Identification of a Phosphothionate Analogue of Lysophosphatidic Acid (LPA) as a Selective Agonist of the LPA3 Receptor*

Yutaka HasegawaDagger , James R. Erickson§, Graham J. Goddard§||, Shuangxing YuDagger , Shuying LiuDagger , Kwai Wa ChengDagger , Astrid EderDagger , Koji Bandoh**, Junken Aoki**, Renata JaroszDagger Dagger , Andrew D. SchrierDagger Dagger , Kevin R. LynchDagger Dagger , Gordon B. MillsDagger , and Xianjun FangDagger §§

From the Dagger  M. D. Anderson Cancer Center, Houston, Texas 77030, § LXR Biotechnology, Richmond, California 94804, the ** University of Tokyo, Tokyo 113-0033, Japan, and the Dagger Dagger  Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia 22908

Received for publication, September 6, 2002, and in revised form, January 17, 2003

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA1 receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA3 receptor-expressing Sf9 cells but poorly in LPA2 receptor-expressing cells. Guanosine 5'-O-(3-[35S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA3 receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA3 but not the LPA1 or LPA2 receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA1/LPA3 receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA3-selective agonist. OMPT binding to the LPA3 receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.

    INTRODUCTION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Lysophosphatidic acid (LPA,1 1-acyl-sn-2-hydroxyl-glycerol-3-phosphate) is an extracellular lipid mediator that can be produced by a number of cell types including platelets, adipocytes, fibroblasts, and ovarian cancer cells (1, 2). The most prominent cellular effects of LPA include stimulation of cell proliferation (3), cell survival (4-7), platelet aggregation (8), smooth muscle cell contraction (9), and tumor cell invasion (10). Recent studies (11-14) on the pathophysiological roles of LPA have revealed abnormalities in LPA production and function in cardiovascular and neoplastic diseases. In particular, LPA is present in elevated levels in ascites of ovarian cancer patients and may thus contribute to the progression of certain types of human cancer (13).

LPA binds and activates G protein-coupled receptors of the endothelial cell differentiation gene (Edg) family (15). The Edg family encodes receptors for both LPA and the related lysophospholipid, sphingosine 1-phosphate (S1P) (15, 16). To date, eight Edg family members have been identified. S1P1/Edg1, S1P3/Edg3, S1P2/Edg5/H218/AGR16, S1P4/Edg6, and S1P5/Edg8/NRG-1 are S1P receptors (16), whereas LPA1/Edg2, LPA2/Edg4, and LPA3/Edg7 are high affinity receptors for LPA (17-19). Although S1P1 has been shown to have the ability to interact with both S1P and LPA, S1P appears to be its preferred ligand at physiological levels (20, 21). In Xenopus oocytes, Guo et al. (22) identified a functional high affinity LPA receptor, PSP24, that purportedly mediates LPA-induced depolarization in Xenopus oocytes. Some recent studies (23) suggest the existence of additional, non-Edg receptors for LPA.

Several groups have studied the structure-activity relationship of LPA to gain a better understanding of the function of particular receptors in the mammalian system and to define the mechanism by which LPA binds to and activates its cognate receptors. Several conclusions can be drawn from these studies, including a strong requirement for a free phosphate (3, 24). The glycerol backbone is important but not essential for optimal functionality of LPA as it can be substituted with certain substructure replacements such as ethanolamine (24-26). In addition, the location, linkage, length, and degree of saturation of the fatty acid side chain and the presence of the hydroxyl moiety at the second carbon are also functional determinants of LPA (24, 27-29). In an effort to create LPA analogues with improved potency and/or receptor selectivity, we characterized an LPA analogue, 1-oleoyl-2-O-methyl-rac-glycero-3-phosphothionate (OMPT). Previous studies of structure-function relationship of LPA analogues have proven difficult to interpret in complex mammalian systems where more than one LPA receptor is present on the cell surface. However, G protein-coupled receptors can be expressed in eukaryotic cell models such as yeast (30) and insect cells (19) to study a receptor in an isolated context and without an endogenous LPA response.

By virtue of these approaches combined with assays in mammalian cells such as guanosine 5'-O-(3-[35S]thio)triphosphate (GTP[gamma -35S]) binding assay (31), we have demonstrated that OMPT, at low concentrations (<= 100 nM), triggers cellular responses through the LPA3 but not the LPA1 or LPA2 receptors. At least 10-fold higher concentrations of OMPT are required to activate the LPA2 receptor. OMPT does not exhibit agonistic effects on the LPA1 receptor at concentrations of 1 µM or higher. In mammalian cells, OMPT coupling to the LPA3 receptor is sufficient to evoke activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases (MAPK). Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.

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Reagents-- LPA (18:1) and S1P were obtained from Avanti Polar Lipids (Alabaster, AL) and Calbiochem, respectively. Indo-1-AM was obtained from Molecular Probes (Eugene, OR). Fetal bovine serum (FBS) and monoclonal antibodies against FLAG M2 and beta -actin were purchased from Sigma. Cell culture reagents were from Invitrogen. The antibody against active, phosphorylated Erk was purchased from Promega (Madison, WI). Anti-HA monoclonal antibody and anti-Erk polyclonal antibody were from Babco (Berkeley, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. GTP[gamma -35S] (1200 Ci/mmol) was purchased from Amersham Biosciences.

Chemistry-- OMPT was synthesized at Oxford Asymmetry (Oxford, UK) under contract with LXR Biotechnology, Inc. Full details of the chemical synthesis are available on request. Briefly, starting with 1-O-benzyl-glycerol, the primary alcohol was converted to a silyl ether, followed by standard methylation of the secondary alcohol to yield a 2-methoxy substituent. The resulting compound was subjected to hydrogenation to cleave the benzyl ether yielding an alcohol that was then acylated using oleoyl chloride. After cleavage of the silyl group with fluoride, the revealed alcohol was reacted with phosphoramidite followed by elemental sulfur. Finally, the isopropyl deprotecting groups were removed to afford the racemic thiophosphonate compound, which was purified chromatographically. The purity of OMPT was nearly 100% as analyzed by electrospray ionization mass spectrometry.

Cell Lines-- Swiss 3T3, C3H10T1/2, and HEK293 were obtained from American Tissue Culture Collections (ATCC, Manassas, VA). The ovarian cancer cell line A2780CP was kindly provided by T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA). HEK293T and PC-3M cells were provided by Dr. Judy White and Dr. Dan Theodorescu at the University of Virginia, respectively. HEK293T cells were cultured in alpha -minimum Eagle's medium supplemented with 1 mM sodium pyruvate and 10% FBS. PC-3M cells were cultured in Dulbecco's modified Eagle's medium and 10% charcoal and dextran-stripped FBS (HyClone). Swiss 3T3 and HEK293 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. C3H10T1/2 was cultured in Basal Eagle's medium containing 10% FBS. A2780CP was maintained in RPMI 1640 supplemented with 10% FBS. These mammalian cell lines were cultured at 37 °C in a humidified atmosphere with 5% CO2. The insect cell line Sf9 from Invitrogen was cultured in Grace's insect medium (Invitrogen) containing 10% FBS at 27 °C. All cell lines were frozen at early passages and used for less than 10 weeks in continuous culture.

Western Blot-- Cells were lysed in SDS sample buffer or ice-cold X-100 lysis buffer (1% Triton X-100, 50 mM Hepes, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 100 mM NaF, 10 mM sodium pyrophosphate, and protein inhibitor mixture (Roche Molecular Biochemicals)). Total cellular protein was resolved by SDS-PAGE, transferred to Immobilon (poly(vinylidene difluoride)), and immunoblotted with antibodies following the protocols provided by manufacturers. Immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham Biosciences) using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad).

Northern Blot-- Poly(A)+ RNA was extracted from cell lines using the FastTrack 2.0 mRNA Isolation kit following the instructions of the supplier (Invitrogen). Northern blotting analyses (4 µg of poly(A)+ RNA for loading) were performed as described previously (32). The human LPA1, LPA2, and LPA3 cDNA were isolated from expression vectors, labeled with [32P]dCTP, and used as probes for hybridization.

Cytoplasmic [Ca2+]i Assay-- After starvation in serum-free medium for 12-24 h, C3H10T1/2 cells were harvested and loaded with 1 µM Indo-1 AM in phosphate-buffered saline (PBS) for 30 min at 37 °C. Cells were washed in PBS and resuspended at 2 × 106 cells/ml in a [Ca2+]i assay buffer (140 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 25 mM Hepes, and 10 mM glucose, pH 7.4). Cytoplasmic [Ca2+]i was determined at an excitation wavelength of 331 nm and an emission wavelength of 410 nm using a fluorescence spectrophotometer (F-4000, Hitachi). Approximately 3 × 106 cells were used for [Ca2+]i determination in a stirred quartz cuvette kept at 37 °C.

PC-3M cells were plated onto opaque, 96-well clear bottom plates at about 80% of confluence. The next day, cells were washed with Hanks' Basal Salts Solution containing 0.1% fatty acid-free BSA and loaded in the same solution with the calcium-sensing dye, Fluo-4AM. Calcium signals were recorded using a fast kinetic fluorometer (FLEXStation, Molecular Devices, Inc.). Excitation was at 485 nm, and records were collected at 525 nm.

For the cytoplasmic [Ca2+]i assay in Sf9, ~2 × 106 cells were infected with recombinant baculovirus engineered to express the LPA2, LPA3, or LPA1/LPA2 chimeric receptor (19, 29). The preparations of the virus and infection of Sf9 cells have been described previously (19). Two days after virus infection, cells were starved and harvested for cytoplasmic [Ca2+]i assays. After cells were loaded with 1 µM Indo-1 AM in PBS for 30 min at 27 °C, cytoplasmic [Ca2+]i was determined at 27 °C in the same [Ca2+]i assay buffer as described above for C3H10T1/2 cells.

Pheromone Response Assays in Yeast-- The JEY5 yeast cells transformed with LPA1 or S1P1/Edg1 was grown in SC media containing 2% galactose lacking uracil to an approximate OD of 0.1 prior to the addition of lipids as described previously (30). LPA and OMPT were stored in chloroform or methanol and dried down under vacuum immediately prior to experiments. Dried LPA and OMPT were resuspended at 20 mM in a solution containing 50 mM NH4HCO3, 104 mM NaCl, 250 µM EDTA, pH 7.6, with sonication until the solution was clear. Cells were grown for 7 h in the presence of lipid agonists, and then 100 µl of yeast culture was mixed with 900 µl of assay buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 0.1 mM MgSO4, pH 7.0, 2.7 ml of beta -mercaptoethanol per liter) plus 50 µl of 0.1% SDS and 3 drops of chloroform. Cells were vortexed for 10 s and incubated for 5 min at 28 °C. O-Nitrophenol-beta -D-galactopyranoside (ONPG, Sigma) (200 µl of 4 mg/ml) was added to allow a 30-min reaction at 28 °C. The assay was stopped by adding 500 µl of 1 M Na2CO3. Color development was measured at A420 and normalized to A600. Units were expressed as Miller units (30).

GTP[gamma -35S] Binding-- The GTP[gamma -35S] assay was performed as described previously (31). Briefly, HEK293T cells were co-transfected by calcium phosphate precipitation with expression vectors encoding one of the receptors for LPA (LPA1, LPA2, and LPA3) or S1P (S1P3/Edg3) and plasmids encoding three G proteins (rat Gi2alpha , cow beta 1, and cow gamma 2). After 48 h, cells were harvested and crude microsomal membranes prepared. Membranes containing 5 µg of protein were incubated in 0.1 ml of GTP-binding buffer (50 mM Hepes, 100 mM NaCl, 10 mM MgCl2, pH 7.5) containing 5 µg of saponin, 0.1% fatty acid-free BSA, 10 µM GDP, 0.1 nM GTP[gamma -35S] (1200 Ci/mmol), and indicated concentrations of LPA or OMPT for 30 min at 30 °C. Membranes were collected using a 96-well Brandel Cell Harvester (Gaithersburg, MD), and bound radionuclide was determined using a Packard TopCount liquid scintillation counter.

Transient Expression of LPA Receptors and HA-Erk1 in HEK293 Cells-- The LPA1 receptor expression vector, pcDNA3-FLAG-LPA1, was provided by Dr. W. H. Moolenaar (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The LPA2 and LPA3 cDNAs were obtained from OVCAR-3 by reverse transcriptase-PCR and inserted into pcDNA3 that had been pre-modified to contain the FLAG M2 DNA sequence. The structures of pcDNA-FLAG-LPA1, pcDNA3FLAG-LPA2, and pcDNA3FLAG-LPA3 were confirmed by sequencing. The HA-Erk1 expression vector (pCEP4-HA-Erk1) was a gift of Dr. M. Cobb (University of Texas Southwestern Medical Center). HEK293 cells in 6-well plates were transfected with 1.5 µg of DNA/well using FuGENE 6 according to the protocol of the supplier (Roche Molecular Biochemicals). The molar ratio of an Edg expression vector to the HA-Erk1 vector was adjusted to 5:1 to ensure co-expression of Edg receptors with HA-Erk1 as described previously (33).

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INTRODUCTION
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OMPT Is a Functional LPA Analogue with Potent Mitogenic Activity-- OMPT was designed and synthesized as part of a screen for Edg receptor-specific agonists. As compared with 18:1 LPA, in OMPT, the hydroxy and the phosphate have been converted to an O-methoxy group and a phosphothionate, respectively (Fig. 1). These modifications were designed to prevent metabolism to phosphatidic acid by LPA acyltransferases (2, 34) and to evade lipid phosphatases (23, 35-37). To determine whether OMPT exhibits agonistic activities, we first assessed the ability of OMPT to increase intracellular calcium change in mammalian cells, a standard assay for LPA activity. As shown in Fig. 2A, both LPA and OMPT were able to trigger [Ca2+]i responses in a dose-dependent manner in C3H10T1/2 cells. However, LPA was more potent than OMPT in this assay. OMPT also induced activation of Erk in C3H10T1/2 cells as revealed by immunoblotting with an Erk phospho-specific antibody (Fig. 2B), demonstrating a generalized ability of OMPT to couple to multiple intracellular signaling cascades. As shown in Fig. 2B, LPA was able to induce significant Erk phosphorylation at 0.01 µM, whereas OMPT had weaker effect at this concentration. At higher concentrations tested, OMPT and LPA were equipotent in the induction of Erk phosphorylation. However, compared with LPA, OMPT stimulated more sustained phosphorylation of Erk in C3H10T1/2 cells as shown in Fig. 2B.


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Fig. 1.   Chemical structures of LPA (oleoyl, 18:1) and OMPT.


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Fig. 2.   OMPT is a functional LPA analogue in mammalian cells. A, OMPT induces intracellular calcium mobilization. C3H10T1/2 cells were grown to subconfluence, starved in serum-free medium, and harvested for cytoplasmic [Ca2+]i assays as detailed under "Experimental Procedures." B, OMPT stimulates MAPK activation. Serum-starved C3H10T1/2 cells were stimulated for 5 min with LPA or OMPT at the indicated concentrations (upper panel) or were treated with LPA or OMPT (1 µM) for the indicated intervals (hour) (lower panel). Phosphorylation of Erk1 and Erk2 was assessed by immunoblotting with a phospho-specific Erk antibody. Immunoblotting for total cellular Erk using phosphorylation-independent Erk antibody was included to show equal loading among samples. C, C3H10T1/2 cells express LPA1 and LPA3 receptor mRNAs. Poly(A)+ RNA was isolated from C3H10T1/2 and the human ovarian carcinoma cell line A2780CP, a control cell line known to express multiple LPA receptors. The expression of Edg LPA receptors was examined by Northern blotting as described under "Experimental Procedures." Transcripts of the LPA1 and LPA3 receptors expressed in C3H10T1/2 cells are marked with arrows. The hamster transcript of the LPA3 receptor in C3H10T1/2 cells is different in size from that in the human cell line A2780CP. D, OMPT is a potent mitogen in C3H10T1/2 cells. The mitogenic activity of LPA and OMPT was measured by [3H]thymidine incorporation as described under "Experimental Procedures." The data represent the means ± S.D. of triplicate assays. Similar results were obtained in three independent experiments.

The activation of these signaling processes by LPA and OMPT in C3H10T1/2 cells is likely mediated by specific LPA receptors. The lack of sufficiently sensitive antibodies to detect endogenously expressed Edg LPA receptors has precluded direct analysis of receptor protein expression in mammalian cell lines. We thus examined mRNA expression of the LPA1, LPA2, and LPA3 receptors in C3H10T1/2 cells. Northern blot analysis showed the presence of detectable levels of LPA1 and LPA3 mRNAs in this cell line (Fig. 2C).

One of the most prominent cellular effects of LPA is the stimulation of cell proliferation, particularly in fibroblasts (3, 38). To determine whether OMPT retains this activity of LPA, we measured the mitogenic responses to LPA and OMPT by [3H]thymidine incorporation in C3H10T1/2 cells (Fig. 2D). Consistent with previous studies (3, 38), high concentrations of LPA are required to stimulate cell proliferation. LPA at 1 µM induced only minimal DNA synthesis as reflected by limited thymidine incorporation (Fig. 2D). A prominent activity was seen only when 10 µM or higher concentrations of LPA were administered. Consistent with the more sustained activation of Erk seen in OMPT-treated cells (Fig. 2B), OMPT exhibited much stronger proliferative activity than LPA (Fig. 2D). The compound stimulated DNA synthesis at ~100-fold lower concentrations than LPA (Fig. 2D). Detectable levels of DNA synthesis were observed as low as 0.01 µM OMPT. The optimal stimulatory activity of OMPT occurred at 1 µM with higher concentrations of OMPT (10 and 20 µM) showing lower activity. Therefore, OMPT constitutes a novel LPA analogue exhibiting potent mitogenic activity at submicromolar concentrations. This greatly enhanced mitogenic activity probably reflects an improved stability, efficacy, and/or receptor selectivity of OMPT.

OMPT Does Not Activate LPA1 in Yeast-- As demonstrated previously, LPA1 can couple to the endogenous yeast heterotrimeric G proteins and activate the pheromone response pathway resulting in activation of a FUS1 promoter fused to the lacZ gene (FUS1::lacZ) stably integrated into the yeast genome as a reporter (30). To determine the receptor specificity of OMPT compared with LPA, we employed this system to ask whether OMPT can activate the LPA1 receptor in yeast. As seen in Fig. 3, LPA induced activation of the lacZ reporter in a dose-dependent manner. In contrast, OMPT did not stimulate the pheromone response as indicated by lack of an increase in lacZ activity, suggesting that OMPT is unable to activate LPA1, at least in the yeast system. With increasing concentrations, OMPT slightly decreases basal levels of lacZ activity (Fig. 3). In untransformed yeast cells (data not shown) or cells transformed to express an S1P receptor (S1P1/Edg1), neither LPA nor OMPT stimulated any significant increase in lacZ activity (Fig. 3). These results suggest that the structural alterations in OMPT lead to loss of the capability to interact functionally with the LPA1 receptor. Although LPA1 couples efficiently to the yeast pheromone response, LPA2 works poorly, and LPA3 does not function in this system (28), precluding the use of the yeast model to study the ligand specificity of LPA receptors other than the LPA1 receptor.


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Fig. 3.   LPA, but not OMPT, stimulates FUS1::lacZ activity in yeast cells expressing the LPA1 receptor. Yeast cells expressing LPA1 (upper panel) or S1P1/Edg1 (lower panel) were grown in SC + 2% galactose in the presence of indicated concentrations of LPA or OMPT. After 7 h, cells were assayed for galactosidase (lacZ) activity as described under "Experimental Procedures." S1P1/Edg1-transformed yeast cells did not respond to LPA or OMPT. Similar results were obtained in three independent experiments.

OMPT Selectively Activates LPA3 to Evoke [Ca2+]i Increases in Sf9 Cells-- To assess the ability of OMPT to activate the LPA2 and LPA3 receptors, we used the Sf9/baculovirus expression system with calcium mobilization as an indicator of receptor activation. Parental Sf9 does not respond to exogenous LPA (39), making it an ideal system for analyzing functions of specific LPA receptors. As described previously, the LPA2 and LPA3 receptors, but not the LPA1 receptor, couple to cytosolic calcium changes in Sf9 cells (39).

After infection with recombinant baculovirus expressing LPA2 or LPA3, Sf9 cells were assessed for LPA- and OMPT-induced calcium mobilization. LPA efficiently elevated intracellular [Ca2+]i in a concentration-dependent manner in Sf9 cells expressing either the LPA2 or LPA3 receptor (Fig. 4). The threshold concentrations of LPA required to induce detectable [Ca2+]i increase in the LPA2 and LPA3 receptor-expressing cells were ~0.1 and 10 nM, respectively. EC50 values were 0.84 nM for the LPA2 receptor-expressing cells and 76 nM for the LPA3 receptor-expressing cells, suggesting that LPA has a higher affinity for the LPA2 receptor than for the LPA3 receptor expressed in Sf9 cells. In contrast, OMPT, at concentrations lower than 100 nM, induced increases in intracellular [Ca2+]i in the LPA3 receptor-expressing Sf9 cells, but not in the LPA2 receptor-expressing cells (Fig. 4). At 10 nM, OMPT was sufficient to induce detectable calcium increases in the LPA3 receptor-expressing cells. Furthermore, OMPT induced modestly greater increases in intracellular [Ca2+]i than LPA in the LPA3 receptor-expressing Sf9 cells (Fig. 4A). In the LPA2 receptor-expressing cells, the threshold concentration of OMPT to initiate detectable intracellular [Ca2+]i increases was >= 100 nM (Fig. 4B). The EC50 value of OMPT in the LPA3 receptor-expressing Sf9 cells was 68 nM (Fig. 4A), whereas >= 100 times higher concentration of OMPT was required to achieve a half-maximal effect in the LPA2 receptor-expressing cells (Fig. 4B). Thus in this insect cell system, OMPT exhibits a strong selectivity for the LPA3 receptor subtype. Previous studies (29) indicated that the inability of the LPA1 receptor to couple to cytosolic changes in Sf9 cells could not be reverted by replacing the C-terminal intracellular domain of LPA1 (amino acids 315-355) with that of LPA2 (amino acids 298-351). In our control experiments, Sf9 cells infected with baculovirus expressing such a chimeric receptor did not respond to either LPA or OMPT, confirming that LPA- or OMPT-induced calcium changes in Sf9 cells are dependent on expression of specific LPA receptors.


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Fig. 4.   OMPT induces calcium mobilization in Sf9 cells through the LPA3 but not the LPA2 receptor. Shown are dose curves of LPA and OMPT-induced [Ca2+]i changes in Sf9 cells infected to express the LPA3 receptor (A), LPA2 receptor (B), or LPA1-LPA2 chimeric receptor (C). In all panels, results are means ± S.E. of four independent experiments.

OMPT Selectively Activates LPA3 in Mammalian Cells-- To determine whether the receptor specificity of OMPT observed in yeast and the insect Sf9 cells could be reproduced in mammalian cells, we performed GTP[gamma -35S] binding assays to compare agonistic activity of LPA and OMPT at each of the LPA receptors. Different from the above analyses in yeast and Sf9 cells, GTP[gamma -35S] binding assay allows analysis of all three LPA receptors in the same system (31). The approach has been used successfully to measure the relative efficacies and potencies of LPA analogues (31, 40, 41). HEK293T cells were co-transfected with LPA1, LPA2, LPA3, or a related S1P receptor S1P3/Edg3 (control) and G protein alpha , beta , and gamma  subunits. GTP[gamma -35S] binding to cell membranes isolated from cells transfected with each of these receptors was determined by incubation with various concentrations of LPA or OMPT. As shown in Fig. 5, neither LPA nor OMPT stimulated GTP[gamma -35S] binding activity in the cells transfected with the S1P3/Edg3 receptor. However, LPA stimulated GTP[gamma -35S] binding activity in the cells transfected with each of the three LPA receptors in a dose-dependent manner (Fig. 5). The EC50 values of LPA in the LPA1, LPA2, and LPA3 receptor-transfected cells were 128, 27, and 196 nM, respectively. In contrast, OMPT efficiently increased GTP[gamma -35S] binding activity only in LPA3 receptor-transfected cells with EC50 value being 276 nM (Fig. 5C). No agonistic activity was seen with OMPT at the LPA1 receptor (Fig. 5A). OMPT, at concentrations >= 1 µM, showed weak agonistic effects on the LPA2 receptor (Fig. 5B), consistent with the results of calcium mobilization assays in Sf9 cells. Together, these results identify OMPT as an LPA3 receptor-specific agonist.


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Fig. 5.   OMPT selectively activates the LPA3 receptor in mammalian cells. GTP[gamma -35S] binding to HEK293T cell membranes in response to LPA and OMPT was measured as detailed under "Experimental Procedures." Cells were transfected with the LPA1 receptor (A), the LPA2 receptor (B), the LPA3 receptor (C), or the control S1P3/Edg3 receptor (D) along with G protein expression constructs. LPA or OMPT was dissolved at 1 mM in aqueous 1.0% fatty acid-free BSA and diluted in aqueous 0.1% BSA. Points are in triplicate and are representative of at least two independent experiments.

OMPT Activates MAPK through LPA3 in Mammalian Cells-- To determine whether activation of the LPA3 receptor by OMPT can be linked to Erk activation in mammalian cells, HEK293 cells were co-transfected with hemagglutinin (HA)-tagged Erk1 (HA-Erk1) along with a control vector or an LPA1, LPA2, or LPA3 receptor expression vector. After serum starvation, transfected cells were stimulated with 0.01, 0.1, or 1 µM LPA or OMPT. Phosphorylation of transfected HA-Erk1 protein of a molecular mass larger than the endogenous Erk1 was revealed by immunoblotting with an anti-phospho-Erk antibody. As demonstrated in Fig. 6A, in control vector-transfected cells, 1 µM LPA or OMPT induced only trace phosphorylation of co-transfected HA-Erk1. Lower concentrations of LPA or OMPT did not lead to significant phosphorylation of HA-Erk1. In cells transfected with each of these LPA receptors, LPA stimulated a dose-dependent phosphorylation of co-transfected HA-Erk1, suggesting that each of these Edg LPA receptors is functionally expressed in the transfected cells and that each receptor can couple to MAPK activation in response to LPA.


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Fig. 6.   OMPT activation of the LPA3 receptor is linked to MAPK activation in mammalian cells. A, HEK293 cells were co-transfected with HA-Erk1, along with an empty vector or a FLAG-tagged Edg expression vector (LPA1, LPA2, or LPA3) as indicated. Two days after transfection, cells were starved for 12 h and stimulated with LPA or OMPT at the indicated concentrations for 5 min. Cell lysates were prepared and analyzed for Erk phosphorylation by immunoblotting with an Erk phospho-specific antibody. The greater molecular weight of transfected HA-Erk1 allows its separation from the endogenous Erk. Expression of transfected HA-Erk1 was verified by immunoblotting with an anti-HA monoclonal antibody. Shown is a representative of three independent experiments. B, expression of FLAG-tagged LPA receptors in transfected cells was confirmed by immunoblotting with anti-FLAG-M2 antibody. To avoid the formation of aggregates of Edg LPA receptors, cell lysates in SDS sample buffer were loaded without boiling.

Despite the ability of LPA to stimulate HA-Erk1 phosphorylation in each LPA receptor-transfected cells, OMPT did not increase HA-Erk1 phosphorylation in LPA1 receptor-transfected cells as compared with control vector-transfected cells (Fig. 6A), demonstrating that OMPT at concentrations up to 1 µM does not activate LPA1 in transfected HEK293 cells. OMPT was also much less efficient than LPA in stimulation of HA-Erk1 phosphorylation in LPA2 receptor-transfected cells in that OMPT at 0.1 µM did not evoke HA-Erk1 phosphorylation. Even at 1 µM, the effect of OMPT was similar to that of 0.1 µM LPA, suggesting that OMPT possesses a dramatically reduced agonistic activity at the LPA2 receptor. In LPA3 receptor-transfected cells, however, OMPT led to higher levels of HA-Erk1 phosphorylation than LPA at all concentrations tested. The effect of OMPT on HA-Erk1 phosphorylation was detectable when only 0.01 µM OMPT was administered. Thus, OMPT, compared with LPA, evoked stronger MAPK phosphorylation via the LPA3 receptor. The data further indicate that OMPT is the LPA3 receptor-selective agonist and that ligation of the LPA3 receptor is capable of inducing MAPK activation.

LPA- and OMPT-induced Calcium Mobilization Is Differentially Inhibited by the LPA1/LPA3-selective Antagonist VPC12249-- We have demonstrated recently (31) that an N-acyl (oleoyl)-serine phosphate-based compound, VPC12249, is an LPA1/LPA3-selective antagonist. The compound blocks binding of LPA to the LPA1 and LPA3 receptors without interfering with the action of LPA2. If OMPT is LPA3-selective ligand lacking agonistic activity toward the LPA1 receptor as supported by the data described above, the cellular responses to OMPT, compared with LPA which activates all LPA receptors, should be more efficiently inhibited by VPC12249 in mammalian cells that express multiple LPA receptors. To test this hypothesis, we examined intracellular calcium responses to LPA and OMPT with or without VPC12249 in the prostate cancer cell line PC-3M, a subclone of PC-3 (42). Both the parental line and PC-3M express mRNA of all three LPA receptors (Refs. 43 and 44 and data not shown). LPA and OMPT induced calcium mobilization in a concentration-dependent manner in PC-3M cells (Fig. 7). In the presence of 10 µM VPC12249, the concentration-response curves for both LPA and OMPT shifted to the right, indicating competition for LPA receptors by the LPA1/LPA3 antagonist. As expected, the calcium response to OMPT, compared with LPA, was more dramatically inhibited by VPC12249. As shown in Fig. 7, VPC12249 led to a more than 5-fold increase in the EC50 concentration of OMPT compared with less than 2-fold increase for the EC50 dose of LPA. The data are compatible with the contention that calcium mobilization by LPA proceeds through a combination of multiple LPA receptors, whereas the response to OMPT is mediated by the LPA3 receptor, a preferred target of VPC12249.


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Fig. 7.   LPA and OMPT-induced calcium mobilization is differentially inhibited by the selective LPA1/LPA3 antagonist VPC12249. Intracellular calcium mobilization in PC-3M cells in response to LPA (left panel) or OMPT (right panel) in the absence or presence of 10 µM VPC12249 was measured as described under "Experimental Procedures." The measurements were converted to percentage values of the maximum response. The EC50 concentrations (M) of LPA and OMPT in the absence and presence of VPC12249 are listed in each panel. Data are means ± S.E. of five replicates, representative of at least two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LPA signals through selected Edg subfamily members that can couple to multiple G proteins to evoke a variety of responses (12). Although it is currently unknown why multiple LPA receptors exist, these receptors might fulfill different functionalities during development, growth, and pathological processes. The exact role of each of the Edg family receptors remains poorly defined. This is due, in part, to a lack of receptor subtype-specific agonists or antagonists of individual LPA receptors. In the present study, we demonstrate that OMPT, a phosphothionate analogue of LPA, is a selective agonist of the LPA3 receptor. Our results derived from different cell models of eukaryotes including yeast, insect, and mammalian cells demonstrate that OMPT exhibits marked selectivity for the LPA3 receptor versus the LPA1 or LPA2 receptors. At low concentrations (<100 nM), OMPT activates LPA3, but not LPA1 or LPA2, allowing linkage of the LPA3 receptor to specific signaling processes such as activation of G proteins, intracellular calcium mobilization, and MAPK activation. At concentrations of 1 µM or higher, OMPT can activate the LPA2 receptor albeit much less potently than LPA. Because we have not detected activation of the LPA1 receptor by OMPT in either yeast or mammalian cells, OMPT may not be able to activate this receptor subtype even at concentrations exceeding 10 µM.

The availability of LPA receptor-selective agonists, such as OMPT, will not only help delineate functions of each LPA receptor but also shed light on the mechanisms for Edg LPA receptor-ligand interactions. OMPT differs from LPA by the replacement of the 2-hydroxyl with a methoxy group and the phosphate by a phosphothionate. The 2'-hydroxyl has been suggested to be critical for high potency interactions between LPA and its receptors (27), and the accurate mimicry of LPA by the ethanolamide-containing compound, N-oleoylethanolamide phosphate, would seem to support the importance of a dissociable proton at the second carbon of both molecules (27, 31). However, this supposition was made prior to the identification of individual LPA/Edg receptors and would seem to hold true only for the LPA1 and LPA2 receptors. For example, LXR 100023, another LPA analogue in which the 2 hydroxyl group is substituted with an sn-2-O-methyl moiety, is only a partial agonist at the LPA1 receptor in the yeast system and inactive at the LPA2 receptor (28). The activity of OMPT at the LPA3 receptor demonstrates that the importance of the 2-hydroxyl group does not extend to this receptor type.

Phosphate is a difficult moiety to mimic chemically. A phosphonate group is an obvious surrogate that has the advantage of being phosphatase-resistant, but phosphonate-containing LPA analogues show sharply reduced potency at all LPA/Edg receptors.2 Some of this lost potency is recovered, however, by substitutions at the alpha -carbon of the phosphonate (23). The activity inherent in OMPT suggests that a different phosphate surrogate, phosphothionate, is a fully accurate phosphate mimetic for at least one LPA receptor. The relative contributions of the methoxy and phosphothionate substitutions to the selectivity of OMPT remain to be determined.

Development of receptor subtype-specific agonists makes it possible to dissect LPA-induced signals in complex mammalian systems where expression of multiple LPA receptors is common. Certain natural LPA species or analogues have been reported previously to exhibit differential agonistic or antagonistic activity toward different LPA receptors. For example, 14:0 (myristoyl) LPA shows strong preference for LPA2 as an agonist (29). Phosphatidates with short fatty acid chains, such as 8:0, function as selective antagonists of the LPA1 and LPA3 receptors, particularly the LPA3 receptor (45). LPA analogues lacking the 2-hydroxyl moiety may also act as receptor subtype-specific agonists as these compounds can retain the potency of LPA to evoke calcium responses but are not as strong as LPA in inhibiting adenylate cyclase (27). By virtue of heterologous desensitization experiments involving LPA, alkenyl-glycerolphosphate (a vinyl ether-linked LPA analogue), and cyclic phosphatidic acid (cPA), Fischer et al. (46) demonstrated that potentially three receptor subclasses exist in 3T3 fibroblasts. One pharmacological class of receptors (presumably LPA1) is predicted to be activated by LPA, alkenyl-glycerolphosphate, and cPA. Indeed, LPA1 is activated in yeast by all three of these compounds.3 In contrast to LPA, cPA inhibits tumor cell invasion and cell proliferation (47, 48). This indicates that cPA does not mimic the full spectrum of LPA functions consistent with cPA acting as a receptor-selective agonist (47-49). In a similar study utilizing heterologous desensitization of calcium mobilization in MDA MB-231 cells, it was found that a naturally occurring serine-based lipid, N-acyl (palmitoyl)-serine phosphoric acid (NASPA) could completely desensitize the cells to subsequent treatment with NASPA (25). However, NASPA could not completely desensitize the ability of LPA to mobilize intracellular calcium indicating the presence of LPA receptors refractory to NASPA presumably due to receptor selectivity of NASPA (25). Furthermore, NASPA and a related lipid, N-palmitoyltyrosine phosphoric acid are potent inhibitors of LPA-induced platelet aggregation (49, 50) as well as LPA-induced chloride currents in Xenopus oocytes (51). More recently, Heise et al. (31) developed a series of NASPA analogues with 2-substituted moieties. A compound with a bulky hydrophobic group (VPC12249) at the sn-2 was found to be a selective antagonist of LPA1 and LPA3 (31) that we have re-evaluated in the present study.

LPA can be metabolized through removal of the fatty acyl chain by lysophospholipid lipases, by acylation of the sn-2 hydroxyl group by LPA acyltransferases such as endophillin, and by hydrolysis of phosphate by lipid phosphate phosphohydrolases (LPPs, formerly phosphatidic acid phosphohydrolases) (2). Expression of LPPs has been shown to antagonize cellular responses to LPA (23, 37). The LPA concentrations required for significant mitogenic effect are usually orders of magnitude higher than those required for LPA-mediated early signaling responses. Hooks et al. (23) have recently suggested that the mitogenic activity of LPA is regulated by membrane-located LPPs and could be mediated by non-Edg LPA receptor(s). These authors have developed LPA analogues with phosphonate head groups that exhibited more potent mitogenic activities than natural LPA (23). In OMPT, the methyl group and phosphothionate should render the compound more resistant to metabolism by acyltransferases or by LPPs. The potentially improved stability of OMPT may account, in part, for the prolonged MAPK activation and the enhanced mitogenic activity we have observed as well as for the ability of OMPT to function in vivo (52). The sustained activation of MAPK is thought to be an important component of proliferative response to growth factors (53). OMPT stimulates significant DNA synthesis at 0.1 µM and even at 0.01 µM, making it a unique LPA analogue of extremely strong mitogenic activity. At these low concentrations, OMPT is not likely to activate LPA receptors rather than the LPA3 receptor. If LPA-induced DNA synthesis is mediated by an Edg receptor, our results would imply that activation of the LPA3 receptor could couple to the proliferative response. This is consistent with our observation that OMPT occupation of the LPA3 receptor is sufficient to activate MAPK, an integral component of the proliferative signaling network. However, involvement of other unidentified receptor(s) or non-receptor-mediated process in OMPT-induced cell proliferation could not be ruled out.

In summary, we have demonstrated that OMPT is an LPA3 receptor-selective ligand with distinct signaling properties. OMPT thus provides a probe for LPA3 receptor function in mammalian systems. The phosphothionate probably renders OMPT resistant to LPPs, thus offering an attractive reagent to study the functions of LPA and the LPA3 receptor in vivo.

    ACKNOWLEDGEMENTS

We are grateful to Dr. W. H. Moolenaar and Dr. G. C. Zondag (The Netherlands Cancer Institute) for the LPA1/Edg2 expression vector; Dr. E. J. Goetzl and Dr. S. An (University of California, San Francisco) for the LPA2/Edg4 and S1P3/Edg3 expression vectors; Dr. T. Hla (University of Connecticut) for the S1P1/Edg1 expression vector; and Dr. M. Cobb (University of Texas Southwestern Medical Center) for the HA-Erk1 expression vector.

    FOOTNOTES

* The work was supported by Atairgin Technologies Contract LS99-225RG, the Lynne Cohen Foundation Ovarian Cancer Research Grant 80095031 (to X. F.), and by National Institutes of Health Grants CA64602 (to G. B. M.), CA88994, and GM052722 (to K. R. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: AGY Therapeutics, South San Francisco, CA.

|| Present address: Genentech Inc., South San Francisco, CA.

§§ To whom correspondence should be addressed: M. D. Anderson Cancer Center, Dept. of Molecular Therapeutics, Box 317, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-745-1134; Fax: 713-745-1184; E-mail: Xianjunfang@mail.mdanderson.org.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M209168200

2 W. L. Santos, R. Jarosz, K. R. Lynch, and T. L. Macdonald, manuscript in preparation.

3 J. R. Erickson, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LPA, lysophosphatidic acid; OMPT, 1-oleoyl-2-O-methyl-rac-glycerophosphothionate; S1P, sphingosine 1- phosphate; Edg, endothelial cell differentiation gene; MAPK, mitogen-activated protein kinase; GTP[gamma -35S], guanosine 5'-O-(3-[35S]thio)triphosphate; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HA, hemagglutinin of the influenza; BSA, bovine serum albumin; Erk, extracellular signal-regulated kinase; cPA, cyclic phosphatidic acid; LPP, lipid phosphate phosphohydrolases; NASPA, N-acyl (palmitoyl)-serine phosphoric acid.

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