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Synthesis of Receptor Replenished at the Cell Surface by De Novo 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 References This article cites 68 articles, 41 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/180/10/6713.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 cytom- etry 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 mo- nensin, 5 ␮g/ml filipin, and 50 ␮g/ml nystatin. Cell sur- face 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).
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