From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, March 14, 2003 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has been demonstrated that cell-surface G protein-coupled receptors mediate the cellular effects of LPA. At least three types of G protein-coupled receptors, Edg-2/LPA1/vzg-1 (7), Edg-4/LPA2 (8), and Edg-7/LPA3 (9), which belong to the Edg (endothelial cell differentiation gene) family, have been identified as specific receptors for LPA. These three G protein-coupled receptors share 5057% amino acid identities. Several experiments have demonstrated that they can mediate adenylyl cyclase inhibition, mitogen-activated protein kinase activation, phospholipase C activation, and Ca2+ mobilization through pertussis toxin-sensitive (Gi/o) and -insensitive G proteins (G12/13 and Gq/11/14) (2, 3). Edg-4/LPA2 and Edg-7/LPA3 have also been shown to activate adenylyl cyclase when they were overexpressed in Sf9 insect cells (9). However, the existence of one or more additional LPA receptors has been implied from the analysis of Edg-2/LPA1(/) Edg-4/LPA2(/) double knockout mice (10) and various pharmacological studies (1113).
During a "de-orphaning" project of G protein-coupled receptors, we found that p2y9/GPR23 responded to LPA. p2y9/GPR23 specifically bound to LPA and mediated LPA-induced adenylyl cyclase stimulation and intracellular Ca2+ mobilization. Although p2y9/GPR23 shares only 2024% amino acid identities with Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3, and the phylogenetic analysis also shows that p2y9/GPR23 is distant from the Edg family (Fig. 1), our results consistently indicate that p2y9/GPR23 is the fourth LPA receptor (LPA4).
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EXPERIMENTAL PROCEDURES |
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Construction of the Phylogenetic TreePeptide sequences of selected G protein-coupled receptors were obtained from GenBankTM and SwissProt. The phylogenetic tree was generated from peptide sequences of selected G protein-coupled receptors, using the all-against-all matching method (available at cbrg.inf.ethz.ch/Server/AllAll.html). The tree was constructed on the basis of point-accepted mutation distances between each pair of sequences estimated by the dynamic programming algorithm.
Cloning of p2y9/GPR23The tBLASTn program was used to search the data base of GenBankTM for orphan G protein-coupled receptors sharing high identities with the human PAF receptor (14). A DNA fragment containing the entire open reading frame of p2y9/GPR23 (GenBankTM accession number NM_005296 [GenBank] ) was first amplified from human genomic DNA by PCR using KOD-Plus (Toyobo, Osaka, Japan) and oligonucleotides (sense primer, 5'-GTCCATAGTGTCAGAGTGGTGAAC-3'; antisense primer, 5'-CATATCTGGACCTGAACACATTTC-3'). The entire open reading frame of p2y9/GPR23 with an additional sequence of hemagglutin (HA)-epitope at the 5'-end was subsequently amplified from the resultant PCR products using KOD-Plus and oligonucleotides (sense primer containing KpnI and HA tag sequences, 5'-GGGGTACCGCCATGTACCCCTACGACGTGCCCGACTACGCCGGTGACAGAAGATTCATT-3'; antisense primer containing XbaI sequence, 5'-GCTCTAGACTAAAAGGTGGATTCTAG-3'). The resultant DNA fragment was digested with KpnI and XbaI and subsequently cloned into the mammalian expression vector pCXN2.1, a slightly modified version of pCXN2 (15) with multiple cloning sites, between KpnI and NheI sites.
Binding AssayRH7777 cells and B103 cells were cultured on collagen-coated dishes in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (Cambrex Co., Walkersville, MD), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Roche Applied Science). Cells were transfected with p2y9/GPR23-pCXN2.1 or empty vector using LipofectAMINE 2000 reagent (Invitrogen). After 24 h of transfection, cells were washed with phosphate-buffered saline three times and serum-starved for 24 h in DMEM supplemented with 0.1% BSA. The cells were washed again with phosphate-buffered saline twice and scraped off. After further washing with binding buffer (25 mM HEPES-NaOH (pH 7.4), 10 mM MgCl2, and 0.25 M sucrose), the cells were suspended in the buffer with additional 20 µM 4-amidinophenylmethylsulfonyl fluoride (Sigma) and a protease inhibitor mixture (Complete, Roche Applied Science), sonicated three times at 15 watts for 30 s, and centrifuged at 800 x g for 10 min at 4 °C. The supernatant was further centrifuged at 105 x g for 60 min at 4 °C, and the resultant pellet was homogenized in ice-cold binding buffer. Binding assays were performed in 96-well plates in triplicates. For Scatchard analysis, 40 µg of the membrane fractions were incubated in binding buffer containing 0.25% BSA with various concentrations of [3H]LPA for 60 min at 4 °C. The bound [3H]LPA was collected onto a Unifilter-96-GF/C (PerkinElmer Life Sciences) using a MicroMate 196 harvester (Packard, Wellesley, MA). The filter was then rinsed ten times with binding buffer containing 0.25% BSA and dried for 2 h at 50 °C. 25 µl of MicroScint-0 scintillation mixture (PerkinElmer Life Sciences) was added per well. The radioactivity that remained on the filter was measured with a TopCount microplate scintillation counter (Packard). Total and nonspecific bindings were evaluated in the absence and presence of 10 µM unlabeled LPA, respectively. The specific binding value (dpm) was calculated by subtracting the nonspecific binding value (dpm) from the total binding value (dpm). For competition assay with related lipids, 20 µg of the membrane fractions were incubated with 5 nM [3H]LPA in the absence or presence of 1 µM of unlabeled 18:1-LPA, 18:1-LPC, 18:1-LPE, 18:1-LPS, 18:1-LPG, 18:1-PA, PAF, S1P, or SPC. For competition assay with structural analogs of LPA, 10 µg of the membrane fractions was incubated with increasing concentrations of unlabeled 18:1-, 18:0-, 1-alkyl-, and 1-alkenyl-LPA in the presence of 2.5 nM [3H]LPA. Before conducting the binding assays, the cell surface expression of p2y9/GPR23 was confirmed by flow cytometric analysis (Epics XL, Beckman Coulter, Fullerton, CA) with anti-HA rat IgG (3F10, Roche Applied Science) and phycoerythrin-labeled anti-rat IgG (Beckman Coulter) as the second antibody.
Reporter Gene AssayPC-12 cells were cultured in DMEM supplemented with 10% horse serum and 5% fetal bovine serum. 2 x 105 cells were transfected with 450 ng of p2y9/GPR23-pCXN2.1 or empty vector, 530 ng of zif 268-firefly luciferase-pGL2 (a kind gift from Dr. T. Naito, Japan Tobacco, Tokyo, Japan), and 20 ng of CMV-Renilla luciferase-pRL (Promega, Madison, WI) using SuperFect (Qiagen, Hiden, Germany), and cultured on collagen-coated 24-well plates for 48 h. Cells were washed three times with DMEM supplemented with 0.1% BSA and cultured in the serum-free DMEM for 12 h, and stimulated with various concentrations of LPA analogs. Firefly and Renilla luciferase activities were measured using PICAGENE Dual Seapansy (Toyo Ink, Tokyo, Japan) and a MiniLumat LB 9506 luminometer (Berthold, Bundoora, Australia). Firefly luciferase values were standardized to Renilla ones.
Ca2+ MeasurementChinese hamster ovary (CHO) cells were cultured in Ham's F-12 (Sigma) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were transfected with p2y9/GPR23-pCXN2.1 or empty vector using LipofectAMINE 2000 reagent. Stably transfected clones were selected with 2 mg/ml G418 (Invitrogen, Carlsbad, CA) and maintained with 0.3 mg/ml G418. Cell surface expression of p2y9/GPR23 was detected by flow cytometric analysis as described above (see Fig. 4A). For the ligand screening assay, clones highly expressing p2y9/GPR23 were seeded in 96-well plates at a density of 4 x 104 cells/well and cultured overnight. They were loaded with 4 µM Fluo-3 AM (Dojindo, Kumamoto, Japan) in HEPES-HBSS buffer (1 x Hanks' balanced salt solution (HBSS) containing 20 mM HEPES-NaOH (pH 7.4), 1 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, and 2.5 mM probenecid) and 0.04% pluronic acid for 1 h at 37 °C, washed twice, and filled with HEPES-HBSS buffer. They were then stimulated with 198 lipids contained in the Bioactive Lipid Library and 17 nucleotides (described under "Materials and Cells") individually, and intracellular Ca2+ mobilization was monitored with a scanning fluorometer (FLEXstation, Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. For examination of dose dependence, CHO cell clones highly expressing p2y9/GPR23 or mock-transfected cells were washed with phosphate-buffered saline three times, serum-starved for 24 h in Ham's F-12 supplemented with 0.1% BSA, washed twice again with phosphate-buffered saline, and then harvested. The harvested cells were further washed with HEPES-Tyrode's buffer (25 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 0.49 mM MgCl2, 12 mM NaHCO3, 0.37 mM NaH2PO4, and 5.6 mM D-glucose), and loaded with 3 µM Fura-2 AM (Dojindo) in HEPES-Tyrode's buffer containing 0.1% BSA (HEPES-Tyrode's BSA buffer) for 1 h at 37 °C. Cells were washed twice and resuspended in HEPES-Tyrode's BSA buffer at a density of 2 x 106 cells/ml. 0.5 ml of the cell suspension was applied to a CAF-100 spectrofluorometer (Jasco, Tokyo, Japan), and 5 µl of various concentrations of 18:1-LPA in HEPES-Tyrode's BSA buffer was added. Intracellular Ca2+ concentration ([Ca2+]i) was measured by the ratio of emission fluorescence of 500 nm by excitations at 340 and 380 nm.
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cAMP AssayCHO cell clones stably expressing p2y9/GPR23 were used to measure cAMP levels. After 24 h of serum starvation, cells were harvested and suspended in HBSS containing 0.1% BSA and 0.5 mM isobutylmethylxanthine. The density was 5 x 106 or 5 x 107 cells/ml for assay in the presence or absence of forskolin, respectively. 20 µl of the cell suspension was applied to 96-well plates and incubated for 20 min at room temperature. The reaction was initiated by adding 10 µl of ligand solution of 18:1-LPA in HBSS-BSA buffer with or without 5 µM forskolin. After 30 min of incubation at room temperature, the reaction was terminated by adding 10 µl of HBSS-BSA buffer containing 4% Tween 20. After centrifugation at 800 x g for 5 min, cAMP contents in the supernatant were measured by a Fusion system (Packard) using an AlphaScreen cAMP assay kit (PerkinElmer Life Sciences). Pretreatment with pertussis toxin (List Biological Laboratories, Campbell, CA) was for 12 h at a concentration of 100 ng/ml. Results were expressed as -fold increases over respective controls.
Quantitative Real-time PCRHuman first strand cDNAs from 16
tissues were purchased from Clontech (Human MTC Panel I and II), whose
concentrations were normalized to the mRNA expression levels of four different
housekeeping genes (-tubulin,
-actin, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and PLA2). The cDNA levels of GAPDH and
p2y9/GPR23 were quantified using a LightCycler apparatus (Roche
Applied Science). The PCR reactions were set up in microcapillary tubes in a
volume of 20 µl, consisting of 2 µl of cDNA solution, 1x FastStart
DNA Master SYBR Green I (Roche Applied Science), 0.5 µM each
sense and antisense primers and 3 mM MgCl2. The PCR
program for GAPDH was as follows; denaturation at 95 °C for 3 min and 50
cycles of amplification consisting of denaturation at 95 °C for 0 s,
annealing at 60 °C for 5 s, and extension at 72 °C for 40 s. The PCR
program for p2y9/GPR23 was as follows: denaturation at 95 °C
for 5 min and 50 cycles of amplification consisting of denaturation at 95
°C for 15 s, annealing at 60 °C for 5 s, and extension at 72 °C
for 6 s. For GAPDH, a human GAPDH control Amplimer Set (Clontech), designed to
amplify a 983-bp fragment was used as primers; sense primer:
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' and antisense primer:
5'-CATGTGGGCCATGAGGTCCACCAC-3'. The primers for
p2y9/GPR23 were designed to amplify a 139-bp fragment; sense
primer: 5'-AAAGATCATGTACCCAATCACCTT-3' and antisense primer:
5'-CTTAAACAGGGACTCCATTCTGAT-3'. The PCR products were detected by
measuring the fluorescence of SYBR Green I, which selectively bound to
double-stranded DNA and emitted the greatly enhanced fluorescence. The cDNA
level of each sample was quantified by Fit Points Method in LightCycler
analysis software. The control cDNA contained in Clontech human MTC Panels and
a linearized p2y9/GPR23-pCXN2.1 were used as standards for GAPDH
and p2y9/GPR23, respectively. In both cases, the quality of PCR
products was assessed by monitoring a fusion step.
Northern HybridizationHuman poly(A)+ RNA samples
from kidney and skeletal muscle were purchased from Clontech. Total RNA of
human megakaryoblastic MEG-01 cells was extracted with Absolutely RNA RT-PCR
Miniprep kit (Stratagene, La Jolla, CA). Poly(A)+ RNA was isolated
from 200 µg of the total RNA using µMACS mRNA isolation kit (Miltenyi
Biotec, Bergisch Gladbach, Germany). For Northern analysis, 2.5 µg of
poly(A)+ RNA was hybridized with [32P]dCTP-labeled
probes of human p2y9/GPR23 and human -actin, as described
previously (16). The washed
membrane was subjected to autoradiography for 4 days (p2y9/GPR23)
or 3 h (
-actin).
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RESULTS |
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Membrane Binding AssayTo characterize the binding of LPA to p2y9/GPR23, we conducted membrane binding assays using [3H]18:1-LPA. RH7777 rat hepatoma cells transiently transfected with p2y9/GPR23 were used, because RH7777 cells were not responsive to LPA (19). As expected, membrane fractions of mock-transfected RH7777 cells displayed low background binding of [3H]LPA (Fig. 2A). [3H]LPA binding to membrane fractions of RH7777 cells was greatly enhanced by p2y9/GPR23 transfection (Fig. 2A). Reverse transcriptase-PCR analyses showed no induction of endogenous receptors (Edg-2/LPA1, Edg-4/LPA2, or Edg-7/LPA3)byp2y9/GPR23 transfection (data not shown). Scatchard analysis indicated a dissociation constant (Kd) of 44.8 nM and a maximum binding capacity (Bmax) of 37.2 pmol/mg of protein (Fig. 2B). Competition analyses revealed that only 18:1-LPA, but not 18:1-LPC, 18:1-LPE, 18:1-LPS, 18:1-LPG, 18:1-PA, PAF, S1P, or SPC competed for [3H]LPA binding to the membrane fractions of p2y9/GPR23-expressing RH7777 cells (Fig. 2C). 18:1- and 18:0-LPA competed for [3H]LPA binding to the membrane fractions of p2y9/GPR23-expressing RH7777 cells with Ki values of 80.0 and 282 nM, respectively (Fig. 2D). 1-Alkyl- and 1-alkenyl-LPA also competed for [3H]LPA binding but rather weakly (Fig. 2D). Because B103 rat neuroblastoma cells were also reported to lack responsiveness to LPA (19), binding assays using B103 cells were performed in the same manner as RH7777 cells. The specific binding activities of p2y9/GPR23-transfected B103 cells were 289, 859, and 2975 dpm/50 µg of protein at 1, 3, and 10 nM [3H]LPA, respectively. Based on the specific activity of [3H]LPA, 2975 dpm/50 µg of protein corresponds to 0.47 pmol/mg of protein. The binding activities of the mock-transfected B103 cells were 79, 197, and 238 dpm/50 µg of protein at 1, 3, and 10 nM [3H]LPA, respectively.
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Reporter Gene AssayWe next performed reporter gene assays to examine the ligand preference of p2y9/GPR23. The transfected PC-12 cells responded to all LPA analogs with a significant increase in the luciferase activity in a dose-dependent manner; 18:1-LPA evoked the highest activity, followed by 18:0-, 16:0-, and 14:0-derivatives (Fig. 3). Compared with 1-acyl-LPAs, 1-alkyl-, and 1-alkenyl-LPA exhibited weaker activities. Mock-transfected PC-12 cells showed no responses to any analogs of LPA (data not shown).
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Effect on Intracellular Ca2+
MobilizationTo characterize the intracellular signals of
p2y9/GPR23, the effect of LPA on intracellular
Ca2+ mobilization was examined in detail using a CAF-100
spectrofluorometer. We established four independent clones of CHO cells stably
expressing p2y9/GPR23 and a polyclonal population of
mock-transfected CHO cells. Cell surface expression of p2y9/GPR23
was confirmed by flow cytometric analysis
(Fig. 4A). The other
three clones showed similar expression patterns. Either in
p2y9/GPR23-expressing or mock-transfected CHO cells, 18:1-LPA
induced an increase in [Ca2+]i in a
dose-dependent manner (Fig.
4B). Although mock-transfected CHO cells displayed a
significant increase in [Ca2+]i, the
increase in [Ca2+]i was enhanced
2-fold by the stable expression of p2y9/GPR23. Similar results
were obtained in all four different clones. The effects of 18:1-LPA were
reproducibly observed, using different batches of the LPA from two companies
(Cayman and Avanti). In B103 cells transiently expressing
p2y9/GPR23, 18:1-LPA at concentrations of 1 and 10 nM
evoked increases in [Ca2+]i by 38
± 5 and 49 ± 5nM (mean ± S.D.; n =
3), respectively. Mock-transfected B103 cells showed no response at 10
nM 18:1-LPA. On the other hand, RH7777 cells were unresponsive to
18:1-LPA both in mock-transfected and in p2y9/GPR23-expressing form
(data not shown).
Effect on cAMP Formation18:1-LPA induced an increase in cAMP levels in p2y9/GPR23-expressing CHO cells either in the absence or presence of 5 µM forskolin (Fig. 5, A and B), and pretreatment of the cells with pertussis toxin further increased the cAMP levels (Fig. 5, A and B). In mock-transfected CHO cells, LPA induced no change or a decrease in cAMP levels in the absence or presence of forskolin, respectively (Fig. 5, A and B), and pretreatment of the cells with pertussis toxin attenuated an LPA-induced decrease in cAMP levels (Fig. 5B).
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Tissue DistributionTo explore the physiological function of p2y9/GPR23 in vivo, it is important to know the tissue distribution of the receptor. By using cDNAs prepared from 16 human tissues as templates, quantitative real-time PCR was performed to estimate the mRNA expression levels. In a set of samples, ovary showed the highest expression of p2y9/GPR23 mRNA, whereas other tissues showed only weak expressions (Fig. 6A). Northern hybridization of human poly(A)+ RNA from kidney, skeletal muscle, and megakaryoblastic MEG-01 cells (20) detected a transcript of about 4.4 kb (Fig. 6C).
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DISCUSSION |
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p2y9/GPR23 was identified as a novel G protein-coupled receptor from an analysis of the expressed sequence tag (EST) data base, and the complete clone was isolated from human genomic DNA (25, 26). The p2y9/GPR23 gene is located on chromosome X, region q13-q21.1, and contains an intronless open reading frame of 1113 bp encoding 370 amino acids (25, 26). However, information is limited regarding the specific ligands, tissue distribution, and biological functions of this orphan receptor.
Membrane fractions of RH7777 cells transiently transfected with
p2y9/GPR23 had a specific binding activity for 18:1-LPA with a
Kd value of 44.8 nM
(Fig. 2, A and
B). Competition assays displayed the binding affinity
with a rank order of 18:1- > 18:0- > 1-alkyl- > 1-alkenyl-LPA
(Fig. 2D). This order
was consistent with that of the luciferase activity: 18:1- > 18:0- >
16:0- > 14:0- > 1-alkyl- > 1-alkenyl-LPA
(Fig. 3). These results
indicate that 1-acyl-LPA is a better ligand for p2y9/GPR23 than
1-alkyl- or 1-alkenyl-LPA, like the previously identified LPA receptors
(Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3)
(27,
28). Furthermore, Im et
al. (29) showed that,
among 1-acyl-LPAs, 18:1-LPA was more active than 18:0-, 16:0-, and 14:0-LPA in
[-35S]GTP binding assay with Edg-4/LPA2 and
Edg-7/LPA3.
Mock-transfected CHO cells displayed an increase in
[Ca2+]i
(Fig. 4, B and
C), possibly due to the presence of endogenous LPA
receptors. Despite this background response of CHO cells, the stable
expression of p2y9/GPR23 significantly enhanced the LPA-induced
Ca2+ response by 2-fold
(Fig. 4B) in four
independent clones. These results strongly suggest that p2y9/GPR23
could elicit an intracellular Ca2+ mobilization, a well
documented cellular effect of LPA
(27).
In mock-transfected CHO cells, LPA induced a decrease in cAMP levels in the presence of forskolin, which was inhibited by pretreatment of the cells with pertussis toxin (Fig. 5B), suggesting the existence of endogenous LPA receptors coupling with pertussis toxin-sensitive G protein (Gi/o). By contrast, LPA induced an increase in cAMP levels in p2y9/GPR23-expressing CHO cells, and pretreatment of the cells with pertussis toxin further potentiated the LPA-induced cAMP accumulation (Fig. 5, A and B). It is, therefore, possible that p2y9/GPR23 is coupled with Gs, and that the effect of LPA on p2y9/GPR23 is unmasked by blocking the pertussis toxin-sensitive signals from endogenous LPA receptors in CHO cells. Muscarinic M2 and somatostatin sst5 receptors are coupled with Gi, inhibiting adenylyl cyclase in CHO cells. However, at higher agonist concentrations, these receptors can also mediate activation of adenylyl cyclase by a mechanism involving Gs activation (30, 31). Conversely, at lower concentrations of LPA, p2y9/GPR23 might inhibit the production of cAMP via Gi, like Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3 (2, 3).
We also found that forskolin facilitated LPA-induced cAMP accumulation in p2y9/GPR23-expressing CHO cells (Fig. 5, A and B). At least ten types of mammalian adenylyl cyclase are at present identified (32). All types of adenylyl cyclase are activated by Gs and by forskolin, and some types of adenylyl cyclase (type II, IV, V, and VI) are synergistically activated in the presence of both Gs and forskolin (32). One possible explanation of our results is that the latter types of adenylyl cyclase might be involved in the LPA-induced cAMP accumulation in p2y9/GPR23-expressing CHO cells. Indeed, type VI and type VII adenylyl cyclases are expressed in CHO cells (33).
The mRNA levels of p2y9/GPR23 were significantly high in ovary (Fig. 6). Various species of LPA such as linoleic, arachidonic, and docosahexaenoic acids were detected from ascites of ovarian cancer patients (24), and they had many effects on the ovarian cancer progression such as cell proliferation, prevention of apoptosis, resistance to cisplatin, and production of vascular endothelial growth factor (34). Consistently, a prominent expression of Edg-4/LPA2 has been shown in primary cultures and established lines of ovarian cancer cells (35). LPA was also found at relatively high concentrations in human ovarian follicular fluid from healthy subjects (36), suggesting the relevance of LPA for normal ovarian functions as well. Tokumura (37) recently described in his review article that LPA increased the intracellular cAMP level in mouse cumulus cells. This phenomenon is consistent with our findings that the activation of p2y9/GPR23 evoked cAMP accumulation in CHO cells. Thus, p2y9/GPR23 might explain some of the pathological and physiological roles of LPA in ovary. It remains to be determined whether the expression of p2y9/GPR23 is modulated in ovarian cancer cells. Although the EST cDNA encoding p2y9/GPR23 was originally isolated from human brain (25, 26), high expression of p2y9/GPR23 was not detected in brain in our study. It is possible that specific types of cells in restricted areas express p2y9/GPR23, which will be examined by in situ hybridization in the near future.
Interestingly, p2y9/GPR23 shares only 2024% amino acid identities with Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3. Phylogenetic analysis also shows that p2y9/GPR23 is far distant from the Edg family (Fig. 1). These facts suggest that p2y9/GPR23 has evolved from ancestor sequences that are different from those of the Edg family. There are several examples of structurally unrelated receptors recognizing the same ligand. Prostaglandin D2 binds to DP and CRTH2 (38) and histamine has four structurally distant receptors, H1H4 (39). In addition, some neurotransmitters and nucleotides have both metabotropic (G protein-coupled) and ionotropic (ion channel) receptors. These examples show a limitation of the ligand search strategy utilizing a structural similarity of receptor.
As described above, there are some reports implying the existence of additional receptors for LPA. It is possible that LPA-induced responses in embryonic fibroblast cells of Edg-2/LPA1(/) Edg-4/LPA2(/) double knockout mice (10) might be mediated by p2y9/GPR23, although we do not have any direct evidence. However, a mitogenic response to LPA in RH7777 cells (12) might be due to the activity of intracellular receptors (40), rather than G protein-coupled receptor, because mock-transfected RH7777 cells exhibited no significant binding to [3H]LPA (Fig. 2A). As to the putative LPA receptor in platelets (11, 13), there may be a receptor other than p2y9/GPR23, because the ligand preference of platelets to 1-alkyl-LPAs is not consistent with that of p2y9/GPR23. Existence of further unidentified LPA receptors is, therefore, expected.
In conclusion, we report here the identification of p2y9/GPR23 as a novel fourth LPA receptor (LPA4). Cells expressing p2y9/GPR23 displayed intracellular Ca2+ mobilization, cAMP accumulation, and luciferase activation. The Kd value of p2y9/GPR23 (45 nM) was equivalent to those of Edg-4/LPA2 (73.6 nM) and Edg-7/LPA3 (206 nM) (9). Although p2y9/GPR23 mRNA was significantly detected in ovary, its biological functions in vivo remain to be determined. Nevertheless, the present findings introduce a further complexity for LPA and its receptors. In addition, our study shows a limitation of the "de-orphaning" strategy based on the receptor structure.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax:
81-3-3813-8732; E-mail:
mame{at}m.u-tokyo.ac.jp.
1 The abbreviations used are: LPA, lysophosphatidic acid; LPC,
lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPS,
lysophosphatidylserine; PLA2, phospholipase A2; PA,
phosphatidic acid; PC, phosphatidylcholine; 14:0, 1-myristoyl; 16:0,
1-palmitoyl; 18:0, 1-stearoyl; 18:1, 1-oleoyl; LPG, lysophosphatidylglycerol;
PAF, platelet-activating factor; S1P, sphingosine 1-phosphate; SPC,
sphingosylphosphorylcholine; BSA, bovine serum albumin; DMEM, Dulbecco's
modified Eagle's medium; HA, hemagglutinin; CHO, Chinese hamster ovary; HBSS,
Hanks' balanced salt solution;
[Ca2+]i, intracellular
Ca2+ concentration; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; EST, expressed sequence tag.
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ACKNOWLEDGMENTS |
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REFERENCES |
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