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The Chemokine Receptor CXCR3 Is Degraded following Internalization and Is Replenished at the Cell Surface by De Novo Synthesis of Receptor This information is current as of September 30, 2021. Andrea Meiser, Anja Mueller, Emma L. Wise, Ellen M. McDonagh, Sarah J. Petit, Namita Saran, Peter C. Clark, Timothy J. Williams and James E. Pease J Immunol 2008; 180:6713-6724; ; doi: 10.4049/jimmunol.180.10.6713 Downloaded from http://www.jimmunol.org/content/180/10/6713

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Chemokine Receptor CXCR3 Is Degraded following Internalization and Is Replenished at the Cell Surface by De Novo Synthesis of Receptor1

Andrea Meiser,2 Anja Mueller,2,3 Emma L. Wise, Ellen M. McDonagh, Sarah J. Petit, Namita Saran, Peter C. Clark, Timothy J. Williams, and James E. Pease4

The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T lymphocytes. We describe in this study the endocytosis of CXCR3 using T lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 down-regulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was Downloaded from dependent upon mRNA transcription, de novo protein synthesis, and transport through the endoplasmic reticulum and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C terminus revealed that internalization and degradation were independent of phosphorylation, ubiquitination, or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C terminus. Although freshly isolated T lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having http://www.jimmunol.org/ significant activity on activated T lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Because CXCR3؉ cells are themselves a source of IFN-␥, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T lymphocyte recruitment. The Journal of Immunology, 2008, 180: 6713–6724.

he chemokine receptor CXCR3 is expressed on a wide sclerosis (11), autoimmune diseases (12), transplant rejection (13, variety of cells including activated T lymphocytes, NK 14), and viral infections (15). Consequently, the mechanisms un- cells, malignant B lymphocytes, endothelial cells, and derlying the regulation of CXCR3 expression at the cell surface are T by guest on September 30, 2021 thymocytes (1–6). Three major CXCR3 ligands, CXCL9, of considerable interest. CXCL10, and CXCL11, have been identified, all of which are The number of receptors on a cell surface results from a balance induced by IFN-␥ and are therefore thought to promote Th1 im- between the rate of internalization and the rate of replacement mune responses (7–9). Recent studies have shown that the CXCR3 (recycling and synthesis of nascent receptor). Following ligand ligands exhibit unique temporal and spatial expression patterns, binding, there are two major routes whereby G protein-coupled suggesting that they have nonredundant functions in vivo. More- receptors (GPCRs),5 as typified by chemokine receptors, are in- over, the CXCR3 ligands share low sequence homology (around ternalized into cells. The first and most well-defined route involves 40% amino acid identity) and exhibit differences in their potencies the binding of arrestin to the phosphorylated receptor, which in and efficacies at CXCR3 with CXCL11 being the dominant ligand turn initiates the internalization process by binding to clathrin. The in several assays (8, 10). CXCR3 and its ligands have been im- receptor-arrestin complex is then sequestered in clathrin-coated plicated as playing an important role in the induction and perpet- pits. This pathway is often considered a default system for degra- uation of several human inflammatory disorders including athero- dation and recycling of receptors (16, 17). The second pathway involves invaginations of the cell membrane known as caveolae and functions independently of clathrin-coated pits (18). Although Leukocyte Biology Section, National Heart and Lung Institute, Faculty of Medicine, the rate of internalization of a receptor is an important factor in Imperial College London, South Kensington Campus, London SW7 2AZ, United determining its level at the cell surface, the rate of recycling and Kingdom the rate of synthesis of new receptors are also important. Until Received for publication March 14, 2008. Accepted for publication March 14, 2008. recently, the mechanisms of the recycling process were poorly The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance understood, and internalized receptors were thought to have sev- with 18 U.S.C. Section 1734 solely to indicate this fact. eral potential fates. The concept of two different classes of receptor 1 This work was supported by Grants PG/2000055 and FS/05/021 from the British (as distinguished by their recycling) has been introduced recently, Heart Foundation, Grant 174240 from the Arthritis Research Campaign, and Project in which class A receptors traffic to recycling endosomes and are Grant 076036/Z/04/Z from the Wellcome Trust. 2 A. Meiser and A. Mueller contributed equally to the study. 3 Current address: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, U.K. 5 Abbreviations used in this paper: GPCR, G protein-coupled receptor; HA, hemag- 4 Address correspondence and reprint requests to Dr. James E. Pease, Leukocyte glutinin; LAMP, lysosome-associated membrane protein; MEF, mouse embryonic Biology Section, Faculty of Medicine, National Heart and Lung Institute, Sir Alex- fibroblast; ER, endoplasmic reticulum; WT, wild type. ander Fleming Building, Imperial College London, South Kensington Campus, Lon- don SW7 2AZ, U.K. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org 6714 INTERNALIZATION AND DEGRADATION OF CXCR3

FIGURE 1. Internalization of CXCR3 by PBMCs and L1.2 transfectants. A and B, Dose-dependent nature and the respective kinetics of ligand-induced CXCR3 internalization in PBMCs as determined by ﬂow cytometry using a speciﬁc anti-CXCR3 mAb. The ligands CXCL11 (f), CXCL10 (F) and CXCL9 (Œ) are shown. C and D, The levels of CXCR3 internalization in PBMCs and L1.2 transfectants, respectively, following

incubation with 50 nM CXCL11 after pretreatment in Downloaded from the presence or absence of 0.4 M sucrose, 50 ␮M monensin, 5 ␮g/ml filipin, and 50 ␮g/ml nystatin. Cell surface CXCR3 levels were measured as described and the -p Ͻ 0.001 com ,ءءء .untreated control (Ⅺ) is shown pared with CXCL11 treatment alone. E, The effect of filipin pretreatment on the specific binding of 125I-

CXCL11 to L1.2 CXCR3 transfectants. Data represent http://www.jimmunol.org/ the mean Ϯ SEM of at least three different experiments. by guest on September 30, 2021

rapidly returned to the cell surface (16). In contrast, class B re- Plasmids encoding the fusion proteins GFP-DIII and GFP-DIII⌬2 were gift ceptors are dephosphorylated in endosomes followed by slow re- of Dr. A. Benmarah (Institute Cochin, Paris, France). cycling back to the plasma membrane. Sequentially, the receptors pass through late endosomes and the Golgi and finally are trans- Cell culture and transient transfection ported back to the cell surface. Another potential fate is that of The murine pre-B cell line L1.2 was maintained as previously described in degradation, which may be perceived to down-regulate receptor RPMI 1640 supplemented medium (22). L1.2 cells stably transfected with expression. To date, protein synthesis has not been shown to play pCDNA3 containing the CXCR3A cDNA hemagglutinin (HA)-tagged at a role in GPCR replenishment (19–21). the N terminus (10) were cultured in the same medium with the addition of 1 mg/ml geneticin (G418) to maintain selection. Mutant CXCR3 constructs In this study we show that CXCR3 is internalized both consti- were generated by site-directed mutagenesis using the QuikChange mu- tutively and following incubation with CXCL11, resulting in deg- tagenesis kit (Stratagene) with the pCDNA3 HA-CXCR3A plasmid as tem- radation of the receptor. We also show that in the absence of de- plate. Transient transfection of L1.2 cells with plasmids was conducted by tectable recycling, cell surface replenishment of CXCR3 is electroporation as previously described (23). Cells were cultured for 24 h in medium supplemented with 10 mM sodium butyrate before use to en- dependent upon de novo protein synthesis. hance cell surface receptor expression. Mouse embryonic fibroblasts (MEFs) derived from both wild-type (WT) mice and mice deficient in Materials and Methods ␤-arrestins 1 and 2 were a gift of Dr. R. Lefkowitz (Duke University Materials Medical Center, Durham NC) and were maintained as previously described (24). Transfection of MEFs was by electroporation as previously described All chemicals unless otherwise stated were purchased from Sigma-Aldrich. (25). Cells were cultured overnight in medium supplemented with 10 mM Chemokines were purchased from PeproTech. Filipin, sucrose, nystatin, sodium butyrate before use to enhance cell surface receptor expression. monensin, and nocodazole were purchased from Sigma-Aldrich or Calbio- For the generation of activated T lymphocytes, PBMC were isolated chem. Brefeldin A, actinomycin D, and bafilomycin A1 were obtained from blood sampled from healthy donors, according to the Royal Brompton from Tocris. Cycloheximide was from ICN Biomedicals. The mouse anti- Hospital Ethics Committee approved protocol as previously described (26). human CXCR3 mAb (clone 49801.111) and the mouse isotype-matched Lymphocytes were separated from monocytes by allowing the latter to control IgG1 (MOPC 21 clone) were obtained from Sigma-Aldrich. The adhere to a tissue culture flask for2hat37°C and were activated by culture anti-HA.11 Ab was from Covance, and the anti-␣-tubulin Ab was from in the presence of 100 IU/ml IL-2 and 2 mg/ml Con A for 7–10 days. Abcam. Secondary Abs were obtained from DakoCytomation. Plasmids Purified T lymphocytes were isolated from whole blood using the Rosette- encoding dominant negative mutants of ␤-arrestin 1 and ␤-arrestin 2 were Sep Human T cell Enrichment Cocktail kit (StemCell Technologies), gifts of Dr. M. Caron (Duke University Medical Center, Durham, NC). which typically gave a population Ͼ95% pure. Nucleofection of purified T The Journal of Immunology 6715

FIGURE 2. Neither clathrin nor arrestins are required for the CXCL11-induced internalization of CXCR3. A–D, CXCL11-induced internalization of CXCR3 in purified T lymphocytes as deduced by flow cytometry following nucleofection in the presence or absence (mock) of a plasmid containing a dominant negative GFP-tagged DIII construct that inhibits clathrin-coated pit assembly. Nucleofection was conducted 24 h before the induction of CXCR3 internalization with 50 nM CXCL11. The percentage of cells is shown in each quadrant and the ex- periment shown is representative of three separate experiments. E, The relative internalization of CXCR3 in WT MEF cells (Ⅺ) and ␤-arrestin 2- or ␤-arrestin 3-deficient MEF cells (f), transiently transfected with CXCR3, following incubation with 50 nM

CXCL11 or buffer alone. Data represent the Downloaded from mean Ϯ SEM of four different experiments. F, The relative internalization of CXCR3 in response to 50 nM CXCL11 or buffer alone in a stable L1.2 transfectant cell line following transfection 24 h previously with either plasmid pcDNA3 (mock) or pcDNA3 containing the V53D and V54D dominant neg- http://www.jimmunol.org/ ative mutants of ␤-arrestin 1 and ␤-arrestin 2. Data represent the mean Ϯ SEM of three ,ءءء p Ͻ 0.01 and ,ءء different experiments p Ͻ 0.001 compared with buffer-treated controls.

lymphocytes with plasmids encoding GFP-DIII and GFP-DIII⌬2 was nomycin D (5 ␮M), bafilomycin A1 (100 nM), or cycloheximide (10 ␮g/ achieved by using an Amaxa nucleofector, according to the manufacturer’s ml) were added to the cells during the recovery phase, following induction by guest on September 30, 2021 instructions, using program U-014, which typically gave around 60% cell of CXCR3 down-regulation by ligand. viability as deduced by staining with the dye ToPro3 (Invitrogen). Internalization assay and flow cytometry analysis Confocal analysis The H9 human T cell lymphoma line was washed in RPMI 1640 and Internalization assays were essentially conducted as previously described resuspended at a concentration of 5 ϫ 106 cells/ml in serum-free RPMI by Sauty et al. (27). Activated T lymphocytes or L1.2-CXCR3 cells were 1640 and were incubated at 37°C with 50 nM CXCL11. Samples were incubated with serum-free medium for1hat37°C and then resuspended in removed either before or at the indicated times following the addition of medium without serum at 5 ϫ 106 cells/ml. Cells were then incubated with CXCL11. Internalization buffer (1% FCS, 1% NaN in PBS) was added to chemokines (50 nM) for various times at 37°C, and washed in ice-cold PBS 3 the samples removed, and tubes were incubated on ice until all time points containing 1% FCS and 1% NaN before flow cytometry analysis. Cell 3 were collected. Each time point was divided into either isotype control or surface-expressed CXCR3 was detected using an anti-CXCR3 Ab and Ab-staining tubes. Cells were washed twice in cold PBS before fixation in FITC conjugated anti-mouse IgG. Samples were quantified on a FACS- 4% paraformaldehyde for 20 min on ice. Cells were then washed in PBS Calibur, and data processed with CellQuest software (version 3.1; BD Bio- and permeabilized in 0.5% saponin buffer containing anti-human lyso- sciences) with dead cells excluded from analysis. The relative CXCR3 some-associated membrane protein (LAMP)-1 Ab (1/20 dilution; BD surface expression was calculated as a percentage using the following: Pharmingen) or an equal concentration of mouse IgG1 isotype control. 100 ϫ (mean channel of fluorescence (stimulated) Ϫ mean channel of After incubation with the primary Ab, cells were washed in saponin fluorescence (negative control))/(mean channel of fluorescence (me- buffer before incubation with goat anti-mouse IgG Alexa Fluor 568 dium) Ϫ mean channel of fluorescence (negative control)). Pilot experi- (1/100 dilution in 0.5% saponin buffer; Invitrogen). Cells were washed ments staining CXCR3 transfectants on ice in either the presence or ab- and then preblocked with mouse IgG before being incubated with sence of 50 nM CXCL11 confirmed that binding of ligand by CXCR3 did mouse anti-human CXCR3 FITC (1/10 dilution in 0.5% saponin; R&D not significantly reduce detection by the primary Ab (data not shown). Systems) or isotype control. Cells were then resuspended in 4% para- Where inhibitors were used, cells were incubated for1hat37°C with formaldehyde and were spun onto poly-L-lysine-coated glass coverslips filipin (5 ␮g/ml), nystatin (50 ␮g/ml), monensin (50 ␮M), sucrose (0.4 M), in 24-well tissue culture plates at 1200 rpm for 5 min. The supernatant or cycloheximide (10 ␮g/ml) before assays of receptor down-regulation was removed and the coverslips washed twice in PBS and once in were performed. deionized water before being removed from the wells, allowed to air Recovery of cell surface receptor levels dry, and mounted onto slides in VectorShield Hardset fluorescence mounting medium (Vector Laboratories). Analysis was conducted by Receptor down-regulation was initiated as described. After 30 min incu- confocal microscopy using a Leica TCS NT confocal microscope with bation with chemokines, the cells were washed three times in medium a ϫ40 oil objective. Image analysis was conducted using Leica LCS without FCS and resuspended in medium without FCS and incubated at Lite software version 2.61 and the images manipulated for presentation 37°C. To remove CXCL11 from endogenous glycosaminoglycans, acti- using Adobe Photoshop version 6.0. vated T lymphocytes were washed once in prewarmed 0.5 M NaCl/RPMI 1640 as previously described (27) then twice in RPMI 1640 and incubated SDS-PAGE and Western blot analysis at 37°C. Samples were taken at different time points and cells were washed in PBS buffer containing 1% FCS and 1% NaN3. Cells were stained with L1.2 transfectants expressing WT CXCR3, and CXCR3-AAA, CXCR3- Abs as described. Where inhibitors were used, brefeldin A (5 ␮M), acti- K324R, CXCR3-⌬4, and CXCR3-⌬34 constructs were washed in RPMI 6716 INTERNALIZATION AND DEGRADATION OF CXCR3 Downloaded from http://www.jimmunol.org/ FIGURE 4. Cell surface expression of CXCR3 is dependant upon functional ER and Golgi. Activated PBMCs (A) and L1.2 CXCR3 cells (B) were incubated with 50 nM CXCL11 for 30 min, washed three times with serum-free medium, and then incubated in serum-free medium for up to 3 h in the presence or absence of actinomycin D (5 ␮M), brefeldin A (5 ␮M), and bafilomycin A1 (100 nM). Cell surface CXCR3 was assessed by flow cytometry. The extent of CXCR3 internalization following the initial 30 min incubation with CXCL11 (Ⅺ) and CXCR3 cell surface level recovery FIGURE 3. CXCR3 cell surface replenishment is dependent upon de (f) of an untreated control after 3 h are presented. Data represent the novo protein synthesis. Activated PBMCs (A), L1.2 CXCR3 transfec- p Ͻ 0.001 by guest on September 30, 2021 ,ءءء .mean Ϯ SEM of at least three independent experiments tants (B), and L1.2 CXCR6 transfectants (C) were incubated for1hin compared with untreated control. serum-free medium in the presence (f) or absence (E)of10␮g/ml cycloheximide. Following incubation with CXCL11 (PBMCs and CXCR3 transfectants) or CXCL16 (CXCR6 transfectants), cells were further incubated with cycloheximide and samples taken at the indi- was used, although at the end of the assay, cells were transferred from the cated time points for the determination of cell surface expression of lower chamber to a white 96-well microtiter plate using a funnel plate CXCR3 by flow cytometry. Data represent the mean Ϯ SEM of at least (NeuroProbe), and cells were detected with CellTiter Glo (Promega). Lu- p Ͻ 0.001 com- minescence was measured using a TopCount microplate scintillation and ,ءءء p Ͻ 0.01 and ,ءء .three independent experiments pared with cycloheximide-treated cells. luminescence counter (PerkinElmer). Data described are expressed as a chemotactic index, relative to migration observed to medium alone. Ligand binding assays 6 1640 and resuspended at 5 ϫ 10 cells/ml in serum-free RPMI 1640 con- 125I-CXCL11 and 125I-CXCL10 were purchased from PerkinElmer Life taining 10 ␮g/ml cycloheximide. Where indicated, 40 ␮M MG132 or 200 Sciences. Ligand binding was performed as previously described using ␮M chloroquine was also added. The cells were preincubated for 30 min centrifugation through oil to separate bound chemokine from free chemo- at 37°C before the addition of 50 nM CXCL11. Samples were taken at the kine (26). Data are presented following the subtraction of nonspecific bind- indicated time points, the cells washed in ice-cold PBS and resuspended in ing, taken as the counts obtained when the labeled chemokine was dis- lysis buffer containing 1% N-dodecyl-␤ D-maltoside, 10% glycerol, 1/1000 placed by a 1000-fold excess of homologous cold chemokine. protease inhibitor cocktail in PBS (28). Equal quantities of cell lysates were separated on 4–12% SDS-PAGE gels and were electrophoretically trans- Data analysis ferred onto a nitrocellulose membrane, which was subsequently blocked with 5% milk in 0.01 M PBS with 0.05% Tween 20. The blots were in- Data were analyzed using Prism 4.0 (GraphPad Software) by ANOVA with dependently probed with either anti-HA (1/1000 dilution; Covance) or anti- Bonferroni’s Multiple comparisons test. ␣-tubulin (1/10,000 dilution; Abcam) as a loading control. Following washing and probing with a secondary HRP-conjugated polyclonal goat Results anti-mouse Ig (1/1000 dilution), blots were developed by ECL (GE Down-regulation of cell surface CXCR3 following incubation Healthcare). with ligand Chemotaxis We initially used activated PBMCs to investigate the process of Chemotaxis assays using either CXCR3 transfectants or purified T lym- CXCR3 down-regulation following incubation of the cells with the phocytes were performed essentially as previously described (10, 29) using three natural ligands described to date for CXCR3, namely ChemoTX plates with a 5-␮m pore size, purchased from NeuroProbe. For CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC. Activated T lymphocyte migration, enumeration was conducted using a hemocytom- eter and cell migration to buffer alone was subtracted from the resulting PBMCs, cultured for 7–10 days with Con A and IL-2, readily data, with individual results expressed as a percentage of the total cells expressed CXCR3 on their cell surface as detected by flow cytom- applied to the filter. For L1.2 transfectant chemotaxis, the same apparatus etry using a specific mAb. Incubation of PBMCs with all three The Journal of Immunology 6717

FIGURE 5. The effects of C-terminal mutation upon CXCR3 function. A, The amino acid identity of the C-terminal CXCR3 mutants analyzed. B, The relative expression levels of the constructs following the transient transfection of L1.2 cells, compared with cell surface staining observed with the WT CXCR3 con- p Ͻ 0.001. C, The relative ,ء .struct chemotactic responses to CXCL11 of the same CXCR3 mutants. D, depicts the internalization of the CXCR3 mutants in response to increasing concentrations of CXCL11. Comparisons Downloaded from were made with untreated transfec- p Ͻ 0.001 and ,ء.tants in each case -p Ͻ 0.01 compared with con ,ءءء trols. E, Constitutive internalization in the absence of ligand of both WT CXCR3 and the ⌬4 construct over a 6-h period following treatment with http://www.jimmunol.org/ cycloheximide to inhibit de novo synthesis of receptor. All data represent the mean Ϯ SEM of at least three experiments. by guest on September 30, 2021

CXCR3 ligands induced a dose-dependent loss of CXCR3 from coated pits (31), whereas internalization via caveolae can be the cell surface (Fig. 1A) as deduced by staining with the same inhibited with filipin or nystatin (32). Monensin is an inhibitor of CXCR3-specific mAb. vesicle acidification, a process essential for the sorting events oc- Notably, CXCL11 was the most efficacious ligand, with a 50 nM curring during endocytosis of GPCRs such as the ␤2-adrenergic concentration of CXCL11 reducing cell surface staining to Ͻ20% receptor (33). We assessed the activity of these inhibitors on of their starting levels. We subsequently examined the kinetics of CXCR3 down-regulation in PBMCs and L1.2 CXCR3 transfec- this response, using 50 nM concentrations of each ligand (Fig. tants (Fig. 1, C and D). Neither filipin nor nystatin had any inhib- 1B). Loss of cell surface CXCR3 occurred rapidly, with optimal itory effect on CXCR3 down-regulation in either cell type, sug- down-regulation observed by 30 min in agreement with a pre- gesting that caveolae are not involved in the endocytosis of vious study (27). Similar data was also obtained using a previ- CXCR3. Although sucrose had little effect on ligand-induced ously described L1.2 cell line stably expressing CXCR3 (data CXCR3 down-regulation in PBMCs (Fig. 1C), it was observed to not shown) with CXCL11 again the most efficacious of the li- significantly reduce the levels of CXCR3 internalization in L1.2 gands, although the maximum level of receptor down-regula- CXCR3 cells following treatment with CXCL11 (Fig. 1D). In L1.2 ϳ tion observed was reduced to 50% of starting levels as we transfectants, monensin treatment significantly reduced CXCL11- have previously observed with L1.2 cells expressing the related induced internalization of the receptor, suggesting that vesicular receptor CCR3 (30). This reduction likely reflects less effica- acidification is necessary for the sorting events occurring follow- cious coupling of the human receptor to murine intracellular ing CXCR3 endocytosis. In PBMCs, monensin had a modest in- machinery in the transfectants. In all subsequent experiments, hibitory effect on CXCR3 endocytosis that did not reach statistical we therefore incubated cells with 50 nM CXCL11 to achieve significance. Because it has been previously demonstrated that optimal CXCR3 down-regulation. cholesterol and lipid rafts are required for the maintenance of chemokine receptor conformation (34, 35), we also sought to examine Pathways of CXCR3 internalization the effects of filipin and nystatin on ligand binding. Although nys- Two major pathways are known by which chemokine receptors are tatin treatment altered the density of CXCR3 transfectants making internalized; either via clathrin-coated vesicles following the clath- them unable be centrifuged through oil in our ligand binding assay rin-mediated endocytic pathway or via caveolae. Hypertonic su- (data not shown) treatment of cells with filipin was observed to crose medium has been shown to block the assembly of clathrin- have little effect on CXCL11 binding (Fig. 1E). 6718 INTERNALIZATION AND DEGRADATION OF CXCR3

FIGURE 6. Internalized CXCR3 trafﬁcs to late endosomes. T lymphocytes were processed for confocal microscopy either in the absence of treatment (A–C) or following incubation with CXCL11 for 15 min (D–F)or60min(G–I). Following processing, T lymphocytes were stained with Abs directed against CXCR3 (green) and LAMP-1 (red) before analysis by confocal microscopy. A, D, and G show the CXCR3 signal. B, E, and H show the LAMP-1 signal. C, F, and I show the two signals overlaid. Scale bar represents 50 ␮m. F, Colocalization of CXCR3 and LAMP-1 signals in cells is highlighted (arrow- Downloaded from heads). Data shown are representative of at least three independent experiments. http://www.jimmunol.org/

CXCR3 can be internalized independently of clathrin and 2-deficient mice. Internalization was then induced by incubation ␤-arrestin 1 and ␤-arrestin 2 with CXCL11 and CXCR3 down-regulation assessed as before by Because sucrose and monensin were without effect on CXCR3 flow cytometry. CXCR3 down-regulation in WT MEFs was sim- internalization in PBMCs we sought to confirm our findings by ilar to that seen in L1.2 transfectants, with CXCL11 reducing cell by guest on September 30, 2021 using an alternative strategy to inhibit clathrin. T lymphocytes surface levels to around 50% of their starting levels (Fig. 2E). In ␤ ␤ were purified from blood and underwent nucleofection either in MEFs from -arrestin 1- and -arrestin 2-deficient mice, CXCR3 buffer alone (mock nucleofection) or in buffer containing plasmid down-regulation in response to ligand was observed, but at a re- encoding a GFP-tagged construct, DIII (DIII transfection). This duced level, with only a 20% reduction of CXCR3 cell surface construct inhibits clathrin-coated pit assembly and therefore clath- levels in response to CXCL11, suggestive of an incomplete re- ␤ rin-dependent internalization (36, 37). Twenty-four hours after quirement for arrestin in the down-modulation process. Simi- nucleofection, cells were harvested and incubated at 37°C either in larly, transfection of L1.2 CXCR3 transfectants with either empty the presence or absence of CXCL11 before staining for CXCR3 plasmid or plasmids encoding the V53D and V54D dominant form ␤ ␤ expression. A significant percentage of mock-nucleofected T lym- of -arrestin 1 and -arrestin 2 were without effect upon CXCR3 phocytes were shown to express CXCR3 (Fig. 2A, top left quad- down-regulation induce by CXCL11 (Fig. 2F). Collectively, the ␤ rant), which was seen to be reduced following CXCR3 treat- data suggest the existence of a -arrestin-independent pathway for ment (Fig. 2C, top left quadrant). Nucleofection of T the endocytosis of CXCR3. lymphocytes led to the identification of two populations of CXCR3-positive cells, a major population not expressing the Inhibition of CXCR3 cell surface replenishment DIII-GFP fusion protein (Fig. 2B, top left quadrant) and a mi- nor population expressing the DIII-GFP fusion protein (Fig. 2B, After the induction of down-regulation by ligand, the recovery of top right quadrant). CXCL11 treatment was seen to signifi- cell surface CXCR3 levels was relatively slow in both PBMCs and cantly reduce the number of cells within both populations (Fig. L1.2 transfectants (Fig. 3, A and B), with only ϳ70–80% recovery 2D, top right and left quadrants). A similar lack of effect upon of the original CXCR3 cell surface levels observed a full 3 h after CXCL11-induced CXCR3 endocytosis was also seen following incubation with CXCL11. This was in contrast to another Th1- nucleofection of T lymphocytes with the control protein GFP- expressed chemokine receptor CXCR6, which showed 100% re- DIII⌬2, which corresponds to the GFP-DIII construct lacking covery of cell surface levels within1hofligand-induced down- all AP-2 binding sites (data not shown). Collectively this sug- regulation (Fig. 3C) and is typical of receptor recycling to the cell gests that ligand-driven endocytosis of CXCR3 in T lympho- surface as described for other chemokine receptors (19, 38, 39). cytes occurs independently of clathrin. The slow recovery of cell surface CXCR3 levels suggested to us The clathrin-dependent pathway for endocytosis of GPCRs typ- that upon ligand-induced internalization, CXCR3 is either slowly ically involves the binding of arrestins to the intracellular face of recycled, as is the case for class B GPCRs such as the vasopressin the phosphorylated receptor. To examine whether CXCR3 inter- type 2 receptor (40) or alternatively, is degraded. In the case of nalization is dependent upon arrestins, we transiently transfected degradation, cell surface replenishment would therefore require de MEFs obtained from both WT and ␤-arrestin 1- and ␤-arrestin novo synthesis of receptor. The Journal of Immunology 6719

FIGURE 7. CXCR3 is degraded following constitutive or CXCL11-induced internalization. L1.2 cells transiently transfected with plasmids encoding CXCR3 were preincubated for 30 min at 37°C with 10 ␮g/ml cycloheximide and an aliquot was reserved (0 h time point). Incubation of the remaining cells was then allowed to proceed in the presence or absence of 50 nM CXCL11 for 3 h. Cell lysates were generated and analyzed by Western blotting using anti-HA mAb (top). Blots were subsequently stripped and reprobed with an anti-␣-tubulin (aT) Ab as a loading control (bottom). A, Both constitutive (untreated) and CXCL11-induced Downloaded from degradation of WT CXCR3 over the 3-h time period as deduced by a loss of immunoreactivity. B, The effects of preincubation of either 40 ␮M MG132 or 200 ␮M chloroquine on CXCL11-induced degradation. C, The CXCL11-induced degradation of WT CXCR3 and C- terminal CXCR3 mutants. Data shown are from one ex- http://www.jimmunol.org/ periment representative of three different experiments. by guest on September 30, 2021

To examine this latter hypothesis, we preincubated the cells for surface CXCR3 levels remained at baseline following incuba- 1 h with cycloheximide, induced CXCR3 internalization with tion with CXCL11 in the presence of actinomycin D, brefeldin CXCL11, and let the cells recover in the presence of cyclohexi- A, or bafilomycin A1 (Fig. 4B). Collectively, the data suggest mide. Treatment with cycloheximide ablated the recovery of that the observed recovery of CXCR3 at the cell surface is CXCR3 in both PBMCs and L1.2 transfectants, whereas recovery dependent upon newly synthesized receptor trafficking through of cell surface CXCR6 levels to 80% of the starting levels was functional Golgi apparatus in the cell, in contrast to chemokine observed at the 2-h point. This latter value was approximately half receptors such as CCR4, CCR5, and CXCR6, which appear to of the level of staining seen at the same time point with CXCR6 be replenished by a recycling mechanism (19, 20). transfectants that had not been treated with cycloheximide, suggesting that both recycling and de novo synthesis cooperate in The role of the CXCR3 C terminus in internalization maintaining CXCR6 cell surface levels in the transfectant system Because the C terminus of several GPCRs has been implicated in used (Fig. 3C). the internalization process, we sought to examine the role of this Thus, in contrast to CXCR6 and other chemokine receptors de- motif in the internalization of CXCR3. Site-directed mutagenesis scribed in the literature (19, 20, 41), cell surface replenishment of of the CXCR3 cDNA was performed to generate four mutant con- CXCR3 is dependent upon de novo protein synthesis. If this pos- structs (Fig. 5A). The first of these mutated a triple LLL motif to tulate is true, then CXCR3 cell surface replenishment should also AAA, thereby losing two potential LL motifs previously reported be dependent upon mRNA transcription and efficient transport to be involved in CXCR2 internalization (43). The second muta- through the endoplasmic reticulum (ER) and Golgi. We therefore tion targeted the sole intracellular lysine residue, K324. Ubiquiti- preincubated PBMCs or L1.2 transfectants for1hinthepres- nation of internalized GPCRs has been shown to target them for ence or absence of actinomycin D (an inhibitor of transcrip- degradation, a process whereby the 74 aa ubiquitin is covalently tion), or brefeldin A and bafilomycin A1, which have been attached to intracellular lysine residues. The two remaining mu- shown to inhibit function of the ER and Golgi apparatus, re- tations introduced premature stop codons within the cDNA, spectively, and therefore inhibit transport of receptors through truncating the receptor by either 4 aa (⌬4 construct) or 34 aa these compartments (41, 42). Internalization was induced with (⌬34 construct). These mutations removed a YXXL motif at the 50 nM CXCL11 and the expression of CXCR3 was monitored extreme C terminus and the entire repertoire of C-terminal at 3 h post-internalization. In PBMCs, cell surface replenish- serine and threonine residues, respectively. The latter construct ment of CXCR3 was also significantly inhibited, although not allowed us to examine the requirement for phosphorylation of reduced to basal levels (Fig. 4A). In L1.2 transfectants, cell CXCR3 in the internalization process. All four mutants were 6720 INTERNALIZATION AND DEGRADATION OF CXCR3

FIGURE 8. CXCR3 function in T lymphocytes is strictly controlled. A–F, A series of experiments to compare CXCR3 expression and function in freshly isolated T lymphocytes (day 0) and T lymphocytes cultured for 10 days in medium supplemented with IL-2 and Con A (day 10). A, The relative expression levels of CXCR3 on T lymphocytes as deduced by flow cytometry using an anti-CXCR3 p Ͻ 0.01. B–D, The relative chemotactic ,ءء .Ab responses of T lymphocytes to increasing concentrations of CXCL9, CXCL10, and CXCL11. E, The relative induction of CXCR3 internalization on Downloaded from freshly isolated T lymphocytes as deduced by flow cytometry, following incubation with 50 nM of -p Ͻ 0.05 com ,ء .CXCL9, CXCL10, or CXCL11 pared with controls. F, The relative levels of specific binding of 0.1 nM 125I-CXCL10 and 125I-CXCL11 by T lymphocytes. All data represent the mean Ϯ http://www.jimmunol.org/ SEM of at least three experiments. by guest on September 30, 2021

transiently expressed in L1.2 cells, and cell surface expression Endocytosed CXCR3 passes through late endosomes or was monitored by flow cytometry. All four mutants trafficked to lysosomes before its degradation the cell membrane, although the ⌬34 mutant was expressed at We subsequently turned our attention to the fate of CXCR3 fol- levels significantly below those of WT CXCR3 (Fig. 5B). Con- lowing its internalization, using confocal microscopy to examine versely, the ⌬4 mutant was consistently expressed at greater intracellular localization of the receptor in permeablized T lym- levels than found in WT CXCR3, although this did not reach phocytes. A predominantly granular intracellular staining pattern significance. All four constructs were able to mediate chemo- for CXCR3 was evident in untreated cells (Fig. 6A), identical with taxis of cells in response to CXCL11, with the typical bell- that previously described by Gasser and colleagues (44). Likewise, shaped responses optima around the 3 nM concentration (Fig. a similar pattern was seen for staining with the late endosome 5C). Likewise, internalization of CXCR3 in response to B CXCL11 was unimpaired by mutation, with the 50 and 100 nM marker LAMP-1 (Fig. 6 ) with little colocalization of signals seen (Fig. 6C). Treatment with CXCL11 for 15 min resulted in clus- concentrations of ligand inducing significant internalization ϩ compared with untreated cells (Fig. 5D). Because the ⌬4 con- tering of LAMP-1 vesicles with apparent colocalization of struct appeared to be expressed at higher levels than WT CXCR3 with LAMP-1 in some but not all cells (Fig. 6, E and F). CXCR3 (Fig. 5B), we postulated that CXCR3 might be inter- This result may reflect either a rapid loss of CXCR3 immunore- nalized constitutively, i.e., in the absence of ligand, and the loss activity following trafficking to lysosomes or the fact that this path- of the four most C-terminal residues might inhibit this process. way is not the sole route of CXCR3 degradation. Little, if any, We subsequently examined the expression of both WT CXCR3 colocalization of CXCR3 with LAMP-1 staining was observed in and the ⌬4 construct over a 6-h period, following pretreatment cells 60 min following treatment with CXCL11, suggesting that with cycloheximide to inhibit de novo synthesis. WT CXCR3 degradation of CXCR3 may be complete by this point (Fig. 6, was seen to be quite rapidly lost from the cell surface in the G–I). absence of ligand, with approximately half of the original cell To further examine CXCR3 degradation, we used Western blot- surface levels of CXCR3 observed after4hofincubation. In ting methodologies. HA-tagged CXCR3 was expressed transiently comparison, internalization of the ⌬4 construct was less effi- in L1.2 cells and following pretreatment with cycloheximide to cacious, with the remaining cell surface levels of mutant recep- inhibit de novo synthesis, the cells were incubated for varying time tor at the 6-h time point significantly greater than receptor lev- periods in the presence or absence of CXCL11. Cell lysates were els of WT CXCR3. then examined by SDS-PAGE, followed by Western blotting. As The Journal of Immunology 6721 can be seen in Fig. 7A, CXCR3 appears as a band of ϳ50 kDa. 47). In this study, we provide several lines of evidence to sug- Following3hofincubation at 37°C, either in buffer alone or gest that the fate of endocytosed CXCR3 is predominantly one supplemented with CXCL11, the band representing CXCR3 was of degradation, with de novo synthesis of CXCR3 required for seen to reduce considerably in intensity, suggestive of a degrada- the recovery of CXCR3 cell surface levels. Indeed, little if any tive fate. Additional pretreatment of CXCR3 transfectants with CXCR3 was seen to reappear at the cell surface when transcrip- either the proteosome inhibitor MG132 or the lysosomal inhibitor tion, translation, or the trafficking of nascent proteins through chloroquine, before treatment with CXCL11, was observed to in- the golgi-ER was perturbed with appropriate pharmacological hibit the degradative process (Fig. 7B). We subsequently examined inhibitors. the panel of four C-terminal CXCR3 mutants to examine the ef- Although the related receptor CXCR4 has been shown to un- fects of mutation upon degradation. Compared with untreated dergo lysosomal degradation in a ubiquitin-dependent manner (47, cells, obvious degradation of each construct was observed, sug- 48), both the lysosome and the proteosome appear to facilitate the gesting that none of the C-terminal motifs we examined are critical degradation of CXCR3, as deduced by sensitivity to both MG132, for targeting CXCR3 for degradation (Fig. 7C). Thus it appears a 26 S proteasome inhibitor (49), and chloroquine, an inhibitor of that CXCR3 is readily degraded in the presence or absence of intralysosomal catabolism (50). Taken in the context of our con- ligand, by pathways involving both the proteosomes and lyso- focal microscopy data, we suggest that internalized CXCR3 traffics somes, and that ubiquitination of CXCR3 is not a fundamental part to late endosomes or lysosomes that may communicate in part with of this process. the proteosome for CXCR3 degradation. Cooperation between Previous reports have detailed the strict control of CXCR3 both the lysosomal and proteosomal pathways has been described mRNA expression in freshly isolated PBMCs. Although a signif- for the degradation of other receptors including the growth hor- Downloaded from icant proportion of freshly isolated PBMCs express CXCR3 at the mone receptor (51) and the IL-2R/IL-2R ligand complex (52). In cell surface, mRNA transcripts remain undetectable and the cells the case of the growth hormone receptor, endocytosis occurs in the are unresponsive to CXCL9 and CXCL10 in assays of chemotaxis absence of receptor ubiquitination but still requires intact protea- and calcium flux (1, 2). This unresponsive phenotype is reversed somal activity, suggesting that an adaptor protein targets the re- following culture of PBMCs for several days in a medium con- ceptor to the proteosome (53). In the case of CXCR3, ubiquitina-

taining IL-2 and a mitogen such as PHA, and correlates with tion of the receptor appears not to be required for either http://www.jimmunol.org/ mRNA induction and increased levels of CXCR3 at the cell sur- internalization or degradation, as mutation of the sole intracellular face. We revisited these data using freshly isolated T lymphocytes lysine residue had little effect upon either process. This may sug- used either immediately after isolation or following culture for 10 gest the existence of an additional motif within the CXCR3 intra- days in medium supplemented with IL-2 and Con A. As previously cellular regions that targets it for degradation by this route. Alter- described for PBMCs, freshly isolated T lymphocytes expressed natively, CXCR3 may be envisaged to interact with an adapter modest levels of cell surface CXCR3, which were significantly protein, which itself undergoes ubiquitination, targeting both pro- up-regulated following 10 days of culture (Fig. 8A). Modest num- teins to the proteosome. Such a process has been described for the bers of freshly isolated cells were seen to migrate in response to lectin Siglec-7, which is targeted to the proteosome as a complex increasing concentrations of CXCL9 and CXCL10, responses with SOCS-3 (54). by guest on September 30, 2021 which were significantly enhanced following 10 days of culture Two main routes have been described for the internalization of (Fig. 8, B and C). In contrast, chemotactic responses of both GPCRs following their activation. The best-characterized pathway freshly isolated and cultured T lymphocytes to CXCL11 were ro- uses clathrin-coated pits. In this pathway the phosphorylated re- bust, notably at the optimal concentration of 10 nM (Fig. 8D). The ceptor is bound by arrestins and located to clathrin-coated pits, greater efficacy of CXCL11 on freshly isolated cells was also ev- where the complex is internalized in vesicles. These vesicles are ident when internalization assays were performed, with CXCL11 subsequently released from the plasma membrane by dynamin and but neither CXCL9 nor CXCL10 inducing significant internaliza- transported to endosomes, where dephosphorylation of the recep- tion of CXCR3 (Fig. 8E). Radioligand binding assays competing tor occurs and resensitized receptor is recycled to the plasma mem- 125I-CXCL10 and 125I-CXCL11 with homologous unlabelled li- brane (55). The clathrin-mediated pathway has been demonstrated gand (Fig. 8F) suggested a greater number of binding sites for for the internalization of other chemokine receptors of the CXC CXCL11 on freshly isolated T lymphocytes than were evident class, notably CXCR1 (56), CXCR2 (57), and CXCR4 (38). A for CXCL10, despite both ligands reporting to have similar second pathway of internalization depends on caveolae (58), cho- nanomolar affinities at CXCR3 (8, 10, 45). Culture for 10 days lesterol rich, highly organized membrane structures that have been in the presence of IL-2 and Con A resulted in a trend for an shown to be involved in the internalization of other GPCRs in- increase in the number of binding sites for both ligands, al- cluding the chemokine receptors CCR4 and CCR5 (19, 20). Al- though this increase was not statistically significant. Thus, de- though caveolae have been described in macrophages (59), there is spite freshly isolated cells expressing CXCR3 at the cell sur- still some debate as to whether lymphocytes contain caveolae (60, face, the responses to CXCR3 ligands appear to be muted, with 61). Evidence for the use of either pathway of receptor internal- only CXCL11 inducing significant biological function. ization is often provided through the overexpression of dominant negative constructs (e.g., arrestin, dynamin, and clathrin mutants) or the use of pharmacological inhibitors to invoke or preclude the Discussion use of a particular pathway (62). In both human PBMCs and an Although there is growing information regarding the mecha- established transfectant system expressing the human ortholog nisms of GPCR internalization and recycling, no data concern- of CXCR3 (10), cell surface levels of CXCR3 were rapidly ing the fate of internalized CXCR3 have been published to date. reduced in a concentration- and time-dependant manner follow- Once internalized, a GPCR can experience one of two fates, ing exposure to ligand. In CXCR3 transfectants, use of inhib- namely dissociation of ligand and recycling of functional re- itors suggested that the pathway mediating ligand-induced en- ceptor back to the plasma membrane or degradation. These fates docytosis did not appear to involve caveolae but involved are not mutually exclusive, as CXCR4 has been shown to un- clathrin. In contrast, treatment of activated PBMCs with hyper- dergo both processes following engagement with ligand (46, tonic sucrose did not inhibit the internalization of CXCR3, and 6722 INTERNALIZATION AND DEGRADATION OF CXCR3 the use of an inhibitor of clathrin-coated pit assembly had no sulted in a total loss of CXCL10 binding. In contrast, CXCL11 can effect upon the down-regulation of CXCR3 in purified T lym- bind to both coupled and uncoupled CXCR3 (45). Supportive of phocytes. Likewise, we found no absolute requirement for ar- this idea, both resting and activated T lymphocytes were observed restin in the internalization process. In MEFs from mice defi- to bind significantly more 125I-CXCL11 than125I-CXCL10. Re- cient in ␤-arrestins 1 and 2 (24), internalization of CXCR3 was cently published data from studies using mice deficient in the G ␣ ␣ ␣ significantly reduced, but not completely abolished, whereas the protein subunits G i2 and G i3 found that although T lympho- ␣ use of dominant negative arrestin mutants was without effect cytes from mice lacking G i2 subunits exhibited no chemotaxis to ␣ upon CXCR3 internalization in our transfectant system. Col- CXCR3 ligands, T lymphocytes from mice lacking G i3 displayed lectively, our results suggest an alternative pathway for the en- significant increases in both migration and GTP␥S binding and docytosis of CXCR3, one that is independent of clathrin or migration as compared with WT T lymphocytes (67). This sug- ␣ arrestin or a combination. This finding is in agreement with a gests that in mice, G i2 subunits are crucial for CXCR3 signaling, ␣ previous study in which CXCL11-induced internalization of and that G i3 subunits can act as intracellular inhibitors of CXCR3 CXCR3 in a transfectant system was found to occur in a dy- function, thereby modulating CXCR3 responsiveness. Examining namin and ␤-arrestin 2-independent manner (28). our findings in the light of these data, we can hypothesize that The cellular motifs controlling ligand-driven internalization up-regulation of CXCR3 itself does not necessarily result in re- and targeting it for subsequent degradation remain elusive. Re- sponsiveness to CXCL10 and that CXCR3 function in the human moval of potential phosphorylation sites in the C terminus by is likely be modulated at the intracellular level by interaction with truncation had no effect on CXCL11-induced internalization, as G proteins. previously described for CXCR3 transfectants in both HEK-293 CXCR3 has previously been reported to be expressed in an in- Downloaded from and 300-19 cell lines (28, 63). Likewise, mutation of the LLL tracellular compartment within T lymphocytes, which can rapidly motif was without effect on CXCL11-induced internalization be mobilized to the cell surface by treatment with arachidonic again in agreement with a study using 300-19 transfectants (28) acid (44). This rapid, transient mobilization of receptor has but in disagreement with a study using HEK-293 transfectants been postulated to enable the cells to respond timely to changes in which some inhibition of CXCL11-induced internalization in the microenvironment in vivo. Such a capacity for increased

was observed (63). This likely reflects differences in the intra- cell surface expression is likely to be counterbalanced by the http://www.jimmunol.org/ cellular machinery to which CXCR3 is coupled in either cell degradative fate of CXCR3 we describe in this study. It is note- system. worthy that the CXCR3 ligands are all readily induced by IFN-␥ Of interest was the finding that CXCR3 is constitutively de- (7–9) and that the Th1-polarized lymphocytes specifically at- graded in the absence of ligand, a robust process that was me- tracted by these chemokines are themselves a source of IFN-␥ diated to a significant extent by a canonical YXX␸ motif at the (68). It can be postulated that such fine tuning of CXCR3 ac- extreme of the C terminus. Because such motifs have been im- tivity by degradation of internalized receptor serves to avoid the plicated in the sorting of transmembrane proteins to endosomes unnecessary amplification of T lymphocyte recruitment in vivo, and lysosomes (64), we hypothesize that the YSGL motif in- which would have undesirable consequences for the host. Gen- teracts with currently unknown intracellular proteins and con- eration of an artificial CXCR3 ligand that promotes the cellular by guest on September 30, 2021 trols the constitutive internalization of CXCR3. Supportive of degradation of CXCR3 in the absence of intracellular signaling our hypothesis, a distal YKKL motif within the C terminus of may represent an alternative strategy for the therapeutic mod- the GPCR PAR1 directs constitutive receptor internalization ulation of CXCR3 with potential benefit in a wide variety of that is clathrin- and dynamin-dependent but independent of ar- disease processes. restins (65, 66). Cell surface levels of CXCR3 are tightly regulated by both con- Acknowledgments stitutive and ligand-driven degradation and the replenishment of We are grateful to Dr. Robert Lefkowitz for providing the WT and ␤-ar- cell surface CXCR3 appears not to be dependent upon recycling as restin-deficient MEF, to Dr. Marc Caron for the provision of plasmids has been shown for other chemokine receptors, but upon de novo encoding arrestin mutants, and to Dr. Alexandre Benmerah for providing synthesis of CXCR3 protein and its subsequent transportation plasmids encoding the DIII and DIII⌬2 constructs. We thank Professor through the Golgi apparatus. To our knowledge, this is the first Mark Marsh, University College London, and Dr. Richard Colvin, Mas- example of a GPCR in which protein synthesis is essential for the sachusetts General Hospital, for helpful discussions. replenishment of the receptor on the cell surface following stim- ulation with ligand. This strict control is in addition to other mech- Disclosures anisms of posttranslational regulation of CXCR3 function. Al- The authors have no financial conflict of interest. though expressing significant amounts of CXCR3 on the cell surface, freshly isolated T lymphocytes were poorly responsive to References the CXCR3 ligands CXCL9 and CXCL10 as previously described 1. Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L. Piali, I. Clark-Lewis, (2), with the exception of the ligand CXCL11. This phenotype was M. Baggiolini, and B. Moser. 1996. Chemokine receptor specific for IP10 and corrected upon activation of the T lymphocytes by prolonged in- mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184: 963–969. cubation with IL-2 and a mitogen such as Con A, a process that 2. Loetscher, M., P. Loetscher, N. Brass, E. Meese, and B. Moser. 1998. Lympho- corresponds with CXCR3 mRNA induction (2), increased cell sur- cyte-specific chemokine receptor CXCR3: regulation, chemokine binding and face expression of the receptor, and the acquisition of robust func- gene localization. Eur. J. Immunol. 28: 3696–3705. 3. Trentin, L., C. Agostini, M. Facco, F. Piazza, A. Perin, M. Siviero, C. Gurrieri, tional responses to all three ligands. CXCL10 and CXCL11 have S. Galvan, F. Adami, R. Zambello, and G. Semenzato. 1999. The chemokine previously been described as allotopic ligands of CXCR3, with receptor CXCR3 is expressed on malignant B cells and mediates chemotaxis. activated T cells expressing a significant population of CXCR3 J. Clin. Invest. 104: 115–121. 4. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, 125 125 molecules that can bind I-CXCL11 but not I-CXCL10 (45). A. E. Koch, B. Moser, and C. R. Mackay. 1998. The chemokine receptors The binding of CXCL10 is thought to be controlled at the level of CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101: 746–754. G protein coupling because treatment of cell membranes with 5. Van Der Meer, P., S. H. Goldberg, K. M. Fung, L. R. Sharer, GTP␥S (guanosine 5Ј-O-(3-thiotriphosphate) or pertussis toxin re- F. Gonzalez-Scarano, and E. Lavi. 2001. Expression pattern of CXCR3, CXCR4, The Journal of Immunology 6723

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