Identification of p2y9/GPR23 as a Novel G Protein-coupled Receptor for Lysophosphatidic Acid, Structurally Distant from the Edg Family*

Kyoko Noguchi, Satoshi Ishii {ddagger} and Takao Shimizu

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA) is a bioactive lipid mediator with diverse physiological and pathological actions on many types of cells. LPA has been widely considered to elicit its biological functions through three types of G protein-coupled receptors, Edg-2 (endothelial cell differentiation gene-2)/LPA1/vzg-1 (ventricular zone gene-1), Edg-4/LPA2, and Edg-7/LPA3. We identified an orphan G protein-coupled receptor, p2y9/GPR23, as the fourth LPA receptor (LPA4). Membrane fractions of RH7777 cells transiently expressing p2y9/GPR23 displayed a specific binding for 1-oleoyl-LPA with a Kd value of around 45 nM. Competition binding and reporter gene assays showed that p2y9/GPR23 preferred structural analogs of LPA with a rank order of 1-oleoyl- > 1-stearoyl- > 1-palmitoyl- > 1-myristoyl- > 1-alkyl- > 1-alkenyl-LPA. In Chinese hamster ovary cells expressing p2y9/GPR23, 1-oleoyl-LPA induced an increase in intracellular Ca2+ concentration and stimulated adenylyl cyclase activity. Quantitative real-time PCR demonstrated that mRNA of p2y9/GPR23 was significantly abundant in ovary compared with other tissues. Interestingly, p2y9/GPR23 shares only 20–24% amino acid identities with Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3, and phylogenetic analysis also shows that p2y9/GPR23 is far distant from the Edg family. These facts suggest that p2y9/GPR23 has evolved from different ancestor sequences from the Edg family.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycero-3-phosphate)1 is a bioactive phospholipid with diverse physiological actions on many cell types (1, 2). LPA induces mitogenic and/or morphological effects on the cells and has been proposed to be involved in biologically important processes, including neurogenesis, myelination, angiogenesis, wound healing, and cancer progression (1, 3). LPA is present in serum at micromolar concentrations (4). LPA is generated mainly by two different pathways; 1) generation of lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS) from membrane phospholipids by phospholipase A2 (PLA2) or phospholipase A1, followed by conversion of these lysophospholipids to LPA by lysophospholipase D (5) and 2) generation of phosphatidic acid (PA) from phosphatidylcholine (PC) by phospholipase D, followed by conversion of PA to LPA by specific classes of PLA2 (6).

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 50–57% 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 20–24% 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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FIG. 1.
Phylogenetic tree constructed for p2y9/GPR23 and selected human G protein-coupled receptors. Values show branch lengths that represent the evolutionary distance between each pair of sequences. The sequence divergence is equal to the sum of each value of branch length. A small dot indicates the weighted centroid of the tree.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Cells—1-Myristoyl (14:0), -palmitoyl (16:0), -stearoyl (18:0), and -oleoyl (18:1)-LPAs, 18:1-LPC, 18:1-LPE, 18:1-lysophosphatidylglycerol (LPG), and 18:1-LPS were purchased from Avanti Polar Lipids (Alabaster, AL). 18:1-LPA was also purchased from Cayman Chemical (Ann Arbor, MI). 1-Alkyl- and 1-alkenyl-LPAs were kind gifts from Dr. R. Taguchi (Nagoya City University, Japan), which were prepared from bovine heart lyso-platelet-activating factor (lyso-PAF) and lyso-plasmalogen, respectively, by using phospholipase D. 18:1-PA and 17 nucleotides (ADP-glucose, ATP, ADP, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, CTP, GDP-fucose, GDP-mannose, GDP, UDP, UDP-N-acetylglucosamine, UDP-galactose, UDP-glucose, UDP-N-acetylgalactosamine, UTP, UDP-glucuronic acid, and GTP) were from Sigma (St. Louis, MO). Sphingosine 1-phosphate (S1P) and the Bioactive Lipid Library were from Biomol Research Laboratories (Plymouth Meeting, PA). Sphingosylphosphorylcholine (SPC) and 1-O-hexadecyl-PAF were from Cayman Chemical. [3H]LPA (1-oleoyl[oleoyl-9,10-3H(N)]LPA, 57 Ci/mmol) was from PerkinElmer Life Sciences (Boston, MA). Bovine serum albumin (BSA) from Serologicals Proteins (Kankakee, IL) was of fatty acid-free and very low endotoxin grade. Other chemical reagents were of analytical grade. RH7777 rat hepatoma cells and B103 rat neuroblastoma cells were kindly provided from Dr. J. Chun (University of California-San Diego, La Jolla, CA). Human megakaryoblastic MEG-01 cells were purchased from the Health Science Research Resources Bank (Osaka, Japan).

Construction of the Phylogenetic Tree—Peptide 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/GPR23—The 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 Assay—RH7777 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 Assay—PC-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+ Measurement—Chinese 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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FIG. 4.
LPA-induced increase in [Ca2+]i in CHO cells. A, cell surface expression of p2y9/GPR23. In a clonal population of CHO cells stably expressing p2y9/GPR23 tagged with HA at N terminus (black area), the higher intensity of fluorescence was detected by flow cytometric analysis than in mock-transfected CHO cells (gray area). This is a representative result of four independent clones, which gave essentially identical patterns. B, dose-response curves. A clonal population of CHO cells stably expressing p2y9/GPR23 was loaded with 3 µM Fura-2 AM, and stimulated with increasing concentrations of 18:1-LPA ({blacksquare}). The results of mock-transfected CHO cells are also shown as a negative control ({square}). Data are means ± S.D. (n = 3) of a representative of four different stable clones. C, representative traces of Ca2+ responses. A clonal population of CHO cells stably expressing p2y9/GPR23 was stimulated with 100 nM 18:1-LPA (right). A result of mock-transfected CHO cells is also shown as a negative control (left).

 

cAMP Assay—CHO 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 PCR—Human 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 ({alpha}-tubulin, {beta}-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 Hybridization—Human 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 {beta}-actin, as described previously (16). The washed membrane was subjected to autoradiography for 4 days (p2y9/GPR23) or 3 h ({beta}-actin).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Screening of Candidate Ligands—Phylogenetic analysis showed that p2y9/GPR23 was most closely related to the orphan receptor p2y5 and relatively close to the functional receptors for nucleotides (P2Y1, P2Y4, and P2Y6) (17) and for lipids (G2A, GPR4, GPR65/TDAG8, GPR68/OGR1, PAF receptor, CysLT1, and CysLT2) (18) (Fig. 1). It was, therefore, supposed that p2y9/GPR23 might interact with lipids or nucleotides. We screened 198 lipids and 17 nucleotides using CHO cells stably expressing p2y9/GPR23 by measuring intracellular Ca2+ mobilization with FLEXstation, a scanning fluorometer. Among them, 18:1-LPA at a concentration of 10 µM induced a significant increase in [Ca2+]i in p2y9/GPR23-expressing cells, but not in p2y5-expressing cells (data not shown).

Membrane Binding Assay—To 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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FIG. 2.
[3H]LPA binding to RH7777 cell membranes. A, [3H]LPA binding to p2y9/GPR23. Membrane fractions of RH7777 cells transiently expressing p2y9/GPR23 (closed symbols) and mock-transfected cells (open symbols) were incubated with increasing concentrations of [3H]18:1-LPA in the presence or absence of 10 µM unlabeled 18:1-LPA. Total binding ({blacksquare} and {square}) and nonspecific binding ({blacktriangleup} and {triangleup}) are presented. Data are means ± S.D. (n = 3). B, Scatchard analysis of the specific binding of [3H]LPA to p2y9/GPR23. C, competition for [3H]LPA binding with related lipids. Membrane fractions of RH7777 cells transiently expressing p2y9/GPR23 were incubated with 5 nM [3H]18:1-LPA in the presence of 1 µM unlabeled lipids. The total amounts of [3H]LPA bound are presented. Data are means ± S.D. (n = 3) of a representative of two independent experiments. D, competition for [3H]LPA binding with structural analogs of LPA. Membrane fractions of RH7777 cells transiently expressing p2y9/GPR23 were incubated with increasing concentrations of unlabeled 18:1- ({blacksquare}), 18:0- ({blacktriangleup}), 1-alkyl- ({triangleup}), and 1-alkenyl- ({triangledown}) LPA in the presence of 2.5 nM [3H]18:1-LPA. The total amounts of [3H]LPA bound are presented. Data are means ± S.D. (n = 3) of a representative of two independent experiments.

 

Reporter Gene Assay—We 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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FIG. 3.
Induction of reporter gene expression by structural analogs of LPA in PC-12 cells. PC-12 cells were transiently transfected with the reporter plasmids containing the zif 268 promoter-driven firefly luciferase gene and the control plasmid containing the cytomegalovirus promoter-driven Renilla luciferase gene together with the p2y9/GPR23 expression plasmid. Cells were stimulated with increasing concentrations of 18:1- ({blacksquare}), 18:0- ({blacktriangleup}), 16:0- (•), 14:0- ({blacktriangledown}), 1-alkyl- ({triangleup}), and 1-alkenyl- ({triangledown}) LPA. The ratios of firefly luciferase activity to Renilla one are shown. Data are means ± S.D. (n = 3) of a representative of two independent experiments.

 

Effect on Intracellular Ca2+ Mobilization—To 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 Formation—18:1-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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FIG. 5.
Effects of LPA on cAMP accumulation in CHO cells. A, dose-response curves. A clonal population of CHO cells stably expressing p2y9/GPR23 was stimulated with increasing concentrations of 18: 1-LPA in the presence of 0.5 mM isobutylmethylxanthine. The cAMP contents are presented ({blacksquare}). The effect of pertussis toxin pretreatment on cAMP accumulation is also shown ({square}). The results of mock-transfected CHO cells are displayed as a negative control ({blacktriangleup}, pertussis toxin-untreated cells; {triangleup}, pertussis toxin-treated cells). cAMP levels are expressed as -fold increases above basal contents. Data are means ± S.D. (n = 4). B, effects of forskolin on cAMP accumulation. Cells were stimulated with 18:1-LPA in the presence of 5 µM forskolin. Assay was performed in the same manner as A. Symbols are expressed as in A. Data are means ± S.D. (n = 4).

 

Tissue Distribution—To 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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FIG. 6.
Expression of p2y9/GPR23 mRNA. A, quantitative realtime PCR in 16 human tissues. Expression levels of p2y9/GPR23 mRNA are presented with the number of cDNA molecules initially involved in 2 µl of the template solutions. Bars show the mean ± S.D. of three independent experiments. B, expression levels of GAPDH mRNA. Quantitative real-time PCR was performed for GAPDH mRNA. Data are presented in arbitrary units. Bars show the means ± S.D. of three independent experiments. C, Northern blot analysis. Human poly(A)+ RNAs (2.5 µg) were electrophoretically separated, transferred to a nylon membrane, and hybridized sequentially with [32P]dCTP-labeled probes of human p2y9/GPR23 and human {beta}-actin.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
LPA is a lipid mediator with diverse physiological activities (1, 3). Many structural analogs of LPA have been identified in mammalian cells and tissues. Most are 1-acyl-LPAs with unsaturated fatty acyl-chains (oleoyl, linoleoyl, and arachidonoyl), and smaller amounts are with saturated fatty acylchains (palmitoyl and stearoyl) (21). Recently, 1-alkyl-, 1-alkenyl-, and 2-acyl-LPAs were also found (2224). LPA has been widely considered to elicit its physiological functions through three types of G protein-coupled receptors, Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3 (2, 3). However, there are some reports implying the existence of an additional LPA receptor(s). First, in the study of Edg-2/LPA1(/) Edg-4/LPA2(/) double knockout mice, some LPA-induced responses, such as inositol phosphate production, adenylyl cyclase inhibition, and stress fiber formation, were absent or severely reduced but still remained at high LPA concentrations in embryonic fibroblast cells (10). They reported that Edg-7/LPA3 was not detected by Northern blotting or reverse transcriptase-PCR in these cells. Second, RH7777 cells that do not express Edg-2/LPA1, Edg-4/LPA2, or Edg-7/LPA3 have a mitogenic response to LPA and LPA analogs (12). Third, LPA-induced platelet aggregation showed different ligand specificities with Edg receptor-mediated response; the platelet response lacks the stereoselectivity (11), requires micromolar concentrations of LPA (11), and displays a distinct ligand selectivity with a preference to 1-alkyl-LPAs (13). Here we identified p2y9/GPR23 as the fourth LPA receptor (LPA4).

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 [{gamma}-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 20–24% 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, H1–H4 (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.


    FOOTNOTES
 
* This work was supported by grants-in-aid from the Ministry of Education, Science, Culture, Sports and Technology of Japan (to T. S. and S. I.), grants-in-aid for Comprehensive Research on Aging and Health, and Research on Allergic Disease and Immunology from the Ministry of Health, Labour, and Welfare, Japan, and grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Kanae Foundation for Life and Socio-medical Science, and the Uehara Memorial Foundation (to S. I.). 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. Back

{ddagger} 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. Back


    ACKNOWLEDGMENTS
 
We thank Dr. T. Yokomizo and H. Hagiya (The University of Tokyo) for vital discussions and critical suggestions. We are also grateful to Dr. J. Miyazaki (Osaka University, Japan) for supplying pCXN2, and H. Shindou for expert technique of binding assay.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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