Cloning and Characterization of Rat Leukotriene B4 Receptor

Biochemical and Biophysical Research Communications 262, 806 – 812 (1999) Article ID bbrc.1999.1284, available online at http://www.idealibrary.com on

Cloning and Characterization of Rat Leukotriene B 4 Receptor Akiko Toda, Takehiko Yokomizo, Kazuyuki Masuda, Akihide Nakao,* Takashi Izumi, and Takao Shimizu 1 Department of Biochemistry and Molecular Biology and *Department of Nephrology and Endocrinology, and Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received July 28, 1999

Leukotriene B 4 (LTB 4) is a potent chemoattractant for neutrophils and eosinophils. cDNAs for LTB 4 receptor (BLT) have been cloned from human, mouse, and guinea pig. Here we report the isolation of BLT from rat genomic library. Rat BLT consists of 351 amino acids with homologies of 80.2, 93.2, and 71.6%, to human, mouse, and guinea pig BLT, respectively. When expressed in human embryonic kidney (HEK)293 cells, rat BLT showed a specific and high-affinity binding to LTB 4 with a Kd value of 0.68 nM (mean, n 5 3). Northern blot analysis showed that BLT is exclusively expressed in polymorphonuclear leukocytes. Furthermore, the expression of BLT was high in proteosepeptone-activated peritoneal macrophages, while the resident macrophages did not show significant expression. The present results suggest important roles of LTB 4 in macrophage recruitment and activation. © 1999 Academic Press

Leukotriene B 4 (LTB 4) is biosynthesized from arachidonic acid by the actions of 5-lipoxygenase and LTA 4 hydrolase (1, 2). LTB 4 plays important roles in the host defense system against infections or invasion of foreign bodies (3–5). The binding of LTB 4 to its specific cell surface receptor (BLT), stimulates a number of leuko1 To whom correspondence should be addressed. Fax: 81-3-38138732. E-mail: [email protected]. The work was supported in part by CREST of JST, Japan, Grantsin-aid from the Ministry of Education, Science, Sports, and Culture of Japan, and the Yamanouchi Foundation for Metabolic Disorders. Abbreviations used: LTB 4, leukotriene B 4; BLT, leukotriene B 4 receptor; GPCR, G-protein-coupled receptor; PMNL, polymorphonuclear leukocytes; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; SSC, saline-sodium citrate solution; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PMSF, phenylmethanesulfonyl fluoride; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; ORF, open reading frame; TM, transmembrane; IL-8, interleukin 8; G-CSF, granulocyte colony-stimulating factor; and LPS, lipopolysaccharide.

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cyte functions, such as adhesion to vascular endothelial cells (6), transendothelial migration (7), chemotaxis (8), release of lysosomal enzymes (9), and production of reactive oxygen species (10). We previously isolated BLT cDNA from retinoic aciddifferentiated HL-60 cells (11). Human BLT is a GPCR with seven TM-spanning domains, and shows low homology to the other known receptors including chemoattractant receptors. BLT cDNAs have been isolated from human (11, 12), mouse (13), and guinea pig (14, 15), and all of them showed subnanomolar Kd values when expressed in mammalian cells. LTB 4 is also reported to participate in several inflammatory diseases including bronchial asthma (16) and inflammatory bowel diseases (17), and the effect of LTB 4 antagonist was evaluated in asthmatic patients (18). Although LTB 4 plays crucial roles in hyperlipidemiainduced renal injury (19), or immune complex-induced lung injury of the rat (20), the BLT in this species has not been isolated. Here we report cloning of rat BLT, the primary structure, binding properties, and tissue distribution. We also found that BLT was induced by the activation of peritoneal macrophages. METHODS Isolation of rat PMNL. Sprague-Dawley (SD) rats were given an intraperitoneal injection of 2% (w/v) solution of casein (100 ml/kg body weight). After 16 h, peritoneal cells were harvested by washing twice with 25 ml of PBS. The cells were stained with a Diff-Quick staining kit (International Reagents Corp.), and more than 95% of the cells were identified as PMNL. Poly (A) 1 RNA was extracted from PMNL using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech). Isolation of partial rat BLT cDNA. cDNA was obtained by RTPCR. The mixture for reverse-transcriptase reaction (50 ml) contained 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2, 1 mg of oligo-dT primer, 0.2 mM dNTPs, 10 mM DTT, 200 units of Superscript II reverse transcriptase (GIBCO BRL), and 3 mg of poly (A) 1 RNA of rat PMNL. The mixture was incubated at 37°C for 60 min, and the reaction was terminated by denaturing the mixture of 75°C for 10 min. A set of degenerative primers (sense; 59-CTCACTGC(ATGC)CC(ATGC)TT(TC)TT(TC)-39, antisense, 59-GCCAGC(ATGC)-

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GC(AG)TA(ACTG)A(AG)-39) corresponding to the 2nd and 7th TM domains of human BLT were used for PCR. PCR reaction mixture contained 1 ml of rat peritoneal PMNL cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2, 0.2 mM dNTPs, 2.5 units of Taq DNA polymerase (TaKaRa) and both primers at the concentration of 30 mM. Three steps of amplification were carried out: 96°C for 2 min, followed by 34 cycles of extension at 72°C for 3 min, followed by 1 cycle at 72°C for 10 min (denaturaion at 96°C for 1 min, and annealing at 50°C for 2 min). A PCR product (690 bps in length) was subcloned into pGEM-T vector (Promega), and the cDNA was sequenced on both strands using a Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and a 373 DNA automated DNA sequencer (Applied Biosystems). Genomic library screening. 1 3 10 6 plaque forming units from rat genomic library (Stratagene: RAT LAMBDA GENOMIC Sprague Dawley) were lifted to Hybond-N 1 nylon membranes (Amersham Pharmacia Biotech), and hybridized with a radiolabelled probe. The hybridization probe was generated by labelling the rat BLT partial cDNA with [ 32P]dCTP (NEN Life Science Products, Inc.) using a Megaprime DNA labelling system (Amersham Pharmacia Biotech). Prehybridizaion was carried out at 42°C for 12 h in a solution containing 6 3 SSC, 10 3 Denhardt’s solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA, followed by a 48 h hybridization in the same buffer with the labelled probe. After hybridization, filters were washed in 1 3 SSC/0.1% SDS at room temperature for 30 min, and in 1 3 SSC/0.1% SDS at 50°C for 1 h. Filters were autoradiographed to Fuji Medical X-ray Films RX-U for 16 h. Tertial screening gave two positive clones, designated pTRB1 and pTRB2. Phage DNAs of these positive clones were purified by liquid lysate method, digested with SacI, and the fragment, positive in Southern blotting using the partial rat BLT cDNA as a probe, was subcloned into SacI site of pBluescript II SK 1 (Stratagene), and designated prBS. Both sense and antisense strands were DNA-sequenced. Expression of rat BLT in HEK-293 cells. A vector for Hemagglutinin (HA)-tagged rat BLT was constructed for the expression in HEK-293 cells. The ORF was amplified by PCR using a sense primer (59-CGGGATCCCGCCATGTACCCCTACGACGTGCCCGACTACGCCGCTGCAAACACTACATCTAC-39) covering a BamHI site, and an antisense primer (59-GGAATTCTCACTTCGAAGATTCAGGAG-39) at the stop codon covering an EcoRI site. The PCR reaction mixture (200 ml) contained 40 ng of pTRB1, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2, 0.2 mM dNTPs, 14 units of Expand High Fidelity Taq DNA Polymerase (Boehringer Mannheim), and primers at 300 nM. Two steps of amplification were carried out 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 2 min. PCR products were digested with BamHI and EcoRI and subcloned into pcDNA3 (Invitrogen), and designated prBLT. The entire sequence of the insert was confirmed by DNA sequencing. HEK-293 cells were maintained in DMEM containing 10% FBS (EQUITECH-BIO), 100 IU/ml penicillin and 100 mg/ml streptomycin. HEK-293 cells seeded on 15 cm plates were transfected with prBLT by lipofection using TRANSFECTAM (GIBCO BRL) according to the manufacturer’s protocol. Membrane preparation and radiolabeled ligand binding. The cells were suspended in a sonication buffer (50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2, 1 mM EDTA-Na 2, 0.25 M sucrose, and 1 mM PMSF), and disrupted by sonication and with a Potter homogenizer. The homogenate was centrifuged for 10 min at 12,000 3 g, and the supernatant was further centrifuged for 60 min at 105,000 3 g. The pellet was suspended in a binding buffer (50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2, and 10 mM NaCl). The concentration of the protein was determined using Bio-Rad Protein Assay (Bio-Rad Laboratories). [ 3H]LTB 4 binding assay was performed using the membrane fraction containing 10 or 20 mg of protein in 200 ml of the binding buffer, and 0.25 nM [ 3H]LTB 4 (NEN Life Science Products, Inc.) with or without 0.25 mM unlabelled LTB 4. The mixture was incubated at room temperature for 60 min by gentle shaking. After incubation,

the samples were filtered rapidly through GF/C glass-fiber filters (Packard) and the filters were washed with 2 ml of the binding buffer. The remaining radioactivities were counted with a Top Count scintillation counter (Packard). Northern blotting analysis. Total RNAs were extracted from various rat tissues using ISOGEN (Nippon Gene). 10 mg of total RNA from each tissue was electrophoresed in a 1% denaturing agarose gel, and transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech). The filter was hybridized with [ 32P]dCTP-labelled ORF of rat BLT, or a human G3PDH cDNA in a hybridization buffer containing 4 3 SSC, 5 3 Denhardt’s solution, 0.2% SDS, 50% formamide, and 200 mg/ml salmon sperm DNA at 42°C for 24 h. The filter was washed in 0.5 3 SSC/0.1% SDS at 65°C for 1 h and autoradiographed for 16 h. Induction of BLT mRNA in rat peritoneal cells. SD rats were given an intraperitoneal injection of 2% (w/v) solution of casein (100 ml/kg body weight) or 10% (w/v) solution of proteosepeptone (70 ml/kg body weight). Peritoneal cells were recovered in 50 ml PBS (30 ml 1 20 ml). Resident cells were recovered by the same procedure. Total RNAs were purified as described above.

RESULTS AND DISCUSSION Cloning of Rat BLT from a Genomic Library To clone rat BLT, a rat genomic library was screened using a partial rat BLT cDNA amplified by PCR. Two identical clones were isolated after tertial screening, and were analyzed by Southern blotting. The restriction map of the clone including rat BLT is shown in Fig. 1A. The length of the insert was 14 kbps, and the entire sequences of the SacI fragment (3 kbps in length) with a putative ORF was determined (Fig. 1B). Hydropathy plot showed that the ORF encodes a protein with seven-TM spanning domains (data not shown). The deduced amino acid sequence contained several consensus residues for GPCRs, such as Cys-92 and Cys169 in the exoloops 1 and 2, which may form a disulfide bond (21). Asp-116, Arg-117, and Ser-118 (DRS) corresponding to the DRY motif at the cytoplasmic side of TM3 (22), and Asn-281, Pro-282, Val-283, Leu-284, and Tyr-285 to be an NPXXY motif (23, 24). Asn-4 and Asn-164 are possible N-glycosylation sites (25, 26). Ser127, Thr-220, Ser-314 and Thr-315 are consensus for the phosphorylation by protein kinase C (27, 28). Blast search (29) showed that this receptor is highly homologous to human (11, 12), mouse (13), and guinea-pig BLT (14, 15) (Fig. 1C). As the genomic DNAs of human and mouse BLT do not contain any introns in the ORF (Kato and Yokomizo, unpublished observations), it is possible that there are no introns in rat BLT ORF. The putative ORF of rat BLT encodes a protein of 351 amino acids, with the identities of 80.2, 93.2 and 71.6% to human (11, 12), mouse (13), and guinea-pig BLT (14, 15), respectively. The amino acid sequence of the putative third intracellular loop is completely in human, mouse, and rat, suggesting that this domain plays important roles in the signal transduction like other GPCRs (30 –32). The amino acid sequences in the TM2 and TM7 are also highly conserved among four species

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FIG. 1. Structure of rat BLT gene. (A) The restriction map of rat genomic clone (pTRB1) which contains rat BLT ORF. (B) Primary structure of rat BLT. The nucleotide sequence of prBS and the deduced amino acid sequence are shown. The putative TM domains are underlined. The putative in frame stop codon in 59 region of ORF is shown by bold letters. The GenBank/EMBL/DDBJ accession number of rat BLT is AB025230. (C) Amino acid alignment of BLT from four species. The putative TM domains are underlined. Amino acids conserved among four species are boxed.

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FIG. 3. Northern blotting of BLT in rat tissues. Lane 1, brain; 2, thymus; 3, lung; 4, heart; 5, liver; 6, spleen; 7, kidney; 8, colon; and 9, casein-induced leukocytes. (a) Hybridization with rat BLT ORF. (b) Hybridization with human G3PDH cDNA. (c) Staining with ethidium bromide.

(21), suggesting that these domains contribute to the recognition of the ligand, as reported for plateletactivating factor receptor (33). [ 3H]LTB 4 Binding Assay To examine if the cloned receptor is a functional BLT, the membrane fractions of HEK-293 cells transfected with the rat BLT were examined for LTB 4 binding. The specific bindings of [ 3H]LTB 4 to the membrane fractions of the cells transfected with pcDNA3 (mock) and prBLT were 68 6 26 and 2,600 6 110 dpm/10 mg of protein (mean 6 S.D., n 5 3), respectively (Fig. 2A). The binding was diminished in the presence of the excess amount of unlabelled LTB 4. The membrane fractions of the HEK cells expressing the rat BLT showed a robust and saturable [ 3H]LTB 4 binding with a Kd value of 0.68 6 0.56 nM and Bmax of 1,245 6 505 fmol/mg membrane protein (mean 6 S.D., n 5 3) (Fig. 2B, C). The Kd value is comparable to the previous reports on LTB 4 binding in rat leukocytes (4.5 nm (34), 2.4 nM (35)). Tissue Distribution of Rat BLT Northern blot analysis using total RNAs from various tissues of the rat revealed that the expression of

FIG. 2. Expression of rat BLT in HEK-293 cells. (A) [ 3H]LTB 4 binding to the membrane fractions of HEK-293 cells transfected with expression vectors. For the total binding, the membrane was incubated with 0.25 nM [ 3H]LTB 4. For the nonspecific binding, the membrane (20 mg of protein) was incubated with 0.25 nM [ 3H] LTB 4 and 0.25 mM unlabeled LTB 4. Filled columns, the total binding; open

columns, the nonspecific binding. The figure shows the mean 6 S.D. from six independent determinations. (B) Binding isotherms of rat BLT. The membrane (10 mg of protein) was incubated with various concentrations of [ 3H]LTB 4. The specific binding was determined as the difference between the total and the nonspecific binding. Open squares, the total binding; open circles, the nonspecific binding; filled circles, the specific binding. The figure shows the mean 6 S.D. from four determinations. (C) Scatchard analysis of the binding data from B.

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BLT was observed exclusively in casein-induced peritoneal cells, but not in other tissues (Fig. 3). The sizes of the transcripts were approximately 2.0 kb and 3.9 kb (Fig. 3), similar sizes as human BLT (11). There are few reports on the LTB 4 binding assay using rat tissues. Although BLT antagonists were effective in several rat animal disease models, such as hyperlipidemia-renal injury (19), immune complex-induced lung injury (20), and splanchnic ischemia and reperfusion (36), we did not detect any positive signals of BLT in these tissues by Northern blot analysis. Further studies including RT-PCR or in situ hybridization of BLT pave the way for understanding of the roles of LTB 4 and its receptor in these animal models. Induction of BLT mRNA in Rat Peritoneal Cells BLT is highly expressed in PMNL (Fig. 3), and the role of LTB 4 is most extensively studied using PMNL (2). Although Cristol et al. (37) reported the LTB 4 binding activity in guinea-pig alveolar macrophages, the presence and roles of BLT in macrophages remain unclear. Thus, we examined the expression of rat BLT using the peritoneal cells collected by several ways. Casein-induced peritoneal cells consisted of more than 80% of PMNL. On the other hand, both resident and proteosepeptone-induced peritoneal cells had a cell population of 98% macrophages and 2% PMNL, as reported (38). Northern blot analysis of total RNA from these cells revealed that both casein-induced and proteosepeptone-induced peritoneal cells express high amount of BLT mRNA, while resident macrophages express negligible amount of BLT mRNA (Fig. 4). Similar inductions were reported using mouse peritoneal cells (13). These result suggest that transcriptional regulation of BLT in response to inflammatory stimuli. IL-8 is one of the chemokines with biological profiles similar to LTB 4. The transcription of two IL-8 receptors, CXCR1 and CXCR2 (39), was regulated by G-CSF stimulation or LPS treatment (40 – 42). We need further information on BLT promoters and in vivo regulation. In summary, we isolated a rat BLT from a genomic library and determined the structure of the putative ORF. The primary structure of BLT is well conserved among human, mouse, guinea-pig and rat, especially in the third intracellular loop and the second and seventh TM domains. The cloned receptor showed a specific and high-affinity [ 3H]LTB 4 binding when expressed in HEK-293 cells. Comparison of the primary structures of BLT from various species is helpful to determine the amino acids responsible for the ligand recognition and signal transduction. The described nformation is useful for the development of anti-inflammatory BLT antagonists. We also showed that BLT expression is upregulated in peritoneal macrophages upon inflam-

FIG. 4. Induction of rat BLT in peritoneal cells. Northern blot analysis of BLT in rat peritoneal cells. Lane 1, resident cells; 2, proteosepeptone-induced cells; and 3, casein-induced cells. (a) Hybridization with rat BLT ORF. (b) Hybridization with human G3PDH cDNA. (c) Staining with ethidium bromide.

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