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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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 G
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), G
q/11, G
i/o,
G
12 and G
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
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
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
-glycerophosphate, 2 mM sodium orthovanadate, 0.5 mM dithiothreitol), and resuspended in kinase buffer
containing 100 µM ATP, 5 µCi of
[
-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]GTP
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]GTP
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 G
subunit-selective antibody. (Santa
Cruz). Nonspecific binding was determined by immunoprecipitation with
nonimmune serum. Bound [35S]GTP
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.
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RESULTS |
[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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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
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.
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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.
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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).
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EDG-8 Coupling to Individual G Proteins--
Coupling of EDG-8 to
endogenous G protein family members was assessed by a
[35S]GTP
S binding assay (30). COOH-terminal
peptide-directed antibodies were used to immunoprecipitate either
specific G
subunits (e.g. G
s,
G
12 or G
13) or G
family members
(e.g. G
i/o or G
q/11). S1P-stimulated [35S]GTP
S binding in the
immunoprecipitates was used as a measure of G protein coupling and activation.
As shown in Fig. 7, negligible
[35S]GTP
S binding was evident for G
s,
G
i/o, G
q/11, and G
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]GTP
S binding to the
G
i/o family of G proteins and G
12 (Fig. 7). Whereas S1P stimulation of EDG-8 activated G
i/o and
G
12, it had no effect on G
s or
G
q/11 (Fig. 7). In agreement with previous reports (20,
23), CHO cells express G
s, G
i/o,
G
q/11, and G
12 proteins (Fig. 7). Thus,
the lack of detectable coupling to G
s or
G
q/11 is not due to the absence of these G proteins in
CHO cells. Moreover, when membranes from CHO cells expressing the
2-adrenergic receptor were incubated with 2 µM isoproterenol, a 2.9 ± 0.4-fold increase in
[35S]GTP
S binding to G
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]GTP
S binding to G
q/11. CHO cells
express only trace levels of G
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
G
13 (30), S1P did not significantly stimulate
[35S]GTP
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]GTP S in the presence of
vehicle or S1P (1 µM). Membranes were extracted, and the
G subunits were immunoprecipitated with subunit-specific antibodies.
Nonspecific binding was determined by immunoprecipitation with
nonimmune serum. S1P-induced [35S]GTP 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 G subunit-specific
antibodies and ECL detection.
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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.
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DISCUSSION |
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]GTP
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. G
12 is a likely candidate G protein for the
inhibitory effects of EDG-8 on ERK activity. Mutationally activated
G
12 and G
13, but not G
q,
G
s, or G
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
G
12 and G
13 stimulate JNK via Cdc42 and
MEKK (60). Whereas expression of the activated G
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
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.