Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region This information is current as of September 27, 2021. Alexander A. Preobrazhensky, Sofya Dragan, Tomonori Kawano, Mikhail A. Gavrilin, Irina V. Gulina, Leena Chakravarty and P. E. Kolattukudy J Immunol 2000; 165:5295-5303; ; doi: 10.4049/jimmunol.165.9.5295 Downloaded from http://www.jimmunol.org/content/165/9/5295 References This article cites 68 articles, 39 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/165/9/5295.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 27, 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region Alexander A. Preobrazhensky, Sofya Dragan, Tomonori Kawano, Mikhail A. Gavrilin, Irina V. Gulina, Leena Chakravarty, and P. E. Kolattukudy1 Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed muta- tions in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological Downloaded from consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamelli- .podium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2؉ influx into cytoplasm Mutations that prevented adenylate cyclase inhibition and Ca2؉ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of http://www.jimmunol.org/ the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational mod- ifications probably have significant biological functions. The Journal of Immunology, 2000, 165: 5295–5303. hemotactic cytokines (chemokines) recruit leukocytes to fated in proteins (6, 11). Several examples are known where sulfa- sites of injury, infection, and inflammation (1–4). Che- tion of tyrosines was shown to be important either for protein-protein C mokines are classified mainly in terms of the patterns of interaction (12–16) or for protein precursor processing (11). Cys residues, which form structurally important disulfide bonds. Monocyte chemotactic protein-1 (MCP-1)2 receptor (CCR2B), by guest on September 27, 2021 Those with an amino acid residue between the N-terminal pair of which is known to play an important role in a variety of diseases Cys residues, called ␣ or CXC chemokines, chemotax neutrophils involving inflammation and/or injury such as wound healing, ath- and nonhemopoietic cells, whereas those without an intervening erosclerosis, and arthritis (1–4), also contains a consensus sulfa- ␤ amino acid residue, called or CC chemokines, chemotax mono- tion domain at the N-terminal region. We noticed that a hexad of cytes, T cells, eosinophils, and NK cells. These two groups con- amino acid residues in this region is conserved in several chemo- stitute the majority of known chemokines. Chemokines specifi- kines, and therefore we suspected it to be functionally important. cally bind to seven transmembrane G protein-coupled receptors To test whether these residues are involved in the biological func- present on the target cells and cause chemotaxis and signal trans- tion of the receptor, site-directed mutagenesis was used. This mu- duction events that are not well understood. tagenesis involved the consensus sulfation site, including tyrosine, A chemokine receptor, CCR5 was recently reported to require the suspected site of sulfation, and the neighboring amino acid tyrosine sulfation for its function as a coreceptor for HIV-1 (5). residue that should be a part of the consensus required for enzy- Tyrosine sulfation of proteins is a modification that widely occurs matic sulfation by protein tyrosine sulfotransferases (6, 11). The in multicellular eukaryotic organisms (6, 7). Recently, two protein results presented here demonstrate that the hexad is important for tyrosine sulfotransferases, PTST-1 and PTST-2, have been cloned ligand binding, lamellipodium formation, chemotaxis, and signal (8–10). The sulfation of tyrosines occurs post-translationally, in the trans part of the Golgi network (6). The most important feature transduction. Structural alterations in this region of the receptor of tyrosine sulfation consensus sequences is the presence of an caused differential effects on lamellipodium formation and chemo- 2ϩ acidic or neutral amino acid residue directly before a tyrosine to be taxis vs Ca influx and adenylate cyclase inhibition, indicating sulfated, although not all theoretical consensus sequences are sul- that the receptor plays a role beyond the known function of causing ␣ ␤␥ dissociation of the trimeric G protein into Gi and G . Our re- sults also show that tyrosine within the hexad is sulfated and that Neurobiotechnology Center and Departments of Biochemistry and Molecular and the mutation in the consensus residue thought to be required for Cellular Biochemistry, Ohio State University, Columbus, OH 43210 sulfation severely inhibits sulfation and consequently inhibits the Received for publication March 14, 2000. Accepted for publication August 9, 2000. biological function of the receptor. Some chemokine receptors, The costs of publication of this article were defrayed in part by the payment of page such as CXCR4, are known to be glycoproteins, although no CC charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. P. E. Kolattukudy, Neurobio- technology Center, Ohio State University, 206 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. E-mail address: [email protected] 2 Abbreviation used in this paper: MCP-1, monocyte chemotactic protein-1. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 5296 SULFATION AND GLYCOSYLATION OF CCR2B chemokine receptor has been shown to be glycosylated. Our stud- once with HEPES containing binding buffer (1 mM CaCl2, 5 mM MgCl2, ies show that CCR2B is a glycoprotein and is sulfated at 0.5% BSA, and 50 mM HEPES, pH 7.2) (24). The final assay volume (250 ␮ ϫ 6 tyrosine 26. l) contained 5 10 transfected and washed cells, various amounts of MCP-1, and 0.02 pmol of iodinated MCP-1 protein. Various transfected cell lines were incubated with labeled and unlabeled MCP-1. Following Materials and Methods incubation, the cells were passed through a Whatman GF/C filter, and Reagents radioactivity on the filter was measured in a Packard COBRA gamma radiation counter (Downers Grove, IL). Dissociation constant (Kd) values The expression vector, pcDNA3 was obtained from Invitrogen (San Diego, for MCP-1 binding to CCR2B and mutated receptor in various cell lines of CA). Human embryonic kidney (HEK293) cells were obtained from Amer- HEK293 were determined using the LIGAND program (25). ican Type Culture Collection (ATCC CRL 1573; Manassas, VA). Lipo- fectamine, G418, penicillin-streptomycin solution, DMEM/F-12, BAC- ϩ TO-BAC baculovirus expression system, and FBS were purchased from Measurement of Ca2 influx induced by MCP-1 Life Technologies (Gaithersburg, MD). MEM/Eagle’s medium was ob- tained from ICN. Anti-Flag M2 mAb was purchased from Eastman Kodak Calcium influx into the cytoplasm was measured in HEK293 cell lines ␮ (Rochester, NY). Mouse IgG1, ␬ (MOPC-21), FITC-conjugated sheep anti- expressing CCR2B or its mutants after incubation with 2 M fura-2/ace- mouse IgG (whole molecule), phalloidin-FITC, chloramine-T, isobutyl- toxymethyl ester for 30 min at 37°C in a CO2 incubator. Fluorescence was methylxanthine, forskolin, QAE-Sephadex A-25, and Igepal CA-630 were measured after the addition of different concentrations