From the Department of Pharmacology and
Neurosciences and Biomedical Sciences Programs, School of
Medicine, University of California, San Diego, La Jolla,
California 92093-0636, the § European Molecular Biology
Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany, and the
¶ Division of Molecular Genetics, National Institute of
Neuroscience, Kodaira, Tokyo 187-8502, Japan
Received for publication, December 22, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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Lysophosphatidic acid (LPA) induces diverse
biological responses in many types of cells and tissues by activating
its specific G protein-coupled receptors (GPCRs). Previously, three
cognate LPA GPCRs (LPA1/VZG-1/EDG-2,
LPA2/EDG-4, and LPA3/EDG-7) were identified in
mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a
high affinity LPA receptor in Xenopus laevis oocytes,
raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two
novel Xenopus genes,
xlpA1-1 and
xlpA1-2, encoding LPA1
homologs (~90% amino acid sequence identity with mammalian
LPA1). Both xlpA1-1 and
xlpA1-2 are expressed in oocytes and
the nervous system. Overexpression of either gene in oocytes
potentiated LPA-induced oscillatory chloride ion currents through a
pertussis toxin-insensitive pathway. Injection of antisense
oligonucleotides designed to inhibit
xlpA1-1 and
xlpA1-2 expression in oocytes
eliminated their endogenous response to LPA. Furthermore,
retrovirus-mediated heterologous expression of
xlpA1-1 or
xlpA1-2 in B103 rat neuroblastoma cells
that are unresponsive to LPA conferred LPA-induced cell rounding
and adenylyl cyclase inhibition. These results indicate that
XLPA1-1 and XLPA1-2 are functional
Xenopus LPA receptors and demonstrate the evolutionary
conservation of LPA signaling over a range of vertebrate phylogeny.
Lysophosphatidic acid
(LPA1;
1-acyl-2-sn-glycerol-3-phosphate) is a simple phospholipid
that exerts hormone- and growth factor-like effects in many organisms
and organ systems. LPA can alter cell fates by inducing proliferation
and differentiation or by preventing apoptosis in many cell types (1).
In addition, LPA can induce cytoskeletal reorganization that leads to
cell rounding and stress fiber formation (1-3).
Biological responses to LPA are elicited by activation of its specific
G protein-coupled receptors (GPCRs). Thus far, three genes
(lpA1, lpA2, and
lpA3) encoding high affinity LPA receptors,
LPA1/EDG-2, LPA2/EDG-4, and
LPA3/EDG-7, have been identified in mammals (reviewed in
Refs. 3 and 4). Biological functions of these receptors have been
characterized by overexpression and/or heterologous expression in
mammalian cells (5-13). All three LPA receptors can mediate adenylyl
cyclase inhibition, increases in intracellular calcium, inositol
phosphate production, and MAP kinase activation. LPA1 and
LPA2 can also induce cell rounding via activation of the
small GTPase, Rho. Pharmacological studies suggest that both
LPA1 and LPA2 couple to at least three types of
G proteins, Gi/o, G12/13, and Gq,
whereas LPA3 couples with Gi/o and
Gq but not with G12/13 (13). Genetic deletion
of LPA1 in mice demonstrated that LPA1 is at
least in part responsible for LPA signaling in vivo and is
essential for normal development (14).
A molecularly different LPA receptor was reported in studies on
Xenopus oocytes (15). Guo et al. (15) isolated a
novel GPCR gene, PSP24, by polymerase chain reaction
(PCR) using degenerate oligonucleotide primers against a
platelet-activating factor receptor. Overexpression of PSP24 in oocytes
potentiated maximal LPA-induced oscillatory chloride ion
(Cl Mouse, human, and Xenopus homologs of PSP24 share Materials--
[ Amplification of Xenopus cDNAs by Reverse
Transcription-PCR--
mRNA was prepared from
Xenopus oocytes using the Oligotex direct mRNA kit
(Qiagen, Valentia, CA) as described by Ferby et al. (20).
First strand cDNA was synthesized from 500 ng of mRNA using
oligo(dT) primers and the SUPERSCRIPT first-strand synthesis system
(Life Technologies, Inc.). This cDNA was used as a template for PCR
with degenerate primers designed toward sequences in transmembrane domains (TMD) II and VII conserved among members of the GPCR family (21). The nucleotide sequence for the TMD II primer is
5'-CCIATGTAYYTITTYYTYWSGAATTCIWSITTI-3', and the sequence for the TMD
VII primer is 5'-AARTCIGGRSWICGISARTAIATSAIIGGRTT-3'. The PCR condition
was 40 cycles of 94 °C for 1 min, 45 °C for 1.5 min, and 72 °C
for 2 min. After electrophoresis on agarose gels, three prominent bands
of the expected size range (400-1300 base pairs (bp)) were recovered
from the gel and re-amplified by PCR under the same conditions. The
final PCR products were cloned into pCR 2.1 using TOPO TA cloning kit
(Invitrogen, Carlsbad, CA) and sequenced.
Cloning of Full-length Xenopus cDNAs--
To obtain
full-length cDNAs for the PCR products, we screened a
Xenopus oocyte cDNA library (a gift from Dr. John
Shuttleworth, University of Birmingham, UK) using a
32P-labeled 560-bp PCR fragment as a probe. Two different
full-length Xenopus LPA receptor isoforms were isolated as
XLPAR-1 and XLPAR-10. XLPAR-1 contains 2053 bp, with 321 bp of 5'
untranslated region and 631 bp of 3' untranslated region followed
by a poly(A) tail. XLPAR-10 contains 1941 bp and lacks a
poly(A) tail. These cDNA sequences were deposited in the EMBL data
base with the accession numbers AJ249843 (XLPAR-1) and AJ249844
(XLPAR-10). In view of their high degree of homology to mammalian
LPA1, they are referred to here as XLPA1-1 and
XLPA1-2 for XLPAR-1 and XLPAR-10, respectively. Nucleic
acid and amino acid alignment was performed using the Clustal W
multiple sequence alignment program found on the Web page of the DNA
Data Bank of Japan.
Northern Blot Analysis--
Tissues were quickly removed from
female Xenopus, and total RNA was isolated from each tissue
using Trizol according to the manufacturer's instructions (Life
Technologies, Inc.). Northern blotting was performed as described
previously (6, 13), and membranes were analyzed with a
Bio-Imaging analyzer BAS2500.
Electrophysiology in Xenopus Oocytes--
Open reading frames
(ORFs) of xlpA1-1 and -2 were
subcloned into BamHI-XhoI sites of the
pBluescript SK(+) vector (Stratagene). Constructs were linearized with
KpnI digestion and used as a template in in vitro
RNA transcription using mMESSAGE mMACHINE Kits (Ambion, Austin, TX).
Xenopus oocyte preparation, cRNA injection, and
electrophysiology were performed as described previously (22). Briefly,
stage V and VI oocytes from adult females were injected with 50 nl of
appropriate cRNA (1 µg/µl) for overexpression and incubated at
16 °C for 3-5 days in modified Barth saline (88 mM
NaCl, 1 mM KCl, 0.33 mM
Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM Hepes (pH 7.4))
before recording. Oocytes were impaled by two microelectrodes filled with 3 M KCl and voltage-clamped at Retrovirus Systems--
The entire ORFs for
xlpA1-1 and -2 were subcloned
into HindIII and XbaI sites of a pFLAG-CMV-1
mammalian expression vector (Eastman Kodak Co.) to introduce
preprotrypsin-leader/FLAG-tag sequences into amino-terminal
extracellular regions of each receptor for immunocytochemical detection
of the receptor proteins. These constructs were then subcloned into
BamHI and XhoI sites of a Moloney murine leukemia
retroviral vector, LZRS-EGFP (24). Sequences of internal
ribosomal entry sites in the vector enable concomitant expression of
EGFP and FLAG-tagged receptors within the single cell (13). The inserts
of the constructs were confirmed by sequencing. Retrovirus supernatants
were prepared using a Phoenix cell line, as described previously
(13).
Functional Assays--
For the cell rounding assay, B103 cells
were seeded onto glass coverslips coated with Cell-Tak (Becton
Dickinson Labware, Bedford, MA) and infected with viral supernatants
(13). After treatment with LPA, cells were fixed with 4% (w/v)
paraformaldehyde and incubated with the blocking solution (0.1% (w/v)
Triton X-100, 0.25% (w/v) bovine serum albumin in phosphate-buffered
saline). EGFP protein was visualized by incubation with anti-GFP
polyclonal antibody, followed by incubation with fluorescein
isothiocyanate-conjugated anti-rabbit IgG antibody (Vector
Laboratories, Burlingame, CA). FLAG-tagged receptor was visualized by
incubating cells with anti-FLAG antibody, followed by incubation with
Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA). Cells were observed with a Zeiss
Axiophot and a Plan-Neofluor × 40 objective (Carl Zeiss,
Thornwood, NY) or a confocal laser-scanning microscope TCS NT and a PL
APO 63 × 1.20 water-immersion objective (Leica, Deerfield, IL).
For stress fiber formation assays, fixed RH7777 cells were
immunostained for FLAG and polymerized actin, as previously described
(5). For measurement of intracellular cAMP contents,
retrovirus-infected B103 cells were stimulated with LPA in the presence
of 1 µM forskolin and 0.5 mM
3-isobutyl-1-methylxanthine. Intracellular cAMP contents were measured
using a cAMP enzyme-immunoassay system (Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
Statistical Analysis--
Data shown are the means ± S.E.
from replicate samples from replicate experiments. Statistical analysis
was performed by Student's t test.
Isolation of xlpA1-1 and xlpA1-2--
To
identify novel GPCRs in Xenopus oocytes, we
performed a PCR-based screen using degenerate oligonucleotide primers
designed against TMD II and VII (6, 21). The PCR amplifications
resulted in three faint bands in the expected size range for the region between TMD II and TMD VII of GPCRs (400-1300 bp) that were
re-amplified and cloned. DNA sequencing identified two fragments with
90% identity in predicted amino acid sequences to those of the
mammalian LPA1 receptor. These PCR fragments were then used
as probes to screen a Xenopus oocyte cDNA library, and
two different cDNAs (xlpA1-1 and
xlpA1-2) were cloned, which encoded GPCRs
consisting of 366 amino acids that differed by 6 amino acids (Fig.
1, A and B). The
comparison of nucleic acid sequences showed that
xlpA1-1 was 96% identical in the
predicted ORF with xlpA1-2, whereas there
was much less identity in their 5' and 3' untranslated regions (69 and
61%, respectively; Fig. 1A). Because the nucleotides that
differ between xlpA1-1 and -2 are distributed throughout the entire ORFs,
xlpA1-1 and -2 were probably
encoded by different genes rather than produced by alternative
splicing.
Sequence alignment of these receptors with mouse and human
LPA1 demonstrated high identity in both nucleic acid and
predicted amino acid sequences. At the nucleic acid level,
xlpA1-1 was 75%
identical to mouse lpA1 and 77% identical to human
lpA1, whereas xlpA1-2
was 78% identical to mouse lpA1 and 77% identical
to human lpA1. A comparison of predicted amino acid
sequences indicated that both clones were 89-90% identical to both
mouse and human LPA1 (Fig. 1B). The amino acids
were least conserved between Xenopus clones and mammalian
LPA1s in the first 24 amino acids of the amino-terminal
regions (5 of 22 amino acids were identical).
Expression of xlpA1-1 and -2 in Xenopus
Tissues--
To examine expression of
xlpA1-1 and -2 in
Xenopus, total RNA from various tissues was isolated and
analyzed by high stringency Northern blot analysis (Fig.
2). The strongest signal was observed in
oocytes, where a band of ~2.2 kb was observed. In addition, brain and
spinal cord samples expressed this 2.2-kb band but also expressed
larger species of ~5.8 and 11 kb (Fig. 2). Because the nucleic acid
sequences of xlpA1-1 differed by only 3%
from xlpA1-2 and were dispersed
throughout the ORFs, attempts to differentiate the forms by Northern
blot analysis were unsuccessful. xlpA1-1
and -2 expression was highest in oocytes, at lower levels in
brain and spinal cord, and below detection in lung, heart, kidney,
liver, muscle, stomach, and intestine.
xlpA1-1 or xlpA1-2 Overexpression in
Xenopus Oocytes--
Application of LPA is known to evoke oscillatory
inward Cl xlpA1-1 or xlpA1-2 Antisense
Oligonucleotide Injection in Xenopus Oocytes--
If
XLPA1-1 and/or XLPA1-2 mediate endogenous LPA
responses, a reduction in receptor expression could result in decreased
Cl Pertussis Toxin Treatment of Oocytes Overexpressing
xlpA1-1 or xlpA1-2--
In
Xenopus oocytes, endogenous LPA-evoked Cl Heterologous Expression of XLPA1-1 or -2 in Mammalian
Cells--
To examine additional signaling properties of
XLPA1-1 and -2 as compared with the known properties of
mammalian LPA1, each was expressed in B103 rat
neuroblastoma cells (19) by infection with receptor-expressing
recombinant retrovirus. The B103 cell line was chosen because it does
not express any known LPA receptors and lacks endogenous responses to
LPA, but does express appropriate
To ascertain whether XLPA1-1 or -2 was expressed in
infected B103 cells, immunohistochemistry was used to identify the
epitope (FLAG)-tagged receptors. FLAG-tagged protein was detected as
punctate labeling in both soma and neurites (Fig.
5, E, H, and K).
Consistent with retroviral mediated protein expression (see
"Experimental Procedures" and Ref. 13), EGFP protein was also
strongly and ubiquitously expressed in all receptor-expressing cells
(Fig. 5, A, D, G, and J). Because the cells
expressing EGFP proteins completely overlapped the cells expressing
FLAG proteins (Fig. 5 and Ref. 13), EGFP fluorescence was used to
monitor the infection efficiency before all functional assays. Typical
infection percentages approximated 70-90%.
Mouse LPA1 mediates LPA-induced cytoskeletal reorganization
including cell rounding in B103 cells and stress fiber formation in
RH7777 rat hepatoma cells (5, 6, 13). To examine whether XLPA1-1 and XLPA1-2 also mediate cell rounding,
B103 cells were infected, treated with various concentrations of LPA
for 15 min, double-immunostained against EGFP and FLAG-tagged proteins,
and observed by fluorescence microscopy. Without treatment, B103 cells had neurites protruding from the cell body (Fig. 5, A, D, G,
and J), and after LPA stimulation, cells infected with
control virus did not change their shapes (Figs. 5C and
6A). In contrast, B103 cells
expressing XLPA1-1 or XLPA1-2 and exposed to
LPA resulted in an increase in rounded cells with retracted neurites
(Figs. 5, F and I, and 6A). The
maximal effects and EC50 values (~10 nM) for
LPA were comparable with those observed for cells expressing mouse
LPA1 (Figs. 5L and 6A). LPA-induced
cell rounding in cells expressing XLPA1-1 or
XLPA1-2 was completely inhibited by pretreatment with a Rho
kinase inhibitor, Y-27632 (29), but not with PTX pretreatment (data not
shown).
As with B103 cells, RH 7777 cells neither express any known LPA
receptors nor show endogenous response to LPA (5). However, heterologous expression of mouse LPA1 in these cells
produces LPA-dependent stress fiber formation (5).
Heterologous expression of XLPA1-1 or XLPA1-2
increased the percentage of cells with stress fibers following LPA
stimulation (1.9% in control cells, 29.1% in
XLPA1-1-expressing cells, and 32.0% in
XLPA1-2-expressing cells). This effect was comparable in
extent and completely indistinguishable from the stress fibers produced
in previous studies of mouse LPA1-expressing cells (5).
Mouse LPA1 mediates LPA-induced inhibition of adenylyl
cyclase in B103 cells (13), TR immortalized neuroblast cells
(6), and HTC4 hepatoma cells (11). Infected B103 cells were incubated with forskolin (1 µM) in the absence or presence of
various concentrations of LPA for 15 min. Intracellular cAMP
content was measured by enzyme immunoassay. Forskolin-induced
cAMP accumulation in B103 cells expressing XLPA1-1 or
XLPA1-2 was inhibited by LPA treatment (Fig.
6B). Maximal inhibition in both was ~32% and was smaller than that observed in cells expressing mouse LPA1
(~63%). However, the EC50 values for inhibition were
comparable among XLPA1-1, XLPA1-2, and mouse
LPA1 (~10 nM). This inhibition was completely blocked by PTX pretreatment (data not shown).
In this study, we identified and characterized two novel
Xenopus GPCRs, XLPA1-1 and XLPA1-2.
Based on nucleotide and amino acid sequence similarities, endogenous
expression in Xenopus tissues, and their function in
both oocytes and mammalian cells, XLPA1-1 and
XLPA1-2 are functional Xenopus homologs of the
mammalian high affinity LPA receptor LPA1.
A comparison of nucleic acid sequences, including divergent
untranslated regions, and predicted amino acid sequences of
xlpA1-1 and
xlpA1-2 indicates that they are derived
from two distinct genes rather than generated by alternative splicing
of a single gene. The existence of multiple genes for a given function is not uncommon for Xenopus laevis (20, 30-33), in part
reflecting the occurrence of genome duplication in Xenopus
that results in a tetraploid (allotetraploid) genome (34).
xlpA1-1 and/or -2 mRNA was
expressed at high levels in oocytes and at lower levels in brain and
spinal cord among the various Xenopus tissues examined.
Alignment of amino acid sequences revealed that both
XLPA1-1 and XLPA1-2 were highly similar to
mammalian LPA1 (~89% identity) (3, 4, 35). By contrast, no signal was detected after Northern blot hybridization with mouse
LPA2 or LPA3 probes under conditions allowing
detection of XLPA1-1 and XLPA1-2 using mouse
LPA1 (data not shown). This result suggests that
Xenopus homologs of mammalian LPA2 or
LPA3 do not exist in the Xenopus tissues
examined or, at least, not those with the same degree of similarity as
between mouse and Xenopus LPA1.
In Xenopus oocytes, endogenous LPA-evoked Cl In addition to Gq activation, both XLPA1s
stimulate Rho (perhaps through G12/13) and Gi
pathways, resulting in cell rounding and stress fiber formation, and
inhibition of cAMP accumulation, respectively. All responses are
similar to those observed directly in previous studies of mammalian
LPA1 (5, 13). The EC50 of LPA for cytoskeletal
changes, as well as adenylyl cyclase inhibition in cells expressing
XLPA1, is below 10 nM, which is comparable with
that of mouse LPA1 (5, 13). These results strongly support the identification of XLPA1-1 and -2 as LPA receptors in
Xenopus.
Xenopus oocytes appear to express both high and low affinity
receptors for LPA based on electrophysiological studies of
LPA-dependent Cl It should be noted that a different gene, PSP24, was
previously reported to encode Xenopus high affinity LPA
receptor, based on the potentiation of LPA-evoked
Cl In summary, we have identified and characterized two high affinity
Xenopus LPA receptors, both of which are similar in
structure and function to mammalian LPA1 receptors. The
existence of XLPA1s provides a more evolutionarily frugal
mechanism for LPA signaling that appears to be conserved from
Xenopus through humans.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) currents, whereas injection of antisense
oligonucleotide against PSP24 inhibited endogenous responses to LPA.
Based on these observations, the authors concluded that PSP24 is a high
affinity receptor for LPA. However, subsequent studies by others were
inconsistent with this conclusion. Heterologous expression of PSP24 did
not mediate LPA responses in yeast, whereas LPA1 did (7).
In addition, mammalian orthologs of PSP24 did not mediate responses to
LPA in assays such as [35S]GTP
S binding,
[3H]LPA binding, MAP kinase activation,
[3H]thymidine incorporation, adenylyl cyclase inhibition,
and increases in intracellular calcium (16, 17).
55%
amino acid sequence identity with one another (18). These PSP24s have
comparatively little amino acid sequence identity (
20%) with members
of the mammalian LPA receptor family (4). They instead show
closest similarity to receptors for a bioactive peptide, cholecystokinin (4). The dissimilarity between mammalian and Xenopus LPA receptors was surprising based on the
phylogenetic conservation of many other GPCRs for a single ligand. This
disparity raises the question of whether Xenopus might use a
fundamentally different LPA receptor system or might also express and
use LPA receptors homologous to those found in mammals. Here, we report identification and characterization of two novel Xenopus
GPCRs that show 89-90% amino acid sequence identity with mammalian
LPA1.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]deoxy CTP was purchased
from PerkinElmer Life Sciences. LPA
(1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was purchased from Avanti Polar Lipids (Alabaster, AL). Pertussis toxin (PTX) was
purchased from Calbiochem (La Jolla, CA). B103 rat neuroblastoma cells
(19) were a gift from Dr. David Schubert (The Salk Institute, La Jolla,
CA). RH7777 rat hepatoma cells were a gift from Dr. Hyam Leffert
(University of California, San Diego, La Jolla, CA). Retrovirus
expression vector (LZRS-EGFP) and Phoenix ecotropic retrovirus
producer cell lines were gifts from Dr. Garry P. Nolan (Stanford
University, Stanford, CA). Y-27632 was a gift from Welfide Pharmaceutical Industries (Saitama, Japan). Trizol and all cell culture
reagents were purchased from Life Technologies, Inc. Anti-GFP antibody was obtained from CLONTECH (Palo Alto,
CA). Forskolin, 3-isobutyl-1-methylxanthine, anti-FLAG M2 monoclonal
antibody, and other reagents were purchased from Sigma, unless
otherwise noted.
50 mV. Only oocytes
with resting potentials of less than
30 mV were used. Oocytes were continuously superfused with Ringer's solution (120 mM
NaCl, 2 mM KCl, 1.8 mM CaCl2, 50 mM Hepes (pH 7.4), 0.1% (w/v) fatty acid-free bovine serum
albumin) in the presence of LPA. For antisense oligonucleotide studies,
injection of oocytes with 50 nl of oligonucleotides (2 µg/µl) was
performed 3-5 days before recording. The antisense oligonucleotides
were designed to complement 11-12 nucleotides 5' and 3' to the
initiation codon for xlpA1-1 or
xlpA1-2. All oligonucleotides were
phosphorothioated near 5' and 3' ends (*) to prevent degradation (23).
Antisense oligonucleotide sequences were
5'-G*A*A*A*GAGAAGCCAUUUUAGC*C*C*A*G-3' for
xlpA1-1 and
5'-G*A*A*A*GCGAAGUCAUUUUAG*C*C*C*A*G-3' for
xlpA1-2. Because the antisense
oligonucleotides differed by only two nucleic acids, a single
sense-orientation oligonucleotide corresponding to the region targeted
for xlpA1-1 and -2 was used as a negative control (5'-C*U*G*G*GCUAAAAUGGCUUCGC*U*U*U*C-3'). For the PTX experiments, Xenopus oocytes were incubated
with 2 µg/ml PTX in modified Barth saline for 48 h before recording.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparative sequences of
xlpA1-1 and
xlpA1-2. A, alignment of
nucleotide sequences of xlpA1-1 and
xlpA1-2 cDNAs. The lower sequence
shows only those residues for xlpA1-2
that differ from xlpA1-1. ORFs are
indicated in black boxes. Gaps are indicated by -. B, alignment of amino acid sequences of XLPA1-1,
XLPA1-2, mouse (m) LPA1, and human
(h) LPA1. Putative TMD I-VII are
overlined. Amino acid residues identical among all the
sequences are indicated by *. Similar amino acid residues found in two
or three sequences are indicated by :. Potential post-translational
modification sites conserved among members of the GPCR family are also
indicated: N-linked glycosylation sites ( ), protein
kinase C/casein kinase II phosphorylation sites with
(S/T)X(R/K) motif (
), palmitoylation sites (X).
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Fig. 2.
Expression of
xlpA1-1 and/or
xlpA1-2 in various Xenopus
tissues. Total RNA samples (10 µg) isolated from females
were analyzed by high stringency Northern blot analyses as compared
with a loading control (ribosomal RNA). Molecular size markers are
indicated on the left in kb.
currents in native Xenopus oocytes,
an indication that oocytes endogenously express LPA receptors (25-27).
To examine whether XLPA1-1 or XLPA1-2 could
function as high affinity LPA receptors, each was overexpressed by cRNA
injection into oocytes, and the Cl
currents in response
to LPA were recorded (Figs. 3 and 4).
Control (diethyl pyrocarbonate-treated
water)-injected oocytes did not show any
response to 3 nM LPA, whereas overexpression of either xlpA1-1 or
xlpA1-2 elicited LPA-induced
Cl
currents at this concentration (data not shown). At
higher LPA concentrations (10 nM), application of LPA on
control oocytes induced small Cl
currents averaging 50 nA
(Figs. 3A and 4A). Overexpression of either
xlpA1-1 or
xlpA1-2 significantly potentiated the
LPA-induced Cl
currents (Figs. 3A and
4A). Sphingosine 1-phosphate, a structurally related
bioactive lysophospholipid, did not evoke Cl
currents in
either control or xlpA1-1- and/or
xlpA1-2-overexpressing oocytes (data
not shown).
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Fig. 3.
LPA-induced Cl currents in
oocytes. A, overexpression of XLPA1-1 or
XLPA1-2. Overexpression of XLPA1-1 or
XLPA1-2 produced by cRNA injection potentiates LPA (10 nM)-induced Cl
current in Xenopus
oocytes. B, oligonucleotide injection. Antisense
oligonucleotides (100 ng/oocyte) designed against
xlpA1-1 or
xlpA1-2 inhibit endogenous responses to
LPA, whereas injection of sense oligonucleotides did not alter LPA
responses. C, effect of PTX treatment. PTX pretreatment does
not inhibit LPA-induced Cl
currents in
xlpA1-1-injected oocytes. A typical trace
is shown in each panel.
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Fig. 4.
Statistical analyses of LPA-induced
Cl currents in oocytes. A, overexpression
of XLPA1-1 or XLPA1-2. B,
oligonucleotide injection. C, effect of PTX treatment. Data
are the means ± S.E. (n = 6-9). **,
p < 0.01; *, p < 0.05 as compared
with controls.
currents in response to LPA. To address this, sense
(as a control) or antisense oligonucleotides were synthesized as
23-24-mers designed to block the initiation codon and thus inhibit
xlpA1-1 and -2 translation.
When these oligonucleotides were injected into oocytes, only injection
of antisense oligonucleotides completely blocked Cl
currents evoked by 10 nM LPA (Figs. 3B and
4B).
currents
have been documented to be unaffected by preincubation with PTX,
consistent with the involvement of Gq pathways (28). Thus,
we further examined whether XLPA1 might produce
PTX-insensitive Cl
currents.
xlpA1-1-injected oocytes were
preincubated with PTX and electrophysiologically examined. As shown in
Figs. 3C and 4C, PTX did not significantly affect
LPA-evoked Cl
currents. This observation demonstrated the
involvement of PTX-insensitive G proteins in XLPA1-mediated
responses in Xenopus oocytes.
subunits of heterotrimeric G
proteins (Gi/o, Gq, and G12/13 subtypes) that can couple with LPA receptors (5, 13). Retroviral infection was used to introduce receptors into B103 cells to permit high efficiency expression and low cytotoxicity compared with conventional transfection methods (13).
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Fig. 5.
Confocal microscopy of cells
heterologously expressing XLPA1-1 and
XLPA1-2. Confocal laser-scanning microscopy of B103
cells infected with control retrovirus (vector control)
(A-C), XLPA1-1-expressing retrovirus
(D-F), XLPA1-2-expressing retrovirus
(G-I), and mouse LPA1-expressing retrovirus
(J-L). Cells were treated with 1 µM LPA for
15 min (C, F, I, and L).
After fixation, cells were double-immunostained for EGFP (fluorescein
isothiocyanate (green) in A, C, D, F, G, I, J,
and L) and FLAG epitope (Cy3 (red) in B, E,
H, and K). Cells expressing LPA1s
(XLPA1-1, XLPA1-2, and mouse LPA1)
showed a marked increase in cell rounding in response to LPA as
compared with controls.
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Fig. 6.
XLPA1-1 and XLPA1-2
mediate cellular LPA responses in B103 cells. A, LPA
concentration-dependent cell rounding in cells
heterologously expressing XLPA1-1 or XLPA1-2,
as compared with positive (mouse LPA1-infected cells) and
negative (vector-only infected cells) controls. Infected B103 cells
were treated with LPA for 15 min, fixed, and immunostained. The number
of rounded cells was expressed as a percentage of EGFP-positive cells
(>200 cells/well). Data are the means ± S.E. (n = 4). B, LPA concentration-dependent inhibition
of cAMP accumulation by XLPA1-1 and XLPA1-2
expression. Infected B103 cells were incubated with forskolin (1 µM) and LPA for 15 min. Forskolin-induced cAMP
accumulation (750.1-1183.6 fmol/well) was expressed as 100%. Data are
the means ± S.E. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
currents have been reported to be mediated through the activation of
Gq and phospholipase C, based on studies using
pharmacological or antisense oligonucleotide approaches (28, 36). In
studies of mammalian LPA1, it was recently reported that
LPA stimulates phospholipase C through Gq pathways (13).
Combined with data from the present study including PTX-insensitive
augmentation of LPA-evoked Cl
currents by
xlpA1, we conclude that endogenous LPA responses probably are mediated by Gq activation via
XLPA1s.
currents (25, 37, 38). Guo
et al. (15) have reported a high affinity site for LPA with
an EC50 of 12 nM and a low affinity site of 1 µM. Here, we observed that overexpression of
XLPA1-1 or -2 potentiated the Cl
currents
evoked by application of low concentrations (3-10 nM) of
LPA. Injection of antisense oligonucleotide designed to inhibit expression of endogenous XLPA1-1 and -2 completely
inhibited the Cl
currents evoked by the application of 10 nM LPA. Combined with the data from heterologous
expression, we conclude that XLPA1-1 and
XLPA1-2 are high affinity LPA receptors in
Xenopus oocytes.
currents in oocytes following its overexpression
(15). Xenopus PSP24 is dissimilar to mammalian LPA
receptors, having less than 20% amino acid sequence identity with
mammalian LPA1 (4). Moreover, heterologous expression of
Xenopus PSP24 in yeast and of mammalian orthologs of PSP24
in mammalian cells does not mediate LPA responses (7, 16). Our data did
not directly address the role of PSP24, because experimental results
focused on XLPA1-1 and XLPA1-2 were sufficient
to account for measured LPA responses in Xenopus oocytes. It
is possible that an indirect relationship exists between
XLPA1-1 and XLPA1-2 and PSP24, although we note
the existence of technical difficulties in examining altered gene
expression or post-translational changes in single, injected, and
electrophysiologically characterized oocytes. The precise mechanism for
LPA-evoked effects related to PSP24 in oocytes remains for future work,
along with the question of what receptor mechanisms mediate the low
affinity LPA interactions, which were not addressed in this study.
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FOOTNOTES |
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* This work was supported by grants from the National Institute of Mental Health (to J. C.), the National Institute of Neuroscience, Japan (to H. K.), and the Uehara Memorial Foundation (to N. F. and I. I.).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.
The nucleotide sequences reported in this paper have been submitted to the EMBL/GenBankTM/EBI Data Bank with accession numbers AJ249843 and AJ249844.
** To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 858-534-2659; Fax: 858-534-8242; E-mail: jchun@ucsd.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M011588200
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ABBREVIATIONS |
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The abbreviations used are:
LPA, lysophosphatidic acid;
GPCR, G protein-coupled receptor;
PCR, polymerase chain reaction;
GTPS, guanosine
5'-O-(thiotriphosphate);
PTX, pertussis toxin;
GFP, green
fluorescent protein;
EGFP, enhanced GFP;
TMD, transmembrane domain(s);
bp, base pair(s);
ORF, open reading frame;
kb, kilobase(s);
Y, cytidine
or thymidine;
W, adenosine or thymidine;
S, guanosine or cytidine;
R, adenosine or guanosine.
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