Lysophosphatidic Acid Stimulates the G-protein-coupled Receptor EDG-1 as a Low Affinity Agonist*

Menq-Jer Lee, Shobha Thangada, Catherine H. Liu, Brian D. Thompson, and Timothy HlaDagger

From the Department of Physiology, University of Connecticut School of Medicine, Farmington, Connecticut 06030

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

EDG-1, an inducible G-protein-coupled receptor from vascular endothelial cells, is a high affinity receptor for sphingosine 1-phosphate (SPP) (Lee, M-J., van Brocklyn, J. R., Thangada, S., Liu, C. H., Hand, A. R., Menzeleev, R., Spiegel, S., and Hla, T. (1998) Science 279, 1552-1555). In this study, we show that lysophosphatidic acid (LPA), a platelet-derived bioactive lipid structurally related to SPP, is an agonist for EDG-1. LPA binds to EDG-1 receptor with an apparent Kd of 2.3 µM. In addition, LPA binding to EDG-1 induces receptor phosphorylation, mitogen-activated protein kinase activation, as well as Rho-dependent morphogenesis and P-cadherin expression. These data suggest that LPA is a low-affinity agonist for EDG-1. Activation of the endothelial receptor EDG-1 by platelet-derived lipids LPA and SPP may be important in thrombosis and angiogenesis, conditions in which critical platelet-endothelial interactions occur.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The G-protein coupled receptor (GPR)1 EDG-1 was originally cloned as an immediate-early gene induced during the morphogenetic differentiation phase of angiogenesis (1). We subsequently demonstrated that the EDG-1 binds to heterotrimeric Gi proteins and stimulates Gi-dependent mitogen-activated protein (MAP) kinase activation (2). Recently, we showed that human embryonic kidney (HEK293) cells stably transfected with the EDG-1 cDNA exhibit enhanced adherens junction-based cell-cell aggregation, exaggerated cadherin expression and were phenotypically differentiated (3). Interestingly, the induction of morphogenetic differentiation was ligand dependent and we demonstrated that serum-borne sphingosine 1-phosphate (SPP) is a highly potent agonist for the EDG-1 GPR. Indeed, SPP binding to EDG-1-transfected cells was reversible, saturable, specific, and exhibited a high-affinity binding site with apparent Kd of 8.1 nM (3). In addition, SPP induces morphogenetic differentiation and P-cadherin expression as well as Gi-dependent MAP kinase activation via the EDG-1 receptor (3). Morphogenesis induced by SPP was inhibited by the C3 exoenzyme which specifically ADP-ribosylates the small G-protein Rho (4). These data allowed us to conclude that EDG-1 is a high-affinity receptor for SPP (3).

SPP, released from activated platelets (5), is recognized as a potent bioactive lipid mediator with multiple biological activities (6-12). LPA, which is also a platelet-derived bioactive lipid mediator (13), is structurally similar to SPP. Like SPP, LPA also regulates a wide range of cellular responses, including proliferation (14-16), platelet aggregation (14-16), stress fiber formation (17), neurite retraction (18, 19), cell rounding (20), tumor cell migration (21), among others. Moreover, it is well documented that LPA regulates these responses by activating specific G-protein-coupled receptors present on the cell surface of numerous cell types (22).

Recently, EDG-2/Vzg-1 was shown to be an LPA receptor involved in cell rounding, Gi-dependent inhibition of adenylyl cyclase (20), and serum response factor induced transcription (23). Prompted by the fact that (i) EDG-2/Vzg-1 is highly related to EDG-1 and (ii) LPA and SPP are structurally and functionally similar, we investigated in this report whether EDG-1 serves as a receptor for LPA.

    EXPERIMENTAL PROCEDURES
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Procedures
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References

Materials-- Phospholipases, fatty acid free BSA, LPA, lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylethanol, aprotinin, leupeptin, and pepstatin were from Sigma; [3H]LPA (specific activity 56.2 Ci/mmol) from NEN Life Science Products Inc., [32P]orthophosphate (specific activity 370 MBq/mmol) and [gamma -32P]ATP (specific activity 3000 Ci/mmol) from Amersham; SPP, sphingomyelin, ceramide, and ceramide 1-phosphate from Biomol; C3 exotoxin, pertussis toxin, and PD 98059 from Calbiochem; anti-P-cadherin antibody was from Transduction Laboratories; anti-M2 antibody from Eastman Kodak; rhodamine-conjugated sheep anti-mouse IgG from Cappel; anti-HA monoclonal antibody, endoglycosylase H, and alkaline phosphatase were from Boehringer Mannheim; G418, LipofectAMINE, and LipofectAMINE Plus reagents were from Life Technologies, Inc.; DMEM, RPMI 1640, and Dulbecco's phosphate-buffered saline (PBS, pH 7.4) were from Mediatech; trypsin EDTA was from JRH Bioscience; fetal bovine serum (FBS) was from HyClone; mouse mammary leukemia virus-reverse transcriptase from Life Technologies, Inc.; RNasin from Promega; Taq DNA polymerase from Cetus; and the FastTrackTM 2.0 mRNA isolation kit from Invitrogen.

Cell Culture, Transfection, and Morphogenetic Differentiation Assay -- Culture of HEK293 cells (ATCC CRL-1573) and transfection of EDG-1 cDNA was performed as described (2, 3). Stably transfected cells were subcloned twice to isolate pure clones. Four independently isolated EDG-1-transfected clones and two vector transfected clones were used in this study. Morphogenetic differentiation assay of EDG-1 expressed cell clones (HEK293-EDG-1) was carried out essentially as described (3). Briefly, cells (7.5 × 105 cells/ml) were plated into 6-well tissue culture plates (Falcon) in DMEM, 10 mM HEPES pH 7.4 containing 10% (v/v) charcoal-stripped lipid-depleted FBS (CFBS) with the indicated supplements. To examine the effect of serum-borne bioactive lipids on differentiation, FBS was treated with specific phospholipases (1 unit/500 µl) at 25 °C for 24 h. As a control, FBS was incubated with heat-denatured (95 °C for 1 h) phospholipases.

The human HEL 92.1.7 erythroleukemia (ATCC TIB-180) cell line was cultured in RPMI 1640 medium supplemented with 10% FBS. The transfection of these cells were performed with LipofectAMINE Plus reagent according to the manufacturer's instructions.

Immunostaining of EDG-1-- HEK293 cells were grown onto fibronectin (10 µg/ml)-coated coverslips, fixed with 4% paraformaldehyde at room temperature for 15 min, washed with PBS, and permeabilized with 0.2% (v/v) Triton X-100 for 5 min. Cells were then incubated with anti-M2 antibody (5 µg/ml in PBS containing 1% BSA) at room temperature for 1 h. After washing with PBS, cells were incubated with rhodamine-conjugated sheep anti-mouse IgG (10 µg/ml in PBS containing 1% BSA) for 30 min at room temperature. The EDG-1 signal was visualized by a Zeiss Axiovert 100TV fluorescence microscope.

Radiolabeled Ligands Binding Assay-- Stably transfected HEK293 cells were plated onto human fibronectin-coated (10 µg/ml, 0.5 ml/well at 37 °C for 1 h) 12-well culture plates (Falcon, 5 × 105 cells/well). Three days later, cells were washed once with binding buffer (0.1% fatty acid-free BSA in PBS, pH 7.4) and the binding assays were conducted with 0.5 ml of binding buffer containing 10 nM [3H]LPA in the presence or absence of cold competitor for 30 min at 4 °C, unless indicated in the text. Cells were then washed three times with binding buffer (0.5 ml each), solubilized with 250 µl of 1 N NaOH, 0.1% (v/v) SDS at room temperature for 1 h, and counted in a liquid scintillation counter. Nonspecific binding, defined as binding which occurred in the presence of 50 µM LPA, was approximately 60% of total binding. [3H]LPA does not bind specifically to the fibronectin-coated plastic surface. Apparent Kd and Bmax values of LPA-binding sites were determined by the homologous competition assay, in which binding was performed in the presence of 10 nM [3H]LPA without or with an increase cold competitor LPA (ranging from 10 nM to 250 µM). Nonspecific binding of [3H]LPA reached a plateau in the presence of 50 µM cold competitor LPA. The data obtained were then transformed into a Scatchard plot or the equation of Cheng and Prusoff (24) to analyze the apparent Kd and Bmax values. Both analyses obtained similar results. To determine the effect of the anti-M2 antibody, cells were preincubated at 4 °C with the indicated concentration of the antibodies for 60 min, and the [3H]LPA binding assay was conducted in the presence of the antibodies. The LPA binding assay of HEL 92.1.7 (1 × 106 cells) was conducted in 250 µl of binding buffer containing 10 nM [3H]LPA without or with competitor LPA at 4 °C for 30 min. Cells were then washed three times with binding buffer, centrifuged (300 × g for 10 min), and the bound [3H]LPA was counted.

EDG-1 Phosphorylation-- Cells were plated in 10% charcoal-stripped FBS for 3 days, labeled with [32P]orthophosphate (300 µCi/4 ml) for 2 h at 37 °C, and stimulated with the indicated ligands for 5 min. Cells were then washed, extracted with buffer A (50 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 1 mM EDTA, 20 mM CHAPS) containing 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µM Na3VO4, 10 µg/ml each of aprotinin, leupeptin, and pepstatin at 4 °C for 1 h. After microcentrifugation (15,000 × g for 10 min), extracts were immunoprecipitated with anti-M2 antibody to isolate the EDG-1 polypeptide (2). The immunoprecipitates were separated on a 10% SDS-PAGE and autoradiographed. The extent of phosphorylation was quantitated by densitometry.

Deglycosylation and Dephosphorylation of EDG-1 GPR-- HEK293-EDG-1 cells were stimulated in the absence or presence of SPP (0.5 µM) for 5 min, and then 1 mg of cellular extracts were immunoprecipitated with anti-M2 antibody as described. Subsequently, the immunoprecipitates were deglycosylated and dephosphorylated with endoglycosidase H and alkaline phosphatase, respectively, according to the manufacturer's instructions. Polypeptides were then separated by 10% SDS-PAGE and immunoblotted with anti-M2 antibody (2).

MAP Kinase Assay-- COS-7 and HEL 92.1.7 cells were co-transfected with (0-2 µg) of EDG-1 plasmid and 0.1 µg of HA-ERK-2 plasmid as described previously (2). The amount of DNA used for transfection was normalized with vector DNA. Thirty hours later, cells were made quiescent in 0.5% FBS/DMEM for 16 h. Following stimulating with 20 µM LPA for 5 min, cell lysates were prepared and immunoprecipitated with the anti-HA monoclonal antibody. The ERK-2 activity of the immune complexes was assayed as described before (2).

Immunoblot Analysis of P-Cadherin Expression-- HEK293-EDG-1 cells were cultured in 10% charcoal-stripped FBS (0.8 × 106 cells/35 mm) containing indicated concentrations of bioactive lipid ligands. The C3 exotoxin was used at 5 µg/ml. Three days later, cells were extracted with buffer A containing 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of aprotinin, leupeptin, and pepstatin at 4 °C for 1 h. After centrifugation (15,000 × g for 10 min), total extracted protein was quantified with the Bradford method (Bio-Rad). Fifty µg of cell extracts were resolved by 10% SDS-PAGE and immunoblotted with anti-P-cadherin antibody or with anti-M2 as described previously (2, 3).

Reverse Transcriptase-Polymerase Chain Reaction and Northern Analysis-- mRNAs were isolated from cultured cells with FastTrackTM mRNA isolation kit 2.0 as per the manufacturer's instructions. The full-length coding region of EDG-1 was labeled with [alpha -32P]dCTP by the random primer method and used as a probe for Northern hybridization analysis. One microgram of isolated poly(A)+ RNA in each lane was electrophoresed on 1% agarose/formaldehyde gel and transferred onto Zeta Probe membrane (Bio-Rad). Membrane was hybridized at 65 °C for 16 h with 32P-labeled probe in Church-Gilbert's solution (7% SDS, 0.5 M Na2HPO4, 0.5% BSA, 40 mM EDTA, 20% formaldehyde). Subsequently, membrane was washed with 2 × SSC, 0.05% SDS for 30-40 min at 65 °C with several changes. After the last wash with 0.1 × SSC, 0.1% SDS for 40 min at 65 °C, the filter was exposed to Kodak X-Omat film and autoradiographed. Total RNA from HUVEC cells (5 µg/lane) was used as a control to show the endogenous EDG-1. For reverse transcriptase-polymerase chain reaction analysis, total RNA from cultured cells was purified as described (1). RNA was then converted to cDNA by treatment with 200 units of mouse mammary leukemia virus-reverse transcriptase in 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 15 mM NaCl, 3 mM MgCl2, 1 unit of RNasin, 0.2 µg of random hexamer primers, 0.8 mM dNTPs and incubated at 37 °C for 1 h. The reaction was terminated by heating at 95 °C for 10 min and diluted to 1 ml with distilled water. Enzymatic amplification was conducted with a 10-µl aliquot of the cDNA mixture. Polymerase chain reaction was performed in 50 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM dNTPs, 0.5 µg of each primers for EDG-2/Vzg-1 and 2.5 units of Taq DNA polymerase. The reaction mixture was heated at 94 °C for 1 min, annealed at 55 °C for 2 min, and extended at 72 °C for 3 min for 30 repetitive cycles. The primers used were 5'-GGTCCAGAACTATGCCGAGAC-3' (sense) and 5'-GCCTCATTGACACCAGCCTGA-3' (antisense) to amplify EDG-2/Vzg-1 mRNA.

Cell Rounding Assay-- HEK293-pCDNA and HEK293-EDG-1 cells (25,000/35-mm culture dish) were starved in 10% CFBS for 24 h, and the media containing the desired agents were added. Phase-contrast photographs of cellular morphology were taken at the indicated times. As controls, other lysophospholipids (i.e. lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylcholine) were used.

    RESULTS
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Procedures
Results
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References

We have established several human embryonic kidney 293 fibroblasts (HEK293) clones which stably express the FLAG epitope-tagged EDG-1 GPR (3). Immunostaining with anti-FLAG antibody (anti-M2) shows that the expressed EDG-1 GPR is predominantly located on the plasma membrane (Fig. 1). Immunostaining of vector-transfected clones did not express immunofluorescence signals.


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Fig. 1.   The expression of EDG-1 receptor on HEK293-EDG-1 cells. The immunostaining of EDG-1 polypeptides with anti-M2 was performed as described under "Experimental Procedures." EDG-1, HEK293-EDG-1 cells; pCDNA, HEK293-pCDNA cells. Note the cell membrane localization of EDG-1 receptor in HEK293-EDG-1. Scale bar, 8.3 µM.

To determine if LPA is an agonist for EDG-1, we employed a radioligand [3H]LPA binding assay on HEK293 stable transfectants. As shown in Fig. 2A, specific [3H]LPA binding was markedly enhanced in four independent HEK293-EDG-1 clones compared with the vector-transfected and parental counterparts. Specific LPA binding was time-dependent and was observed in both EDG-1-transfected and vector-transfected cells. However, EDG-1-transfected cells bound significantly more [3H]LPA (Fig. 2B). Specific LPA binding was competed by unlabeled LPA but not by other lysophospholipid and sphingolipid analogs (Fig. 2C). Moreover, specific [3H]LPA binding was suppressed by the anti-M2 antibody in a dose-dependent manner only in HEK293-EDG-1 cells (Fig. 2D). This implies that LPA binds directly to the EDG-1 GPR. Furthermore, LPA binding was saturable; Scatchard analysis of [3H]LPA binding on HEK293-EDG-1 cells indicated two types of binding sites; apparent Kd = 178 nM, Bmax = 313 fmol/105 cells as well as apparent Kd = 2.3 µM, Bmax = 6.23 pmol/105 cells (Fig. 2E). In contrast, there is only one type of binding site detected on HEK293 parental cells with an apparent Kd = 212 nM and Bmax = 420 fmol/105 cells (Fig. 2E). To determine whether the N-terminal FLAG tag affect the binding properties of EDG-1, competition of [3H]LPA binding was compared between pDNFE (FLAG epitope-tagged at N-terminal) and pEGFPEDG-1 (green fluorescent protein tagged at C-terminal) (3) transfected human HEL92.1.7 erythroleukemia cells. These cells were chosen since they exhibit minimal endogenous LPA responsiveness and barely detectable expression of EDG-1 mRNA in Northern analysis (data not shown). As shown in Fig. 2F, LPA binding was significantly enhanced in pDNFE and pEGFPEDG-1 transfected cells and cold competition studies showed similar IC50 values (2.5 and 1.8 µm, respectively). Together, these results strongly suggest that EDG-1 GPR is a low affinity receptor with an apparent Kd of 2.3 µM for LPA.


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Fig. 2.   LPA Binds to EDG-1. A, specific [3H]LPA binding on four different HEK293-EDG-1 clones, HEK293-pCDNA, and parental HEK293 cells. B, time dependence of specific LPA binding. C, the competition of specific LPA binding on HEK293-EDG-1 cells with various lipid analogs (20 µM each). Nonspecific LPA binding was defined as binding in the presence of 50 µM LPA. D, indicated concentration of the antibody (µg/ml) was preincubated with the cells for 1 h at 4 °C and [3H]LPA binding was performed as described. mIgG, irrelevant mouse IgG. E, Scatchard plot of [3H]LPA binding to HEK293-EDG-1 and HEK293-pCDNA cells. Note that there are two types of LPA-binding sites in HEK293-EDG-1 cells, whereas only a single type of binding site in parental HEK293 cells. All panels are derived from a representative experiment which was repeated at least two times. Experiments on panels B-E were conducted on HEK293-pCDNA and HEK293-EDG-1 (#1) clones. F, homologous competition of [3H]LPA binding on HEL 92.1.7 cells which were transiently transfected with N-terminal FLAG-tagged (EDG-1) or C-terminal green fluorescent protein-tagged (EDG-1GFP) EDG-1 cDNAs. Note that the N-terminal FLAG tag does not alter the LPA binding properties of EDG-1.

Although unlabeled LPA competed effectively with [3H]LPA binding (IC50 congruent  3 µM), unlabeled SPP competed less effectively. At 100 µM SPP, 10 nM [3H]LPA binding was competed ~47% (Fig. 3A). However, a 15-min pretreatment at 37 °C with LPA or SPP, followed by wash out and [3H]LPA binding analysis at 4 °C indicated that both LPA and SPP inhibited subsequent LPA binding to HEK293-EDG-1 cells (Fig. 3B). This suggests that both LPA and SPP are capable of binding to EDG-1.


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Fig. 3.   Interaction of SPP and LPA with EDG-1. A, competition of [3H]LPA binding by LPA and SPP in HEK293-EDG-1 cells. B, SPP desensitizes [3H]LPA-binding sites. HEK293-EDG-1 cells were treated with the indicated concentrations of lipids for 15 min at 37 °C, washed, and the [3H]LPA binding was conducted with 10 nM radiolabeled LPA as described. Note that both LPA and SPP, in a dose-dependent manner, inhibited [3H]LPA-binding sites in HEK293-EDG-1 cells. SPM, sphingomyelin (10 µM); LPS, 10 µM.

Next, we investigated whether LPA can signal via the EDG-1 receptor. As shown previously (2, 3), the activation of EDG-1 transduces two distinct intracellular signaling pathways: the Gi-linked MAP kinase pathway and Rho-dependent morphogenetic differentiation (3). LPA is known to activate MAP kinase via a Gi-coupled mechanism (25). We therefore tested whether LPA binding to EDG-1 also activates MAP kinase. The EDG-1 cDNA was co-transfected into COS-7 cells with the HA epitope-tagged ERK-2 construct (2) and stimulated with LPA. As shown in Fig. 4A, LPA-induced MAP kinase activity was greatly potentiated by EDG-1 expression. LPA induced approximately 2-3-fold increase of MAP kinase activity in the absence of EDG-1 whereas it induced more than a 10-fold increase in MAP kinase activity when EDG-1 was overexpressed. This assay was also conducted in HEL92.1.7 cells which do not respond to LPA. LPA treatment markably activates MAP kinase in HEL 92.1.7 cells overexpressed EDG-1 GPR, and this activation is completely inhibited in the presence of pertussis toxin (Fig. 4B). Together, these data show that LPA activates MAP kinase via the EDG-1 GPR and Gi pathway.


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Fig. 4.   LPA-induced MAP kinase activation in EDG-1-transfected cells. A, upper panel, COS-7 cells were co-transfected with varying amounts (0-2 µg) of EDG-1 expression vector and a fixed amount of HA-ERK-2 plasmid (0.1 µg), stimulated with LPA for 5 min, and HA-ERK-2 activity was assayed as described. In a parallel experiment, extracts (50 µg) of transfected cells were immunoblotted with anti-HA (middle panel) or anti-M2 (lower panel) to measure the expression of HA-ERK-2 or EDG-1 polypeptides, respectively. B, HEL 92.1.7 cells (8 × 106 cells) were transfected with either control (control) or EDG-1 (EDG-1) vectors (4 µg), plus HA-ERK-2 plasmid (0.4 µg). Twenty-four hours later, cells were starved in serum-free medium in the presence or absence of pertussis toxin (200 ng/ml) for overnight. Subsequently, cells were stimulated with LPA (2.5 µM) for 5 min and MAP kinase activity was measured. Autoradiogram shows the phosphorylated myelin basic protein. Fold activation was derived from the densitometric scans of the autoradiograph.

Ligand binding to GPR results in the rapid activation of intracellular signaling components, which is accompanied by the phosphorylation of the cytosolic domains of the receptor (26). Many cytosolic kinases, such as the beta -adrenergic receptor kinase family, participate in the ligand-activated receptor phosphorylation responses (27). Such responses are thought to be important in GPR desensitization (26, 27). We next investigated if LPA is capable of inducing phosphorylation of EDG-1 GPR. As shown in Fig. 5A, LPA stimulated EDG-1 phosphorylation whereas the biologically inactive lipids such as lysophosphatidylinositol did not. SPP, which is a high affinity agonist for EDG-1, induces EDG-1 phosphorylation more potently. Also, FBS, which contains LPA and SPP, stimulates the EDG-1 phosphorylation. The deduced molecular mass of nascent EDG-1 polypeptide is approximately 42 kDa. Interestingly, ligand stimulation results in immunoreactive EDG-1 polypeptides which migrate in SDS-PAGE at the approximate 52-kDa position (Fig. 5, A and B). This is most likely due to the combination of glycosylation and phosphorylation of EDG-1, since deglycosylation or dephosphorylation of EDG-1 results in mobility shift to the approximate 42-kDa position (Fig. 5B).


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Fig. 5.   Ligand-induced EDG-1 phosphorylation. A, HEK 293 cells (EDG-1 or pCDNA) were made quiescent in 10% CFBS for 3 days, metabolically labeled with [32P]orthophosphate, and stimulated for 5 min with the indicated lipids (20 µM) or 10% FBS. Cell extracts were immunoprecipitated with the anti-M2 antibody and analyzed by SDS-PAGE and autoradiography. CFBS, 10% FBS; FBS, 10%; LPI, lysophosphatidylinositol; SPM, sphingomyelin. pCDNA, control HEK293-pCDNA cells; EDG-1, HEK293-EDG-1 cells. B, glycosylation and phosphorylation of EDG-1 receptor. EDG-1 polypeptides were immunoprecipitated from HEK293-EDG-1 cells stimulated without or with 0.5 µM SPP for 5 min. The EDG-1 immunoprecipitates were then deglycosylated and dephosphorylated with endoglycosidase H (Endo H) or alkaline phosphatase (AP). Note that ligand stimulation results in the phosphorylation and glycosylation of EDG-1 which migrates at approximately 52 kDa (**, indicated by arrow), whereas deglycosylated/dephosphorylated EDG-1 migrates at approximately 42 kDa (*, indicated by arrow). P, control HEK293-pCDNA cells; EDG-1, HEK293-EDG-1 cells.

The activation of EDG-1 specifically induces Rho-dependent morphogenetic differentiation and up-regulation of the P-cadherin polypeptide (3). We therefore examined if LPA is capable of inducing HEK293-EDG-1 cell differentiation. As shown in Fig. 6A, LPA induced morphogenetic differentiation in HEK293-EDG-1 cells and not in vector-transfected counterparts. LPA-induced morphogenetic differentiation was suppressed by co-incubation with the anti-M2 antibody, which binds to the N-terminal FLAG epitope on the EDG-1 receptor (Fig. 6A); and irrelevant antibodies did not influence morphogenetic differentiation. Other control lysophospholipids (i.e. lysophosphatidylserine (LPS), lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylcholine) at doses ranging from 1 to 50 µM were ineffective. In addition, phospholipase B treatment of FBS, which hydrolyzes the sn-1 fatty ester bond of phospholipids, destroyed the differentiation inducing activity of FBS (Fig. 6B). In contrast, phospholipase A2 or heat-denatured phospholipase B treatment of FBS did not influence the differentiation inducing activity. These data support the notion that LPA is an important component of FBS in inducing EDG-1-dependent morphogenetic differentiation.


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Fig. 6.   LPA induces morphogenetic differentiation in HEK293-EDG-1 cells. A, HEK293-EDG-1 (left column) and HEK293-pCDNA (right column) cells were plated in the indicated medium (DMEM plus indicated supplements) and phase-contrast photographs of cellular morphology are shown. Control, 10% lipid-depleted, charcoal-stripped FBS (CFBS); LPA, 5-50 µM LPA in CFBS; anti-M2, anti-FLAG antibody (50 µg/ml) in 20 µM LPA; mIgG, irrelevant mouse IgG (50 µg/ml) in 20 µM LPA; LPS, 20 µM. Note that LPA induced morphogenetic differentiation specific in HEK293-EDG-1 cells. The scale bar represents 86 µm. B, Phospholipase B treatment destroys the FBS-derived differentiation inducing activity. Active, FBS was incubated with 1 unit of phospholipase A2 (PLA2) or phospholipase B (PLB) at 25 °C for 16 h; boiled, boiled enzymes (95 °C, 30 min) were used as controls. Treated FBS were used at the concentration of 10% (v/v) in the morphogenetic differentiation assay using HEK293-EDG-1 cells as described. The scale bar represents 92 µm. C, HEK293-EDG-1 cells were plated in DMEM containing 10% CFBS supplemented with 20 µM LPA. For C3 treatment, cells were pretreated for 48 h with 5 µg/ml toxin prior to the morphogenetic assay. The scale bar represents 85 µm. D, top panel, HEK293-EDG-1 cells were treated with indicated concentrations of LPA in medium containing 10% charcoal-stripped FBS (CFBS). Bottom panel, HEK293-EDG-1 cells were plated in 20 µM LPA in 10% CFBS. Some wells were received indicated amounts of anti-M2 antibody or the mouse IgG (mIgG) (µg/ml) or the C3 exotoxin. Cell extracts were analyzed for the P-cadherin expression by immunoblot analysis. Data are from a representative experiment which was repeated at least two times.

In addition, LPA-induced morphogenetic differentiation was blocked by pretreating the cells with the C3 exotoxin (Fig. 6C). Also, LPA treatment, in a dose-dependent manner up-regulated P-cadherin levels, which is inhibited by C3 exotoxin (Fig. 6D). Furthermore, consistent with the inhibition of [3H]LPA binding by anti-M2 (Fig. 2D), the ability of LPA to induce P-cadherin in HEK293-EDG-1 cells was specifically blocked by the anti-M2 antibody, but not by an irrelevant antibody (Fig. 6D). Together, these data strongly argue that LPA binding to EDG-1 is able to activate the Rho pathway to regulate P-cadherin polypeptides expression.

Recently, EDG-2/Vzg-1, which is highly related to EDG-1, was shown to bind to LPA and transduce signals for cell rounding via a non-Gi/Go signaling pathway (20). As shown in Fig. 7A, EDG-2/Vzg-1 mRNA was detected in parental HEK293 cells and in a variety of other cell types including human endothelial cells, fibroblasts, but not vascular smooth muscle cells. Previously we showed that human endothelial cells contain high levels of the EDG-1 mRNA but other cell types contain low but detectable levels of EDG-1 mRNA (1). HEK293 cells contain extremely low levels of EDG-1 mRNA; poly(A)+ Northern analysis (Fig. 7B) indicates undetectable levels of EDG-1 mRNA in parental and HEK293-pCDNA cells (Fig. 7B). In contrast, HEK293-EDG-1 cells contain extremely high levels of transfected EDG-1 mRNA. LPA-induced cell rounding was observed in HEK293-pCDNA as well as HEK293-EDG-1 cells (Fig. 7C). These observations are also consistent with a previous report (20) that EDG-2/Vzg-1 confers LPA-induced cell rounding at nanomolar concentrations of LPA and that LPA binds to EDG-2/Vzg-1. In contrast, LPA-induced morphogenetic differentiation was observed only in HEK293-EDG-1 cells (Fig. 6), suggesting that LPA/EDG-1 interaction specifically induces cell differentiation.


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Fig. 7.   LPA induces cell rounding in both HEK293-EDG-1 and HEK293-pCDNA cells. A, expression of EDG-2/Vzg-1 mRNA. Reverse transcriptase-polymerase chain reaction analysis of EDG-2/Vzg-1 mRNA of cultured cells. E, human endothelial cells; S, human vascular smooth muscle cells; F, human fibroblasts; ecv, immortalized human endothelial cells; 293, HEK293 cells; Mr, 1-kilobase ladder DNA marker. The amplified band at 380 base pairs is reflective of the EDG-2/Vzg-1 mRNA expression (indicated by arrowhead). B, Northern blot analysis of EDG-1 mRNA. Top panel, poly(A)+ RNAs (1 µg) from HEK293 parental, HEK293-pCDNA, and HEK293-EDG-1 cells were analyzed for EDG-1 mRNA expression. Total RNA (5 µg) from HUVEC cells were used as endogenous EDG-1 message control. Bottom panel, a parallel filter was blotted with [32P]cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to show the equivalent RNA loading. C, induction of cell rounding by LPA treatment. HEK293-pCDNA and HEK293-EDG-1 cells were treated with 1 µM LPA for 2 min. Phase-contrast photographs of cellular morphology indicates that LPA induces rapid cell rounding in both cell types. This phenomenon was previously shown to be mediated by EDG-2/Vzg-1 (20). Arrows indicate cellular processes and extensions which retracted after LPA treatment. NT, cells before LPA treatment. As controls, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylcholine were unable to induce cell rounding for up to 10 min. The scale bar represents 12.5 µm.

    DISCUSSION
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In this report, we show that: (i) LPA binding is enhanced in EDG-1-transfected cells and the enhancement is suppressed specifically by anti-FLAG antibody (Fig. 2, A-D). (ii) LPA is able to activate MAP kinase via the EDG-1 GPR (Fig. 4). (iii) LPA induces rapid phosphorylation of EDG-1 receptor (Fig. 5). (iv) LPA induces, in a Rho-dependent manner, morphogenetic differentiation and P-cadherin expression in HEK293-EDG-1 (Fig. 6). Collectively, these data indicate that LPA directly binds to EDG-1 and activates EDG-1-mediated signaling events, and therefore suggests that EDG-1 can serve as a plasma membrane-localized receptor for the serum-borne bioactive lipid LPA.

Furthermore, Scatchard analysis indicates that there are two types of LPA-binding sites present in HEK293-EDG-1 cells (apparent Kd values = 178 nM and 2.3 µM), whereas a single type of binding site present in parental HEK293 cells (apparent Kd = 212 nM) (Fig. 2E). It is possible that the Bmax values for LPA-binding sites may be an overestimated due to the lipophilic nature of LPA. Nevertheless, these data suggest that EDG-1 is a low affinity receptor for LPA. In agreement with this notion, micromolar LPA is required to induce EDG-1-mediated differentiation (Fig. 6A), P-cadherin induction (Fig. 6D), and MAP kinase activation (Fig. 4). SPP is more potent than LPA in inducing EDG-1 signaling (3). The physiological concentration of LPA in serum was estimated to be approximately 5 µM (28). Therefore, the apparent Kd is well within in vivo concentrations. Thus, EDG-1 might be activated by LPA under physiological or pathophysiological conditions.

Phospholipase B treatment (which is specific for glycerol-based lipids and not sphingoid-based lipids) destroyed the differentiation inducing activity of serum (Fig. 6B), suggesting that LPA is an important component of serum required for EDG-1-dependent morphogenetic differentiation. However, it requires 20-40 times more LPA to mimic the effect induced by 10% serum (Fig. 6A). Moreover, we were unable to completely reconstitute the differentiation inducing activity of serum by adding 1 µM LPA and SPP together (data not shown). Thus, there may be unidentified factor(s) present in serum which may induce EDG-1-mediated differentiation synergistically with serum-borne SPP and LPA, or stabilize the serum-borne SPP and LPA. In agreement, a recent report suggest that LPA induced Rho activation and cytoskeletal changes require the activity of the EGF receptor (29).

Data presented in this report suggest that EDG-1, a high affinity receptor for SPP (3), is a low affinity receptor for LPA. Intriguingly, the binding of [3H]LPA was competed poorly by unlabeled SPP (Fig. 3A). However, pretreatment of HEK293-EDG-1 at 37 °C for 15 min with SPP or LPA, but not with control lipids (e.g. LPS and SPM), reduced the subsequent [3H]LPA binding in a dose-dependent manner (Fig. 3C). Previously, we have shown that SPP treatment resulted in EDG-1 GPR trafficking into intracellular compartments (3). One plausible explanation for these observations is that SPP and LPA bind to EDG-1 at distinct sites. However, this assumption awaits definitive assignment of binding sites by site-directed mutagenesis studies of EDG-1. A common binding site for SPP/LPA was described in human platelets (11). It should be noted that EDG-1 possesses distinct properties than the platelet SPP/LPA receptor. First, SPP and LPA bind to a common site on platelets (11), whereas SPP and LPA may bind to two distinct sites on EDG-1. Second, EDG-1 has a single type of binding site for SPP (apparent Kd = 8.1 nM), whereas platelets have two types of binding sites for SPP (Kd = 110 nM and 2.6 µM).

Recently, EDG-2/Vzg-1, which is highly related to EDG-1 (40% sequence identity over 309 residues), was shown to bind to LPA and transduce signals for cell rounding via a non-Gi/Go pathway (20). Two independent reports confirmed that EDG-2/Vzg-1 indeed respond to LPA (23, 30). Indeed, we observed that parental HEK293 cells express the EDG-2/Vzg-1 mRNA (Fig. 7). Also, specific [3H]LPA binding (Fig. 2) and LPA-induced cell rounding (Fig. 7) was observed in these cells. Therefore, the high affinity LPA-binding site in HEK293 may be encoded by EDG-2/Vzg-1 mRNA. In this report, we show that LPA-induced morphogenetic differentiation and P-cadherin expression was observed only in HEK293-EDG-1 cells, indicating that these signals are EDG-1 specific. Collectively, these data support the concept that multiple receptors exit for LPA to mediate its pleiotropic functions (22).

LPA and its structural analogs such as SPP activate multiple signaling pathways: such as Gi-dependent MAP kinase activation (22, 31), Rho-dependent stress fiber, and focal adhesion assembly (17). Involvement of these signaling pathways in EDG-1 induced morphogenetic differentiation was determined by treating with pertussis toxin, an inhibitor of Gi (32), PD 98059, a selective inhibitor of MAP kinase kinase (33), and C3 exoenzyme, an inhibitor of the small G-protein Rho (4). However, EDG-1-induced morphogenetic differentiation was blocked by C3 exoenzyme (Fig. 6C) but not by pertussis toxin or PD 98059,2 suggesting a specific requirement for Rho-regulated signaling pathways. The small G-protein Rho binds to several protein kinases and regulate gene expression (34), cytoskeletal structure (35), and cell-cycle (36). Our finding that LPA/EDG-1/Rho signaling regulates P-cadherin expression is novel; however, the molecular details of the pathway from EDG-1 to P-cadherin expression via Rho remains to be defined.

Both SPP and LPA are released by activated platelets (5, 13). EDG-1 was originally cloned as an immediate-early gene from phorbol ester-induced differentiating endothelial cells (1). Indeed, we named this orphan receptor, endothelial differentiation gene (EDG)-1 because of our original hypothesis that this receptor mediates the morphogenetic differentiation of endothelial cells into capillary-like structures (1). Since platelet activation and thrombus formation usually accompany angiogenesis (37), SPP and LPA released from platelets may activate endothelial cells to undergo angiogenesis via the EDG-1 receptor. However, whether coupling of EDG-1 GPR signaling with cadherin-based signaling events is a mechanism to achieve regulated morphogenesis, a critical event in endothelial cell differentiation awaits future study. A recent report indicated LPA stabilized endothelial junctions and decreased permeability (38). Also, the role of EDG-1 and its ligands SPP and LPA in the vascular system needs to be addressed in the future.

In conclusion, our data suggest that EDG-1 is a low affinity LPA receptor. Furthermore, our data support the concept that multiple receptors exist for LPA each of which regulate specific biological functions. Third, EDG-1 represents a GPR which interacts with both LPA and SPP, albeit at different affinities.

    ACKNOWLEDGEMENTS

We thank Drs. Sarah Spiegel and James R. van Brocklyn for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK45659 and HL49094 (to T. H.).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.

Dagger To whom correspondence should be addressed: Dept. of Physiology, MC3505, University of Connecticut School of Medicine, 263 Farmington Ave., Farmington, CT 06030. E-mail: hla{at}sun.uchc.edu; Fax: 860-679-1269.

The abbreviations used are: GPR, G-protein-coupled receptor; EDG-1, endothelial differentiation gene-1; MAP kinase, mitogen-activated protein kinase; FBS, fetal bovine serum; CFBS, charcoal-stripped FBS; SPP, sphingosine 1-phosphate; SPC, sphingosylphosphoryl choline; SPM, sphingomyelin; LPA, lysophosphatidic acid; LPS, lysophosphatidylserine; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid .

2 M.-J. Lee, S. Thangada, C. H. Liu, B. D. Thompson, and T. Hla, unpublished observations.

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Discussion
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