Nrg-1 Belongs to the Endothelial Differentiation Gene Family of G Protein-coupled Sphingosine-1-phosphate Receptors*

Renae L. MalekDagger , Rachelle E. Toman§, Lisa C. Edsall§, Sylvia WongDagger , Jeffrey ChiuDagger , Catherine A. Letterle||, James R. Van Brocklyn||, Sheldon Milstien**, Sarah Spiegel§, and Norman H. LeeDagger DaggerDagger

From the Dagger  Department of Molecular and Cellular Biology, Institute for Genomic Research, Rockville, Maryland 20850, the § Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, the || Department of Pathology, Ohio State University, Columbus, Ohio 43210, the  Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, D.C. 20007, and the ** Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892

Received for publication, May 10, 2000, and in revised form, October 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The previously cloned rat nerve growth factor-regulated G protein-coupled receptor NRG-1 (Glickman, M., Malek, R. L., Kwitek-Black, A. E., Jacob, H. J., and Lee N. H. (1999) Mol. Cell. Neurosci. 14, 141-52), also known as EDG-8, binds sphingosine-1-phosphate (S1P) with high affinity and specificity. In this paper we examined the signal transduction pathways regulated by the binding of S1P to EDG-8. In Chinese hamster ovary cells heterologously expressing EDG-8, S1P inhibited forskolin-induced cAMP accumulation and activated c-Jun NH2-terminal kinase. Surprisingly, S1P inhibited serum-induced activation of extracellular regulated protein kinase 1 and 2 (ERK1/2). Treatment with pertussis toxin, which ADP-ribosylates and inactivates Gi, blocked S1P-mediated inhibition of cAMP accumulation, but had no effect on c-Jun NH2-terminal kinase activation or inhibition of ERK1/2. The inhibitory effect of S1P on ERK1/2 activity was abolished by treatment with orthovanadate, suggesting the involvement of a tyrosine phosphatase. A subunit selective [35S] guanosine 5'-3-O-(thio)triphosphate binding assay demonstrates that EDG-8 activated Gi/o and G12 but not Gs and Gq/11 in response to S1P. In agreement, EDG-8 did not stimulate phosphoinositide turnover or cAMP accumulation. The ability of S1P to induce mitogenesis in cells expressing the EDG-1 subfamily of G protein-coupled receptors is well characterized. In contrast, S1P inhibited proliferation in Chinese hamster ovary cells expressing EDG-8 but not empty vector. The antiproliferative effect, like S1P-mediated ERK1/2 inhibition, was orthovanadate-sensitive and pertussis toxin-insensitive. Our results indicate that EDG-8, a member of the EDG-1 subfamily, couples to unique signaling pathways.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lysophospholipids sphingosine-1-phosphate (S1P)1 and lysophosphatidic acid (LPA) are endogenous ligands of the EDG family of G protein-coupled receptors (GPCRs) (1-3). Presently, eight EDG receptors have been cloned and are divided into two or three homology clusters or subfamilies (4-13). EDG-1, -3, -5, and -8 exhibit high sequence homology to one another and have high affinity for S1P (14-17). EDG-2, -4, and -7 form a second cluster and are high affinity receptors for LPA (1, 5, 8, 18). EDG-6 displays intermediate homology between the two clusters and binds S1P with moderate to high affinity (7, 19).

S1P-activated EDG receptors are coupled to multiple effector pathways, including activation/inhibition of adenylyl cyclase, stimulation of phosphoinositide (PI) hydrolysis, mobilization of Ca2+ (20-26), induction of DNA synthesis (27-29), and stimulation of the MAP kinase family members ERK1/2, SAPK/JNK, and p38 (17, 20-24). For example, activation of EDG-1, -3, or -5 by S1P leads to a pertussis toxin (PTX)-sensitive stimulation of ERK1/2 (17, 20-22, 24), suggesting the involvement of Gi/o proteins. In contrast, EDG-5 stimulates both stress-activated protein kinases, SAPK/JNK and p38, in a PTX-insensitive manner (23), indicating that EDG receptor coupling to MAP kinase family members involves PTX-sensitive and -insensitive G proteins. In addition to the differential coupling of G proteins and EDG receptors to MAP kinases, there exists differential coupling to the adenylyl cyclase-cAMP and phospholipase C-Ca2+ systems (20-26). EDG-1 couples exclusively to Gi, whereas EDG-3 and EDG-5, in addition to Gi, couple to Gq/11 and G13 (25, 30).

Although the biological functions of the EDG-1 subfamily of receptors are not well understood, binding of S1P to EDG-1 and -5 has been recently shown to regulate migration (15, 17, 25, 29) and angiogenesis (29, 31). More recently, EDG-1, -3, and -5 have been linked to induction of cell proliferation (32). Interestingly, EDG-8 is closely related to EDG-5, exhibiting 40-44% sequence identity. Edg-8, which was initially cloned from rat pheochromocytoma cells as the nerve growth factor-regulated orphan GPCR nrg-1 (6, 33), has been shown to be in close proximity to edg-5 on rat chromosome 8 (6), possibly because of a gene duplication event. Thus, it is of great interest to examine the signaling pathways regulated by S1P binding to EDG-8 and its potential biological consequences. Unexpectedly we found that EDG-8 is mainly coupled to Gi/o and G12 and inhibits, rather than stimulates, ERK activation in CHO cells. Moreover, S1P markedly inhibits proliferation in CHO cells overexpressing EDG-8 in a Gi/o-independent manner.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
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Materials-- CHO cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in Ham's F-12 medium containing 10% charcoal-treated dialyzed fetal bovine serum (Life Technologies, Inc.) as described previously (33-35). Dialyzed serum treated with activated charcoal removes all contaminating lipids such as S1P (33-35) and was used in all experiments involving serum stimulation. Pertussis toxin, isobutylmethylxanthine, and forskolin were from Sigma. S1P and Galpha s polyclonal antibody were from Calbiochem (San Diego, CA). Lipids other than S1P, which was from Biomol (Plymouth Meeting, PA), were from Avanti Polar Lipids (Birmingham, AL). Phospho-JNK MAP kinase (G-7), c-Myc (9E10), and HA probe (Y-11), Galpha q/11, Galpha i/o, Galpha 12 and Galpha 13 antibodies and GST-ATF-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-ERK1/2, phospho-p38, ERK1/2, SAPK/JNK, and p38 MAP kinase antibodies and Elk-1 and c-Jun fusion proteins were from New England Biolabs (Beverly, MA). Goat anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Upstate Biotechnology (Lake Placid, NY).

Transfection of Cells with HA-tagged EDG-8-- A polymerase chain reaction strategy was used to insert a 9-amino acid HA epitope sequence (YPYDVPDYASL) into the COOH terminus of EDG-8. The sense and antisense polymerase chain reaction primers were 5'-aaaaaagcttCAAGGTTCGCATAT AGACCAG-3' and 5'aaaactcgagCTAAGCATAATCTGGAACATCATATGGATAGTCTGTAGCATCAGGCACCAG-3', respectively. The sense primer has 21 nucleotides of 5'-untranslated region from the rat EDG-8 cDNA (6). The antisense primer has 27 nucleotides coding for the HA epitope and 7 codons from the COOH terminus of EDG-8 (bold type). Polymerase chain reaction products were cloned into the HindIII and XhoI cloning sites of the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA), generating HAedg8pcDNA3. The fidelity of the plasmid construct was verified by sequencing. CHO cells were either transiently (2 days) or stably transfected with HAedg8pcDNA3 using LipofectAMINE 2000 (Life Technologies, Inc.). Expression of EDG-8 in transfected cells was verified by immunoblotting using the HA polyclonal antibody.

[32P]S1P Binding-- [32P]S1P was prepared as described previously (36). CHO cells seeded at a density of 75,000 cells/cm2 were transiently transfected with HAedg8pcDNA (1 µg/well) and incubated with the indicated concentrations of [32P]S1P. Unlabeled lipid competitors were added as 4 mg/ml fatty acid-free bovine serum albumin complexes, and bound [32P]S1P was quantitated by scintillation counting as described previously (36).

Measurement of cAMP Production-- CHO cells were grown in 12-well plates, transiently transfected with HAedg8pcDNA3 or the human beta 2-adrenergic receptor cDNA in pSVL (37), washed with phosphate-buffered saline, and pretreated with 0.5 mM isobutylmethylxanthine for 20 min at 37 °C. To measure inhibition of cAMP formation, EDG-8-expressing cells were incubated with 10 µM forskolin in the presence of the indicated concentrations of S1P for 20 min, and cAMP levels were measured as described (38). Measurement of stimulation of cAMP accumulation was performed as above in the absence of forskolin. cAMP accumulation in beta 2-adrenergic receptor-expressing cells was induced with isoproterenol (2 µM; 20 min) and served as a positive control.

Measurement of PI Turnover-- CHO cells were grown in 12-well plates, transiently transfected with HAedg8pcDNA3 or Rm1pcDNA3 (rat M1 muscarinic receptor cDNA in pcDNA3; Ref. 38), and washed with phosphate buffered saline and pretreated with 10 mM LiCl for 30 min at 37 °C. EDG-8-expressing cells were incubated with the indicated concentrations of S1P for 20 min, and PI turnover was measured as described (38). PI turnover in M1 muscarinic receptor-expressing cells was stimulated with carbachol (1 mM; 20 min) and served as a positive control.

SDS-PAGE and Immunoblotting-- Transiently transfected CHO cells were grown in 35-mm tissue culture dishes, treated with the indicated lipids, and harvested/lysed in 95 °C SDS-PAGE sample buffer as described previously (34). Proteins were separated on 10% polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Blots were probed with phosphorylation state-specific MAP kinase primary antibodies. Goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies allowed detection of proteins by the ECL Plus Detection System (Amersham Pharmacia Biotech). Fluorograms were quantitated by densitometry. Blots were stripped, reprobed with primary antibodies that recognize MAP kinases independently of phosphorylation state, and quantitated. Data are expressed as the means ± S.E. of n independent experiments.

Immunocomplex Kinase Assay-- Transiently transfected CHO cells were treated with the indicated lipids, washed in ice-cold phosphate-buffered saline, lysed in IP Buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P-40, 1% Triton X-100, 2 mM sodium orthovanadate, 20 µg/ml aprotinin, 5 µg/ml leupeptin, 50 mM sodium fluoride), and disrupted by aspiration through a 21-gauge needle (35). Cell debris was removed by centrifugation. Supernatants were incubated with MAP kinase antibody for 2 h at 4 °C. Immunocomplexes were precipitated with IP Buffer equilibrated protein A-agarose (Sigma) for 2 h at 4 °C, washed three times with IP Buffer, washed twice with kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 25 mM sodium beta -glycerophosphate, 2 mM sodium orthovanadate, 0.5 mM dithiothreitol), and resuspended in kinase buffer containing 100 µM ATP, 5 µCi of [gamma -32P]ATP. For measurement of ERK1/2, SAPK/JNK, or p38 MAP kinase activities, immunocomplexes were incubated with 2 µg of Elk-1 fusion protein, c-Jun fusion protein, or GST-ATF-2 substrate, respectively. Reactions were incubated 30 min at 30 °C and terminated by the addition of 2× SDS-PAGE loading buffer. Proteins were separated on a 10% SDS-PAGE gel and analyzed by autoradiography.

[35S]GTPgamma S Binding Assay-- CHO cells transfected with HAedg8pcDNA or vector alone were harvested and homogenized in ice-cold 20 mM HEPES, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin by 15 passages through a 25-gauge needle. Homogenates were centrifuged at 1,000 × g for 5 min, and the resulting supernatant was centrifuged at 25,000 × g for 30 min to obtain the membrane fraction. Membranes (20 µg of protein/assay point) were resuspended in 50 µl of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 µM GDP and 3 mM MgCl2 and incubated with 1 µM S1P or vehicle for 10 min at 30 °C. Subsequently, 30 nM [35S]GTPgamma S (1300 Ci/mmol; PerkinElmer Life Sciences) was added, and membranes were incubated for an additional 10 min at 30 °C. Incubations were terminated by adding 500 µl of ice-cold 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM MgCl2, 0.5% Nonidet P-40 (Calbiochem), 10 µg/ml aprotinin, 100 µM GDP, and 100 µM GTP. After 30 min, solubilized extracts were incubated for 1 h at 4 °C with 10 µl of a Galpha subunit-selective antibody. (Santa Cruz). Nonspecific binding was determined by immunoprecipitation with nonimmune serum. Bound [35S]GTPgamma S was quantitated by scintillation spectrometry. Nonspecific binding typically represented 10-20% of total binding and was subtracted from the latter.

Cell Proliferation Assays-- Cell proliferation was measured as previously described (39). CHO cells (5,000 cells) were transfected with HAedg8pcDNA or vector alone and plated in 12-well plates containing Ham's F-12 medium supplemented with 10% charcoal-treated dialyzed fetal bovine serum. After 18 h (t = 0), cells were washed twice with Ham's F-12 and grown in Ham's F-12 without serum to measure proliferative effects following S1P treatment or Ham's F-12 with 10% charcoal-treated dialyzed fetal bovine serum to measure the inhibitory effects of S1P on proliferation. S1P and inhibitors were added at the indicated concentrations. After 48 h, cells were washed with phosphate-buffered saline, fixed with 70% ethanol for 10 min, and stained with crystal violet. Incorporated dye was dissolved in 0.1 M sodium citrate in 50% ethanol, pH 4.2, and the absorbance was measured at 540 nm. In parallel experiments, cells were trypsinized and counted in a hemocytometer. Cell numbers in four random microscopic fields were counted from each well. Measurements from the two methods gave identical results. Each determination represents the mean ± S.E. of three to four individual wells.


    RESULTS
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ABSTRACT
INTRODUCTION
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[32P]SIP Binds to EDG-8-- Many cell lines commonly employed for heterologous expression of receptor genes, including COS and HEK293 cells, respond to S1P (40). In contrast, previous reports have demonstrated that native CHO cells, while expressing detectable levels of EDG-5 mRNA (41), do not bind or readily respond to S1P (20, 23, 25). In agreement with these studies, we found that native CHO cells transfected with vector exhibited negligible binding of [32P]S1P, as shown by the absence of significant correlation between B and B/F in the Scatchard plot (Fig. 1, left panel). Therefore, CHO cells were subsequently employed for the analysis of EDG-8 function (Tables I and II). Following transient transfection with an expression vector for HA-tagged EDG-8, CHO cells specifically bound [32P]S1P with high affinity (Kd = 6 ± 4 nM; n = 3 independent experiments; Fig. 1, left panel). This Kd value is in excellent agreement with a recent study performed in HEK293T cells (14), demonstrating that EDG-8 is indeed a high affinity receptor for S1P in diverse cell types. The level of expression of EDG-8 in CHO cells based on [32P]S1P binding was comparable with that seen in these cells transfected with EDG-1, -3, and -5 (25). In agreement with a recent report in HEK293T cells (14), only unlabeled S1P and dihydro-S1P effectively competed with [32P]S1P for binding to EDG-8 (Fig. 1, right panel), whereas LPA, sphingosine, and SPC had no significant effects.



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Fig. 1.   S1P binding to EDG-8. Specific binding of [32P]S1P to CHO cells transiently transfected with EDG-8 () or Vector (). Left panel, cells were incubated with increasing concentrations of [32P]S1P in the absence or presence of 10 µM unlabeled S1P to define nonspecific binding. A representative Scatchard transformation of the binding data is shown where B is the specific [32P]S1P binding (fmol/106 cells) and F is the unbound concentration of [32P]S1P (nM). The binding dissociation constant (Kd) and maximal binding capacity (Bmax) are 10 nM and 225 fmol/106 cells, respectively. There was no significant correlation between B and B/F in vector-transfected cells. The correlation coefficient in vector and EDG-8-transfected cells was 0.06 and 0.9, respectively. Displacement [32P]S1P binding studies. Right panel, transiently transfected CHO cells were incubated with 1 nM [32P]S1P in the absence or presence of 1 µM of the indicated lipid. The amount of bound [32P]S1P was determined as described under "Experimental Procedures" and is plotted as the percentage of total binding. Results are the means ± S.D. of three independent experiments. Con, control cells with no competitive lipid; dh-S1P, dihydro-S1P; Sph, sphingosine. An asterisk indicates statistically significant difference compared with cells incubated with [32P]S1P alone as determined by Student's t test (p < 0.05).


                              
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Table I
EDG receptor effector pathways


                              
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Table II
Differential coupling of EDG receptors to heterotrimeric G proteins

Effect of EDG-8 on cAMP Formation-- Previously it has been shown that S1P treatment of EDG-transfected CHO cells increases cAMP content in a PTX-insensitive manner with the following rank order of efficacy: EDG-5 > EDG-3 EDG-1 (25). Treatment of CHO cells transiently expressing EDG-8 with S1P (1 to 1000 nM), however, had no effect on intracellular cAMP content (Table I), whereas isoproterenol (2 µM) caused a robust 6-fold increase in cAMP levels in CHO cells expressing the beta 2-adrenergic receptor. To assess the ability of EDG-8 to mediate inhibition of cAMP accumulation, cAMP levels were increased by treatment with forskolin. Addition of S1P resulted in a dose-dependent, PTX-sensitive, decrease in forskolin-induced cAMP accumulation (Fig. 2A), whereas in empty vector-transfected cells, S1P, even at a concentration as high as 1 µM, had no significant effect on forskolin-induced cAMP accumulation (Fig. 2B). Similar to S1P, dihydro-S1P (1 µM) effectively inhibited cAMP formation, whereas low concentrations of LPA, sphingosine, and SPC (10 to 100 nM) had no significant effect, and only small (22%) inhibition was found at a high concentration of LPA (1 µM) (Fig. 2B).



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Fig. 2.   Inhibition of cAMP accumulation by EDG-8. CHO cells transiently transfected with EDG-8 were pretreated with saline vehicle or PTX (100 ng/ml, 18 h). Cells were pretreated with 0.5 mM isobutylmethylxanthine for 30 m and incubated with the indicated concentrations of S1P (A) or related lipids (B) in the presence of 10 µM forskolin. Results are the means ± S.E. of three to six independent experiments. An asterisk indicates statistically significant difference compared with forskolin treatment in the absence of S1P as determined by Student's t test (p < 0.05). SPC, sphingosylphosphorylcholine; dh-S1P, dihydro-S1P; Sph, sphingosine.

Effect of EDG-8 on PI Turnover-- In CHO cells transfected with EDG-1, -3, or-5, S1P increases intracellular Ca2+ concentrations caused by the activation of phospholipase C (25). In contrast, S1P, at concentrations ranging from 1 nM to 1 µM, did not significantly stimulate PI turnover in EDG-8-expressing cells (Table I), whereas carbachol (1 mM) produced a marked 5 ± 0.9-fold increase in PI turnover in cells transiently expressing the M1-muscarinic receptor known to stimulate phospholipase C via Gq/11 (n = 3 independent experiments).

EDG-8 Mediates Repression of Serum-activated ERK1/2 in a PTX-insensitive but Phosphatase-dependent Manner-- Because several EDG receptors have been reported to activate ERK1/2 (17, 20, 21, 23, 42), we investigated whether S1P-mediated stimulation of EDG-8 led to a similar response. The serine/threonine kinases ERK1/2, SAPK/JNK, and p38 belong to the MAP kinase superfamily, share sequence homology, and are activated upon phosphorylation of homologous threonine and tyrosine residues by dual specificity MAP kinase kinases. Thus, the phosphorylation state of a MAP kinase family member reflects its activation state (43-47).

In serum-starved EDG-8-expressing CHO cells, S1P did not activate ERK1/2 as measured by either Western blot analysis with phosphorylation state-specific antibodies or immunocomplex kinase assay of whole cell lysates (Table I). Even after prolonged incubations of up to 60 min, S1P had no significant effect on ERK activation. Surprisingly, induction of ERK1/2 phosphorylation by serum was repressed in a time-dependent fashion by S1P (1 µM) in CHO cells expressing EDG-8 (Fig. 3A). This inhibitory response was insensitive to PTX (Fig. 3A). Furthermore, serum-induced ERK1/2 phosphorylation was not inhibited by S1P in CHO cells transfected with empty vector (Fig. 3A). Similar to S1P, dihydro-S1P inhibited ERK1/2 phosphorylation by ~40% (Fig. 3B). In agreement with their inability to bind to EDG-8, LPA, SPC, and sphingosine had no effects on ERK phosphorylation induced by serum (Fig. 3B). Similar results were obtained by immunocomplex kinase assays (Fig. 4). Once more, S1P but not LPA repressed serum activation of ERK1/2 by ~50-60%.



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Fig. 3.   EDG-8 mediates repression of ERK1/2 phosphorylation. A, CHO cells transiently transfected with vector (V) or EDG-8 were pretreated with vehicle or PTX (100 ng/ml, 18 h). After serum stimulation (10%; 2 h), cells were incubated with 1 µM S1P for the indicated times. A representative Western blot is shown. Relative phospho-ERK1 and phospho-ERK2 levels were normalized to total ERK2 and plotted as the means ± S.E. of three independent experiments. Expression of EDG-8 in transfected cells was verified using an anti-HA antibody. B, CHO cells transiently transfected with EDG-8 were treated with 1 µM of the indicated lipids (60 min). ERK1/2 phosphorylation was detected in cell lysates using a phosphorylation-specific ERK antibody. EDG-8 expression was monitored using an anti-HA antibody. A representative Western blot is shown from experiments that were repeated at least twice. dh-S1P, dihydro-S1P; Sph, sphingosine.



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Fig. 4.   EDG-8-mediated repression of ERK1/2 activity is dependent on phosphatase activity. A, representative autoradiogram is shown from three to four independent experiments. CHO cells transiently transfected with vector (V) or EDG-8 were pretreated with vehicle, okadaic acid (5 µM; 60 min) or orthovanadate (0.1 mM; 6 h). After serum stimulation (10%; 2 h) cells were incubated with the indicated lipids (1 µM, 60 min). ERK1/2 MAP kinase was immunoprecipitated with an anti-ERK1/2 MAP kinase antibody, and an immunocomplex kinase assay was performed as described under "Experimental Procedures" using an Elk-1 fusion protein as a substrate. EDG-8 expression was verified using an anti-HA antibody. B, ERK activity was expressed as a percentage of vehicle-treated cells (mean ± S.E. of n = 3-4). An asterisk indicates statistically significant difference when compared with untreated EDG-8-transfected cells as determined by Student's t test (p < 0.05). A # indicates statistically significant difference when compared with OA as determined by Student's t test (p < 0.05). OA, okadaic acid; OV, orthovanadate.

In contrast, in CHO cells transiently expressing EDG-1, -3, or -5, S1P did not inhibit serum-induced phosphorylation of ERK1/2 as determined by Western blot analysis, even in cells pretreated with PTX (Table I). Identical results were noted by immunocomplex kinase assays (Table I).

Although the binding of S1P to EDG-1, -3, and -5 did not inhibit ERK activation, this is not unprecedented. It has previously been demonstrated that the GPCR, angiotensin II type 2 (AT2) receptor, inhibits serum-activated ERK1/2 in several cell types (48, 49). This effect was abolished by inhibition of protein phosphatases. Similarly, the ability of S1P to inhibit serum-activated ERK 1/2 in EDG-8-expressing CHO cells was blocked by orthovanadate and okadaic acid, albeit to a lesser extent (Fig. 4). Neither okadaic acid (5 µM) nor orthovanadate (0.1 mM) alone had a significant effect on serum-stimulated ERK1/2 activation (Fig. 4).

EDG-8 Activates SAPK/JNK but Not p38 MAP Kinase-- In serum-starved CHO cells transiently expressing EDG-8, S1P induced a time- (Fig. 5A) and dose-dependent (EC50 ~100 nM; data not shown) activation of JNK as measured by an increase in JNK phosphorylation. Increased JNK phosphorylation was apparent as early as 1 min after treatment with S1P (3 ± 0.2-fold over basal; n = 3 independent experiments) and was sustained for at least 60 min. Remarkably, only the p54 isoform of JNK appears to be phosphorylated in a PTX-independent manner, suggesting that EDG-8 may regulate specific JNK isoforms. S1P-stimulated JNK phosphorylation was not observed in parental CHO cells transfected with empty vector (Fig. 5A). This phosphorylation was associated with a 2.8-fold increase in JNK activity (Fig. 6). Similar to S1P, dihydro-S1P also stimulated p54 JNK phosphorylation 2.4-fold, whereas other lysophospholipids did not have any significant effects (Fig. 5B). In agreement with the Western analyses (Fig. 5B), in vitro kinase assays demonstrated that LPA does not stimulate JNK activity in EDG-8-expressing CHO cells (Fig. 6). In contrast to its effect on JNK activity in EDG-8 overexpressing CHO cells, S1P has no significant effect on p38 phosphorylation (data not shown; 1 µM S1P; 1-60 min). Immunocomplex kinase assays also confirmed the inability of S1P/EDG-8 to induce p38 activity, whereas the positive control arsenite (50 µM; 60 min) potently activated p38 in CHO cells (data not shown).



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Fig. 5.   Stimulation of JNK phosphorylation. A, CHO cells transiently transfected with vector (V) or EDG-8 were serum starved (18 h), pretreated with vehicle or PTX (100 ng/ml, 3 h) and then incubated with 1 µM S1P for the indicated times. A representative Western blot is shown. Relative phospho-p54 levels were normalized to total JNK and plotted as the mean ± S.E. of three independent experiments. EDG-8 expression was verified using an anti-HA antibody. B, CHO cells transiently transfected with EDG-8 were treated with 1 µM of the indicated lipids (60 min). Activation of JNK was determined using a phosphorylation state-specific antibody. An anti-HA antibody was used to monitor EDG-8 expression. A representative Western blot is shown from experiments that were repeated at least twice. dh-S1P, dihydro-S1P; Sph, sphingosine.



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Fig. 6.   EDG-8 activates JNK. Inset, representative autoradiogram is shown from three to four independent experiments. CHO cells transiently transfected with vector (V) or EDG-8 were incubated with the indicated lipids (1 µM, 60 min). JNK MAP kinase was immunoprecipitated with an anti-JNK MAP kinase antibody, and an immunocomplex kinase assay was performed as described under "Experimental Procedures" using a c-Jun fusion protein as a substrate. EDG-8 expression was verified using an anti-HA antibody. JNK activity was expressed as a percentage of vehicle-treated cells (mean ± S.E. of n = 3-4).

EDG-8 Coupling to Individual G Proteins-- Coupling of EDG-8 to endogenous G protein family members was assessed by a [35S]GTPgamma S binding assay (30). COOH-terminal peptide-directed antibodies were used to immunoprecipitate either specific Galpha subunits (e.g. Galpha s, Galpha 12 or Galpha 13) or Galpha family members (e.g. Galpha i/o or Galpha q/11). S1P-stimulated [35S]GTPgamma S binding in the immunoprecipitates was used as a measure of G protein coupling and activation.

As shown in Fig. 7, negligible [35S]GTPgamma S binding was evident for Galpha s, Galpha i/o, Galpha q/11, and Galpha 12 in empty vector-transfected CHO cells treated with S1P. In EDG-8-expressing cells, however, S1P treatment produced a 2-2.5-fold increase in [35S]GTPgamma S binding to the Galpha i/o family of G proteins and Galpha 12 (Fig. 7). Whereas S1P stimulation of EDG-8 activated Galpha i/o and Galpha 12, it had no effect on Galpha s or Galpha q/11 (Fig. 7). In agreement with previous reports (20, 23), CHO cells express Galpha s, Galpha i/o, Galpha q/11, and Galpha 12 proteins (Fig. 7). Thus, the lack of detectable coupling to Galpha s or Galpha q/11 is not due to the absence of these G proteins in CHO cells. Moreover, when membranes from CHO cells expressing the beta 2-adrenergic receptor were incubated with 2 µM isoproterenol, a 2.9 ± 0.4-fold increase in [35S]GTPgamma S binding to Galpha s was observed (n = 2 independent experiments). Likewise, treatment of membranes from M1-muscarinic receptor-expressing CHO cells with carbachol (1 mM; 10 min) resulted in a 2.3 ± 0.1-fold (n = 3 independent experiments) increase in [35S]GTPgamma S binding to Galpha q/11. CHO cells express only trace levels of Galpha 13, for which no activation could be discerned between EDG-8- (1.3 ± 0.2-fold; n = 3 independent experiments) and vector-transfected cells (1.1 ± 0.2-fold; n = 3 independent experiments). Even in CHO cells expressing EDG-5, a known activator of Galpha 13 (30), S1P did not significantly stimulate [35S]GTPgamma S binding to this G protein (1.2 ± 0.2-fold; n = 3 independent experiments).



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Fig. 7.   S1P promotes activation of Gi/o and G12 via EDG-8. Membranes from CHO cells expressing EDG-8 were incubated with [35S]GTPgamma S in the presence of vehicle or S1P (1 µM). Membranes were extracted, and the Galpha subunits were immunoprecipitated with subunit-specific antibodies. Nonspecific binding was determined by immunoprecipitation with nonimmune serum. S1P-induced [35S]GTPgamma S binding was quantitated by scintillation spectrometry and expressed as a percentage of vehicle (percentage of control). Data represent the means ± S.E. of four to five independent experiments. An asterisk indicates statistically significant difference when compared with empty vector-transfected cells as determined by Student's t test (p < 0.05). Inset, membranes from lysates of EDG-8-expressing cells were analyzed by immunotransfer blotting using Galpha subunit-specific antibodies and ECL detection.

EDG-8 Inhibits Cell Proliferation in a PTX-insensitive and Orthovanadate-sensitive Manner-- S1P stimulates cell proliferation via EDG-3, EDG-5, and possibly EDG-1 (27, 29, 42). Thus, it was of great interest to examine whether binding of S1P to EDG-8 also regulates mitogenic pathways. To assess induction of proliferation, stably transfected cells plated in 12-well plates containing serum-free medium were counted after treatment with 1 µM S1P for 48 h. Cell numbers from both control vector and EDG-8 transfectants did not increase in response to S1P (data not shown), suggesting that S1P does not behave as a mitogen at EDG-8.

Interestingly, the proliferative response of EDG-8-transfected CHO cells was 62% lower than vector-transfected cells following mitogenic stimulation with 10% serum for 48 h (Fig. 8). This observation was corroborated by independent experiments, demonstrating that EDG-8-transfected CHO cells exhibit about a 3-fold reduction in [3H]thymidine incorporation compared with untransfected or vector-transfected cells (data not shown). Taken together, our findings suggest that EDG-8 contains intrinsic activity and inhibits serum-stimulated cell proliferation. Furthermore, addition of 1 µM S1P significantly inhibited serum-stimulated proliferation of EDG-8-transfected cells (Fig. 8). The anti-proliferative effect induced by S1P binding to EDG-8 was dose-dependent with an EC50 value around 20 nM (data not shown). In contrast, 1 µM S1P treatment did not inhibit proliferation of vector-transfected cells stimulated with 10% serum (Fig. 8). Because biochemical evidence indicates that EDG-8 couples to Gi/o, we investigated the possibility that Gi/o proteins may be involved in the antiproliferative response induced by S1P. To this end, cells were pretreated with PTX. PTX was completely ineffective in abolishing S1P-induced anti-proliferation (Fig. 8). In contrast, treatment of EDG-8-transfected cells with orthovanadate (1 µM) significantly impaired S1P-induced antiproliferative effects, suggesting that this response is dependent on a protein-tyrosine phosphatase (Fig. 8). Neither PTX nor orthovanadate had deleterious effects on CHO overexpressing EDG-8 cells as demonstrated by normal attachment and survival of the cells after 48 h of treatment (data not shown).



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Fig. 8.   EDG-8 inhibition of proliferation is orthovanadate-sensitive and PTX-insensitive. EDG-8- or vector (V)-transfected CHO cells were plated at low density and washed after 18 h. Cells were then cultured with 10% charcoal-treated dialyzed fetal bovine serum in the presence of 1 µM S1P or vehicle (t = 0). The inhibitors PTX (100 ng/ml) and orthovanadate (2 µM) were added 3 h prior to S1P treatment. After 48 h of S1P or vehicle addition, cell proliferation was quantitated as described under "Experimental Procedures." Data represent the means ± S.E. of three to six independent experiments and are expressed as fold increase in cell number following S1P or vehicle treatment. An asterisk indicates statistically significant difference when compared with transfected cells not treated with S1P as determined by Student's t test (p < 0.05). OV, orthovanadate.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that EDG-8, originally cloned as a GPCR termed NRG-1 (6), binds and is activated by S1P when heterologously expressed in CHO cells. CHO cells were selected to characterize EDG-8 because this cell line does not readily respond to S1P in the absence of exogenous EDG gene transfer (20, 23, 25). Furthermore, many studies relating to EDG-1, -3, and -5 signaling have been performed in CHO cells, thereby providing a common background for direct comparison to EDG-8 signaling (Tables I and II). The affinity of S1P for EDG-8 (Kd = 6 nM) is similar to previously reported affinity values obtained for EDG-1, -3, and -5 (Kd = 8-26 nM) (15, 25). Based upon the lysophospholipid ligands that were tested for their ability to displace [32P]S1P binding, the pharmacological profile of EDG-8 closely resembles other S1P-binding EDG-1 receptors. Moreover, similar to our previous results with EDG-1, -3, and -5, SPC (15, 36) did not compete with [32P]S1P for binding to EDG-8. This is not surprising because recently a unique receptor for SPC, known as ORG1, has been identified (50). In contrast, others have previously demonstrated that SPC activates endogenous Gi in HEK293 cells heterologously expressing EDG-1 (30) and induces Ca2+ mobilization in CHO cells transfected with EDG-5 (23). Taken together, the binding data presented here establish that EDG-8 is a specific S1P and dihydro-S1P receptor.

EDG-8 resembles EDG-1 by inhibiting adenylyl cyclase via a PTX-sensitive mechanism in CHO cells, implicating Gi/Go proteins in the signaling process. [35S]GTPgamma S binding assays corroborate the coupling of EDG-8 with Gi. Interestingly, LPA at a relatively high concentration (1 µM) was able to weakly inhibit cAMP formation in CHO cells expressing EDG-8. The ability of LPA to weakly inhibit cAMP formation but not displace [32P]S1P binding in EDG-8-transfected cells (present study) is reminiscent of findings that LPA does not displace [32P]S1P binding in EDG-1-transfected cells but promotes ERK1/2 activation (51). It has been proposed that EDG-1 contains distinct sites for S1P and LPA (51). Our data with EDG-8 supports a similar two-site model. However, this hypothesis awaits definitive characterization of the binding sites by site-directed mutagenesis. Notwithstanding, our displacement binding studies suggest that EDG-8 is not an LPA receptor. Identical results were obtained in HEK293T cells overexpressing EDG-8 (14). Indeed, it has been convincingly demonstrated that LPA is the high affinity ligand for EDG-2, -4, and -7 (1, 5, 8).

Although CHO cells transfected with EDG-1, -3, or -5 can activate ERK in response to S1P in a PTX-sensitive and Ras-dependent manner (17, 20, 23, 24), we found instead that S1P inhibited ERK activity in EDG-8-transfected CHO cells. Although the majority of GPCRs studied to date (including the EDG receptors) appear to couple positively with ERK1/2 (52), examples of GPCR-mediated repression of ERK1/2 activity have been reported (48, 49). In cultured neurons, AT1 receptors activate ERK1/2, whereas AT2 receptors inhibit serum-activated ERK1/2 (48). Considering that the MAP kinases are important in growth, differentiation, and apoptosis (52), it has been proposed that the antagonistic modulation of ERK activity by different receptors within the same gene family acts as a molecular counterbalance system (48). Hence, the ability of EDG-1, -3, and -5 to stimulate ERK1/2 activity and EDG-8 to repress it parallels the AT1/AT2 receptor system. The finding that expression of EDG-8 but not EDG-1 or EDG-3 in PC12 cells is chronically down-regulated by growth factors (6) provides a potential mechanism for fine tuning this counterbalance system. Such a scenario has been demonstrated in the AT receptor family, where AT2 receptor expression is up-regulated following cellular injury to antagonize AT1 signaling (53).

The exact physiological role of ERK inhibition by GPCRs remains to be elucidated. EDG-1, -3, and -5 have been demonstrated to mediate PTX-sensitive cell proliferation (28, 42) in an apparent ERK-dependent manner (27). Moreover, in endothelial cells, growth factor- and S1P-induced DNA synthesis and ERK activation are inhibited by PD98059, an inhibitor of ERK signaling (27, 54). Interestingly, EDG-8 mediates antiproliferative effects in a PTX-insensitive and orthovanadate-sensitive manner. These observations suggest that the binding of S1P to EDG-8 might negatively regulate cellular proliferation by reducing ERK1/2 activity via a protein-tyrosine phosphatase. Analogously, AT1 receptors promote cell proliferation and ERK activation, whereas AT2 receptors exert antiproliferative effects and suppress ERK activation (48, 49, 55).

Several studies have linked protein serine/threonine phosphatases and/or protein-tyrosine phosphatases to AT2 receptor-mediated repression of serum-activated ERK1/2 (48, 49, 53, 56). Depending on the cell type, the protein phosphatases SHP-1, PP2A, and MKP-1 have each been implicated in inhibition of ERK1/2 activity. The decrease in ERK1/2 activity by AT2 receptors in cultured neurons occurs through a PTX- and okadaic acid-sensitive pathway, implicating the involvement of PP2A (48). However, in N1E-115 neuroblastoma cells, AT2 receptors act through a PTX-insensitive and orthovanadate-sensitive pathway, suggesting the involvement of SHP-1 (49). Inhibition of ERK1/2 activity by S1P in CHO cells overexpressing EDG-8 was PTX-insensitive, orthovanadate-sensitive, and only weakly inhibited by okadaic acid. Future studies are warranted to define the exact nature of the phosphatase involved in EDG-8-mediated inhibition of the ERKs.

To determine whether potential inhibitory effects on ERK1/2 activity by activation of EDG-1, -3, or -5 might be masked by the interactions of these receptors with Gi (resulting in ERK1/2 activation), we treated CHO cells with PTX. Regardless of which EDG receptor was expressed, PTX treatment did not facilitate S1P-mediated repression of serum-activated ERK1/2. These findings were not totally unexpected because EDG-1 appears to couple only to Gi (30), and the inhibitory ERK response elicited by EDG-8 is PTX-insensitive. In addition, EDG-3 and EDG-5 are capable of coupling to PI turnover and Ca2+ mobilization (25) via Gq/11 (30), both signaling events that can lead to activation of the ERKs (57-59). EDG-8 does not couple to Gq/11, and thus, to PI turnover or Ca2+ mobilization, which likely explains why this receptor does not mediate ERK activation if it is assumed that coupling of EDG-8 to other G protein(s) may mask the Gi/ERK activation pathway. Galpha 12 is a likely candidate G protein for the inhibitory effects of EDG-8 on ERK activity. Mutationally activated Galpha 12 and Galpha 13, but not Galpha q, Galpha s, or Galpha i2, have been shown to inhibit EGF stimulation of ERK activity in COS-7 cells in a Ras- and Raf-independent manner (60). Furthermore, mutationally activated Galpha 12 and Galpha 13 stimulate JNK via Cdc42 and MEKK (60). Whereas expression of the activated Galpha 12 mutant in HEK293 cells does not appreciably stimulate ERK2 activity, JNK activity is increased by this mutant and involves the small GTPases Rac and Cdc42 (61). These findings are consistent with this study, demonstrating that both ERK inhibition and JNK activation by EDG-8 are PTX-insensitive.

It is of interest that EDG-8 appears to selectively activate the p54 JNK isoform. Other GPCRs, such as the alpha 1A-adrenergic receptor, differentially activate specific JNK isoforms (62). Another member of the EDG family, EDG-5, has been shown to activate JNKs in a PTX-insensitive manner (23). In that study only the p46 JNK isoform activity was measured.

In contrast to the PTX-insensitive S1P-induced activation of p38 in CHO-EDG-5 cells (23), an increase in p38 activity was not observed in S1P-treated CHO overexpressing EDG-8 transfectants. The lack of p38 activation in edg8-transfected CHO cells is another distinguishing feature of the signaling pathways mediated by EDG-5 and EDG-8 receptors.

While this manuscript was in review, Im et al. (14) described the recloning of NRG-1 (EDG-8) from rat brain. In that report, EDG-8 was shown to bind S1P, inhibit forskolin-stimulated cAMP accumulation in rat hepatoma Rh7777 cells, and couple to Gi in Xenopus laevis oocytes, in agreement with our data. In conclusion, we have demonstrated fundamental differences in G protein coupling between the four known high affinity S1P receptors, EDG-1, -3, -5, and-8. Furthermore, we have identified differences in effector pathways that are potentially related to differences in G protein coupling.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants NS352321 (to N. H. L.) and CA61774 (to S. S.) and by funds from the Department of Pathology of the Ohio State University (to J. R. V.).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 Dagger To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Inst. for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850. Tel.: 301-838-3529; Fax: 301-838-0208; E-mail: nhlee@tigr.org.

Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M003964200


    ABBREVIATIONS

The abbreviations used are: S1P, sphingosine-1-phosphate; LPA, lysophosphatidic acid; SPC, sphingosylphosphorylcholine; GPCR, G protein coupled receptor; PTX, pertussis toxin; CHO, Chinese hamster ovary; EDG, endothelial differentiation gene; MAP, mitogen-activated protein; ERK, extracellular regulated protein kinase; PI, phosphoinositide; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; HA, hemagglutinin; AT1, angiotensin II type 1; AT2, angiotensin II type 2.


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RESULTS
DISCUSSION
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