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INTRODUCTION |
The sphingolipid metabolite sphingosine 1-phosphate
(SPP)1 is emerging as a
member of a new class of lipid second messengers (1, 2). SPP is
mitogenic in diverse cell types (3-6) and suppresses programmed cell
death or apoptosis (7-10). Various stimuli, including platelet-derived
growth factor and serum (6, 11), nerve growth factor (NGF) (8, 12),
vitamin D3 (13), activation of protein kinase C (14, 15) or
protein kinase A (10), cross-linking of the Fc
R1 (16) or Fc
R1
(17) receptor by antigens, and binding of carbachol to m2 and m3
muscarinic acetylcholine receptors (18), increase cellular levels of
SPP by activation of sphingosine kinase. Moreover, competitive
inhibitors of sphingosine kinase eliminate the formation of SPP and
selectively block cellular proliferation induced by platelet-derived
growth factor and serum (11, 19), the cytoprotective effects of protein kinase C and cAMP activators (7, 10), NGF (8), and vitamin D3 (13) as well as Fc
R1-, Fc
R1-, and muscarinic
acetylcholine receptor-mediated calcium signaling (16, 17),
further supporting a role for endogenous SPP in cell growth,
survival, and calcium mobilization. In addition, microinjected SPP
mobilizes calcium from internal sources (18) and is mitogenic for Swiss
3T3 fibroblasts (20), indicating that SPP acts intracellularly to
regulate calcium homeostasis and proliferation.
Several other responses to SPP are mediated through cell surface
receptors, including platelet activation (21), inhibition of melanoma
cell motility (22), activation of Gi protein-gated inward
rectifying K+ channels in atrial myocytes (23), and
Rho-dependent neurite retraction and cell rounding of
N1E-115 neurons (24) and PC12 cells (25). We recently identified the G
protein-coupled receptor endothelial differentiation gene-1 (Edg-1) as
a high affinity receptor for SPP (26). In response to SPP, Edg-1
inhibits adenylyl cyclase (20), activates the mitogen-activated protein
kinase Erk2 (26) through a pertussis toxin-sensitive mechanism, and causes morphogenetic differentiation through a pertussis
toxin-insensitive mechanism that requires the small GTPase Rho (26).
Several other responses to SPP, including mobilization of intracellular
Ca2+, activation of phospholipase D, and tyrosine
phosphorylation of p125FAK (20) are not mediated through
Edg-1. Collectively, these data led us to suggest that SPP is a
prototype of a new class of lipid second messengers that can also act
as first messengers (20).
Several receptors related to Edg-1 have been cloned, including the
lysophosphatidic acid (LPA) receptors Edg-2 (27-29) and Edg-4 (30) as
well as Edg-3 (31) and H218/AGR16 (32, 33). H218 and Edg-3 are widely
expressed in adult tissues, particularly in cardiovascular and nervous
systems. However, the ligands, signal transduction pathways, and
physiological functions of these receptors are still not known.
Recently, Edg-3 and H218 were shown by An et al. (34) to
confer responsiveness to SPP of a serum response element-driven reporter gene when expressed in Jurkat cells and to allow
SPP-stimulated 45Ca2+ efflux in
Xenopus oocytes, suggesting that Edg-3 and H218 may also be
functional receptors for SPP. However, no direct binding data were
presented in this study, and therefore it is not clear at present
whether Edg-3 and H218 are bona fide SPP receptors. Moreover,
sphingosine and sphingosylphosphorylcholine (SPC) were almost as
effective as SPP in Jurkat T cells. Since SPP and sphingosine may also
act intracellularly, it was of interest to determine the specificities
and affinities of Edg-3 and H218 for these lipids using our recently
developed binding assay (20). In this report, we show that both Edg-3
and H218 bind SPP with high affinity and remarkable specificity.
Moreover, we have identified H218 as the receptor that mediates cell
rounding and neurite retraction in response to SPP.
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EXPERIMENTAL PROCEDURES |
Materials--
SPP, sphinganine 1-phosphate (dihydro-SPP),
sphingosine, N,N-dimethylsphingosine, and
C2-ceramide were purchased from Biomol Research Laboratory Inc.
(Plymouth Meeting, PA). SPP was determined to be greater than 99% pure
by thin layer chromatography analysis. C8-ceramide 1-phosphate was from
Calbiochem. SPP-phosphonate and SPP-homophosphonate were synthesized as
described by Tarnowski (35). Cyclic SPP was purchased from Alexis
Biochemicals (San Diego, CA). Other lipids were purchased from Avanti
Polar Lipids (Birmingham, AL). Serum and medium were obtained from
Biofluids (Rockville, MD).
Cell Culture--
Human embryonic kidney cells (HEK293; ATCC
CRL-1573) were grown in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml
streptomycin. Rat pheochromocytoma PC12 cells (ATCC CRL-1721) were
maintained in RPMI medium supplemented with 10% heat-inactivated horse
serum and 5% fetal bovine serum (8).
Cloning and Expression of edg-1, edg-3, and
H218--
edg-1, edg-3, and H218
genes were amplified by PCR from human and rat genomic DNA,
respectively. The primers were designed to add a
BamHI site at the 5'-end and a XhoI site at the
3'-end as follows: 5'-GAGGGATCCGGGCCCACCAGCGTCCCGCTG-3' and
5'-GAGCTCGAGCTAGGAAGAAGAGTTGACGTTTCC-3' for edg-1;
5'-GAGGGATCCGCAACTGCCCTCCCGCCGCGT-3' and
5'-GAGCTCGAGTCAGTTGCAGAAGATCCCATTCTG-3' for edg-3;
5'-GAGGGATCCGGCGGTTTATACTCAGAGTAC-3' and
5'-GAGCTCGAGTCAGACCACTGTGTTGCCCTC-3' for H218. PCR
products were cloned into the pcDNA3 vector (Invitrogen, Carlsbad,
CA) containing a myc epitope tag at the 5'-end (a generous gift of Dr. Peter Burbelo). The resulting plasmids were transfected into HEK293 cells using Lipofectamine Plus (Life Technologies, Inc.)
according to the manufacturer's instructions at a 4:1 ratio with pCEFL
GFP, which encodes green fluorescent protein (a generous gift of Dr.
Silvio Gutkind). The cells were then grown for 2 days to allow
expression of receptors before experiments were performed. Transfection
efficiencies were typically 30-35% for HEK293 cells and 10% for PC12 cells.
Western Blotting--
HEK293 cells were transfected as described
above and lysed in PBS containing 1% CHAPS, 1 mM
phenylmethylsulfonyl fluoride, and a 10 µg/ml concentration each of
leupeptin and aprotinin for 1 h at 4 °C. Lysates were
centrifuged, and equal amounts of protein from the supernatant were
separated by SDS-polyacrylamide gel electrophoresis. Proteins were
transferred to nitrocellulose membranes (Bio-Rad) and probed with
monoclonal anti-c-Myc 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). Bands were visualized with Super Signal chemiluminescent reagent
(Pierce) using horseradish peroxidase-conjugated anti-mouse IgG.
SPP Binding Assay--
[32P]SPP was synthesized
enzymatically using partially purified sphingosine kinase (36) as
described previously (20). The specific activity of
[32P]SPP was 6 × 106 cpm/pmol. Cells
were incubated with the indicated concentration of
[32P]SPP in 200 µl of binding buffer (20 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 15 mM NaF, 2 mM deoxypyridoxine, 0.2 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin and leupeptin) for 30 min at 4 °C.
Unlabeled lipid competitors were added as 4 mg/ml fatty acid-free bovine serum albumin complexes (3). Cells were washed twice with 200 µl of ice-cold binding buffer containing 0.4 mg/ml fatty acid-free
bovine serum albumin and resuspended in phosphate-buffered saline, and
bound [32P]SPP was quantitated by scintillation counting
(20).
Cell Rounding Assay--
HEK293 cells were plated at 2 × 105 cells/well in 12-well dishes coated with polylysine and
transfected 2 days later with the indicated receptor expression
plasmids together with pCEFL GFP, as described above. Following
transfections, cells were incubated in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum or 10% serum that had been
stripped with activated charcoal to remove lipids (26). Cells were then
washed and placed in serum-free medium for the indicated times and then
treated with vehicle or with 100 nM SPP. Cells were fixed
in 4% paraformaldehyde containing 5% sucrose for 20 min at room
temperature and photographed using a Nikon Eclipse TE200 inverted
fluorescence microscope connected to a Sony DKC5000 digital camera.
Cells expressing GFP and GFP-expressing cells displaying rounded
morphology were counted. At least three different fields were scored
with a minimum of 300 cells scored.
For neurite retraction assays, PC12 cells were transfected as above,
incubated overnight, and then split into 6-well dishes in RPMI
containing 10% heat-inactivated horse serum and 5% fetal bovine
serum. Cells were transferred to serum-free medium, and NGF (100 ng/ml)
was added to induce differentiation. After 48 h, vehicle or 100 nM SPP was added for the indicated time, cells were fixed
with 4% paraformaldehyde containing 5% sucrose, and the morphology of
transfected cells was evaluated by a blinded observer using a
fluorescence microscope. Cells that had flattened, irregularly shaped
cell bodies with neurite outgrowths were scored as differentiated.
Cells with spherical shape lacking any neurite extensions and filopodia
were scored as rounded. At least three different fields were scored for
differentiated and rounded cells with a minimum of 100 cells scored.
Cell rounding was expressed as the percentage of round shaped cells
among total green fluorescent cells.
Staining of Apoptotic Nuclei--
Fixed cells were washed with
phosphate-buffered saline and then treated with bisbenzimide
trihydrochloride (24 µg/ml in phosphate-buffered saline; Hoechst dye
33258; Calbiochem) for 10 min. Stained cells were examined with an
inverted fluorescence microscope. Transfected cells were marked by the
expression of green fluorescent protein, and apoptotic cells were
distinguished by condensed, fragmented nuclear regions using an
ultraviolet filter. A minimum of 300 cells were scored.
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RESULTS |
Sphingosine 1-Phosphate Binds to H218 and Edg-3--
Quantitative
binding studies with lysolipid agonists such as SPP and LPA have been
hampered by the lipophilic nature of the ligand, resulting in very high
levels of nonspecific binding (for example, see Ref. 37). However,
recently we have developed a sensitive assay for binding of SPP to cell
surface receptors that overcomes these problems (20). To determine if
the Edg-3 and H218 G protein-coupled receptors, which share 48 and 45%
homology, respectively, with Edg-1, were capable of specifically
binding SPP, HEK293 cells were transiently transfected with pcDNA3
expression plasmids containing H218 or edg-3 open
reading frames with c-myc tags fused in frame at the N
terminus. HEK293 cells were selected for these studies, since they have
no specific SPP binding. Although no edg-1 mRNA
expression can be detected by Northern analysis, barely detectable
expression can be seen by RT-PCR. Additionally, a low level of
edg-3 mRNA was detected by RT-PCR; however, no product
was seen using primers specific for H218 (data not shown). Western analysis confirmed that an approximately 43-kDa protein containing the c-Myc tag was expressed in cells transfected with edg-3 or H218 but not with the pcDNA3myc
vector or in untransfected cells (Fig.
1A). H218- and
Edg-3-expressing HEK293 cells display dramatically increased specific
binding of [32P]SPP in comparison with untransfected and
vector-transfected cells (Fig. 1B). HEK293 cells expressing
H218 and Edg-3 had similar binding affinities for
[32P]SPP with KD values of 27 and 23 nM, respectively (Fig. 2).

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Fig. 1.
SPP binds to H218 and Edg-3 transiently
expressed in HEK293 cells. A, HEK293 cells were either not
transfected (untrans.) or transiently transfected with
pcDNA3myc (vector), pcDNA3myc containing the
edg-3 coding sequence, or the H218 coding
sequence as described under "Experimental Procedures." Western
blotting was performed using an antibody specific for the c-Myc epitope
tag. B, binding of 1 nM [32P]SPP
to untransfected HEK293 cells and cells transiently transfected with
pcDNA3myc vector alone or containing H218 or
edg-3 coding sequence was determined as described under
"Experimental Procedures." Total binding is in the absence of
unlabeled competitor, and nonspecific binding is in the presence of 1 µM unlabeled SPP. Results are means ± S.D. of
triplicate determinations.
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Fig. 2.
SPP binding to H218 and Edg-3. Specific
binding of [32P]SPP (defined as binding in the absence of
unlabeled competitor minus binding in the presence of excess unlabeled
ligand) to HEK293 cells transiently transfected with plasmids encoding
H218 (A) and edg-3 (B) was
determined as described under "Experimental Procedures." Results
are means ± S.D. of triplicate determinations.
KD values were determined by hyperbolic curve
fitting with Delta Graph 4.0 for Macintosh.
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Several other lipids that are structurally related to SPP, especially
SPC and LPA, often have similar effects to SPP, and it has been
suggested that in some instances they may share the same receptor (21,
38). Moreover, recently it has been suggested that LPA is a low
affinity agonist for Edg-1 (39). Therefore, the specificity of binding
to H218 and Edg-3 for a variety of lipid analogs of SPP was tested.
Similar to our previous results with Edg-1 (20), only unlabeled SPP and
dihydro-SPP, which lacks the 4-trans-double bond present in
SPP, effectively competed with [32P]SPP for binding to
cells expressing either H218 or Edg-3 (Fig. 3, A and B).
Sphingosine had a small but statistically significant effect on SPP
binding to both Edg-3 and H218, while ceramide had a similarly small
effect on binding to H218 only. Interestingly, the homophosphonate
analog of SPP, which differs from SPP in that the oxygen atom at the
1-position is replaced by a carbon atom, competed for binding to Edg-1
as effectively as did unlabeled SPP (Fig. 3C), although it
had only a slight effect on binding of SPP to Edg-3 and no effect on
binding to H218 (Fig. 3, A and B). The other
lipids tested, including both LPA and SPC, do not compete for binding
of SPP to any of the three Edg family receptors (Fig. 3 and Ref.
20).

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Fig. 3.
Specificity of binding to H218 and
Edg-3. HEK 293 cells transiently transfected with plasmids
encoding H218 (A), Edg-3
(B), or HEK293 (C) cells stably expressing Edg-1
(HEK293-edg-1 cells) were incubated with 1 nM
[32P]SPP in the absence (control) or in the presence of 1 µM of the indicated lipids, and the amount of bound
[32P]SPP was determined as described under
"Experimental Procedures." cSPP, cyclic SPP;
C8 SPP, 8-carbon chain length SPP; ceramide,
C2-ceramide; cer-1-P, N-octanoyl ceramide
1-phosphate. Results are means ± S.D. of triplicate
determinations. The asterisk indicates statistically
significant differences from control in the absence of unlabeled
competitor as determined by Student's t test
(p < 0.05).
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H218 Mediates Sphingosine 1-Phosphate-induced Cell
Rounding--
Cell rounding is one of the most profound biological
responses induced by low (nanomolar) concentrations of SPP that is
thought to be mediated by a cell surface receptor (24). HEK293 cells transfected with H218 and edg-3 exhibited marked
changes in cell morphology and increased numbers of rounded cells in
comparison with vector- and edg-1-transfected cells (Fig.
4). To quantitate the magnitude of this
response, the percentage of transfected cells, indicated by expression
of green fluorescent protein (GFP), which displayed rounded morphology,
was determined. Expression of H218 or Edg-3 increased rounding of
HEK293 cells, while Edg-1 did not have a significant effect (Fig.
5A). Since the loss of cell
attachment can lead to death by apoptosis (40-42), the percentage of
transfected cells that displayed apoptotic characteristics was also
determined. Expression of H218 or Edg-3 also increased the percentage
of transfected cells HEK293 cells that displayed fragmented nuclei
characteristic of apoptosis (Fig. 5B). Edg-1 expression led
to a significant but much smaller increase. Although this response was
seen in the absence of exogenously added SPP, these cells were grown in
the presence of serum, which has been shown to contain high levels of
SPP (43).

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Fig. 4.
Effect of overexpression of Edg-3 and H218 on
cell morphology. HEK293 cells were transiently transfected with
pcDNA3myc (A), pcDNA3myc containing the
edg-1 coding sequence (B), the edg-3
coding sequence (C), or the H218 coding sequence
(D) as described under "Experimental Procedures" and
cultured for 2 days in medium containing 10% fetal bovine serum. Cells
were fixed, and phase contrast images of cellular morphology are shown.
Similar results were obtained in two separate experiments.
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Fig. 5.
Correlation of cell rounding and apoptosis in
HEK293 cells expressing Edg-3 and H218. Cells were transfected as
in Fig. 4 and then fixed as described under "Experimental
Procedures." A, Total GFP-expressing cells and
GFP-expressing cells displaying rounded morphology were counted.
B, cells were also stained with Hoechst dye 33258, and total
GFP-expressing cells and GFP-expressing cells displaying fragmented
nuclei indicative of apoptosis were counted. Data are means ± S.D. of triplicate determinations. The asterisk indicates
statistically significant differences from vector-transfected control
as determined by Student's t test (p < 0.05).
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To determine whether binding of SPP to these receptors mediates cell
rounding, transfected HEK293 cells were grown in charcoal-stripped serum, which contains no detectable
SPP.2 Under these conditions,
HEK293 cell morphology was similar in all cells in the absence of SPP
(Fig. 6). Treatment with 100 nM SPP induced modest cell rounding in vector-transfected
and Edg-1-expressing HEK293 cells. However, SPP induction of cell
rounding was dramatically increased by expression of H218, whereas
expression of Edg-3 appeared to only slightly enhance the rounding
effect of SPP. To quantify this response, the number of rounded cells,
expressed as a percentage of the transfected cells identified by
expression of GFP, was determined (Fig.
7A). Expression of all three
receptors modestly enhanced the percentage of rounded cells in
comparison with vector-transfected controls. Treatment with SPP further
enhanced cell rounding to a small degree in Edg-3-expressing cells and
by approximately 2-fold in H218-expressing cells. Expression of these
Edg receptors had a similar effect on apoptosis (Fig.
7B). Edg-1 expression slightly increased apoptosis, and this
effect was not enhanced by SPP. Edg-3 expression led to a more
pronounced increase in apoptosis. H218 expression was again the most
effective, leading to a significant increase in apoptosis, which was
further enhanced by SPP treatment.

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Fig. 6.
Induction of cell rounding by H218 in
response to SPP. HEK293 cells were transiently transfected with
pcDNA3myc or pcDNA3myc containing the edg-1,
edg-3, or the H218 coding sequences together with
pCEFL GFP; grown overnight in the presence of delipidated serum; and
then changed to serum-free medium for an additional day. Cells were
treated with vehicle or with 100 nM SPP for 3 h and
fixed, and phase contrast images of cellular morphology are shown.
Similar results were obtained in two separate experiments.
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Fig. 7.
Enhancement of cell rounding and apoptosis in
HEK293 cells overexpressing H218 in response to SPP. Cells were
transfected and treated as described in the legend to Fig. 6.
A, total GFP-expressing cells and GFP-expressing cells
displaying rounded morphology were counted. , control; , 3-h SPP
treatment. B, cells were also stained with Hoechst dye
33258, and total GFP-expressing cells and GFP-expressing cells
displaying fragmented nuclei indicative of apoptosis were counted. Data
are means ± S.D. of triplicate determinations. The
asterisk indicates statistically significant differences
from edg receptor-transfected cells in the absence of SPP as
determined by Student's t test (p < 0.05).
The open diamond indicates statistically
significant differences compared with vector-transfected cells in the
absence of SPP.
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H218 Mediates Sphingosine 1-Phosphate-induced Neurite
Retraction--
SPP has previously been shown to induce rapid
retraction of developing neurites and transient rounding of the cell
body in PC12 cells (25) and in N1E-115 neuronal cells (24). However, the putative cell surface receptor mediating these effects has not yet
been identified. PC12 cells express H218 (33), and it has been
suggested that H218 is involved in regulation of some of the early
steps in neuronal differentiation, including axonal outgrowth (44). In
agreement, we found by RT-PCR that PC12 cells express H218
but not edg-3 or edg-1 (data not shown). Thus, it was of interest to examine whether H218 might be the cell surface receptor responsible for neurite retraction induced by SPP.
Approximately 50% of PC12 cells transfected with vector alone have
long processes when cultured in serum-free medium in the presence of
NGF. This morphology remained unchanged when cells were treated with
SPP for 10 min. However, expression of H218 and Edg-3, but not Edg-1, decreased NGF-induced differentiation in the absence of added SPP, with
the strongest effect observed with H218 (Fig.
8). Expression of these receptors results
in changes in morphology leading to retraction of neurites and soma
rounding, producing cells with a spherical appearance (Fig. 8). As in
HEK293 cells, H218 was the most potent receptor for this response. The
addition of nanomolar concentrations of SPP to NGF-differentiated PC12
cells overexpressing the different Edg family receptors further
enhanced this response. This is in contrast to the lack of effect of
Edg-1 expression on cell rounding in HEK293 cells. Flattened cells
start to round up rapidly after the addition of SPP, with rounding
being complete within 10 min. As in HEK293 cells, rounding of
Edg-expressing PC12 cells in response to SPP was correlated with death
by apoptosis; however, increased apoptosis in SPP treated
Edg-expressing cells was not seen until 24 h of treatment (Fig.
8C). Since SPP treatment did not lead to increased apoptosis
during the first 3 h, SPP-induced cell rounding is unlikely to be
a result of cell death.

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Fig. 8.
Effects of Edg receptors and SPP on neurite
retraction and cell rounding in NGF-differentiated PC12 cells.
PC12 cells transfected with plasmids encoding Edg receptors and pCEFL
GFP were cultured in serum-free medium containing NGF as described
under "Experimental Procedures" and then treated with 100 nM SPP for 10 min or 3 h. A, total
GFP-expressing cells with flat differentiated morphology and
GFP-expressing cells displaying rounded morphology were counted. Data
are means ± S.D. of triplicate determinations. B,
cells were examined by fluorescence microscopy to visualize GFP. Each
panel shows two fields containing typical cells from
vector-transfected and H218-expressing cells. C, after
treatment without or with SPP for the indicated time, cells were
stained with Hoechst dye 33258, and total GFP-expressing cells and
GFP-expressing cells displaying fragmented nuclei indicative of
apoptosis were counted. Data are means ± S.D. of duplicate
determinations. The asterisk indicates statistically
significant differences from edg receptor-transfected cells
in the absence of SPP as determined by Student's t test
(p < 0.05). The open diamond
indicates statistically significant differences compared with
vector-transfected cells in the absence of SPP.
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DISCUSSION |
The G protein-coupled Edg family of receptors is now known to
include numerous members. Edg-3 and H218 are nearly 50% homologous to
the known SPP receptor Edg-1. In contrast, Edg-2/Vzg-1 and Edg-4, both
of which have been shown to act as receptors for LPA (27-30), are only
31 and 27% homologous to Edg-1, respectively. The binding data
presented in this report establish that, similar to our previous
studies on Edg-1 (26), Edg-3 and H218 are also bona fide receptors for
SPP. The affinities of these receptors for SPP are similar: 8 nM (26), 23 nM, and 27 nM for
Edg-1, Edg-3, and H218, respectively. Moreover, our data showing lack of competition for SPP binding to Edg-1, Edg-3, and H218 by LPA suggest
that there may be two subfamilies of Edg receptors, one specific for
SPP and the other for LPA (27-29). We propose that these receptors
should thus be named based on their ligand binding specificities. Thus,
Edg-1, H218, and Edg-3 should be named SPPR1, SPPR2, and SPPR3,
respectively, and Edg-2/Vzg-1 and Edg-4 should be named LPAR1 and LPAR2.
SPC and LPA are structurally similar to SPP and often induce similar
biological responses. Binding of SPP to platelets is inhibited by LPA,
and LPA-induced platelet aggregation is prevented by pretreatment with
SPP, suggesting that in platelets, LPA and SPP bind to a common
receptor (21). SPC and SPP apparently share a receptor in atrial
myocytes, since they both activate a K+ conductance in
these cells and cause heterologous desensitization (38). However,
neither SPC nor LPA compete for binding of radioactively labeled SPP to
any of the three receptors investigated in this study. Therefore, it is
likely that there are other, as yet unidentified, Edg family members
that do not show such high ligand specificity and are more promiscuous.
Interestingly, expression of Edg-3 or H218 in Jurkat cells allows
activation of SRE-driven gene transcription by SPP, SPC, and
sphingosine (34). Since neither SPC nor sphingosine compete for binding
of SPP to Edg-3 or H218, the reason for this is unclear. However,
because these sphingolipid metabolites also have intracellular actions
(20, 45, 46), the possibility exists that their effects are partially
mediated via intracellular targets. A recent study demonstrated that
LPA can bind to the Edg-1 receptor with an apparent
KD of 2.3 µM, resulting in receptor
phosphorylation and extracellular signal-regulated kinase activation as
well as Rho-dependent morphogenesis and P-cadherin expression (39). Moreover, the binding of labeled LPA was competed only
poorly by unlabeled SPP. However, it should be noted that SPP
desensitizes [3H]LPA binding to Edg-1 (39), while our
studies indicate that LPA does not desensitize [32P]SPP
binding to Edg-1. These results suggest that there may be two different
binding sites on Edg-1: one that binds SPP with high specificity in the
nanomolar concentration range and one that binds LPA with low affinity
in the micromolar concentration range.
Although Edg-1, Edg-3, and H218 bind SPP with high specificity, there
are some differences in the binding of SPP analogs. The homophosphonate
analog of SPP binds to Edg-1 as well as does SPP, but it only competes
weakly for SPP binding to Edg-3 and not at all for H218. In addition,
the short chain C8 analog of SPP slightly inhibits SPP binding to Edg-1
but is completely ineffective for Edg-3 and H218. Thus, although these
receptors are highly homologous and bind SPP with similar affinities,
their binding sites may not be identical. SPP analogs with different
specificities for the different SPP receptors should be useful to
determine which receptors mediate specific biological responses to SPP.
HEK293 cells transiently expressing Edg-3 or H218 receptors exhibited a
rounded morphology when grown in the presence of serum. However,
removal of lipids from serum by charcoal stripping prevented this
effect on morphology. Serum contains high levels of SPP (400 nM) (43), and charcoal stripping reduces SPP to
undetectable levels.2 In agreement, the addition of SPP
markedly increased cell rounding in cells expressing H218 and to a
lesser extent in Edg-3-expressing cells. Moreover, cells transiently
transfected with H218, when cultured in serum-free medium,
also exhibited a small but significant increase in cell rounding. It is
possible that H218 is partially activated in the absence of ligand when
the receptor is overexpressed, a phenomenon commonly observed for G
protein-coupled receptors (47). Our results indicate that H218 may be
the unidentified cell surface receptor that was previously suggested in
several studies to be responsible for SPP-induced cell morphology
alterations and remodeling of the actin cytoskeleton (48, 49).
Apoptosis of mammalian cells can occur by default unless the cell
receives certain survival signals (50, 51). Loss of cell attachment
induces apoptosis in certain cell types due to the loss of
integrin-mediated signaling, which normally suppresses apoptosis,
and this phenomenon has been termed anoikis (40-42). Thus, detachment
of HEK293 cells or PC12 cells following SPP-induced rounding could lead
to death by anoikis. The close correlation of apoptotic appearing cells
with rounded cells (Figs. 5 and 7) supports this possibility. However,
it cannot be concluded that SPP normally induces apoptosis by binding
to these cell surface receptors, since overexpression of receptors
could result in a robust, nonphysiological signal. It has been reported
that nanomolar concentrations of SPP induce neurite retraction and
rounding in differentiated PC12 cells (25) and N1E-115 neuroblastoma
cells (24). In both of these studies, it was postulated that the SPP effect was mediated through a putative cell surface receptor and not by
intracellular actions, since microinjected SPP had no effect (24),
whereas SPP immobilized to glass beads was effective (25). In this
study, we found that expression of H218, and to a lesser extent Edg-3,
in rat pheochromocytoma PC12 cells caused a decrease in NGF-induced
neurite outgrowth and increased the fraction of cells with rounded
morphology. MacLennan et al. (33) demonstrated that PC12
cells express H218. In agreement, we detected H218
expression in PC12 cells by RT-PCR analysis; however, we were not able
to detect expression of edg-1 or edg-3. Thus, it
is likely that SPP-induced neurite retraction in PC12 cells is mediated
through H218. Interestingly, treatment of NGF-differentiated PC12 cells
overexpressing any of the Edg receptors with SPP for as little as 10 min caused further neurite retraction and cell rounding, although the
effect was most marked with H218. This is in contrast to the moderate
efficacy of Edg-3 and the lack of effect of Edg-1 on rounding in HEK293 cells. Therefore, it appears that all three receptors are capable of
coupling to signaling pathways leading to rounding depending on the
cellular context.
H218 is expressed in the cardiovascular system (32) and in the brain
during embryogenesis, where its expression is temporally regulated such
that high levels of expression are found in neuronal cell bodies during
early stages of differentiation and in axons during their outgrowth
(44). This led to the suggestion that H218 plays an important role in
neuronal development and may steer axons by regulating their growth and
inhibiting their extension (44). Thus, SPP synthesized by target
tissues could help guide axons by regulating axon extension or
stabilization through binding to H218.
In summary, we have shown that the G protein-coupled receptors H218 and
Edg-3 are high affinity, specific receptors for SPP and that binding of
SPP to these receptors induces cell rounding. H218 is clearly the most
efficacious mediator of this response, although Edg-3 is also
effective. Since these receptors are widely expressed in most cells and
tissues, important questions that should be addressed in the future are
their roles in mediating various biological responses to SPP. Thus, SPP
might play a role during normal brain development or after traumatic
injury by acting through H218 and possibly Edg-3 to affect neuritogenesis.