and 8 M urea for 5 min to stop reactions, frozen on dry ice, and stored at -70°C until submitted to SDS-PAGE.
Flow cytometric analysis of surface expression
Cells were stained using a mAb against CXCR4 (12G5; PharMingen, San Diego, CA) or murine isotype-control antibody, followed by fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse immunoglobulin G (IgG; Fc-specific; Sigma Chemical Co.). Gating on live cells was assisted by using propidium iodide at 5 µg/ml. Ten thousand events were collected per sample and analyzed using the FL-1 (FITC channel) on a FACScan (BD Biosciences) with Cell Quest Software. 
Mean fluorescence channels (MFC) were calculated by subtracting the isotype-control mAb MFC from the experimental values.
Analysis of syncytium formation
Untreated or treated lymphocytes (IL-4+Dex or IL-4+Dex+lactacystin) were cocultured with the human lymphoid cell line TF228.1.16, which stably expresses gp160 from HIV-1 IIIB/BH10 (X4, T-tropic; a gift from Zdenka L. Jonak, SmithKline Beechham Pharmaceuticals, King of Prussia, PA) [20
]. One hundred-thousand cells in 100 µl each cell type were added to wells of 96-well plates, and each assay was set up in triplicates. Syncytium formation was measured after 2.5–4 h at 37°C.
Sample preparation for two-dimensional (2-D)-PAGE
Elutriated MO were lysed in BRIJ lysis buffer and frozen at -70°C. Lysates from several individuals were pooled to obtain enough material for each experiment. For each sample, 24 ml 1 x 107 elutriated MO/ml was added to 300 µl packed beads conjugated to OKT4. After mixing overnight at 4°C, beads were washed and eluted with 300 µl lysing solution that contained 7 M urea, 2 M thiourea, 4% CHAPS, 1% MEGA-10, 1% octyl-ß-glucopyranoside, 40 mM Tris-HCL, 50 mM DTT, 2 mM tributyl phosphine, and 0.5% (v/v) Pharmalytes.
2-D-PAGE
First-dimension isoelectric focusing was performed on a Pharmacia Immobiline immobilized pH gradient (IPG) dry-strip system as described by the manufacturer. Precast, immobilized pH gradient strips (18 cm pH 3–10 nonlinear) were used for the first-dimensional separation and focused for 75,000 voltage h at room temperature using a discontinous gradient. After focusing, the IPG strips were incubated for 15 min at room temperature with equilibration solution I, which contained 5 ml Tris-HCl, pH 6.8, 18 g urea, 15 ml glycerol, 5 ml 20% SDS, 100 mg DTT, and double-distilled water to 50 ml. The IPG strips were incubated for 15 min at room temperature with equilibration II solution, which is identical to solution I except that 5 g iodoacetamide replaces the DTT, and it contains 10 mg bromophenol blue. The equilibrated strips were loaded onto a second dimension 9% slab gel for separation of proteins based on molecular weight (MW) by SDS-PAGE. After electrophoresis for 4 h at 30 mA/gel [21
, 22
], samples were transferred to nitrocellulose, and immunoblots were prepared as described previously for 1-D gels. To visualize protein before reacting blot with antibody, SYPRO Ruby-red blot stain (Molecular Probes, Eugene, OR) was used, and the fluorescence of the blots was visualized using a Typhoon 8600 gel imager (Molecular Dynamics, Sunnyvale, CA; and see ref. [23]). Prior to immunoblotting with antiubiquitin, blots were boiled in water as described [23
].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To confirm the identity of these proteins as CXCR4, immunoprecipitation with anti-CXCR4 mAb was done before immunoblotting. Several mAb were tried, and cross-linking of mAb 4G10 and mAb 173 to the same beads was found to precipitate CXCR4 most efficiently. When CXCR4 was immunoprecipitated from lymphocytes and blotted with anti-CXCR4 antibodies, only the 47- and 101-kDa bands were detected (Fig. 1 , lane 5). No bands were detected when immunoprecipitation was done with control antibodies (data not shown). These are very close to the predicted MW for a monomer and dimer of glycosylated CXCR4. However, the apparent size of the 101-kDa species did not change even in the presence of urea or under reducing conditions, indicating that it may not be a CXCR4 dimer. The 61- to 62-kDa CXCR4 species in WCL from MO, macrophages, and lymphocytes was not precipitated very well by the two mAb used in our study, and only a very faint band at 62 kDa was detected (lane 5). However, the entire ladder of bands seen in blots of lymphocyte WCL could be precipitated with anti-CXCR4 from thymocytes, which express much higher levels of CXCR4 (see Fig. 7 , lane 5). The 62-kDa isoform was reproducibly coprecipitated with CD4 (see ref. [12 ], and data below).
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The 62-kDa CXCR4 molecules that coprecipitate with CD4 from MO lysates are ubiquitinated
The appearance of a ladder of CXCR4 bands 8.5-kDa apart in lymphocyte WCL suggested that the CXCR4 could be modified by the addition of ubiquitin, an 8.5-kDa protein that covalently attaches to lysine residues and can also form chains with itself [24
]. CXCR4 has multiple lysine residues in the intracellular loops and cytoplasmic tail, which could be modified by ubiquitin [3
]. Furthermore, the molecular size of the 62-kDa isoform of CXCR4 that is prevalent in MO and coprecipitates with CD4 could be accounted for by the addition of two ubiquitin molecules to the 45-kDa protein. To further analyze the 62-kDa species, CXCR4 was coprecipitated with CD4 from MO WCL, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-CXCR4 or antiubiquitin antibodies. The 62-kDa species in immunoblots of 1-D gels reacted with anti-CXCR4 but did not react with antiubiquitin antibodies (Fig. 3a
, lane 1, and data not shown). Subsequently, 2-D gels were used to enhance the sensitivity of detection, as three times more protein can be loaded on 2-D gels. The 62-kDa protein in immunoblots of 2-D gels reacted with anti-CXCR4 and antiubiquitin rabbit antibodies, albeit with different intensities (Fig. 3b)
. The isoelectric point (PI) ranged from 6 to 8, which is close to the predicted PI of eight for ubiquitinated CXCR4. Precipitation of CD4 was confirmed by running on a 1-D gel and blotting with anti-CD4 (data not shown, and see ref. [12
]). CD4 could not be seen on the same 2-D gels, as it has a very basic PI (greater than 9.5) [25
].
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Effects of treatment with the proteasome inhibitor lactacystin on CXCR4 in WCL from lymphocytes and MO
The ladder of bands seen in the anti-CXCR4 blot of Jurkat cell and macrophage WCL and its decrease over time in the mixing experiments suggested that the high MW forms of CXCR4 might be ubiquitinated. Modification by polyubiquitination chains that contain at least four ubiquitin molecules can efficiently target cytosolic and membrane proteins for degradation by proteasomes [24
, 27
, 28
]. In addition, monoubiquitination and multi-monoubiquitination have also been reported to target proteins for degradation by proteasomes, albeit much less efficiently [29
].
To determine if the high MW CXCR4 species are ubiquitinated, we studied the effects of overnight incubation with lactacystin, a specific proteasomal inhibitor. In lymphocytes, there was a fivefold increase in the intensity of the 101-kDa isoform after overnight incubation in the presence of lactacystin (Fig. 5 , lanes 1 vs. 2). Similarly, in MO, a sevenfold increase in the intensity of the 101-kDa band was observed (Fig. 5 , lane 3 vs. 4). These results indicate that the 101-kDa form of CXCR4 is degraded by proteasomes or that its expression is indirectly controlled by some other proteins that are degraded by proteasomes.
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Effects of lysosomal inhibitors on CXCR4
Although 26S proteasomes degrade ubiquitinated substrates in the cytosol, it has recently been reported that they also play a role in targeting ubiquitinated surface proteins to lysosomes for degradation [11
, 30
31
32
]. To determine if CXCR4 is constitutively degraded in lysosomes, the effects of the lysosomal inhibitors monensin and ammonium chloride were tested. Treatment of MO with either of these compounds resulted in significant increases in the intensity of the 101-kDa band (Fig. 8
, lanes 2 and 4), similar to that observed after treatment with lactacystin (Fig. 5) . Therefore, it is likely that agonist-induced surface down-regulation [11
] and constitutive turnover of receptors involve polyubiquitination or multiple monoubiquitination of CXCR4 molecules resulting in lysosomal sorting.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We showed here and in a previous study [12 ] that the 62-kDa form of CXCR4, which is the predominant species in MO, was efficiently coprecipitated with CD4, indicating that it constitutively associates with CD4. This is possibly a result of ubiquitin-induced, conformational changes in CXCR4, as in T-lymphocyte lines, efficient coprecipitation of the 45-kDa form of CXCR4 with CD4 requires pretreatment with the HIV-1 envelope (gp120) at 37°C [2 ].
As the constitutive association of the 62-kDa form of CXCR4 indicated conformational changes, it was not too surprising to find a change in the recognition by the mAb used for immunoprecipitation. The conformational changes may lead lead to "masking" of epitope-recognized mAb but did not abrogate recognition by our polyclonal antibody in the immunoblot that recognizes many epitopes. Based on what is known at this time, ubiquitination most likely occurs in the intracytoplasmic portion of the molecule, but it is well-known that changes in this portion of the molecule of 7-TM receptors can lead to major conformational changes in the entire molecule, including the extracellular portion.
Recently, Baribaud et al. [33 ] also concluded that there are multiple conformations of surface CXCR4 based on the differential reactivity of a panel of mAb against CXCR4 on several different cell lines. However, the relationship between the CXCR4 species observed in our experiments and the conformational epitopes described in the other study remains to be determined.
The finding of constitutive ubiquitination of CXCR4 is significant in light of recent findings that ubiquitination plays an important role in the sorting of membrane proteins to various cellular compartments including the cell surface [30
]. In several studies with other 7-TM proteins, such as the ß2-adrenergic receptor and µ and opioid receptors, ubiquitination of surface molecules was shown to play an important role in agonist-induced internalization as well as in basal receptor turnover [14
, 32
, 34
]. In addition, monoubiquitination of CXCR4 following SDF-1 binding in CEM cells was recently reported by Marchese and Benovic [11
]. These authors found that the lysine residues within a degradation motif in the carboxyl terminus of CXCR4 (SSLKILSKGK) underwent agonist-promoted ubiquitination, which was not required as an internalization signal but rather as a lysosomal-sorting signal. We did not observe an effect of lysosomal inhibitors on the steady-state level of a 62-kDa species of CXCR4 in primary cells, which could mean that these molecules are not marked for degradation in lysosomes. However, an external agonist, which is known to induce internalization, was not added in our study. Spontaneous endocytosis of CXCR4 has been observed in cell lines stably transfected with a chimeric CXCR4-green fluorescent protein construct [7
], but this may not occur as rapidly in primary cells. If the majority of 62-kDa CXCR4 molecules were ubiquitinated on the cell surface, they may eventually be degraded in lysosomes upon internalization. Perhaps we did not observe protection by lysosomal inhibitors, as internalization of this isoform may not have occurred rapidly enough to allow delivery of a significant proportion of the molecules to the lysosomal compartment. Alternatively, ubiquitination of newly synthesized CXCR4 molecules may occur in an intracellular compartment rather than on the cell surface. It it interesting that it was reported that another 7-TM protein Gap1 permease is transported from the trans-Golgi network (TGN) to the cell surface if it is unmodified or monoubiquitinated. In contrast, multiubiquitination of Gap1 targets it to the multivesicular body for subsequent delivery to lysosomes [30
, 35
, 36
]. In an analogous manner, the mono- or diubiquitinated 62-kDa isoform of CXCR4, whose expression is not altered by lysosomal or proteasomal inhibitors, may be delivered to the cell surface from the TGN rather than being marked for degradation. In a previous study, this was the only species of CXCR4 isolated from the surface of MO by biotinylating whole cells and precipitating biotinylated proteins with streptavidin-sepharose beads [12
].
The expression of the 101-kDa form of CXCR4 in MO and lymphocytes was sensitive to proteasomal inhibitors as well as lysosomal inhibitors, indirectly indicating that it is ubiquitinated. The appearance of multiple bands 8.5-kDa apart in the WCL of lymphocytes as well as the sequential intensity shift of these bands in the mixing experiments suggest that a fraction of CXCR4 molecules is multiubiquitinated. Based on the MW, we predict that the upper bands are polyubiquitinated. As ubiquitin forms chains of varying length, the number of ubiquitin units per molecule of CXCR4 may vary. Thus, in the antiubiquitin immunoblots, CXCR4 appears as a "smear." The same pattern of reactivity of antiubiquitin antibodies was demonstrated by Chaturvedi et al. [14 ] in immunoblots of the opioid receptor.
Not surprisingly, the 62-kDa isoform was not affected in the mixing experiment, as the deubiquitinating enzyme activity was found in MO extracts where the 62-kDa protein is prevalent. These data suggest that proteasomes and lysosomes play a role in the degradation of the 101-kDa form. The reactivity of CXCR4 immunoblots from lymphocytes and thymocytes with antiubiquitin mAb further supports this conclusion. As we previously found that the 101-kDa form of CXCR4 is expressed on the cell surface of macrophages [12
], multiubiquitination may act as a signal for proteasomal-assisted lysosomal degradation of surface CXCR4, even in the absence of agonist binding, thus contributing to the steady-state turnover and control of surface density. Alternatively, the protein could be multiubiquitinated in an intracellular compartment. Ubiquitination has been shown to be a lysosomal-sorting signal for some amino acid permeases subsequent to modification in the TGN [30
, 35
, 36
]. In addition, at least some of the protection from degradation by lactacystin may have occurred in the endoplasmic reticulum, which is a well-known site for degradation of polyubiquitinated membrane proteins including the human opioid receptors (also 7-TM GPCRs) [24
, 34
].
Changes in expression and conformation of CXCR4 could affect the susceptibility of a given cell to infection by T-tropic HIV-1 strains, which only use CXCR4 as a coreceptor. Such changes may also indirectly influence infection by M-tropic HIV-1, which uses CCR5 as a coreceptor, as the two coreceptors compete with each other for interaction with CD4 [37 ]. In addition, changes in conformation may alter the effectiveness of vaccines and small drugs that target the coreceptors. Thus, it is crucial to understand the heterogeneity and functional diversity of CXCR4. Ubiquitination may play a role in the constitutive intracellular transport, turnover, and conformation of CXCR4. Our earlier findings together with the current data suggest that monoubiquitination may enhance the role of CXCR4 as an HIV-1 coreceptor by inducing constitutive association with CD4 in MO, perhaps by altering its conformation, but polyubiquitination of CXCR4 may decrease its steady-state surface expression in resting cells.
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ACKNOWLEDGEMENTS |
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Received April 18, 2002; revised August 19, 2002; accepted August 22, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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