Abstract
Aim:
Human CXCR3, a seven-transmembrane segment (7TMS), is predominantly expressed in Th1-mediated responses. Interferon-γ-inducible protein 10 (IP-10) is an important ligand for CXCR3. Their interaction is pivotal for leukocyte migration and activation. Tyrosine sulfation in 7TMS is a posttranslational modification that contributes substantially to ligand binding. We aimed to study the role of tyrosine sulfation of CXCR3 in the protein's binding to IP-10.
Methods:
Plasmids encoding CXCR3 and its mutants were prepared by PCR and site-directed mutagenesis. HEK 293T cells were transfected with plasmids encoding CXCR3 or its variants using calcium phosphate. Transfected cells were labeled with [35S]-cysteine and methionine or [35S]-Na2SO3 and then analyzed by immunoprecipitation to measure sulfation. Experiments with 125I-labeled IP-10 were carried out to evaluate the affinity of CXCR3 for its ligand. Calcium influx assays were used to measure intercellular signal transduction.
Results:
Our data show that sulfate moieties are added to tyrosines 27 and 29 of CXCR3. Mutation of these two tyrosines to phenylalanines substantially decreases binding of CXCR3 to IP-10 and appears to eliminate the associated signal transduction. Tyrosine sulfation of CXCR3 is enhanced by tyrosyl protein sulfotransferases (TPSTs), and it is weakened by shRNA constructs. The binding ability of CXCR3 to IP-10 is increased by TPSTs and decreased by shRNAs.
Conclusions:
This study identifies two sulfated tyrosines in the N-terminus of CXCR3 as part of the binding site for IP-10, and it underscores the fact that tyrosine sulfation in the N-termini of 7TMS receptors is functionally important for ligand interactions. Our study suggests a molecular target for inhibiting this ligand-receptor interaction.
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Abbreviations
- CXCR3:
-
CXC receptor 3
- C3aR:
-
C3a receptor
- C5aR:
-
C5a receptor
- CCR5:
-
CC chemokine receptor 5
- 7TMS:
-
7-transmembrane segment
- GPCR:
-
G-protein-coupled receptor
- ECL:
-
extracellular loop
- HIV:
-
human immunodeficiency virus
References
Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, Clark-Lewis I, et al. Chemokine receptor specific for IP-10 and mig: structure, function, and expression in activated T-lymphocytes. J Exp Med 1996; 184: 963–9.
Thomas SY, Hou R, Boyson JE, Means TK, Hess C, Olson DP, et al. CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells. J Immunol 2003; 171: 2571–80.
Romagnani P, Annunziato F, Lasagni L, Lazzeri E, Beltrame C, Francalanci M, et al. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J Clin Invest 2001; 107: 53–63.
Luster AD . Chemokines-chemotactic cytokines that mediate inflammation. N Engl J Med 1998; 338: 436–45.
Sørensen TL, Tani M, Jensen J, Pierce V, Lucchinetti C, Folcik VA, et al. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J Clin Invest 1999; 103: 807–15.
Zhao DX, Hu Y, Miller GG, Luster AD, Mitchell RN, Libby P . Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cells alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasulopathy and acute rejection. J Immunol 2002; 169: 1556–60.
Medoff BD, Wain JC, Seung E, Jackobek R, Means TK, Ginns LC, et al. CXCR3 and its ligands in a murine model of obliterative bronchiolitis: regulation and function. J Immunol 2006; 176: 7087–95.
Hancock WW, Gao W, Csizmadia V, Faia K, Shemmeri N, Luster AD . Donor-derived IP-10 initiates development of acute allograft rejection. J Exp Med 2001; 193: 975–80.
Luster AD, Greenberg SM, Leder P . The IP-10 chemokine binds to a specific cell surface heparin sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J Exp Med 1995; 182: 219–31.
Arenberg DA, Kunkel SL, Polverini PJ, Morris SB, Burdick MD, Glass MC, et al. Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med 1996; 184: 981–92.
Luster AD, Leder P . IP-10, a CXC chemokine, elicits a potent thymus-dependent antitumor response in vivo. J Exp Med 1993; 178: 1057–65.
Niehrs C, Huttner WB, Carvallo D, Degryse E . Conversion of recombinant hirudin to the natural form by in vitro tyrosine. Differential substrate specifities of leech and bovine tyrosylprotein sulfotransferases. J Biol Chem 1990; 265: 9314–8.
Rosenquist GL, Nicholas HB Jr . Analysis of sequence requirements for protein tyrosine sulfation. Protein Sci 1993; 2: 215–22.
Farzan M, Mirzabekov T, Kolchinsky P, Wyatt R, Cayabyab M, Gerard NP, et al. Tyrosine sulfation of the amino-terminus of CCR5 facilitates HIV-1 entry. Cell 1999; 96: 667–76.
Farzan M, Babcock GJ, Vasilieva N, Wright PL, Kiprilov E, Mirzabekov T, et al. The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry. J Biol Chem 2002; 277: 29484–9.
Preobrazhensky AA, Dragan S, Kawano T, Gavrilin MA, Gulina IV, Chakravarty L, et al. Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region. J Immunol 2000; 165: 5295–303.
Fong AM, Alam SM, Imai T, Haribabu B, Patel DD . CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion. J Biol Chem 2002; 277: 19418–23.
Farzan M, Schnitzler CE, Vasilieva N, Leung D, Kuhn J, Gerard C, et al. Sulfated tyrosines form the docking site of the human C5a anaphylatoxin receptor. J Exp Med 2001; 193: 1059–66.
Gao J, Choe H, Bota D, Wright PL, Gerard C, Gerard NP . Sulfation of Tyrosine 174 in the human C3aR is essential for binding of C3a anaphylatoxin. J Biol Chem 2003; 278: 37902–8.
Xanthou G, Williams TJ, Pease JE . Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation. Eur J Immunol 2003; 33: 2927–36.
Clark-Lewis I, Mattioli I, Gong JH, Loetscher P . Structure-function relationship between the human chemokine receptor CXCR3 and its ligands. J Biol Chem 2003; 278: 289–95.
Choe H, Li W, Wright PL, Vasilieva N, Venturi M, Huang CC, et al. Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120. Cell 2003; 114: 161–70.
Sui G, Soohoo C, Affarel B, Gay F, Shi Y, Forrester WC . A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 5515–20.
Xia L, Ramachandran V, McDaniel JM, Nguyen KN, Cummings RD, McEver R . N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin. Blood 2003; 101: 552–9.
Swillens S . Interpretation of binding curves obtained with high receptor concentrations: practical aid for computer analysis. Mol Pharmacol 1995; 47: 1197–203.
Kehoe JW, Bertozzi CR . Tyrosine sulfation: a modulator of extracellular protein-protein interactions. Chem Biol 2000; 7: R57–61.
Huttner WB . Tyrosine sulfation and the secretory pathway. Annu Rev Physiol 1988; 50: 363–76.
Baeuerle, PA, Huttner, WB . Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster. J Biol Chem 1985; 260: 6434–39.
Choe H, Moore MJ, Owens CM, Wright PL, Vasilieva N, Li W, et al. Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC). Mol Microbiol 2005; 55: 1413–22.
Gerard C, Gerard NP . The pro-inflammatory seven transmembrane segment receptors of the leukocyte. Curr Opin Immunol 1994; 6: 140–5.
Lu B, Humbles A, Bota D, Gerard C, Moser B, Soler D, et al. Structure and function of the murine chemokine receptor CXCR3. Eur J Immunol 1999; 29: 3804–12.
Colvin RA, Campanella GS, Sun J, Luster AD . Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function. J Biol Chem 2004; 279: 30219–27.
Lasagni L, Francalanci M, Annunziato F, Lazzeri E, Giannini S, Cosmi L, et al. An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor-4. J Exp Med 2003; 197: 1537–49.
Bundgaard JR, Vuust J, Rehfeld JF . New consensus features for tyrosine O-sulfation determined by mutational analysis. J Biol Chem 1997; 272: 21700–5.
Seibert C, Cadene M, Sanfiz A, Chait BT, Sakmar TP . Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence. Proc Natl Acad Sci USA 2002; 99: 11031–6.
Colvin RA, Campanella GS, Manice LA, Luster AD . CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis. Mol Cell Biol 2006; 26: 5838–49.
Liu J, Louie S, Hsu W, Yu KM, Nicholas HB Jr, Rosenquist GL . Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease. Am J Respir Cell Mol Biol 2008; 38: 738–43.
Elshourbagy NA, Korman DR, Wu HL, Sylvester DR, Lee JA, Nuthalaganti P, et al. Molecular characterization and regulation of the human endothelin receptors. J Biol Chem 1993; 268: 3873–9.
Crump MP, Gong JH, Loetscher P, Rajarathnam K, Amara A, Arenzana-Seisdedos F, et al. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1. EMBO J 1997; 16: 6996–7007.
Gether U, Kobilka BK . G protein-coupled receptors. II. Mechanism of agonist activation. J Biol Chem 1998; 273: 17979–82.
Barnes PJ, Shapiro SD, Pauwels RA . Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 2003; 22: 672–88.
Acknowledgements
This work was supported in part by grants from the National Natural Science Foundation of China (No 30470767), the Beijing Natural Science Foundation (No 7072063), and the Ministry of Education of China (No NCET 06-0156).
We are grateful to Dr Hyeryun CHOE for helpful encouragement and plasmids, Dr Paulette L WRIGHT for critical reading and editing of this manuscript, Drs Wei CUI and Ding-hua LIU for assistance with FACS analysis and Dr Zhi-ying ZHAO for help in performing binding experiments.
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Gao, Jm., Xiang, Rl., Jiang, L. et al. Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10. Acta Pharmacol Sin 30, 193–201 (2009). https://doi.org/10.1038/aps.2008.24
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DOI: https://doi.org/10.1038/aps.2008.24
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