From LXR Biotechnology Inc., Richmond, California
94804 and ¶ University of Tennessee College of Medicine,
Department of Physiology and Biophysics, Memphis, Tennessee 38163
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ABSTRACT |
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We have functionally expressed the human cDNA
encoding the putative lysophosphatidic acid (LPA) receptor Edg-2
(Vzg-1) in Saccharomyces cerevisiae in an attempt to
determine the agonist specificity of this G-protein-coupled receptor.
LPA activated the pheromone response pathway in S. cerevisiae
expressing Edg-2 in a time- and dose-dependent manner
as determined by induction of a pheromone-responsive
FUS1::lacZ reporter gene. LPA-mediated activation
of the pheromone response pathway was dependent on mutational
inactivation of the SST2 gene, the GTPase-activating protein for the yeast G protein (the GPA1
gene product). This indicates that, in sst2
yeast
cells, Edg-2 can efficiently couple to the yeast heterotrimeric
G-protein in response to LPA and activate the yeast mitogen-activated
protein kinase pathway. The Edg-2 receptor showed a high degree of
specificity for LPA; other lyso-glycerophospholipids, sphingosine
1-phosphate, and diacyl-glycerophospholipids did not activate
FUS1::lacZ. LPA analogs including a cyclic
phosphoester form and ether-linked forms of LPA activated
FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ,
suggesting a role for the side chain in ligand binding or receptor
activation. These results indicate that Edg-2 encodes a highly specific
LPA receptor.
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INTRODUCTION |
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The lysophospholipid lysophosphatidic acid
(LPA,1
1-acyl-sn-glycero-3-phosphate) has been shown to be an
important extracellular signaling molecule in a variety of systems (1).
LPA has been shown to induce mitogenesis in certain cell types, exert
an anti-mitogenic effect in other cells types, activate platelets,
activate MAP kinase, stimulate ion transport, block apoptosis, and
induce morphological changes (Refs. 2-14; for a recent review on these
functions, see Refs. 1 and 15). Recently, two putative receptors for
LPA have been identified, suggesting that functionally different LPA receptors may exist that dictate the particular cellular response of
LPA (16-19). Most cell types respond to LPA, making it difficult to
characterize the receptor dependence of a particular response to LPA
since the response cannot be solely attributed to a single LPA
receptor. In particular, it is difficult to assess ligand binding
specificity of an LPA receptor because other lipid receptors may exist
with overlapping ligand specificity. We have therefore used the yeast
Saccharomyces cerevisiae to study the human LPA receptor
Edg-2 (also called Vzg-1). S. cerevisiae contains no endogenous LPA receptors and is therefore a potentially useful organism
in which to functionally express LPA receptors and analyze their ligand
specificity. Other mammalian receptors have been functionally expressed
in S. cerevisiae including the somatostatin receptor, the
A2a adenosine receptor and the 2-adrenergic
receptor (20-22).
S. cerevisiae contains a heterotrimeric G-protein that is
activated by mating factor binding to a specific receptor (23) (for
review, see Ref. 24). Upon stimulation by an occupied receptor, the subunit of the heterotrimeric G-protein (G
, the
GPA1 gene product; Ref. 25) becomes bound to GTP and
dissociates from the
dimer (26). In yeast, it is the
dimer that transduces the signal to Ste11 (the MEKK equivalent; Ref.
27) and Ste7 (the MEK equivalent; Ref. 28). The active GTP-bound
G
is inactivated by hydrolysis of GTP to GDP at which
time, G
can reassociate with G
and
attenuate the signal (25, 26, 29, 30). The yeast MAP kinases, Fus3 and
Kss1, activate a transcriptional activator, the STE12 gene
product (31). Activated Ste12 in turn activates the transcription of
several mating factor-inducible genes such as FUS1 (32). To
study the Edg-2 receptor using the yeast G-protein/MAP kinase system, a
strain was used that has a mutation in the FAR1 gene. This
mutation has the effect of uncoupling the MAP kinase cascade from cell
cycle arrest, allowing the yeast to continue growing during MAP kinase
activation (33, 34). The strain used also contains the bacterial
lacZ gene fused to the mating pheromone-inducible promoter
from the FUS1 gene, which allows quantification of the
receptor-ligand interaction. We mutationally inactivated
SST2 gene in this strain to increase the sensitivity of the
strain to G-protein activation. The SST2 gene encodes a GTPase-activating protein for the G
subunit (35). By
inactivating the SST2 gene product, G
remains
in the GTP-bound state longer and thus increases the steady-state
concentration of the signal transducing
dimer.
In this report, we show that Edg-2 expressed in yeast efficiently couples to the endogenous heterotrimeric G-protein in response to LPA. Edg-2 does not respond to other lyso-glycerophospholipids or to diacyl-glycerophospholipids such as phosphatidic acid or to sphingosine 1-phosphate, two phospholipids reported to share receptors with LPA in certain cell types (36, 37). The results are consistent with Edg-2 being a functional, specific LPA receptor.
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MATERIALS AND METHODS |
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Strains and Plasmids--
SY2069 (Mata,
far1-bad3, HIS3 :: pFUS1::HIS3,
mfa2-1::pFUS1::lacZ, ura3, leu2, ade1, arg4,
trp1; a gift from Mathais Peter and Ira Herskowitz, University of
California, San Francisco) was used to derive an
sst2
strain for subsequent studies. SST2 was
disrupted using pBC14 (35) (kindly provided by C. Steffan and K. Blumer, Washington University, St. Louis, MO). pBC14 was digested with
NcoI and transformed into SY2069 by lithium acetate using
the URA3 gene for selection. Ura+ colonies were
grown in nonselective medium (YEPD) and plated onto medium containing
5-fluoroorotic acid (5-FOA, Sigma). 5-FOA is metabolized by the
URA3 gene product into a toxic metabolite, thus only
phenotypically Ura3
cells can grow in the presence of
5-FOA (35). 5-FOA-resistant isolates were screened for increased
sensitivity to
-factor (see below). One such
sst2
strain was named JEY5 and used in all
subsequent studies. Yeast were grown in SC + 2% galactose or 2%
glucose medium lacking uracil to maintain selection of the plasmid.
Polymerase Chain Reaction, Subcloning, and Yeast
Transformation--
The Edg-2 coding region was amplified by RT-PCR
using Pfu DNA polymerase under conditions described by the
supplier (Stratagene). The template for RT-PCR was cDNA (5 ng) that
was reverse transcribed from human fetal brain total RNA
(CLONTECH) using SuperScript II Reverse
Transcriptase as described by the supplier (Life Technologies, Inc.). 1 µM each of the following primers: FP,
5-GCGATAGGATCCATCATGGCTGCCATCTCTACTTC-3
, and RP,
5
-GCGATACTCGAGCTAAACCACAGAGTGATCATTGC-3
, were used for RT-PCR.
The primers were designed based on the human edg-2 cDNA sequence submitted to GenBank by Zondag and Moolenaar (accession no.
Y09479) and included restriction site extensions for subcloning into
the pYEUra3 vector (Stratagene). This placed the cDNA under the
control of a galactose-inducible promoter (UASgal). The resulting plasmid was used to transform JEY5 by the lithium acetate method.
Oligonucleotide Synthesis and DNA Sequencing-- RT-PCR primers and DNA sequencing primers were synthesized by the phosphoramidite method with an Applied Biosystems model 394 synthesizer, purified by polyacrylamide gel electrophoresis and desalted on Sep-Pak C18 cartridges (Waters Associates, Milford, MA). The edg-2 cDNA was sequenced in pYEUra3 by the dideoxy chain termination method using the T7 Sequenase 7-deaza-dGTP sequencing kit as described by the supplier (Amersham Life Science).
LacZ Assays in Response to Phospholipids--
JEY5+pJE15 was
grown on SC medium containing either 2% galactose or 2% glucose
lacking uracil to an approximate optical density of 0.1-0.5 before the
addition of lipid or -factor. LPA and other glycerophospholipids
(Avanti Polar Lipids) were dissolved in chloroform and dried down under
argon immediately before experiments and resuspended in BBS/EDTA (50 mM NH4HCO3, 104 mM
NaCl, 250 µM EDTA·2Na, pH 7.63) at 20 mM
with sonication until the solution was clear. Sphingosine 1-phosphate
(Matreya) was resuspended in ethanol/water (9:1) pH 3.0 immediately
before use. Cyclic LPA and ether-linked forms of LPA were synthesized
in G. Tigyi's laboratory.2
Fatty acid-free bovine serum albumin was obtained from Sigma and used
at 0.1 mg/ml in BBS/EDTA. Cells were grown for the indicated time (7 h
for dose-response experiments) before assaying. 100 µl of yeast
culture were then added to 900 µl of assay buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 0.1 mM MgSO4, pH 7.0, plus 270 µl of
-mercaptoethanol/liter) plus 50 µl of 0.1% SDS + three drops of
chloroform. Cells were vortexed for 10 s and incubated for 5 min
at 28 °C. 200 µl of 4 mg/ml
o-nitrophenol-
-D-galactopyranoside (Sigma)
was added, and the reaction was incubated for 30 min at 28 °C. The
assay was stopped by the addition of 500 µl of 1 M Na2CO3. Color development was measured at
A420 and normalized to
A600. Units were expressed as Miller units.
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RESULTS |
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Edg-2 Expressed in Yeast Efficiently Couples to the Endogenous
Heterotrimeric G-protein in a Time- and Dose-dependent
Manner--
Yeast contain multiple MAP kinase cascades that are
functionally analogous to the mitogen-activated protein kinase cascade in mammalian systems (38-41). A schematic of the S. cerevisiae pheromone response pathway and the relevant genetic
components are shown in Fig. 1. The
parental yeast strain, SY2069, contains the FUS1 promoter
fused to lacZ and HIS3 integrated into different chromosomal loci and carries a far1 allele.
The FAR1 gene product is required for cell cycle arrest after exposure to mating pheromone (see Fig. 1). By mutating this gene,
the cells are able to grow in the presence of activated MAP kinase. In
addition, a null mutation in the SST2 gene was created (see
"Materials and Methods") because it has been previously reported
that the somatostatin receptor can efficiently couple to the endogenous
yeast heterotrimeric G-protein after mutationally inactivating the
SST2 gene (20). We created a null allele in the
SST2 gene using pBC14 (see "Materials and Methods" for
detailed description of the null allele construction) and tested the
resultant strains for the supersensitive phenotype by assaying
lacZ activity in response to
-factor (data not shown).
One sst2
strain was named JEY5 and used in
subsequent experiments.
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Edg-2 Responds Selectively to LPA and Not to Other Lyso-glycerophospholipids or to Diacyl-glycerophospholipids-- Yeast do not have endogenous receptors for phospholipids that couple to the pheromone response pathway, so it was of interest to determine the agonist specificity of Edg-2 using this system. Lysophosphatidylethanolamine (LPE), -serine (LPS), -glycerol (LPG) and -choline (LPC) and sphingosine 1-phosphate were tested over the same dose range as LPA for activation of FUS1::lacZ. As shown in Fig. 4A, no other lyso-glycerophospholipids or sphingosine 1-phosphate activated FUS1::lacZ as well as LPA at concentrations up to 200 µM, the highest concentration tested due to toxicity. The results of a similar experiment testing the effects of the diacyl-glycerophospholipids is seen in Fig. 4B. In this experiment, no diacyl-glycerophospholipid activated Edg-2.
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Dependence of Acyl-chain Length and Structural Variations of LPA on Edg-2 Activation-- Since Edg-2 appears to show specificity for LPA, the dependence of the acyl-chain length on Edg-2 activation was investigated. Five forms of LPA that varied in chain length and degree of saturation were tested: 18:1 (oleoyl), 18:0 (stearoyl), 16:0 (palmitoyl), 10:0 (capryl), and 6:0 (caproyl). Fig. 5A shows that forms of LPA with chain length of 16 carbons and greater activated Edg-2 but with a preference to the 18:1 (oleoyl) form. The shorter 10:0 and 6:0 forms were inactive. Two structural analogs of LPA were then tested: an 18:1 and a 16:0 2,3-cyclic phosphate form (cLPA) and an 18:1 ether-linked form. cLPA is a stable intermediate of phospholipase D metabolism and may have biological activities distinct from LPA (43). The data shown in Fig. 5B shows that cLPA activates FUS1::lacZ with similar potency to LPA, whereas ether-linked 18:1 LPA was slightly less active than the 18:1 cyclic and ester-linked forms. As with the 18:1 forms, the 16:0 ester and cyclic forms were equally active although less active than the 18:1 forms, probably due to solubility.
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DISCUSSION |
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In this report, we show that the edg-2 gene product, a putative LPA receptor also reported as vzg-1, couples to the yeast heterotrimeric G-protein and activates a MAP kinase when bound to LPA. The response to LPA was quantitated by using the lacZ gene fused to the FUS1 promoter, a mating pheromone-inducible gene promoter. The yeast strain used in this report was able to grow in the presence of activated G-protein due to a mutation in the FAR1 gene. This mutation has the phenotypic effect of uncoupling G-protein/MAP kinase activation from cell cycle arrest.
The response to LPA is dose- and time-dependent and
requires that the SST2 gene be mutationally inactivated.
SST2 encodes a GTPase-activating protein for the
GPA1 gene product, the G subunit required for
mating pheromone signal transduction. The effect of inactivating
SST2 is that Gpa1 remains in the GTP-bound state longer and
thus permits signaling through the
dimer to proceed at a higher
rate, resulting in a higher signal from the receptor. The response of
Edg-2 to LPA in this yeast-based assay is markedly reduced compared
with the LPA response in mammalian cells. Our results show that LPA
activate FUS1::lacZ with an
EC50 of 20-30 µM. In mammalian
systems, 1 µM LPA is sufficient to induce tyrosine
phosphorylation and depolarize membranes in Rat-1 fibroblasts (6, 11).
This discrepancy is most likely due to receptor/G-protein coupling as
suggested in Fig. 3C. It may be possible to increase the
response by using a mammalian G
i subunit or a
Gpa1/G
i chimera expressed in place of the endogenous
GPA1 gene product (20). However, the response of the
FUS1::lacZ reported is specific to cells
expressing the Edg-2 receptor as yeast expressing other related
receptors such as Edg-1, Edg-3, and H218 as well as the unrelated LPA
receptor, PSP24, did not respond to LPA (data not shown).
This yeast cell-based assay for Edg-2 was used to determine the agonist specificity of the receptor for different forms of LPA as well as other glycerophospholipids. The advantage of this system is that yeast contain few G-protein-coupled receptors. It is therefore a simple task to show that the response of the Edg-2 receptor to a particular phospholipid is dependent on the expression of the receptor, since it is expressed from a galactose inducible promoter. This is in contrast to mammalian cells in which the identity and distribution of LPA and other glycerophospholipids receptors is unclear. Our results show that Edg-2 specifically responds to LPA. Edg-2 does not respond to other lyso-glycerophospholipids or to diacyl-glycerophospholipids, in particular phosphatidic acid or to the related lipid messenger sphingosine 1-phosphate. These results are consistent with the response of neocortical neuroblasts, which express the Edg-2 receptor and do not respond to LPC, LPE, LPG, or phosphatidic acid (16). In platelets, LPA shares a surface receptor with sphingosine 1-phosphate (37), and, in human monocytes, phosphatidic acid and LPA both act as chemoattractants and cross-desensitize one another, suggesting that they act through a common receptor (36). Based on the result of our assay, neither of these effects are mediated by the Edg-2 receptor.
Other results show that the acyl-chain length does have an effect on the ability of LPA to activate FUS1::lacZ. It is most likely that the short chain forms of LPA do not bind to the receptor as, in competition experiments, the 6:0 caproyl form did not attenuate the ability of the 18:1 oleoyl form to activate FUS1::lacZ (data not shown). A cyclic phosphoester form of LPA activated FUS1::lacZ as well as ester-linked LPA, whereas an ether-linked form was less active. Interestingly, in A431 cells, the ether-linked LPA was less potent at activating calcium mobilization than was the corresponding ester-linked form (44). Our results demonstrate that expression of Edg-2 in yeast faithfully reconstitutes many of the key properties of an LPA receptor. We conclude that Edg-2 encodes an LPA receptor. This yeast cell-based system should prove useful for studying LPA analogs as well as identifying novel agonists and antagonists of this LPA receptor.
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ACKNOWLEDGEMENTS |
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We thank Vaclav Prochazka and Frank Bassham for sequencing the edg-2 plasmid construction and synthesizing the oligonucleotides used in this report, Samuil Umansky and Jerilyn Beltman for critical reading of the manuscript, Chris Steffan and Kendal Blumer for providing the SST2 deletion plasmid pBC14, and Mathias Peter and Ira Herskowitz for providing SY2069.
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FOOTNOTES |
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* 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.
§ To whom correspondence should be addressed: LXR Biotechnology Inc., 1401 Marina Way S., Richmond, CA 94804. Tel.: 510-412-9100; Fax: 510-412-9109; E-mail: jerickson{at}lxr.com.
Student in the Contra Costa College Exchange Program in
Biotechnology.
1 The abbreviations used are: LPA, lysophosphatidic acid; LPE, lysophosphatidylethanolamine; LPS, lysophosphatidylserine; LPC, lysophosphatidylcholine; LPG, lysophosphatidylglycerol; cLPA, cyclic LPA; BBS, bicarbonate-buffered saline; MAP, mitogen-activated protein; 5-FOA, 5-fluoroorotic acid; RT-PCR, reverse transcription-polymerase chain reaction.
2 K. Liliom, D. Fischer, G. Sun, D. Miller, J. Tseng, D. Desiderio, M. Seidel, J. Erickson, and G. Tigyi, manuscript in preparation.
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REFERENCES |
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