Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2 This information is current as of September 23, 2021. Alon Zaslaver, Rotem Feniger-Barish and Adit Ben-Baruch J Immunol 2001; 166:1272-1284; ; doi: 10.4049/jimmunol.166.2.1272 http://www.jimmunol.org/content/166/2/1272 Downloaded from References This article cites 61 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/166/2/1272.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2 Alon Zaslaver, Rotem Feniger-Barish, and Adit Ben-Baruch The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37°C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction Downloaded from in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemo- kine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the http://www.jimmunol.org/ ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period. The Journal of Immunology, 2001, 166: 1272–1284. hemokines are small chemotactic cytokines that mediate which consist of the major cellular infiltrate in the course of acute inflammatory processes, hematopoiesis and angiogene- inflammation. Several investigations demonstrated that neutrophil C sis, and play a crucial role in AIDS pathogenesis (1–4). responses could be regulated and desensitized in processes involv- Chemokine-mediated responses are induced by the activation of ing chemokine receptor internalization (12–14). Chemokine recep- specific G protein-coupled receptors (GPCR),2 which are ex- tor internalization and recycling were also shown to be involved in by guest on September 23, 2021 pressed on target cells (5). In similarity to other members of the the regulation of HIV-1 infectivity, as illustrated by the ability of GPCR superfamily, the ability of chemokine receptors to transmit the RANTES antagonist aminooxypentane-RANTES to induce a intracellular signals may be rapidly attenuated, and is tightly reg- highly potent inhibition of HIV-1 entry to target cells. The ami- ulated by different mechanisms, including receptor phosphoryla- nooxypentane-RANTES-mediated inhibition was demonstrated to tion-dependent G protein-uncoupling and receptor internalization be a direct result of its ability to induce elevated levels of CCR5 (6–9). Agonist-induced internalization of GPCR depletes the internalization, and to prevent its recycling back to the plasma mem- plasma membrane of receptors for the agonist and, therefore, may brane, thereby removing a key element of the fusion complex (15). contribute to the desensitization of the functions mediated through Recent interest in the mechanisms regulating ligand-induced these receptors (6–9). Moreover, if the ligand that induced recep- GPCR internalization has led to observations suggesting that some tor internalization is removed, the receptors may be dephosphory- GPCR undergo endocytosis via clathrin-coated pits, in a process lated, recycled back to the plasma membrane and resensitized upon that involves the binding of arrestins to the receptors (8, 9, 16, 17). a subsequent exposure to the same ligand (9–11). Other factors that are potential regulators of the internalization and Chemokine-induced receptor internalization and recycling were recycling processes of GPCR are yet to be defined. In that regard, shown to play a significant role in the regulation of inflammatory elucidation of the involvement of actin filaments and/or microtu- processes, as indicated for example by studies on neutrophils, bules in ligand-induced GPCR intracellular trafficking is of major importance. The plasma membrane is functionally integrated with the cell “cortex” that consists of actin-based cytoskeleton (18). Department of Cell Research and Immunology, George S. Wise Faculty of Life Sci- Therefore, the trafficking of vesicles at the plasma membrane may ences, Tel-Aviv University, Tel-Aviv, Israel necessitate the active rearrangement of actin filaments, which may Received for publication April 10, 2000. Accepted for publication October 20, 2000. then be followed by actin-assisted vesicle budding and fusion at The costs of publication of this article were defrayed in part by the payment of page the plasma membrane. Moreover, microtubules may also play a charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. direct role in trafficking of receptors, and were shown to facilitate 1 Address correspondence and reprint requests to Dr. Adit Ben-Baruch, Department of transport along the trans-golgi network plasma membrane pathway Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel-Aviv (19–23). University, Tel-Aviv 69978, Israel. E-mail address: [email protected] Although of potentially major importance for the regulation of 2 Abbreviations used in this paper: GPCR, G protein-coupled receptors; CSB, cell ligand-induced receptor intracellular trafficking, the involvement sorter buffer; CXCR, CXC chemokine receptor; ELRϩ-CXC, ELR-expressing CXC; GCP-2, granulocyte chemotactic protein-2; HEK 293, human embryonal kidney 293; of cytoskeleton elements in GPCR trafficking was very minimally PTx, pertussis toxin; WT, wild-type. studied so far. Of high significance is the fact that the contribution Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1273 of such elements to the regulation of trafficking of chemokine re- with high affinity (31, 38). Control transfections were performed with the ceptors was not addressed in any manner. In that respect, our study vector (pRc/CMV) alone, and the resulting cells did not specifically bind is focused on the elucidation of the role of cytoskeleton elements IL-8, GCP-2, or Abs specific for human CXCR1 or CXCR2 (29, 31, 38, 39). Parental and stable CXCR1-expressing 300-19 pre B cells (kindly do- in the ligand-induced internalization and recycling processes of nated by Dr. M. Wolf, Theodor Kocher Institute, University of Bern, Bern, CXC chemokine receptor (CXCR)1 and CXCR2, two closely re- Switzerland) were grown in RPMI 1640 medium, supplemented with 10% lated receptors that mediate the migration of neutrophils to inflam- FCS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 250 ng/ml amphoter- matory sites in response to ELR-expressing CXC (ELRϩ-CXC) icin, 2 mM L-glutamine, 1ϫ non essential amino acids (all purchased from Biological Industries, Beit Haemek, Israel), and 0.05% mM 2-ME (Sigma, chemokines (1, 5, 24). The use of CXCR1 and CXCR2 allowed us Rehovot, Israel). The DNA for CXCR1 transfection was expressed in a to compare between two chemokine receptors that have similar SR-␣-puro vector. Puromycin (1 ␮g/ml; Sigma) was used for selection. No general characteristics, but nevertheless are differentially regulated expression of human CXCR1 or CXCR2 was detected on the parental at multiple levels, including their internalization properties 300-19 cells, as determined by monoclonal mouse anti-CXCR1 or anti- (25–37). CXCR2 Abs (R&D Systems,