Molecular Cloning and Characterization of Another Leukotriene B4 Receptor (2000)

Leukotriene B4 is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B4, for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B4, named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B4 induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B4 in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B4 receptor will eventually lead to a better understanding of the classification of leukotriene B4 receptors and reconsideration of the pathophysiological role of leukotriene B4. Leukotriene B4 is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B4, for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B4, named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B4 induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B4 in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B4 receptor will eventually lead to a better understanding of the classification of leukotriene B4 receptors and reconsideration of the pathophysiological role of leukotriene B4. leukotriene B4 G protein-coupled receptor expression sequence tag open reading frame rapid amplification of cDNA ends reverse transcription-polymerase chain reaction hydroxyeicosatetraenoic acids dihydroxyeicosatetraenoic acids transmembrane Chinese hamster ovary minimal essential medium bovine serum albumin kilobase(s) Leukotriene B4(LTB4)1 and cysteinyl leukotrienes (LTC4, LTD4, and LTE4) derived from arachidonic acid metabolism are synthesized in a sequence, first by 5-lipoxygenase followed by LTA4 hydrolase or LTC4 synthase, respectively (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2320) Google Scholar). 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Commun. 1996; 223: 679-684Crossref PubMed Scopus (72) Google Scholar, 18Heath H. Qin S. Rao P. Wu L. LaRosa G. Kassam N. Ponath P.D. Mackay C.R. J. Clin. Invest. 1997; 99: 178-184Crossref PubMed Scopus (436) Google Scholar). In 1997, a cDNA clone was isolated for BLT, a human LTB4 receptor, from retinoic acid-differentiated HL-60 cells (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar), and in 1998, the mouse orthologue was isolated (20Huang W.W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar). Both receptors were predicted to contain a seven-transmembrane-spanning domain, and the recombinant human and mouse BLT showed specific binding of LTB4. We searched for novel chemoattractant receptors using a computational survey of public dbEST and genomic data bases and identified numerous sequences that encode structural characteristics common to GPCRs. One clone, JULF2, is predicted to be a novel GPCR encoding a 358-amino acid glycoprotein that is 36.6% identical to the human BLT receptor. We report evidence that JULF2 is an additional receptor for LTB4. Analysis of its unique pharmacological features will aid in the classification of LTB4 receptors, while preliminary data on its tissue distribution suggest that the pathophysiological role of JULF2 should be reassessed. Using the TBLASTN algorithm, dbEST, and genomic data bases (National Center for Biotechnology Information, NCBI/National Institute of Health) were queried with the amino acid sequence of BLT. One genomic draft sequence (GenBankTM accession number AL096870) derived from chromosome 14 was predicted to encode BLT and a novel GPCR, located approximately 4 kb upstream of the BLT gene. The novel GPCR had with some similarity to BLT. Primers were generated from the novel GPCR sequence, and the full-length cDNA was amplified from Human Leukocyte Marathon-ready cDNA library and Human Spleen Marathon-ready cDNA library (CLONTECH) by 5′- and 3′-rapid amplification of the cDNA ends (RACE) (21Matsumoto M. Nomura T. Momose K. Ikeda Y. Kondou Y. Akiho H. Togami J. Kimura Y. Okada M. Yamaguchi T. J. Biol. Chem. 1996; 271: 27217-27220Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The amplified RACE products were sequenced directly and a 1077-base pair open reading frame (ORF) for AB044402 (JULF2) was found. Using primers 5′-AAGGATGTCGGTCTGCTACCGTCCCCCA-3′ and 5′-GCTGTCAAAGGTCCCATTCCGGAC-3′ determined from the RACE products, the ORF was amplified from human spleen cDNA under previously described high GC conditions (22Matsumoto M. Hidaka K. Tada S. Tasaki Y. Yamaguchi T. Mol. Brain Res. 1995; 29: 157-162Crossref PubMed Scopus (147) Google Scholar). The JULF2 cDNA was cloned into an expression vector plasmid, pEF-BOS (dhfr) (23Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar), using the XbaI sites (pEF-JULF2). pEF-JULF2 or vector alone (mock) was transfected into CHO (dhfr−) cells using FuGENE6 (Roche Molecular Biochemicals). CHO cells expressing JULF2 were selected using a selection medium deficient in nucleotides (100 nm methotrexate). CHO cells expressing JULF2 were plated in 24-well plates at 1 × 105 cells/well and incubated for 24 h. The cells were washed with α-MEM medium containing 1 mm 3-isobutyl-1-methylxanthine (Sigma) and 0.1% (w/v) bovine serum albumin. Cells were treated with appropriate ligands and 1 μm forskolin (dissolved in medium). After incubation at 37 °C in 5% CO2 for 30 min, intracellular cAMP was extracted with 20% (v/v) perchloric acid for 30 min. The extracted cAMP was measured using an enzyme-linked immunoassay kit (Amersham Pharmacia Biotech). For pertussis toxin sensitivity, cells were pretreated for 20 h with 50 ng/ml pertussis toxin (Sigma). Calcium mobilization studies were conducted using Fluo 3-loaded CHO cells and a microtiter plate-based assay, using FLIPR (Molecular Devices). CHO cells were grown to confluence and allowed to adhere to the FLIPR microtiter plates, growth medium was removed and replaced with 1 μmFluo-3AM fluorescent indicator dye (Molecular Probes) in Hanks' balanced salt solution containing 10 mm HEPES, 200 μm CaCl2, 0.1% (w/v) BSA, and 2.5 mm probenecid. After incubation for 1 h (37 °C, 5% CO2), the cells were washed four times with the same buffer. Initially, fluorescence was read every 1 s for 1 min and then every 6 s for the following minute. Agonist was added after 10 s and the concentration-response curves were obtained by calculating the maximal fluorescence counts above background after addition of each concentration of agonist. Northern membranes containing 2 μg poly(A)+ RNA from different human tissues were purchased from CLONTECH. Northern analysis of JULF2 transcripts was performed under high stringency conditions as described previously (24Matsumoto M. Hidaka K. Tada S. Tasaki Y. Yamaguchi T. J. Neurochem. 1996; 66: 915-919Crossref PubMed Scopus (89) Google Scholar). A JULF2 probe was prepared by PCR, using primers to position 22–615 in the nucleotide sequence (GenBankTMaccession number AB044402). After stripping the Northern blot, it was reprobed with a β-actin control (supplied byCLONTECH). The membrane was exposed to x-ray film with an intensifying screen for autoradiography. Human mononuclear cells, neutrophils and eosinophils were isolated from the heparinized blood of healthy volunteers using Ficoll-Paque (Amersham Pharmacia Biotech) technique and CD16 microbeads (MACS reagents, Miltenyi Biotech). Total RNA preparations and cDNA syntheses were performed as described previously (25Matsumoto M. Kamohara M. Sugimoto T. Hidaka K. Takasaki J. Saito T. Okada M. Yamaguchi T. Furuichi K. Gene (Amst.). 2000; 248: 183-189Crossref PubMed Scopus (67) Google Scholar). We quantified JULF2 and BLT mRNA with the Prism 7700 Sequence Detector (Applied Biosystems) using primer sets (5′-CTCTGACCGCTTTCGTGC-3′ and 5′-CAGCGTCACGCTGTAGCA-3′ for JULF2; 5′-GCCCAAGGCACCTGGA-3′ and 5′-ACTCCGCAGACATAGTGACACA-3′ for BLT) and fluorescent-labeled probes (5′-(FAM)-TCCTTTCGGGCTGATGCTCGG-(TAMRA)-3′ for JULF2; 5′-(FAM)-TTGGACTGGCTGGTTGCCGC-(TAMRA)-3′ for BLT). RT-PCR was carried out in a 25-μl reaction mixture prepared with a TaqMan PCR core reagent (Applied Biosystems) containing an appropriately diluted cDNA solution, 0.2 μm of each primer and 0.1 μm of the probe. To obtain a calibration curve, we amplified a known amount of human genomic DNA using the same method as above. The number of mRNA copies per 1 μl of total RNA was calculated using the equation as described previously (26Medhurst A.D. Harrison D.C. Read S.J. Campbell C.A. Robbins M.J. Pangalos M.N. J. Neurosci. Methods. 2000; 98: 9-20Crossref PubMed Scopus (230) Google Scholar). Human β-actin mRNA was also measured as an internal control. Polycarbonate filters with 8-μm pores (Neuroprobe) were coated with 10 μg/ml fibronectin (IWAKI) in phosphate-buffered saline overnight at 4 °C. A dry-coated filter was sandwiched in a 96-blind well chamber (Neuroprobe), with ligands in the lower wells and the CHO cells (200 μl, 2 × 105 per well) in the top wells. The ligand solution and cell suspension were prepared in the same buffer (α-MEM medium containing 0.1% (w/v) BSA). After incubation at 37 °C in 5% CO2 for 4 h, the filter was disassembled, and the cells on the filter were fixed with methanol and stained with a Diff-Quik staining kit (International Reagents Corp.). Cells were then scraped from the upper side of the filter. The number of cells that had migrated to the lower side was determined by measuring optical densities at 595 nm, using a 96-well microplate reader. For pertussis toxin sensitivity, cells were pretreated for 20 h with 50 ng/ml pertussis toxin before seeding. LTB4, LTC4, LTD4, LTE4, 20-hydroxy-LTB4, 20-carboxy-LTB4, 6-trans-LTB4, 5(S)-HETE, (±)12-HETE, 5-oxo-HETE, (±)5,6-DiHETE, (±)8,9-DiHETE, (±)11,12-DiHETE, (±)14,15-DiHETE, 8(S),15(S)-DiHETE were purchased from Cayman Chemical. The human JULF2 gene was identified using a combination of genomic data base screening and 5′- and 3′-RACE. Screening of genomic data bases, using the human BLT sequence as a query, revealed a genomic draft-sequence (GenBankTMaccession number AL096870) encoding a novel GPCR. After identification of a corresponding mRNA in the human placenta by RT-PCR, the ORF was amplified by 5′- and 3′-RACE from human spleen and PBLs derived cDNA. The ORF encodes a unique putative GPCR protein of 358 amino acids. This receptor was designated JULF2. Sequence comparison revealed that JULF2 shares a high level of amino acid sequence similarity with human BLT (36.6%) (Fig. 1), compared with other GPCRs. Genomic sequence analysis revealed that the JULF2 gene is present as a single copy in the human genome and has no intron in its ORF. Interestingly, both JULF2 and BLT genes map to chromosome 14 with JULF2 gene located upstream, adjacent of the BLT gene and is transcribed in the same direction. To identify possible ligands for JULF2, changes in intracellular cAMP accumulation and calcium mobilization were monitored following addition of various bioactive lipids in CHO cells transfected with JULF2 cDNA. Among the lipoxygenase metabolites tested, LTB4 and its derivatives, such as, LTB3, LTB5, and 12-epi-LTB4, but not cysteinyl leukotrienes (LTC4, LTD4 and LTE4) nor 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), induced significant inhibition of forskolin-stimulated intracellular cAMP accumulation (Fig.2 a, TableI). CHO-JULF2 cells responded to LTB4 > 12-epi-LTB4 > LTB5 > LTB3, with EC50 (median effective concentration) values of 8.1, 16.8, 24.3, and 48.3 nm, respectively. Cells transfected with the vector alone did not respond to stimulation by LTB4 (Fig.2 b). Interestingly, LTB4 metabolites, 20-hydroxy-LTB4, 20-carboxy-LTB4, and 6-trans-LTB4, did not elicit specific cAMP changes in CHO-JULF2 cells. Additionally, the DiHETE series, consisting of four diastereomeric dihydroxy acids with varying locations of were also not of leukotrienes for human cells expressing JULF2 were treated with ligands and 1 μm forskolin (37 °C in 5% CO2 for 30 then the extracted cAMP was measured by an enzyme-linked The EC50 values were determined from the concentration-response in a CHO cells expressing JULF2 were treated with ligands and 1 μm forskolin (37 °C in 5% CO2 for 30 then the extracted cAMP was measured by an enzyme-linked The EC50 values were determined from the concentration-response BLT is known to intracellular calcium by stimulation (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, W.W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar, Y. Stocco R. Sawyer N. Greig G.M. Kargman S. O'Neill G.P. Shimizu T. T. Metters K.M. Abramovitz M. J. Pharmacol. 1999; PubMed Scopus Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google calcium mobilization responses was not CHO-JULF2 cells were treated with LTB4 in the and the of or not inhibition was by treatment with 50 ng/ml of pertussis the of as shown in of JULF2 mRNA in human tissues was using Northern A significant of JULF2 was detected at kb in the human the peripheral blood and the ovary a of kb was detected in the and the at In the peripheral and were detected at mRNA expression was in the spleen than in the peripheral an mRNA was detected in the peripheral leukocytes. Previous have indicated that BLT is highly expressed and is to human peripheral blood leukocytes (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, W.W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google Scholar, A. T. K. A. Izumi T. Shimizu T. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar). The expression of JULF2 and BLT in the of leukocytes from peripheral were using RT-PCR shown in JULF2 mRNA was detected in both mononuclear and of BLT mRNA expression was to the leukocytes. was a in the expression JULF2 and BLT in human mononuclear cell To the chemotactic of CHO-JULF2 cells, we performed a using a a chemotactic a with the maximal at 30 In cells, the maximal was previously at nm (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google Scholar). was by of the cells with 50 ng/ml pertussis toxin for 20 h. shown in 4 other lipoxygenase metabolites did not CHO-JULF2 cell for 12-epi-LTB4, which indicated of the induced in CHO cells not We have identified a novel receptor, JULF2, a functional GPCR for LTB4. The amino acid sequence of JULF2 the to BLT with 36.6% amino acid including for and showed to JULF2 as well as to BLT. The JULF2 gene is located 4 kb upstream of the BLT gene on human chromosome that both genes have been generated by a during an and that both receptors constitute an unique GPCR JULF2 shares features with GPCR J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), including sites in the domain, and in the first and second which a is an in an in in and in and and the highly in JULF2 unique characteristics from other such as a the or the and a the found in the in the amino of the second This of the is that JULF2 have unique structural of intracellular cAMP accumulation and of cell were induced by LTB4 in CHO cells. Both were by pertussis BLT, no calcium mobilization was found in CHO-JULF2 cells following LTB4 treatment in or in of or Previous studies have indicated that BLT has to and to specific (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, Y. Stocco R. Sawyer N. Greig G.M. Kargman S. O'Neill G.P. Shimizu T. T. Metters K.M. Abramovitz M. J. Pharmacol. 1999; PubMed Scopus Google Scholar), JULF2 is thought to to the studies for the ligand screening of JULF2 revealed that the ligand of JULF2 and BLT on and JULF2 showed no or (±)12-HETE, BLT, for which is a potent (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google and a J.F. Y. S. D. C. Biophys. 1987; PubMed Scopus Google Scholar). Furthermore, CHO-JULF2 cells responded to LTB4, 12-epi-LTB4, LTB5, and LTB3, but not to 20-hydroxy-LTB4, or that the ligand of JULF2 be as it was by the the and the of the and the of in position 20 of J. Exp. Med. 1984; PubMed Scopus Google of the LTB4 receptor and showed that the high class of LTB4 receptor is present at and the class of LTB4 receptor is present at sites on human leukocytes. also that the high was to and the to different functional responses of neutrophils to LTB4 D.W. J. Clin. Immunol. 1984; Full Text PDF PubMed Scopus Google Scholar). were by H.J. Pettipher E.R. Breslow R. Conklyn M.J. C.A. G.P. Salter E.D. D.J. Jr., L.A. K. J. Pharmacol. Exp. 1995; Google Scholar, H.J. Breslow R. Conklyn M.J. G.P. K. J. Pharmacol. 1996; PubMed Scopus Google using the specific BLT Both of the LTB4 receptor are thought to be the same GPCR, but it has not been the recombinant BLT on the different functional data that the of CHO-JULF2 cells was at 30 nm of LTB4, of that for BLT (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google Scholar). The EC50 of inhibition of in cells was nm, in cells it was nm K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google Scholar). suggest the that JULF2 be the previously identified of LTB4. The BLT gene has been cloned in the human (19Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar), the mouse (20Huang W.W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar), the A. T. K. A. Izumi T. Shimizu T. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar), and the Y. Stocco R. Sawyer N. Greig G.M. Kargman S. O'Neill G.P. Shimizu T. T. Metters K.M. Abramovitz M. J. Pharmacol. 1999; PubMed Scopus Google Scholar, K. T. Izumi T. Shimizu T. Biochem. J. 1999; PubMed Scopus Google Scholar). BLT were found to be to the peripheral leukocytes. The of JULF2 mRNA was different from that of human BLT. mRNA of JULF2 was detected in the human spleen and the peripheral leukocytes. In JULF2 mRNA was detected in a mononuclear leukocyte in comparison with BLT which was barely data suggest that JULF2 be the LTB4 receptor in mononuclear mainly the lymphocytes, as described by A. Acad. Sci. S. A. 1984; 81: PubMed Scopus Google Scholar), and BLT the LTB4 receptor in The of distribution JULF2 and BLT suggest the for of the of LTB4 in the of related In we have cloned a second functional JULF2, from the genomic data JULF2 ligand and tissue distribution compared with BLT. was no pharmacological or evidence that the of LTB4 were an with receptors that to the superfamily of GPCRs. The discovery of the JULF2 gene will a for screening of JULF2 which lead to the of the role of the pathophysiological LTB4. to this are in We Nagata for pEF-BOS and for We also and S. for in this