The murine cysteinyl leukotriene 2 (CysLT2) receptor. cDNA and genomic cloning, alternative splicing, and in vitro characterization.

Two classes of cysteinyl leukotriene receptor, CysLT(1) and CysLT(2), have been identified and pharmacologically characterized in human tissues. Although the CysLT(1) receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT(2) receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT(2) receptor (mCysLT(2)R) from heart tissue. mCysLT(2)R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT(2)R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5'-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT(2)R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT(2)R with a rank order of potency leukotriene C(4)(LTC(4) ) = LTD(4)>>LTE(4). [(3)H]LTD(4) binding to membranes expressing mCysLT(2)R could be effectively competed by LTC(4) and LTD(4) and only partially inhibited by LTE(4) and BAYu9773. The identification of mCysLT(2)R will be useful for establishing CysLT(2)R-deficient mice and determining novel leukotriene functions.

Two classes of cysteinyl leukotriene receptor, CysLT 1 and CysLT 2 , have been identified and pharmacologically characterized in human tissues. Although the CysLT 1 receptor mediates the proinflammatory effects of leukotrienes in human asthma, the physiological roles of CysLT 2 receptor are not defined, and a suitable mouse model would be useful in delineating function. We report here the molecular cloning and characterization of the mouse CysLT 2 receptor (mCysLT 2 R) from heart tissue. mCysLT 2 R cDNA encodes a protein of 309 amino acids, truncated at both ends compared with the human ortholog (hCysLT 2 R). The gene resides on the central region of mouse chromosome 14 and is composed of 6 exons with the entire coding region located in the last exon. Two 5-untranslated region splice variants were identified with the short form lacking exon 3 as the predominant transcript. Although the overall expression of mCysLT 2 R is very low, the highest expression was detected in spleen, thymus, and adrenal gland by ribonuclease protection assay, and discrete sites of expression in heart were observed by in situ hybridization. Intracellular calcium mobilization in response to cysteinyl leukotriene administration was detected in human embryonic kidney 293T cells transfected with recombinant mCysLT 2 R with a rank order of potency leukotriene C 4 (LTC 4 4 and BAYu9773. The identification of mCysLT 2 R will be useful for establishing CysLT 2 R-deficient mice and determining novel leukotriene functions.

) ‫؍‬ LTD 4 > > LTE 4 . [ 3 H]LTD 4 binding to membranes expressing mCysLT 2 R could be effectively competed by LTC 4 and LTD 4 and only partially inhibited by LTE
Cysteinyl leukotrienes (CysLT)-C 4 , D 4 , and E 4 have long been known as primary mediators in inflammatory diseases such as asthma and allergic rhinitis (1). These CysLTs are produced predominantly by eosinophils, mast cells, and macrophages in response to a variety of stimuli, including some antigens, which activate the 5-lipoxygenase pathway (2). The major actions of CysLTs in asthma include bronchoconstriction, bronchial hyperresponsiveness, increased pulmonary vascular permeability, parenchyma edema, and impaired mucus clearance (3).
Two types of CysLT receptor, CysLT 1 and CysLT 2 , have been identified to mediate CysLTs effects (4,5). Both are G proteincoupled receptors. Although pharmacological studies alone have provided valuable information, the recent molecular cloning and functional studies of both human (h) and mouse (m) CysLT 1 R (6 -10) and human CysLT 2 R (11-13) have provided new avenues for study of leukotriene biological functions. CysLT 1 R and CysLT 2 R bind their cognate ligands with high affinity. The human receptor subtypes are only 31% identical at the protein sequence level. They are located on different chromosomes; hCysLT 1 R is on Xq13-Xq21 and hCysLT 2 R on 13q14. The rank order affinities of CysLTs for CysLT 1 R and CysLT 2 R determined in transfected HEK 293 cells and COS cells are LTD 4 Ͼ LTC 4 Ͼ LTE 4 , and LTD 4 ϭ LTC 4 Ͼ Ͼ LTE 4 , respectively, correlating well with what was determined previously pharmacologically (4). Although CysLT 1 R can be inhibited specifically by the specific antagonists zafirlukast, montelukast, pranlukast, and MK-571, these compounds are unable to compete for binding at CysLT 2 R, and no specific antagonist for this subtype has been identified to date (14). BAYu9773 is an antagonist at both receptors and also a partial agonist for CysLT 2 R. The tissue expression patterns of these two receptors are different, indicating distinct roles in mediating leukotriene effects.
In this study we report the molecular cloning of mCysLT 2 R, alternative transcripts, genomic structure and chromosomal mapping, tissue localization, and CysLT 2 R functional characterization. cDNA Cloning of the Murine CysLT 2 Receptor-The mCysLT 2 R coding region corresponding to transmembrane domain III-VII was PCR amplified using degenerate primers based on the sequence similarity between human and rat CysLT 2 R (sense primer: 5Ј-GTCTTATTCYTT-RTATGTCAACATG; antisense primer: 5Ј-CTCYCCAGCAAARTAAT-AG; R ϭ A or G, Y ϭ T or C). Full-length cDNA was cloned from mouse heart total RNA using a CLONTECH SMART RACE cDNA amplification kit according to the manufacturer's protocol. The gene-specific * This work was supported by National Institutes of Health Grant HL58464 (to C. D. F.) and by the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Materials-LTB
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF331658.
Determination of the mCysLT 2 R Gene Structure-The whole gene structure for mCysLT 2 R was determined by PCR. The following primer sets were used to clone each intron starting from the 5Ј-end of the gene. In set 1 for intron 1, the sense primer was 5Ј-AGGGCGACCGAAGGC-AGAGGCAC, and the antisense primer was 5Ј-CCATTCATTTGGGAA-CATCCATTGGC. In set 2 for introns 2 and 3, the sense primer was 5Ј-GGAAGAGAGAAGAGTCCATCTGAGAAGGCATACTTC, and the antisense primer was 5Ј-TTCCAAGTCTTCTCTTTAGCCAATGATACA-CATCC. In set 3 for intron 4, the sense primer was 5Ј-TGTCCTGGG-GAAGGATGTGTATCATTGGC, and the antisense primer was 5Ј-GC-CATTATATGTCCTTTGGAGTTCAAACCATTGC. In set 4 for intron 5, the sense primer was 5Ј-CTGTTAGAGACCACTTGTAGCAATGG, and the antisense primer was 5Ј-GCTCTAGACTATAGATGAACTTTGCTG-AATATAGCCC.
Determination of Alternative Splicing Pattern in Different Tissues Using RT-PCR-Total RNAs from various mouse tissues were reverse transcribed using the Superscript First-strand Synthesis System for RT-PCR (Life Technologies, Inc.) and PCR performed to characterize the alternative splicing pattern of mCysLT 2 R mRNA. Primer sets 5Ј-AGGGCGACCGAAGGCAGAGGCAC and 5Ј-GCCATTATATGTCCTT-TGGAGTTCAAACCATTGC were used to amplify a fragment representing the short mRNA transcript lacking exon 3. A fragment representing the long mRNA transcript containing exon 3 was amplified by two rounds of PCR. The first round PCR used primer sets 5Ј-CTTTTACATGTACCTGAGAGTAATATG and 5Ј-AAGGCTCCCCA-GAAAAATATTATCAGG. The nested PCR was performed with 5Ј-TG-TACCTGAGAGTAATATGAATGAAGAAATAAG and 5Ј-ATCTTATCT-CTTGGACAACTATTCTTCACTC. Both fragments were verified by sequencing. ␤-Actin cDNA amplification was used as an internal control with primer sets 5Ј-TGGAATCCTGTGGCATCCATG and 5Ј-AACGCA-GCTCAGTAACAGTCC.
Ribonuclease Protection Assay-The mCysLT 2 R tissue expression pattern was analyzed by ribonuclease protection assay using an RPA III TM kit (Ambion). Total RNA was prepared from various mouse tissues using TriZOL reagent (Life Technologies, Inc.). The coding region of mCysLT 2 R subcloned into pcDNA3 vector at EcoRI/XbaI sites was linearized at the unique PmlI site. [␣-32 P]UTP (800 Ci/mmol, PerkinElmer Life Sciences)-labeled antisense RNA probe was synthesized by in vitro transcription using SP6 RNA polymerase (Ambion) in the presence of ribonuclease inhibitor at 37°C for 1 h. DNase I was added after transcription, and the mixture was incubated at 37°C for 30 min to remove template. The 280-bp labeled probe was gel purified. Total RNA (10 g) from different tissues was mixed with 7 ϫ 10 4 cpm probe and coprecipitated with NH 4 OAc and EtOH. The precipitated pellets were resuspended in hybridization buffer and incubated at 42°C overnight. A mixture of RNase A/RNase T1 diluted in digestion buffer was added to hybridization tubes and incubated for 30 min at 37°C to yield a 263-bp protected fragment. RNase was inactivated, and samples were precipitated and resuspended in gel loading buffer II, denatured, and electrophoresed in a 5% denaturing polyacrylamide urea gel. Radiolabeled probes were detected by exposure to x-ray film. Yeast tRNA served as a negative control.
Chromosomal Localization of the mCysLT 2 R Gene by Interspecific Mouse Backcross Mapping-Interspecific backcross progeny were generated by mating (C57BL/6J ϫ Mus spretus) F 1 females and C57BL/6J males as described (15). 205 N 2 mice were used to map the Cyslt2r locus. DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (16). All blots were prepared with Hybond N ϩ nylon membrane (Amersham Pharmacia Biotech). The probe, a 1.1-kb fragment of mouse cDNA from the 5Ј-untranslated region (UTR), was labeled with [␣ 32 P]dCTP using a random primed labeling kit (Stratagene); washing was done to a final stringency of 1.0 ϫ SSCP, 0.1% SDS, 65°C. Fragments of 9.0 and 3.7 kb were detected in BamHIdigested C57BL/6J DNA, and fragments of 8.0 and 3.7 kb were detected in BamHI-digested M. spretus DNA. The presence or absence of the 8.0-kb BamHI M. spretus-specific fragment was followed in backcross mice.
A description of the probes and RFLPs for the loci linked to Cyslt2r including hr and Dct has been reported previously (17). Recombination distances were calculated using Map Manager, version 2.6.5. The gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
Cell Culture and Transient Expression of the mCysLT 2 Receptor-HEK 293 cells or HEK 293T cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml Fungizone at 37°C in a humidified atmosphere with 5% CO 2 . The coding region of mCysLT 2 R was subcloned into pcDNA3 vector (Invitrogen). HEK 293 cells were transiently transfected with pcDNA3-mCysLT 2 R using Fugene 6 (Roche), and HEK 293T were Radioligand Binding Assay-HEK 293 cell membranes were prepared as described previously (18). Briefly, cells were harvested 48 h after transfection. The cells were dispersed in ice-cold buffer A (10 mM HEPES, 2 mM EDTA, 0.37 mg/ml protease inhibitor mixture (Roche), pH 7.4) by Dounce B homogenization followed by nitrogen cavitation at 1,100 p.s.i. for 15 min. The cell homogenate was centrifuged at 10,000 ϫ g for 10 min, and the supernatant was centrifuged at 100,000 ϫ g for 30 min. The resulting pellet was subjected to a final Dounce A homogenization. For competition studies, membrane preparations (100 g of protein) were incubated with 0.5 nM [ 3 H]LTD 4 together with different concentrations of reagents in buffer containing 10 mM HEPES, 20 mM CaCl 2 , pH 7.4, in the presence of 20 mM L-penicillamine at room temperature for 1 h. The reactions were stopped by ice-cold wash buffer (10 mM HEPES, 0.01% bovine serum albumin, pH 8.0), and the bound ligand was captured on Whatman GF/B filters. Radioactivity was quantitated by liquid scintillation counting. Specific binding was determined by subtracting nonspecific binding, measured as the radioligand binding in the presence of 1 M LTD 4 , from total binding. Data were analyzed by nonlinear regression using PRISM software. 2 Receptor cDNA, Splice Variants, and Gene-We sought to identify and characterize the mouse CysLT 2 R. Previous data suggested that rat may not possess a functional CysLT 2 R because no evidence of signaling could be obtained from the cloned sequence, and its deduced structure was truncated relative to the human receptor (11). We designed a pair of degenerate primers based on the sequence similarity between human and rat CysLT 2 R, and PCR was carried out using mouse genomic DNA as template. A single band of 590 bp corresponding to transmembrane domains III-VII was obtained. GSP derived from the fragment's sequence were used to perform both 5Ј-and 3Ј-RACE from mouse heart cDNA. Sequence analysis and assembly of the three products identified a contiguous 1762-bp cDNA. This mouse CysLT 2 R cDNA has an open reading frame of 930 bp, a 620-bp 5Ј-UTR, and a 212-bp 3Ј-UTR.

Identification of Murine CysLT
The deduced protein sequence has 309 amino acids with a calculated molecular mass of 35.3 kDa (Fig. 1A). The sequence identities between human and mouse, and rat and mouse are 65 and 84%, respectively. The deduced protein sequence alignment among the three species is shown in Fig. 1B. Interest- Introns are shown as dark gray lines. The gene is drawn to scale, except intron 3 is truncated as indicated by //. ingly, the mouse and rat sequences are both truncated by 16 and 21 residues at the respective N and C termini. Hydrophobicity analysis reveals that each of the proteins has seven transmembrane-spanning domains and six hydrophilic loops, characteristic of G protein-coupled receptors (not shown).
The mCysLT 2 R gene organization was determined by a series of PCRs using primer sets designed from the cDNA sequence. The gene is composed of six exons and five introns and spans around 20 kb. The coding region is intronless with all introns located in the 5Ј-UTR and each exon-intron junction following the GT-AG rule (Fig. 1C).
A mouse cDNA sequenced from adult male tongue by the RIKEN Mouse Gene Encyclopedia Project (19; GenBank accession AK008997) is identical to our cDNA sequence except that a 57-bp fragment in the middle of the 5Ј-UTR is missing. This missing fragment corresponds to the entire exon 3. Hence two potential transcripts were identified: a long form containing all 6 exons and a short form lacking exon 3 (see below).
Expression and Alternative Splicing Pattern of the mCysLT 2 R Gene in Mouse Tissues-Initial Northern blot analysis of poly(A) RNA from various mouse tissues failed to find a detectable hybridization band indicating low level expression of mCysLT 2 R. A more sensitive ribonuclease protection assay was tested subsequently. By this technique, the highest expression was found in spleen, thymus, and adrenal gland with weaker expression in kidney, brain, and buffy coat (primarily peripheral blood leukocytes). mCysLT 2 R expression was not apparent in lung, liver, heart, aorta, skeletal muscle, uterus, and ovary by this technique (Fig. 2A).
A CysLT 2 R mRNA signal was detected in mouse heart using amplified in situ hybridization. CysLT 2 R mRNA was detected selectively on larger cells concentrated at the apical portions of the ventricle. This pattern is suggestive of expression on conducting Purkinje cells (Fig. 3C) and is consistent with the CysLT 2 R expression seen in human heart (11). A CysLT 2 R signal was also noted on endothelial cells surrounding cardiac vessels (Fig. 3, A and B). No signal was detected using sense control oligonucleotides (Fig. 3D).
RT-PCR was used to delineate the alternative splicing pattern of mCysLT 2 R. A 270-bp fragment representing the short splice variant was amplified in some tissues with a primer set spanning exon 3 from first strand cDNA. However, an expected 327-bp fragment containing exon 3 representing the long transcript failed to appear by this approach. A primer derived from exon 3 in combination with a downstream primer was used to amplify a fragment containing part of exon 3, which was followed by a second PCR using a nested primer within exon 3 and additional nested downstream primer. Only by two rounds of PCR could a 266-bp band containing part of exon 3 be detected (Fig. 2B). Neither the first primer set nor nested primer set alone was able to amplify a specific PCR product (data not shown). The short splice variant is predominant in every tissue expressing mCysLT 2 R, whereas the long transcript expression is trivial compared with the short transcript.
Chromosomal Localization of mCysLT 2 R-The mouse chromosomal location of Cyslt2r was determined by interspecific backcross analysis using progeny derived from matings of ((C57BL/6J ϫ M. spretus) F 1 ϫ C57BL/6J) mice. This interspe- cific backcross mapping panel has been typed for more than 3,200 loci that are well distributed among all of the autosomes as well as the X chromosome (15). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse cDNA Cyslt2r probe. The 8.0-kb BamHI M. spretus RFLP (see "Experimental Procedures") was used to follow the segregation of the Cyslt2r locus in backcross mice. The mapping results indicated that Cyslt2r is located in the distal region of mouse chromosome 14 linked to hr and Dct. Although 162 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 4), up to 166 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are: centromere: hr 3 7/166 3 Cyslt2r 3 37/162 3 Dct. The recombination frequencies (expressed as genetic distances in centiMorgans Ϯ the S.E.) are hr The distal region of mouse chromosome 14 shares regions of homology with human chromosomes 8p and 13q (summarized in Fig. 4), consistent with the assignment of CysLT 2 R to 13q14 in humans.
Intracellular Calcium Mobilization in Response to Cys-LTs-To detect the function of mCysLT 2 R, intracellular calcium mobilization was measured by FLIPR assay upon agonist activation in HEK 293T cells transiently transfected with pcDNA3-mCysLT 2 R vector. The cells responded to LTC 4 , LTD 4 , and LTE 4 with marked, dose-dependent elevations of intracellular Ca 2ϩ (Fig. 5). LTC 4 and LTD 4 were equipotent agonists for this receptor with EC 50 values of 13.4 and 14.4 nM, respectively. LTE 4 behaved as a partial agonist. LTB 4 failed to elicit a significant Ca 2ϩ response at concentrations up to 3 M. A Ca 2ϩ mobilization response to CysLTs using hCysLT 2 R in parallel gave comparable elevations. However, CysLTs seemed to have higher efficacy at the mCysLT 2 R, but the difference was not significant (p Ͼ 0.1). The EC 50 values of these reagents for both mouse and human CysLT 2 R are shown in Table I. pcDNA3 vector-alone transfected cells did not respond to leukotrienes. BAYu9773 could elicit a partial Ca 2ϩ mobilization response by itself. Pretreatment with BAYu9773 decreased the response to LTC 4

and LTD 4 (data not shown).
Radioligand Binding Characterization-Cell membranes from HEK 293 cells transiently transfected with pcDNA3-mCysLT 2 R were used for radioligand binding assays. [ 3 H]LTD 4 bound specifically to these membranes. Specific binding represented ϳ60% of the total binding, whereas membranes from vector-alone transfected cells showed no specific binding (data not shown). Competition assays with CysLTs, LTB 4 , CysLT 1 Rspecific antagonist MK-571, and partial agonist/antagonist BAYu9773 were performed in three separate experiments with three different membrane preparations (Fig. 6). The rank order potency of leukotriene agonists to compete with [ 3 H]LTD 4 (0.5 nM) binding was LTC 4 ϭ LTD 4 Ͼ BAYu9773 Ͼ LTE 4 with IC 50 values of 8.5, 17.6, 325, and 1,985 nM, respectively. LTB 4 and MK-571 did not compete with [ 3 H]LTD 4 even at 10 M.

DISCUSSION
In this report we have described the cloning and functional characterization of the mouse CysLT 2 receptor. Contrary to previous data that implicate a nonfunctional rat CysLT 2 R (11), our functional experiments with mCysLT 2 R in parallel with the human and rat homologs revealed that all three CysLT 2 Rs had similar responses to CysLTs in raising intracellular calcium levels ( Fig. 5 and data not shown).
Both mouse and rat CysLT 2 R are truncated by 16 and 21 residues at the respective N and C termini relative to the human sequence. However, this truncation is not significant in terms of ligand binding and intracellular signaling because the binding affinity of CysLTs to mCysLT 2 R is similar to hCysLT 2 R, and the ligand-induced intracellular Ca 2ϩ mobilization is similar in both intensity and EC 50 values in each of the human, rat, and mouse CysLT 2 R-transfected cell preparations. The significance of the extra sequences in hCysLT 2 R merits further investigation.
We report the first determination of a CysLT 2 R gene structure. It is composed of 6 exons and 5 introns with all introns located in the 5Ј-UTR and the coding region intronless like many other G protein-coupled receptor genes. We identified two splice variants for mCysLT 2 R mRNA. The 57-bp exon 3 is spliced out from the short transcript. Even though the long transcript was isolated during the initial cloning step from heart cDNA, later RT-PCR results revealed that the short transcript was the predominant form in every tissue where the mCysLT 2 R gene was expressed. Thus, PCR using a primer set spanning exon 3 could only amplify the short transcript because of the relative rareness of the long transcript. The mCysLT 1 R gene also has long and short splice variants (8,9) resulting in two mCysLT 1 receptors that differ at the N terminus. However, because the alternative splicing of mCysLT 2 R occurs in the 5Ј-UTR region there should be no difference in protein structure. The significance of the alternative splicing remains to be clarified. The long transcript expression pattern is different from the short form, indicating that exon 3 might play some role in transcriptional or translational regulation.
Previously, CysLT 2 R was described pharmacologically in guinea pig trachea and ileum, ferret trachea and spleen, sheep bronchus, and pulmonary arteries and veins and vasculature of various species (4, 20 -23). hCysLT 2 R mRNA was identified mainly in heart, spleen, adrenal gland, and various regions of the brain. Despite the identification of CysLT 2 R expression sites, its function is not well defined. We cloned the mCysLT 2 R cDNA from mouse heart, but this tissue exhibits very low overall expression as detected by Northern blotting, ribonucle-ase protection assay, and RT-PCR. These results are consistent with mCysLT 2 R expression limited to Purkinje and endothelial cells by tyramide-enhanced in situ hybridization. CysLTs were shown to reduce coronary blood flow and myocardial contractility in guinea pig and rat (24). Human CysLT 1 R was cloned from vascular endothelial cells, and LTD 4 -induced vascular leakage was shown in mice (10). The identification of CysLT receptors in both human and mouse Purkinje and endothelial cells indicates multifunctional roles for these receptors in modulating cardiovascular function.
Consistent with hCysLT 2 R distribution, we found the relative highest mCysLT 2 R expression in spleen and adrenal gland. Thymus also has high expression, and kidney, brain, and buffy coat leukocytes show weaker evidence of expression. CysLT 2 R may also mediate pulmonary vessel relaxation possibly through nitric oxide release (22). CysLT 2 R might also take part in the neuroendocrine regulation of hormones (25) and identification of its expression in many brain regions and adrenal gland (Fig. 2; 11) might broaden our current perspective  of CysLTs as inflammatory mediators toward other important physiological roles. CysLTs were able to elicit intracellular Ca 2ϩ mobilization in CysLT 2 R-transfected HEK 293T cells in a dose-dependent manner, indicating that CysLT 2 R couples to the G q/11 family as does CysLT 1 R. Coupling of CysLT 2 R to other members in this family was also tested, but only G q signaled effectively (data not shown). Pertussis toxin did not affect the CysLT-induced Ca 2ϩ response in CysLT 1 R-transfected cells (7). However, it could partially block the response in U937 cells (26), which might only express CysLT 1 R because the binding of CysLTs could be totally inhibited by CysLT 1 R-specific antagonists (18), indicating the potential for differential CysLT-mediated G protein coupling. Whether CysLT 2 R might also couple to the G i/o class in certain tissues in unknown. mCysLT 2 R binds CysLTs in a rank order of affinity LTC 4 ϭ LTD 4 Ͼ Ͼ LTE 4 , as does hCysLT 2 R. BAYu9773 works at this receptor as both antagonist and partial agonist. Pretreatment with BAYu9773 partially abolished cellular reaction to LTC 4 and LTD 4 , indicating desensitization of the receptor. With cloned CysLT 2 receptors available, it should be feasible to develop new pharmaceutical agents specific to CysLT 2 R.
In summary, we identified mCysLT 2 R as a functional CysLT receptor that is derived from 5Ј-UTR alternative transcripts, and which functions similar to hCysLT 2 R. The establishment of a CysLT 2 R-deficient mouse model would potentially yield new insights into biological and pathophysiological roles of this receptor.