Identification of CCR6, the Specific Receptor for a Novel Lymphocyte-directed CC Chemokine LARC
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14893
ISSN1083-351X
AutoresMasataka Baba, Toshio Imai, Miyuki Nishimura, Mayumi Kakizaki, Shin Takagi, Kunio Hieshima, Hisayuki Nomiyama, Osamu Yoshie,
Tópico(s)T-cell and B-cell Immunology
ResumoLiver andactivation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nm. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1α, MIP-1β, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in speen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4+ and CD8+ T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4+ and CD8+ T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6. Liver andactivation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nm. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1α, MIP-1β, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in speen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4+ and CD8+ T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4+ and CD8+ T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6. The chemokines are a group of structurally related approximately 70–90-amino acid polypeptides involved in leukocyte recruitment and activation (1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Google Scholar, 2Ben-Baruch A. Michiel D.F. Oppenheim J.J. J. Biol. Chem. 1995; 270: 11703-11706Google Scholar). The chemokines are grouped into two main subfamilies, CXC and CC, on the basis of the arrangement of the N-terminal two conserved cysteine residues. One amino acid separates the two cysteines in the CXC chemokines while the two cysteines are adjacent in the CC chemokines. Most CXC chemokines are potent neutrophil attractants while most CC chemokines recruit monocytes and also lymphocytes, basophils, and/or eosinophils with variable selectivity. Recently, a novel lymphocyte-specific chemotactic cytokine, lymphotactin/SCM-1, 1The abbreviations and trivial names used are SCM-1single C motif 1G-proteinheterotrimeric guanine nucleotide-binding regulatory proteinCXCRCXC chemokine receptorIL-8interleukin 8IP-10interferon γ-inducible protein 10Migmonokine induced by interferon γSDF-1stroma derived factor 1PBSFpre-B cell stimulatory factorCCRCC chemokine receptorMIPmacrophage inflammatory proteinRANTESregulated on activation normal T cell expressed and secretedMCPmonocyte chemoattractant proteinTARCthymus and activation-regulated chemokineLARCliver and activation-regulated chemokineSEAPsecreted alkaline phosphataseFCSfetal calf serumPBMCperipheral blood mononuclear cellsNKnatural killerBSAbovine serum albuminEBI1EBV-induced gene 1BLR1Burkitt's lymphoma receptor 1IL-2interleukin 2PHAphytohemagglutininHBSSHank's balanced salt solutionPBLperipheral blood leukocytes has been reported, which carries only the second and the fourth of the four cysteine residues conserved in all other chemokines (3Kelner G.S. Kennedy J. Bacon K.B. Kleyensteuber S. Largaespada D.A. Jenkins N.A. Copeland N.G. Bazan J.F. Moore K.W. Schall T.J. Zlotnik A. Science. 1994; 266: 1395-1399Google Scholar, 4Yoshida T. Imai T. Kakizaki M. Nishimura M. Yoshie O. FEBS Lett. 1995; 360: 155-159Google Scholar). This may suggest the existence of the C type chemokine subfamily. single C motif 1 heterotrimeric guanine nucleotide-binding regulatory protein CXC chemokine receptor interleukin 8 interferon γ-inducible protein 10 monokine induced by interferon γ stroma derived factor 1 pre-B cell stimulatory factor CC chemokine receptor macrophage inflammatory protein regulated on activation normal T cell expressed and secreted monocyte chemoattractant protein thymus and activation-regulated chemokine liver and activation-regulated chemokine secreted alkaline phosphatase fetal calf serum peripheral blood mononuclear cells natural killer bovine serum albumin EBV-induced gene 1 Burkitt's lymphoma receptor 1 interleukin 2 phytohemagglutinin Hank's balanced salt solution peripheral blood leukocytes The specific effects of chemokines on leukocytes are known to be mediated by a family of seven-transmenbrane G-protein-coupled receptors (5Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Google Scholar, 6Premack B. Schall T.J. Nat. Med. 1996; 2: 1174-1178Google Scholar). In humans, four CXC chemokine receptors and five CC chemokine receptors have been cloned and defined for their ligand specificity. They are CXCR1 for IL-8 (7Holmes W.E. Lee J. Kuang W.-J. Rice G.C. Wood W.I. Science. 1991; 253: 1278-1280Google Scholar); CXCR2 for IL-8 and other CXC chemokines with the ELR motif (8Murphy P.M. Tiffany H.L. Science. 1991; 253: 1280-1283Google Scholar, 9Lee J. Horuk R. Rice G.C. Bennett G.L. Camerato T. Wood W.I. J. Biol. Chem. 1992; 267: 16283-16287Google Scholar, 10Geiser T. Dewald B. Ehrengruber M.U. Clark-Lewis I. Baggiolini M. J. Biol. Chem. 1993; 268: 15419-15424Google Scholar); CXCR3 for IP-10 and MIG (11Loetscher M. Gerber B. Loetscher P. Jones S.A. Piali L. Clark-Lewis I. Baggiolini M. Moser B. J. Exp. Med. 1996; 184: 963-969Google Scholar); CXCR4 for SDF-1/PBSF (12Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Google Scholar, 13Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J.-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Bggiolini M. Moser B. Nature. 1996; 382: 833-835Google Scholar); CCR1 for MIP-1α, RANTES, and MCP-3 (14Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Google Scholar, 15Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Google Scholar, 16Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Google Scholar, 17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J.-L. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Google Scholar); CCR2 for MCP-1, MCP-3, and MCP-4 (17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J.-L. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Google Scholar, 18Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Google Scholar, 19Franci C. Wong L.M. Van Damme J. Proost P. Charo I.F. J. Immunol. 1995; 154: 6511-6517Google Scholar, 44Garcia-Zepeda E.A. Combadiere C. Rothenberg M. Sarafi M.N. Lavigne F. Hamid Q. Murphy P.M. Luster A.D. J. Immunol. 1996; 157: 5613-5626Google Scholar); CCR3 for Eotaxin, RANTES, MCP-3, and MCP-4 (20Kitaura M. Nakajima T. Imai T. Harada S. Combadiere C. Tiffany H.L. Murphy P.M. Yoshie O. J. Biol. Chem. 1996; 271: 7725-7730Google Scholar, 21Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Google Scholar, 22Ponath P.D. Qin S. Post T.W. Wang J. Wu L. Gerard N.P. Newman W. Gerard C. Mackay C.R. J. Exp. Med. 1996; 183: 2437-2448Google Scholar, 23Uguccioni M. Loetscher P. Forssmann U. Dewald B. Li H. Lima S.H. Li Y. Kreider B. Garotta G. Thelen M. Baggiolini M. J. Exp. Med. 1996; 183: 2379-2384Google Scholar, 44Garcia-Zepeda E.A. Combadiere C. Rothenberg M. Sarafi M.N. Lavigne F. Hamid Q. Murphy P.M. Luster A.D. J. Immunol. 1996; 157: 5613-5626Google Scholar); CCR4 for TARC (24Power C.A. Meyer A. Nemeth K. Bacon K.B. Hoogewerf A.J. Proudfoot A.E.I. Wells T.N.C. J. Biol. Chem. 1995; 270: 19495-19500Google Scholar, 25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar); CCR5 for RANTES, MIP-1α, and MIP-1β (26Samson M. Labbe O. Mollereau C. Vassart G. Parmentier M. Biochemistry. 1996; 35: 3362-3367Google Scholar, 27Raport C.J. Gosling J. Schweickart V.L. Gray P.W. Charo I.F. J. Biol. Chem. 1996; 271: 17161-17166Google Scholar, 45Combadiere C. Ahuja S.K. Tiffany H.L. Murphy P.M. J. Leukocyte Biol. 1996; 60: 147-152Google Scholar). Furthermore, there are a growing number of putative chemokine receptors whose ligands remain to be identified. Recently, we have identified a novel CC chemokine, LARC (liver and activation-regulatedchemokine), and mapped its gene to chromosome 2q33-q37 (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). Expression of LARC mRNA was detected mainly in the liver among various human tissues and also induced in several human cell lines by phorbol 12-myristate 13-acetate. LARC was chemotactic for lymphocytes but not for monocytes. LARC fused with the secreted alkaline phosphatase (LARC-SEAP) bound specifically to lymphocytes with aKd of 0.4 nm. Notably, the binding of LARC-SEAP was competed only by LARC and not by other chemokines so far tested (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). These results indicated the presence of a class of receptors specific for LARC on lymphocytes. In the present study, we have demonstrated that an orpan receptor GPR-CY4 2Deposited by L. L. Lautens, W. Modi, and T. I. Bonner. is the LARC receptor that is selectively expressed on lymphocytes. Human hematopoietic cell lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS). 293/EBNA-1 cells were purchased from Invitrogen (San Diego, CA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Peripheral blood leukocytes were fractionated by surface markers as described previously (25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar, 29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar). In brief, peripheral blood mononuclear cells (PBMC) were isolated from venous blood obtained from healthy adult donors using Ficoll-Paque (Pharmacia, Uppsala, Sweden). Monocytes, B cells, and T cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD14, FITC-conjugated anti-CD19, and FITC-conjugated anti-CD3, respectively, and positively selected from PBMC by using MACS (Miltenyi Biotec, Bergisch, Germany). CD16+ CD3− and CD56+CD3− cells with appropriate forward and side scatters were sorted on a FACStar Plus (Beckton Dickinson, Mountain View, CA) as natural killer (NK) cells. CD4+ T cells and CD8+ T cells were purified from PBMC by negative selection using Dynabeads (Dynal, Oslo, Norway) after incubation with anti-CD16, -CD14, -CD20, and -CD8, or -CD16, -CD14, -CD20, and -CD4, respectively. Granulocytes were obtained from the pellet fraction of Ficoll-Paque gradient by dextran sedimentation and hypotonic lysis of erythrocytes. The purity of each cell population was always >95% as determined by flow cytometry or by staining with Diff-Quik (Baxter Scientific Products, McGaw Park, IL). Recombinant LARC, TARC, Eotaxin, and MCP-1 were produced by using a baculovirus expression system and purified as described previously (20Kitaura M. Nakajima T. Imai T. Harada S. Combadiere C. Tiffany H.L. Murphy P.M. Yoshie O. J. Biol. Chem. 1996; 271: 7725-7730Google Scholar, 28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar, 29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar). MIP-1α and MIP-1β were purchased from Pepro Tech (Rocky Hill, NJ). LARC fused with the secreted form of alkaline phosphate tagged with six histidine residues, LARC-SEAP(His)6, was prepared and purified as described previously (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). In brief, the LARC cDNA was subcloned into the SEAP(His)6 vector (pDREF-SEAP(His)6-Hyg)(28), making the expression vector pDREF-LARC-SEAP. 293/EBNA-1 cells were transfected with pDREF-LARC-SEAP using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) and cultured for 3–4 days in DMEM containing 10% FCS. The culture supernatants were centrifuged, filtered (0.45 μm), added to 20 mm HEPES, pH 7.4, and 0.02% sodium azide, and stored at 4 °C. The concentration of LARC-SEAP was determined by a sandwitch-type enzyme-linked immunosorbent assay as described previously (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). Cells stably expressing CCR1 (14Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Google Scholar, 15Gao J.-L. Kuhns D.B. Tiffany H.L. McDermott D. Li X. Francke U. Murphy P.M. J. Exp. Med. 1993; 177: 1421-1427Google Scholar, 16Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Google Scholar, 17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J.-L. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Google Scholar), CCR2B (17Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J.-L. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Google Scholar, 18Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2752-2756Google Scholar, 19Franci C. Wong L.M. Van Damme J. Proost P. Charo I.F. J. Immunol. 1995; 154: 6511-6517Google Scholar), CCR3 (20Kitaura M. Nakajima T. Imai T. Harada S. Combadiere C. Tiffany H.L. Murphy P.M. Yoshie O. J. Biol. Chem. 1996; 271: 7725-7730Google Scholar, 21Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Google Scholar, 22Ponath P.D. Qin S. Post T.W. Wang J. Wu L. Gerard N.P. Newman W. Gerard C. Mackay C.R. J. Exp. Med. 1996; 183: 2437-2448Google Scholar, 23Uguccioni M. Loetscher P. Forssmann U. Dewald B. Li H. Lima S.H. Li Y. Kreider B. Garotta G. Thelen M. Baggiolini M. J. Exp. Med. 1996; 183: 2379-2384Google Scholar), CCR4 (24Power C.A. Meyer A. Nemeth K. Bacon K.B. Hoogewerf A.J. Proudfoot A.E.I. Wells T.N.C. J. Biol. Chem. 1995; 270: 19495-19500Google Scholar, 25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar), CCR5 (26Samson M. Labbe O. Mollereau C. Vassart G. Parmentier M. Biochemistry. 1996; 35: 3362-3367Google Scholar, 27Raport C.J. Gosling J. Schweickart V.L. Gray P.W. Charo I.F. J. Biol. Chem. 1996; 271: 17161-17166Google Scholar, 45Combadiere C. Ahuja S.K. Tiffany H.L. Murphy P.M. J. Leukocyte Biol. 1996; 60: 147-152Google Scholar), V28/CMKBRL1 (30Raport C.J. Schweickart V.L. Eddy Jr., R.L. Shows T.B. Gray P.W. Gene. 1995; 163: 295-299Google Scholar, 31Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; 14: 673-680Google Scholar), GPR-CY42(GenBankTM accession number U45984), GPR-9–63(GenBankTM accession number:U45982), EBI1 (32Birkenbach M. Josefsen K. Yalamanchili R. Lenoir G. Kieff E. J. Virol. 1993; 67: 2209-2220Google Scholar), and BLR1 (33Dobner T. Wolf I. Emrich T. Lipp M. Eur. J. Immunol. 1992; 22: 2795-2799Google Scholar) were described previously (25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar). In brief, the expression plasmids based on pDREF-Hyg (29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar) were transfected into Raji cells by electroporation and into 293/EBNA-1 cells by LipofectAMINE (Life Technologies, Inc.). After selection with 250 μg/ml hygromycin for 1 to 2 weeks, drug-resistant cells were pooled and used for experiments. K562 cells were transfected with the expression plasmids based on pCAGG-Neo (25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar) by electroporation. After selection with 800μg/ml G418 for 1–2 weeks, clones expressing transfected receptors at high levels were selected by binding assays and/or Northern blot analysis. This was carried out as described previously (25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar). In brief, cells were suspended at 3 × 106 cells/ml in Hank's balanced salt solution (HBSS) containing 1 mg/ml of bovine serum albumin (BSA) and 10 mmHEPES, pH 7.4, (HBSS-BSA) and incubated with 1 μmfura-PE3-AM (Texas Fluorescence Labs) at room temperature for 1 h in the dark. After washing twice with HBSS-BSA, cells were suspended in HBSS-BSA at 2.5 × 106 cells/ml. 2 ml of the cell suspension in a quartz cuvette was placed in a luminescence spectrometer (Perkin-Elmer LS 50B) and fluorescence was monitored at 340 nm (λex1), 380 nm (λex2) and 510 nm (λem) every 200 ms. To determine EC50, a dose-response curve was generated in each experiment by plotting data as percent maximum response. Cell migration was determined by using a 48-well microchemotaxis chamber as described previously (29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar). In brief, each chemo-attractant was diluted in Hepes-buffered RPMI 1640 supplemented with 1% BSA and placed in lower wells (25 μl/well). Cells suspended in RPMI 1640 with 1% BSA at 2 × 106cells/ml were placed in upper wells (50 μl/well). Upper and lower wells were separated by a polyvinylpyrrolidone-free polycarbonate filter with 8-μm pores precoated with type IV collagen. Incubation was carried out at 37 °C for 4 h in 5% CO2, 95% air. Filters were removed, washed, and stained with Diff-Quik. Migrated cells were counted in five randomly selected high-power fields (400 ×) per well. All determinations were done in triplicate. This was carried out as described previously (25Imai, T., Baba, M., Nishimura, M., Kakizaki, M., Takagi, S., and Yoshie, O. (1997) J. Biol. Chem. , 272, in press.Google Scholar, 28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar, 29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar). In brief, for displacement experiments, 2 × 105 cells were incubated for 1 h at 16 °C with 1 nm of SEAP(His)6 or LARC-SEAP(His)6in the presence of increasing concentrations of unlabeled chemokines in 200 μl of RPMI 1640 containing 20 mm Hepes, pH 7.4, 1% BSA, and 0.02% sodium azide. For saturation experiments, cells were incubated for 1 h at 16 °C with increasing concentrations of LARC-SEAP(His)6 in the presence or absence of 1 μm unlabeled LARC. After incubation, cells were washed five times and lysed in 50 μl of 10 mm Tris-HCl, pH 8.0, and 1% Triton X-100. Samples were heated at 65 °C for 10 min to inactivate cellular phosphatase. After brief centrifugation to remove cell debris, alkaline phosphatase activity in 10 μl of lysate was measured by chemiluminescent assay as described previously (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). All determinations were done in duplicate. The binding data were analyzed by the LIGAND program (34Munson P. Rodbard D. Anal. Biochem. 1980; 107: 220-239Google Scholar). This was carried out as described previously (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar, 29Imai T. Yoshida T. Baba M. Nishimura M. Kakizaki M. Yoshie O. J. Biol. Chem. 1996; 271: 21514-21521Google Scholar). In brief, total RNA was prepared by using Trizol® reagent (Life Technologies, Inc.). RNA samples were separated by electrophoresis on a 1% agarose gel containing 0.66 mformaldehyde, blotted onto a filter membrane (Hybond N+) (Amersham Japan, Tokyo). Multiple tissue Northern blots and immune blots were purchased from CLONTECH (Palo Alto, CA). Hybridization was carried out with probes labeled with 32P using Prime It II kit (Stratagene, La Jolle, CA) at 65 °C in QuickHyb solution (Stratagene). After washing at 55 °C with 0.2 × SSC and 0.1% SDS, filters were exposed to x-ray films at −80 °C with an intensifying screen. To examine interaction of LARC with each cloned receptor, we measured LARC-induced calcium mobilization in a panel of K562 cells stably expressing the five known CCRs (CCR1-CCR5) and five orphan chemokine receptors, V28/CMKBRL1 (31Combadiere C. Ahuja S.K. Murphy P.M. DNA Cell Biol. 1995; 14: 673-680Google Scholar, 32Birkenbach M. Josefsen K. Yalamanchili R. Lenoir G. Kieff E. J. Virol. 1993; 67: 2209-2220Google Scholar), EBI1 (33Dobner T. Wolf I. Emrich T. Lipp M. Eur. J. Immunol. 1992; 22: 2795-2799Google Scholar), BLR1 (34Munson P. Rodbard D. Anal. Biochem. 1980; 107: 220-239Google Scholar), GPR-CY42(GenBankTM accession number U45984), and GPR-9–6 3Deposited by L. L. Lautens, H. L. Tiffany, J.-L. Gao, W. Modi, P. M. Murphy, and T. I. Bonner. (GenBankTM accession number U45982). As shown in Fig. 1 A, LARC specifically induced calcium flux in K562 cells expressing GPR-CY4 with complete desensitization against a rapid successive treatment with LARC. LARC did not induce calcium flux in parental K562 cells or those expressing CCR1, CCR2B, CCR3, CCR4, CCR5, or four other orphan receptors. On the other hand, MIP-1α, MIP-1β, MCP-1, eotaxin, or TARC did not induce calcium flux in K562 cells expressing GPR-CY4 (not shown). These chemokines, however, properly induced calcium flux in K562 cells expressing their respective CCRs even after treatment with LARC (Fig. 1 A). Similar results were obtained by using a panel of 293/EBNA-1 cells stably expressing these cloned receptors (data not shown). As shown in Fig. 1 B, 293/EBNA-1 cells stably expressing GPR-CY4 responded to LARC in calcium mobilization with an EC50 of ∼50 nm. These results clearly demonstrated that LARC was a specific functional ligand for GPR-CY4. Previously, we showed that LARC induced chemotaxis in freshly isolated peripheral blood lymphocytes with a maximal effect at 1 μg/ml (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). We therefore examined whether LARC was capable of inducing migration of 293/EBNA-1 cells stably expressing GPR-CY4. As shown in Fig.2 A, LARC induced migration in cells stably expressing GPR-CY4 with a typical bi-modal dose-response curve with a maximum effect at 1 μg/ml and an EC50 of ∼100 ng/ml (∼12 nm). LARC did not induce migration in cells transfected with the vector alone. A checkerboard-type analysis revealed that the migration of GPR-CY4-transfected 293/EBNA-1 cells toward LARC was mostly chemotactic (Fig. 2 B). Previously, we showed that LARC-SEAP(His)6 specifically bound to a single class of receptors expressed on lymphocytes with a Kd of 0.4 nm (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). Importantly, the binding of LARC-SEAP(His)6 was competed only by LARC and not by any other chemokines so far tested, indicating that the LARC receptor is not shared by other chemokines (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). We therefore examined the binding of LARC-SEAP(His)6 to a panel of Raji cells stably expressing GPR-CY4 and other cloned receptors. LARC-SEAP(His)6 bound specifically to cells expressing GPR-CY4 but not to parental cells or those expressing five CCRs or four other orphan receptors (data not shown). As shown in Fig.3 A, the binding of LARC-SEAP(His)6 to GPR-CY4 was saturable when increasing concentrations of LARC-SEAP(His)6 were incubated with Raji cells expressing GPR-CY4. The Scatchard analysis revealed aKd of 0.9 nm and 28,800 sites/cell (Fig.3 B). Unlabeled LARC fully competed with LARC-SEAP(His)6 for GPR-CY4 with an IC50 of 3.4 nm (Fig. 3 C). Furthermore, no other CC chemokines, MCP-1, RANTES, MIP-1α, MIP-1β, or TARC, were capable of competing with LARC-SEAP(His)6 for GPR-CY4 (Fig.3 D). These binding characteristics were highly consistent with those obtained from the endogenous class of LARC receptors expressed on lymphocytes (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). We have shown that the endogenous class of LARC receptors is expressed selectively on lymphocytes (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). Therefore, we examined the expression pattern of GPR-CY4 in various human tissues and leukocyte subsets by Northern blot analysis (Fig. 4). When blots for various tissues were hybridized with the 32P-labeled GPR-CY4 cDNA probe (Fig. 4 A), GPR-CY4 mRNA was found to be expressed strongly in the spleen and weakly in the lymph nodes. Weak expression was also detected in the testis (larger transcripts), small intestine, and PBL. Notably, the mRNA expression was very low, if any, in the liver where the LARC transcripts were mainly detected (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar). When blots specific for various lymphoid tissues were probed, GPR-CY4 mRNA was detected strongly in the spleen, lymph nodes, and Appendix, and weakly in the fetal liver (Fig. 4 B). When the same lymphoid tissue blots were rehybridized with the32P-labeled LARC cDNA probe, LARC mRNA was detected moderately in the appendix and weakly in the lymph nodes, PBL, and fetal liver (Fig. 4 B). Thus, the constitutive expression of GPR-CY4 and that of LARC overlap partly in the secondary lymphoid tissues. We then examined the expression of GPR-CY4 mRNA in various leukocyte subsets. T cells (both CD4+ and CD8+T cells) and B cells were clearly positive, whereas NK cells, monocytes, or granulocytes were virtually negative even though some RNA loading differences were noted (Fig. 5 A). We also examined the expression of GPR-CY4 mRNA in various human hematopoietic cell lines. Only a T cell line, Hut102, weakly expressed GPR-CY4, whereas other T cell lines (Molt-4, Jurkat, and Hut78), monocytic cell lines (THP-1 and U937), B cell lines (Raji and Daudi), an erythroleukemia cell line (K562), a promyelocytic cell line (HL-60), a basophilic cell line (KU812), and a megakaryocytic cell line (MEG-1) were virtually negative (not shown). Collectively, the expression of GPR-CY4 is mostly limited in the secondary lymphoid tissues and also in T and B lymphocytes. The expression pattern of GPR-CY4 is thus highly consistent to the lymphocyte-selective expression of the endogenous LARC receptor described previously (28Hieshima K. Imai T. Opdenakker G. Van Damme J. Kusuda J. Tei H. Sakaki Y. Takatsuki K. Miura R. Yoshie O. Nomiyama H. J. Biol. Chem. 1997; 272: 5846-5853Google Scholar).Figure 5Selective expression of GPR-CY4 mRNA in lymphocytes. A, expression of GPR-CY4 mRNA in human peripheral blood leukocyte subsets. Total RNA samples were prepared from freshly isolated CD4+ T cells (T4), CD8+ T cells (T8), total T cells (T), B cells (B), NK cells (NK), monocytes (Mo), and granulocytes (Gr), separated by agarose gel electrophoresis (5 μg/lane), blotted onto filters, and hybridized with t
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