A Novel Hepatointestinal Leukotriene B4 Receptor
2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês
10.1074/jbc.m004512200
ISSN1083-351X
AutoresSuke Wang, Eric L. Gustafson, Ling Pang, Xudong Qiao, Jiang Behan, Maureen Maguire, Marvin Bayne, Thomas M. Laz,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoLeukotriene B4 (LTB4) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB4 exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB4 in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB4 elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B4 receptor has been recently identified (LTB4-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB4-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB4-R1. Expression of LTB4-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB4-R2 cDNA displayed high affinity (Kd = 0.17 nm) for [3H]LTB4. Radioligand competition assays revealed high affinities of the receptor for LTB4 and LTB5, and 20-hydroxy-LTB4, and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB4 receptor antagonists, 14,15-dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302, had high affinity for LTB4-R1 but not for LTB4-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB4 functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB4 in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments. Leukotriene B4 (LTB4) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB4 exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB4 in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB4 elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B4 receptor has been recently identified (LTB4-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB4-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB4-R1. Expression of LTB4-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB4-R2 cDNA displayed high affinity (Kd = 0.17 nm) for [3H]LTB4. Radioligand competition assays revealed high affinities of the receptor for LTB4 and LTB5, and 20-hydroxy-LTB4, and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB4 receptor antagonists, 14,15-dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302, had high affinity for LTB4-R1 but not for LTB4-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB4 functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB4 in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments. leukotriene B4 leukotriene B4 receptors 1 and 2 inflammatory bowel disease 6-(6-(3-hydroxy-1,5-undecadienyl)-2-pyridinyl)-1,5-hexanediol G-protein-coupled receptor transmembrane open reading frame polymerase chain reaction base pair(s) Dulbecco's modified Eagle's medium fetal calf serum bovine serum albumin pertussis toxin untranslated repeat kilobase(s) hydroxyeicosatetraenoic acid 12-oxo-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid Leukotriene B4(LTB4)1 is derived as a product of eicosanoid metabolism and is a pro-inflammatory lipid mediator that potently stimulates neutrophil chemotaxis to sites of inflammation (1Samuelsson B. Dahlen S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. 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Considerable efforts have been devoted in the development of antagonists targeting the cell surface receptors, by screening compounds with radioligand binding assays utilizing membrane preparations from cells such as neutrophils. Potential treatments of various inflammatory conditions with these antagonists have been recently illustrated in human and animal models (11Jackson W.T. Fleisch J.H. Prog. Drug Res. 1996; 46: 115-168PubMed Google Scholar, 12Weringer E.J. Perry B.D. Sawyer P.S. Gilman S.C. Showell H.J. Transplantation. 1999; 67: 808-815Crossref PubMed Scopus (23) Google Scholar, 13Taylor B.M. Crittenden N.J. Bruden M.N. Wishka D.G. Morris J. Richards I.M. Sun F.F. Prostaglandins. 1991; 42: 211-224Crossref PubMed Scopus (16) Google Scholar, 14Kishikawa K. Tateishi N. Maruyama T. Seo R. Toda M. Miyamoto T. Prostaglandins. 1992; 44: 261-275Crossref PubMed Scopus (82) Google Scholar, 15Showell H.J. Conklyn M.J. Alpert R. Hingorani G.P. Wright K.F. Smith M.A. Stam E. Salter E.D. Scampoli D.N. Meltzer S. Reiter L.A. Koch K. Piscopio A.D. Cortina S.R. Lopez-Anaya A. Pettipher E.R. Milici A.J. Griffiths R.J. J. Pharmacol. Exp. Ther. 1998; 285: 946-954PubMed Google Scholar). Extensive studies of LTB4 and the search for the molecular identity of its receptors have resulted in the recent cloning of a LTB4 receptor (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar) (LTB4-R1). This protein is a cell surface receptor and belongs to the G-protein-coupled receptor superfamily containing seven membrane-spanning domains. The LTB4 receptor binds LTB4 with high affinity, which in turn leads to intracellular signaling and chemotaxis. Among the major tissues tested, the receptor is expressed abundantly only in peripheral leukocytes (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar). In this report, we describe the identification of a novel LTB4 receptor (LTB4-R2) that shares homology with LTB4-R1, and the finding that the two receptors are genetically linked. This novel receptor is highly expressed in several peripheral tissues such as liver, spleen, and intestine and binds LTB4 with high affinity. The ligand-receptor interaction activates the receptor leading to intracellular signal transduction. 3H-labeled LTB4 (∼200 Ci/mmol) was purchased from PerkinElmer Life Sciences. Human Marathon-ready cDNAs and RACE kit were fromCLONTECH. The 293-EBNA cell line was obtained from Invitrogen. Leukotrienes and other ligands were purchased from Sigma Chemicals and Cayman Chemical Co. 14,15-Dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302 were purchased from BIOMOL Research laboratories, Inc. (Plymouth Meeting, PA). Oligonucleotides were custom-synthesized by Life Technologies, Inc. Their sequences are: oligo347, 5′-ctaccacgcagtcaaccttctgcag; oligo348A, 5′-caccggaaggggccttggcgaagct; oligo348B, 5′-tgctctacgtcttcaccgctggaga, oligo358, 5′-gccgccaccatgtcggtctgctaccgtcc; oligo417, 5′-gccgccaccatgaacactacatcttctgcagc; oligo418, 5′-gtgcgcctccttccaccaggcctag; MM311, 5′-ctgacagcaggatgatagcca; 63U, 5′-tttttgttagtttgaggggaag; 480L, 5′-gcagaagggccgcctccattcc; and oligo359, 5′-gcaggttgtagggtctgctgtca. DNA sequencing was performed using the Big Dye Terminators sequencing agents (Applied Biosystems). The amino acid sequences of known G-protein-coupled receptors were used to conduct a BLAST search against expressed sequence tag data bases. The search identified a 397-base sequence as a putative GPCR fragment (HDPYA90R, see Fig. 1 A). A phylogenetic analysis (Wisconsin Package, Genetics Computer Group, Madison, WI) suggested that the sequence was related to a leukotriene receptor covering transmembrane domains 3 through 6. Further computational survey of public data bases identified contiguous sequences that resulted in a composite 2451-base sequence that contained a 888-base sequence at the 3′-end, which appeared to be a portion of an open reading frame of a GPCR (Met to TM6, see Fig. 1 A). A 3′-RACE was performed to obtain the missing 3′-portion of the putative open reading frame (ORF) by PCR using the Marathon RACE kit for PCR reactions and human liver Marathon-ready cDNA (CLONTECH) as a template. Primary PCR using oligo347 and AP1 (35 cycles of 94 °C for 30 s and 68 °C for 3 min), secondary PCR using primers AP2 and oligo348A (35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 2 min), and tertiary PCR using primers AP2 and oligo348B (35 cycles of 94 °C for 30s, 65 °C for 30 s, and 72 °C for 2 min) resulted in a ∼400-base pair (bp) 3′-RACE product (see Fig. 1 A). To obtain a full ORF, a 5′-primer (oligo358) containing the ATG codon in the 888-base sequence and a 3′-primer (oligo359) containing the putative stop codon in the 3′-RACE sequence were generated. A PCR with this pair of primers and human liver cDNA as a template (35 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 2 min) yielded a PCR product of ∼1.1 kb (SP9030, Fig. 1 A). A genomic clone containing both LTB4-R2 (SP9030) and LTB4-R1 receptors was obtained by PCR screening a human PAC PCRable DNA pool (Genome Systems, St. Louis, MO) with primers 63U and 480L. PCR was performed using PCR Supermix (Life Technologies, Inc) with a thermal cycling of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s (35 cycles). The size of the intron was determined by PCR using Supermix HiFidelity (Life Technologies) with primers oligo347 and MM311 and the PAC DNA as a template (94 °C for 30 s, 55 °C for 30 s, and 68 °C for 5 min). The resulting PCR product (∼4.0 kb) was gel-purified with a Qiaex II gel extraction kit (Qiagen) and partially sequenced (∼600 bp) at each of the two ends of the fragment. COS-7 cells grown in DMEM/10% FCS at 80–90% confluency were transfected with SuperFect agent (Qiagen) at 20 μg of DNA/150-mm plate. 48 h after transfection, medium was changed to Opti-MEM or DMEM/Opti-MEM (1:1)/5%FCS. 72 h after transfection, the cells were washed with 20 ml of phosphate-buffered saline without Ca2+/Mg2+ and incubated with 10 ml of 10 mm Hepes, pH 7.4, 0.5 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin at room temperature for 30 min. The cells were scraped off the plate and vortexed. The cell suspension was then centrifuged at 13,000g at 4 °C for 15 min. The pellets were resuspended in 1.8 ml of 50 mm Tris-Cl, pH 7.5, and vortexed. The membranes were homogenized with a 23-gauge needle. The protein concentration of the membrane preparations was determined with the BCA agents (Pierce). For saturation binding, 150 μl of binding assay buffer (30 mm Hepes, pH 7.4, 10 mm CaCl2, 10 mm MgCl2, 0.05% fatty acid free BSA (w/v), kept cold on ice) containing 24 μg of membranes were mixed with 50 μl of binding assay buffer containing 0 or 1 μm leukotriene in 2% (v/v) Me2SO. [3H]LTB4 (PerkinElmer Life Sciences, 50 nm) was added to the assays at increasing concentrations. The reactions were incubated for 1 h at 4 °C with slow rotation. Binding solutions were filtered through Multiscreen FB filters (Millipore) presoaked with 50 μl of binding assay buffer for 1 h at room temperature, and the filters were washed twice with 100 μl of 50 mm Tris-Cl, pH 7.5 (ice-cold). Fifty μl of Microscint fluid was added to the filters and counted for the bound radioligands. For radioligand competition assays, 160 μl of binding assay buffer containing membranes were mixed with 20 μl of binding assay buffer containing various concentrations of competing compounds in 6% Me2SO (v/v). A final 20 μl of binding assay buffer containing 1 μl of [3H]LTB4(PerkinElmer Life Sciences, 50 nm, final concentration 0.25 nm) in 6% (v/v) Me2SO was added to start the binding reaction. The final concentration of ethanol as the solvent in the stock LTB4 solution was 0.5% (v/v). Binding data were analyzed with non-linear regression software (Prism; GraphPad, San Diego, CA). 293-EBNA cells grown in DMEM/10% FCS at 80–90% confluency were transfected with the SuperFect agent (Qiagen). On the next day the cells were trypsinized off the plate and washed with phosphate-buffered saline without Ca2+/Mg2+. The cells were then seeded at a cell density of 35,000 cells/100 μl of medium into 96-well plates precoated with poly-d-lysine (Becton Dickinson). On the third day, the medium was removed, and 100 μl of Hanks' balanced salt solution (without phenol red) containing 4 μm of Fluo-3, AM (Molecular Probes), 20 mmHepes, pH 7.4, 0.1% (w/v) BSA, and 250 mm probenecid were added, and the cells were incubated at 37 °C, 5% CO2for 1 h. The cells were then washed three times with 150 μl of buffer containing Hanks' balanced salt solution, 40 mmHepes, pH 7.4, and 250 mm probenecid. One hundred μl of the wash buffer was added after the final wash, and Ca2+flux was measured with a Fluorometric Imaging Plate Reader (Molecular Devices) after addition of 40 μl of the buffer containing appropriate concentration of ligands. 293 cells were transfected with plasmid DNA and the SuperFect agent. Forty-eight hours post-transfection, 100 ng/ml pertusis toxin (PTX) was added to the cells, which were incubated at 37 °C overnight. Immediately prior to assay, the cells were released from the plate by the cell dissociation buffer (Sigma), and pelleted by centrifuge at 2000 rpm, 5 min, at 4 °C in a 10-ml Ficon tube. The cells were resuspended and seeded at 50,000 cells/50 μl of stimulation buffer/per well of FlashPlate. Fresh LTB4 was prepared in Hanks'-HEPES buffer (Hanks' balanced salt solution with 10 mm HEPES, pH 7.4, and 0.2% BSA (w/v), filtered, and stored at 4 °C). Fifty μl of LTB4 plus or minus 10 μm forskolin was added to the FlashPlate. Standard cAMP ranging from 0 to 1000 pmol/ml were arranged in the same plate. The plate was incubated on a rotating shaker at room temperature for 0.5 h. At the end of the incubation, the assay was terminated by addition of 100 μl of the adenylyl cyclase activation FlashPlate detection mixture (PerkinElmer Life Sciences). The FlashPlate was covered and gently agitated on a shaker for 3–5 h. After the development, the FlashPlate was counted on a 96-well counter. A standard curve was prepared, and the counts were converted to mass (pmol of cAMP/ml). Cyclic AMP production data were analyzed with non-linear regression. Hybridization to Northern blots and dot blots (CLONTECH) was carried out using a PCR-generated 440-bp DNA fragment from the 5′-untranslated region of the open reading frame (ORF) of LTB4-R2. The DNA fragment was random prime-labeled with [32P]dCTP, and the blots were hybridized for 14 h in ExpressHyb (CLONTECH) containing ∼2 million cpm/ml radiolabeled probe. The following day the blots were washed according to the manufacturer's protocol and exposed to Kodak Biomax MS film for 6 days at −70 °C. The films were analyzed for relative expression levels using the MCID M4 image analysis system (Imaging Research, Ontario, Canada). For the known receptor LTB4-R1, a 425-bp fragment corresponding to nucleotides 49–474 of the published LTB4 open reading frame (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar) was generated by PCR. The Northern blot and dot blot analyses were carried out with the random-prime [32P]dCTP-labeled fragment, hybridized overnight at 65 °C with multiple tissue dot blots and Northern blots (CLONTECH). Sequence analysis of the 1.1-kb PCR product resulting from multiple RACE amplification steps identified a putative ORF of 1077 bp (Fig. 1 A), which encodes a protein of 358 amino acids (Fig. 1 B). Hydrophobicity analysis of the 358-amino acid sequence suggested that there are seven transmembrane-spanning regions. BLAST analysis with the amino acid sequence against the GenBank™ data base revealed homology of the amino acid sequence to the human leukocyte LTB4 receptor (LTB4-R1, 42%) (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar) (Fig. 1 B), the human CRTH2 (32%) (17Nagata K. Tanaka K. Ogawa K. Kemmotsu K. Imai T. Yoshie O. Abe H. Tada K. Nakamura M. Sugamura K. Takano S. J. Immunol. 1999; 162: 1278-1286PubMed Google Scholar), and the human somatostatin receptor SSTR4 (27%) (18Rohrer L. Raulf F. Bruns C. Buettner R. Hofstaedter F. Schule R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4196-4200Crossref PubMed Scopus (213) Google Scholar, 19Hein J. Methods Enzymol. 1990; 183: 626-645Crossref PubMed Scopus (349) Google Scholar). The amino acid sequence of the receptor is only distantly related (∼18% homology) to the recently cloned leukotriene D4receptor (20Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (885) Google Scholar, 21Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (304) Google Scholar). The high amino acid sequence homology to LTB4-R1 and the presence in all seven transmembrane domains of conserved amino acid motifs indicated that this was a G-protein-coupled receptor. Thus the novel receptor was tentatively termed as LTB4-R2. Alignment of the cDNA of LTB4-R2 with the ORF of LTB4-R1 revealed that the 5′-untranslated region (UTRs) of the transcript (GenBank™ accession number D89079 (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar)) is identical to the coding region at the 3′-portion and the immediate downstream sequence in the 3′-UTR of the ORF of LTB4-R2. This analysis suggests that portions of both LTB4 receptors could exist on a single mRNA and the two LTB4 receptors are in the same chromosomal region. A genomic clone containing coding regions of LTB4-R2 and LTB4-R1 was obtained (PAC clone 159K10) by PCR screening of a human PAC library using primers 63U and 480L in the coding region of LTB4-R2 (Fig. 2 A). Direct sequencing of the PAC clone revealed no intervening sequences in the coding region of either receptor, thus both receptors are encoded by intronless ORFs (Fig. 2 A). A second PCR using primers oligo347 and MM311 revealed a single ∼3.6-kb intron 3′ downstream of LTB4-R2 and 5′ upstream of LTB4-R1 (Fig. 2 A). GenBank™ entry AL096870 is a genomic sequence from chromosome 14. This fragment contains both the LTB4-R1 and LTB4-R2 genes, as determined by BLAST.Figure 2Genomic organization of the LTB4-R2/LTB4-R1 locus and various mRNAs. A, the genomic organization of the LTB4-R2/LTB4-R1 locus. The portion of the genomic sequence contains both LTB4-R2 and LTB4-R1 receptors and is identical to AL096870. Thesolid bar connecting LTB4-R2 and LTB4-R1 indicates the 3.6-kb intron that is located at nucleotide 1142 of the LTB4-R2 mRNA. B, the alignment of the LTB4-R2 transcript with the genomic sequence shown in A (AL096870). Relative to the reverse compliment of AL096870, LTB4-R2 spans from nucleotides 121,344 to 122,255. The bent dashed line indicates the absence of the intron. C, the alignment of the transcriptD89079 with the genomic sequence. Relative to the reverse compliment ofAL096870, D89079 spans nucleotides 122,183 to 122,491 and nucleotides 126,318 to 127,630 (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar, 22Raport C.J. Schweickart V.L. Chantry D. Eddy Jr., R.L. Shows T.B. Godiska R. Gray P.W. J. Leukoc. Biol. 1996; 59: 18-23Crossref PubMed Scopus (82) Google Scholar). D, the alignment of U33448 (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar, 22Raport C.J. Schweickart V.L. Chantry D. Eddy Jr., R.L. Shows T.B. Godiska R. Gray P.W. J. Leukoc. Biol. 1996; 59: 18-23Crossref PubMed Scopus (82) Google Scholar) with the genomic sequence. Relative to the reverse compliment ofAL096870, U33448 spans nucleotides 125,709 to 127,808. E, the alignment of D89078 with genomic sequence. Relative to the reverse compliment of AL096870, D89078 spans nucleotides 123,995 to 125,702 and nucleotides 126,321 to 127,620.View Large Image Figure ViewerDownload (PPT) The genomic sequence containing both LTB4-R2 and LTB4-R1 was compared with several mRNA transcripts of LTB4-R2 and LTB4-R1 (Fig. 2, B–D). The 3′-untranslated sequence of LTB4-R2 is identical to the 5′-end of the intron, suggesting that this transcript contains only LTB4-R2 (Fig. 2 B). There were three GenBank™ entries that contained LTB4-R1 mRNAs: D89078, D89079, and U33448. D89079 (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar) contains the coding sequences of both LTB4-R2 and LTB4-R1 but does not have the 3.6-kb intron (Fig. 2 C). U33448 (22Raport C.J. Schweickart V.L. Chantry D. Eddy Jr., R.L. Shows T.B. Godiska R. Gray P.W. J. Leukoc. Biol. 1996; 59: 18-23Crossref PubMed Scopus (82) Google Scholar) contains only the coding region of LTB4-R1, and the 5′-untranslated sequence is identical to the 3′-end of the 3.6-kb intron sequence (Fig. 2 D). D89078 also contains only the coding region of LTB4-R1; however, the 5′-untranslated region is identical to the middle portion of the 3.6-kb intron (Fig. 2 E). Dot blot and Northern blot analyses were performed to determine the expression of LTB4-R2 mRNA in human tissues. A dot blot containing mRNAs from 56 human tissues (CLONTECH) was hybridized to a 440-bp fragment derived from the 5′-UTR of LTB4-R2 cDNA. The highest expression of LTB4-R2 was detected in liver followed by small intestine, spleen, and fetal liver (20–40% of that of liver; Fig. 3 A). Adrenal gland and pituitary had expression levels between 10 and 20% of those in the liver. All of the other 49 tissues expressed LTB4-R2 at 10% of or less than that of the liver level (Fig. 3 A), including peripheral leukocytes in which LTB4-R1 is highly expressed (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar). Using the same fragment as probe to hybridize a Northern blot, a mRNA of approximately 1.6 kb with high abundance was seen in the liver and a weak band in kidney (Fig. 3 B). No expression was detected in heart, brain, placenta, skeletal muscle, and pancreas. Employing a quantitative PCR method (Taqman, PE Biosystems) with 27 cDNA preparations generated primarily from human fetal and diseased tissues as templates, the highest expression of LTB4-R2 was again detected in fetal small intestine, Crohn's colon, fetal liver, and fetal lung (not shown). As is the case for the novel LTB4 receptor, the LTB4-R1 mRNA appears to be widely distributed in human tissues based on the results of the dot blot. LTB4-R1 is most abundant in immune-related tissues, including spleen, peripheral blood leukocytes, and bone marrow. Although there is also low expression of the LTB4-R1 mRNA in liver, it is not as prominent as that for LTB4-R2. The Dot blot data for LTB4-R1 are consistent with the Northern blot analysis shown by Yokomizo et al. (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (848) Google Scholar) in which high expression was seen in peripheral leukocytes and low or no mRNA was detected in other tissues. Radioligand binding assays were performed to directly test the ability of LTB4-R2 to bind LTB4. The ORF of LTB4-R2 was cloned in expression vector pCR3.1 to form construct pCR3.1-LTB4-R2. COS-7 cells were transfected with the construct, and membranes were prepared for a [3H]LTB4 binding assay. As shown in Fig. 4 A, specific binding was observed with the membranes prepared from cells transfected with pCR3.1-LTB4-R2; in contrast, no specific binding was seen with membranes prepared from cells transfected with vector alone. Because serum used in the cell cultures may carry low concentrations of LTB4 (23Seggev J.S. Wiessner J.H. Thornton Jr., W.H. Edes T.E. Ann. Allergy Asthma Immunol. 1995; 75: 365-368PubMed Google Scholar), radioligand binding assays were performed in parallel with membranes prepared from cells grown for the last 24 h in either serum-free (Opti-MEM) or medium containing 5% (v/v) FCS. No difference in the abilities of the two membrane preparations to bind [3H]LTB4 were found (Fig. 4 A), indicating that the serum used in the experiments did not affect ligand-receptor interaction through potential effects of either desensitization or receptor down-regulation. Saturation radioligand binding assays employing membranes from cells cultured in serum yielded a Kd of 0.17 ± 0.07 nm andBmax of 70 ± 8 fmol/mg of membrane protein (n = 3) (Fig. 4 B). SimilarKd and Bmax values were obtained when membranes were prepared from cells cultured in serum-free medium: Kd = 0.21 ± 0.06 nm andBmax = 64 ± 7 fmol/mg of protein (not shown). The pharmacological profiles of LTB4-R2 and LTB4-R1 were compared in radioligand competition assays using [3H]LTB4 as the radioligand and a number of unlabeled leukotrienes, leukotriene analogs, leukotriene receptor antagonists, and 5-lipoxygenase products as competitors (TableI). LTB4, LTB5, and an LTB4 metabolite, 20-hydroxy-LTB4, have high affinities for both LTB-R2 (Ki ≤ 41 nm and relative affinity ≤ 18) and LTB4-R1 (Ki ≤ 3.7 nm and relative affinity ≤ 5.3) receptors (Table I and Fig. 5). A 5-lipoxygenase product, 15(S)-HETE, and an arachidonic acid derivative, 12-oxo-ETE, displayed moderate affinities for LTB4-R2 (relative affinity = 67–73) but had low binding affinities for LTB4-R1 (Ki > 1000 nm) (Table I and Fig. 5). The affinities of the three LTB4receptor antagonists (14,15-dehydro-LTB4 (24Shimazaki T. Kobayashi Y. Sato F. Iwama T. Shikada K. Prostaglandins. 1990; 39: 459-467Crossref PubMed Scopus (15) Google Scholar), LTB4-3-aminopropylamide (25Young R.N. Zamboni R. Rokach J. Prostaglandins. 1983; 26: 605-613Crossref PubMed Scopus (13) Google Scholar, 26Goldman D.W. Gifford L.A. Marotti T. Koo C.H. Goetzl E.J. Fed. Proc. 1987; 46: 200-203PubMed Google Scholar), and U-75302 (27Lin A.H. Morris J. Wishka D.G. Gorman R.R. Ann. N. Y. Acad. Sci. 1988; 524: 196-200Crossref PubMed Scopus (19) Google Scholar)) were relatively lower for LTB4-R2 (Ki = 473–5434 nm) than for LTB4-R1 (Ki = 5.1–27 nm) (Table I). Another LTB4 metabolite, 20-carboxy-LTB4, bound LTB4-R1 with high affinity (Ki = 20 nm) but bound LTB4-R2 with much lower affinity (Ki > 1000 nm) (Table I).Table IPharmacological profiles of the LTB4-R2 and LTB4-R1 receptors determined with radioligand competition assaysLigandLTB4-R2LTB4-R1KiKi/Ki(LTB4)KiKi/Ki(LTB4)nmnmLTB42.3 ± 1.110.7 ± 0.41LTB59.4 ± 4.843.7 ± 3.05.320-Hydroxy-LTB441 ± 12180.54 ± 210.815(S)-HETE173 ± 8073>18,000>25,71412-Oxo-ETE155 ± 7467>1,000>1,42914,15-Dehydro-LTB4473 ± 20120527 ± 338LTB4-3-aminopropylamide1,227 ± 6805335.1 ± 0.57.3U-753025,434 ± 1,3202,36225 ± 43620-Carboxy-LTB4>1,000>43520 ± 2296-trans-LTB4 (transstereoisomer of LTB4)>1,000>435336 ± 154805-Oxo-ETE>1,000>435>1,000>1,4295(S)-HETE>3,000>1,304>20,000>28,571(±)5-HETE>1,000>435>1,000>1,42920-HETE>1,000>435>10,000>14,2858(R)-HETE>1,000>435>30,000>42,8575,6-Dehydroarachidonic acid>1,000>435>1,000>1,429Lipoxin-A41,400 ± 1106091,440 ± 1902,057Montelukast>10,000>4,350>10,000>14,285Zafirlukast>10,000>4,350>7,124>10,177LTC3>4,000>1,739>14,000>20,000LTC4>9,000>3,913>1,900>2,714
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