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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2624-2629
IMMUNOBIOLOGY
From the Department of Biochemistry and Molecular Biology,
Georgetown University Medical Center, Washington, DC; and Max
Delbrück Center for Molecular Medicine, Department of Tumor
Genetics and Immunogenetics, Berlin, Germany.
EDG-6 is a recently cloned member of the endothelial differentiation
gene (EDG) G protein-coupled receptor family that is expressed in
lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to
the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and
EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4
suggested that its ligand may be a lysophospholipid or
lysosphingolipid. We examined the binding of [32P]SPP to
HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding
of [32P]SPP was saturable, demonstrating high affinity
(KD = 63 nmol/L). Binding was also specific for SPP, as
only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans
double bond at the 4 position, potently displaced radiolabeled SPP. LPA
did not compete for binding of SPP at any concentration tested, whereas
sphingosylphosphorylcholine competed for binding to EDG-6, but only at
very high concentrations. In addition, SPP activated extracellular
signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis
toxin-sensitive manner. These results indicate that EDG-6 is a high
affinity receptor for SPP, which couples to a Gi/o protein,
resulting in the activation of growth-related signaling pathways.
(Blood. 2000;95:2624-2629)
Sphingosine-1-phosphate (SPP) is a metabolite of
complex sphingolipids that acts as both a second messenger and as a
high-affinity ligand for cell surface receptors.1 SPP is
produced by sphingosine kinase that is activated in response to a
variety of signals, including mitogens such as platelet-derived growth
factor (PDGF) and serum,2 G protein-coupled receptor
agonists such as carbachol,3 the cytokine
TNF- However, because several responses to SPP are at least partially
inhibited by pertussis toxin (PTX), which
adenosine diphosphate (ADP) ribosylates and specifically
inactivates Gi/o proteins, and some require very low
concentrations of SPP, it has been suggested that G protein-coupled
cell surface receptors (GPCRs) might also be involved (reviewed in
Speigel et al12). In agreement, a family of GPCRs, known as
the endothelial differentiation gene (EDG) receptors, which
specifically bind SPP or the related lipid, lysophosphatidic acid
(LPA), has recently been identified.13-18 The EDG family
can be divided into 2 subfamilies based on amino acid sequence
homology. The subfamily consisting of EDG-1, EDG-3, and EDG-5 display
40% to 45% sequence identity to each other and only 30% to 35%
identity to the members of the other subfamily EDG-2 and EDG-4 (Figure 1). EDG-1, EDG-3, and EDG-5 have been shown
to be SPP receptors,13-16 whereas EDG-2, EDG-4, and EDG-7
are LPA receptors.17-19
Recently, a new member of the EDG family was cloned and named
EDG-6.20 EDG-6 is expressed in lymphoid and hematopoietic tissue as well as the lung.20 Interestingly, EDG-6 does not clearly belong to either the SPP or the LPA subfamily of EDG receptors, as it displays a similar degree of homology to all 5 of the previously identified members. Thus, it was unclear whether EDG-6 is likely to be
a receptor for SPP, LPA, or another related lysophospholipid. In this
paper, we show that SPP binds specifically to EDG-6 and activates the
mitogen-activated protein kinase (MAPK) signal transduction pathway.
Materials
Cell culture and transfection
Fluorescence-activated cell sorter (FACS) analysis The human EDG-6 receptor was N-terminal tagged with an HA-epitope (peptide sequence: MGYPYDVPDYAGGP) and C-terminal tagged with a c-myc-epitope. Construction, expression, and flow cytometry analysis were performed as described.21 The N-terminal HA-epitope tag was detected with a fluorescein isothiocyanate (FITC)-labeled anti-HA antibody.SPP binding assay [32P]SPP was synthesized enzymatically using recombinant sphingosine kinase as previously described.22 Transfected cells were incubated with the indicated concentration of [32P]SPP in 200 µL binding buffer (20 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 15 mmol/L NaF, 2 mmol/L deoxypyridoxine, 0.2 mmol/L phenyl-methyl-sulfonyl-fluoride [PMSF], 1 µg/mL aprotinin and leupeptin) for 30 minutes at 4°C. Unlabeled lipid competitors were added as 4 mg/mL fatty acid-free bovine serum albumin (BSA) complexes. Cells were washed twice with ice cold binding buffer containing 0.4 mg/mL fatty acid-free BSA, resuspended in phosphate-buffered saline (PBS), and bound [32P]SPP was quantitated by scintillation counting.14Extracellular signal-regulated kinase activation Cells were seeded in 60-mm plates and transfected the following day with EDG-6 expression plasmid and HA-tagged extracellular signal-regulated kinase (Erk)2 (at a 2:1 ratio of HA-Erk2 to EDG-6) with Lipofectamine Plus. After 2 days, cells were treated as indicated and lysed by the addition of 0.5 mL lysis buffer containing 25 mmol/L 4(-2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.3 mol/L NaCl, 1.5 mmol/L MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 0.5 mmol/L dithiothreitol (DTT), 20 mmol/L -glycerophosphate, 0.2 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L
Na3VO4, 1 mmol/L PMSF, and 10 µg/mL leupeptin
for 10 minutes on ice. Lysates were centrifuged 15 minutes at 4°C.
Anti-HA (2 µg) (Santa Cruz Biotechnology) was then added to lysates
(800 µg protein) and incubated 2 hours at 4°C with rocking.
Protein A/G Sepharose beads (Santa Cruz Biotechnology) (20 µL) were
added and the incubation continued for an additional hour. The beads
were pelleted and washed 3 times in lysis buffer and twice in kinase
buffer (12.5 mmol/L HEPES pH 7.4, 10 mmol/L MgCl2, 0.5 mmol/L DTT, 12.5 mmol/L -glycerophosphate, 0.5 mmol/L NaF, 0.5 mmol/L Na3VO4). The kinase assay was initiated
by resuspending the beads in 50 µL of kinase buffer containing 50 µmol/L adenosine triphosphate (ATP), 0.5 mg/mL myelin
basic protein (MBP), and 5 000 dpm/pmol  -[32P]ATP,
and incubating 20 minutes at 30°C. The reaction was stopped by the
addition of 12 µL of 6 ×-concentrated Laemmli sample buffer and the samples were boiled 5 minutes, separated on 12%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to
nitrocellulose. Nitrocellulose membranes were stained with Ponceau S
(Sigma) to visualize protein bands and then exposed to film for
autoradiography. Radioactivity was measured in a scintillation counter
after cutting the radioactive bands. In some experiments, for the
determination of Erk 1/2 phosphorylation, 15 µg of clarified whole
cell lysate were resolved by 10% SDS-PAGE, and Erk 1/2 phosphorylation
was detected by protein immunoblotting with rabbit polyclonal
phospho-specific MAPK IgG (1:1 000, Promega), followed by horseradish
peroxidase (HRP)-conjugated goat antirabbit IgG (1:10 000; Amersham
Pharmacia Biotech) as a secondary antibody. Erk 1/2 phosphorylation was detected by ECL (Amersham Pharmacia Biotech). Nitrocellulose membranes were stripped and reprobed using rabbit polyclonal anti-Erk2 IgG (Santa
Cruz Biotechnology) to confirm equal loading.
Elk1-dependent transcription of a luciferase reporter For the detection of Elk1-dependent transcription, the PathDetect in vivo signal transduction pathway reporting system (Stratagene) and the Dual-Luciferase reporter assay system (Promega) were used. CHO-K1 cells were seeded in 6-well plates and cotransfected the following day with 50 ng of the fusion activator plasmid pFA-Elk (Stratagene), 500 ng of the firefly luciferase reporter vector pFR-Luc (Stratagene), 100 ng of the Renilla luciferase control reporter vector (Promega), and 50 ng of 1 of the plasmids, pcDNA3.1(+) or pcDNA3.1(+) containing the human EDG-6 receptor or the human EDG-1 receptor. After 30 to 40 hours, cells were washed with PBS and serum-free medium was added. Two to 3 hours later, the cells were stimulated with SPP and incubated for an additional 5 to 6 hours. The medium was removed and the cells were incubated in 300 µL passive lysis buffer (Promega) for 30 minutes at room temperature. Luminescence of 20 µL aliquots was measured with the Berthold Luminat LB 9507 for 10 seconds after injection of 50 µL each of luciferase assay buffer II and Stop & Glo buffer (Promega).
Overexpression of EDG-6 in HEK293 cells Figure 1 shows a phylogenetic tree depicting the relationship of EDG-6 to other EDG family members, as well as the next most closely related group of receptors, the cannabinoid receptors. The LPA-receptors EDG-2 and EDG-4, as well as the SPP-receptors EDG-1, EDG-3, and EDG-5, are clearly distinct from each other and form their own subgroups. Within these 2 EDG-subgroups, EDG-6 has a slightly higher homology to the first and the seventh transmembrane domains of the SPP subgroup than to the LPA subgroup. EDG-6 has a 44% overall identity to EDG-1, 46% to EDG-3, and 42% to EDG-5, and less homology to the LPA subgroup, 39% to EDG-4 and 37% to EDG-2.20 These homologies suggest that EDG-6 could be an SPP receptor. To examine this possibility, we transfected HEK293 cells, which do not express EDG-6, as determined by RT-PCR (data not shown), and have no detectable binding sites for SPP,9,14 with C-terminal c-myc epitope tagged or N-terminal HA-tagged human EDG-6 cDNA. Expression of EDG-6 protein on the cell surface was examined by flow cytometric analysis with anti-c-myc or anti-HA antibodies. FACS analysis showed that intact HEK293 cells transfected with EDG-6-myc were indistinguishable from untransfected cells (Figure 2A). However, when EDG-6-myc-transfected HEK293 cells were permeabilized, a distinct shift in the fluorescence was detected, indicating that permeabilization allowed access of the c-myc antibody to the C-terminus that is located at the cytoplasmic tail of EDG-6 (Figure 2B). HEK293 cells transfected with N-terminal HA-tagged EDG-6 showed a shifted fluorescence without permeabilization (Figure 2C), indicating cell surface expression of HA-tagged EDG-6, as the N-termini of G protein-coupled receptors are located extracellularly. Interestingly, this peak was also slightly enhanced by cell permeabilization (Figure 2D), suggesting that some HA-tagged EDG-6 may be expressed intracellularly.
Binding of SPP to EDG-6 Having established that human EDG-6 is expressed on the surface of transiently transfected HEK293 cells, it was of interest to determine whether SPP binds to EDG-6. In agreement with previous reports,9,13,14 no specific SPP binding was detected in HEK293 cells transfected with the vector alone, whereas HEK293 cells transfected with c-myc-tagged human EDG-6 (HEK293-EDG-6) displayed dramatically increased binding of [32P]SPP, which was competed by 1000-fold molar excess of either unlabeled SPP or dihydro-SPP to a level similar to that seen in untransfected cells (Figure 3). Neither SPC nor LPA effectively competed with [32P]SPP for binding to HEK293-EDG-6 cells at 1000-fold excess (1 µmol/L). A computer curve fitting of binding isotherms indicated that SPP binding to EDG-6 was saturable and displayed moderately high affinity (KD = 63 nmol/L) (Figure 4).
Binding of SPP to EDG-6 activates Erk
EDG-6 displays high homology (37%-46% amino acid identity) to the
previously identified members of the EDG family of
G protein-coupled receptors. Although by homology it cannot be
clearly grouped with either the EDG-1, EDG-3, and EDG-5 subfamily,
which bind SPP, or the EDG-2 and EDG-4 subfamily, which bind LPA,
evidence presented here indicates that EDG-6 is an SPP receptor. SPP
binds specifically to EDG-6 expressed on HEK293 cells, although with a
lower affinity (63 nmol/L) than to EDG-1 (8 nmol/L),13
EDG-3 (23 nmol/L), or EDG-5 (27 nmol/L).14 Thus, residues
conserved among EDG-1, EDG-3, and EDG-5 but not in EDG-6 may contribute
to the increased affinity of SPP binding. Nevertheless, the affinity of
EDG-6 for SPP is high enough to indicate that SPP could be a
physiologically relevant ligand for EDG-6, as the concentration of SPP
in plasma and serum is about 200 nmol/L and 500 nmol/L,
respectively.28
We thank Dr S. Milstien for helpful discussion.
Submitted August 11, 1999; accepted January 3, 2000.
Supported by research grants from the National Institutes of
Health (GM43880 and CA61774) to S.S. and a postdoctoral fellowship (1 F32 GM19209-01A1) to J.V.B.
J.R.V.B. and M.H.G. contributed equally to this study.
Reprints: Sarah Spiegel, Department of Biochemistry and
Molecular Biology, Georgetown University Medical Center, 353 Basic
Science Bldg, 3900 Reservoir Rd, NW, Washington, DC 20007; e-mail: spiegel{at}bc.georgetown.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
,
RI antigen receptor.
Nature.
1996;380:634-636
