From the Division of Cellular Biochemistry
and Centre for Biomedical Genetics, and ¶ Division of Cell
Biology, The Netherlands Cancer Institute,
1066 CX Amsterdam, The Netherlands
Received for publication, October 3, 2002
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ABSTRACT |
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Lysophosphatidic acid (LPA) is a serum-borne
phospholipid that activates its own G protein-coupled receptors present
in numerous cell types. In addition to stimulating cell proliferation,
LPA also induces cytoskeletal changes and promotes cell migration in a
RhoA- and Rac-dependent manner. Whereas RhoA is activated via G Lysophosphatidic acid
(LPA)1 is a bioactive
lysophospholipid that acts on its cognate G protein-coupled receptors
(GPCRs) to induce a host of cellular responses, ranging from rapid
morphological changes to stimulation of cell proliferation and survival
(1-3). Extracellular LPA is produced following platelet activation
and, hence, is an active constituent of serum (4, 5). LPA is also found
in conditioned media from cultured cells (6, 7), and its levels are
elevated in body fluids from cancer patients (8, 9). Three distinct G
protein-coupled receptors for LPA have been identified to
date, termed LPA1, LPA2, and LPA3
(previously Edg2, Edg4, and Edg7, respectively), with
LPA1/Edg2 being the first identified and most widely
expressed subtype (3, 10). Although much is known about LPA signaling
and its cellular responses, the unique biological properties and
specific signaling pathways of each individual LPA receptor remain to
be fully characterized. LPA serves as the prototypic GPCR agonist that
activates the mitogenic Ras-ERK1/2 cascade via Gi (11) and
evokes rapid contractile responses, such as cell rounding and neurite
retraction, via G Although LPA action is most often associated with cell
proliferation and morphological changes, less attention has been paid to the effects of LPA on cell motility and migration. Cell migration is
fundamental to many normal and pathophysiological processes and plays a
central role not only in embryonic development but also in the
progression of tumors from a non-invasive to an invasive and metastatic
phenotype. LPA stimulates the invasion of tumor cells across a
monolayer of normal cells (14, 16) and promotes wound healing both
in vitro and in vivo (17, 18). Interestingly, a
migratory response to LPA is also observed in Dictyostelium discoideum amoebae (19), suggesting the possible existence of as-yet-unidentified LPA receptors in invertebrates. However, it remains
unclear how LPA receptors signal cell migration. In general, cell
migration is driven by signaling pathways controlled by the three Rho
GTPases, RhoA, Rac1, and Cdc42, acting in a coordinate fashion (20).
Rac1 regulates lamellipodia protrusion and forward movement; Cdc42
establishes cell polarity, and RhoA mediates actomyosin-driven cytoskeletal contraction and stress fiber formation but also detachment of the rear end of migrating cells (20, 21).
The mechanisms by which LPA and other GPCR agonists activate RhoA have
been studied in some detail and are reasonably well understood. These
studies have shown that RhoA is activated via G Cell Culture--
All cells were cultured in DMEM
containing 10% fetal calf serum. Mouse embryo fibroblasts
(MEFs) and Tiam1-deficient MEFs derived from homozygous
knockout mice were isolated from 14-day embryos according to standard
procedures, as described (25).
Expression Constructs, Retroviral Transduction, and
Transfections--
The LPA1/Edg2 cDNA was isolated
from a human brain cDNA library (Stratagene) and its sequence
submitted to GenBankTM (accession number U78192).
C-terminally FLAG- or HA-tagged LPA1 receptor cDNAs
were cloned into the retroviral vector LZRS-IRES-Neo (26), and
recombinant viruses were produced by transfection of retroviral
cDNA constructs into Phoenix packaging cells (27). B103 cells were
infected with virus-containing supernatants in the presence of 4 µg/ml Polybrene, followed by G418 selection. Myc-tagged N17Rac,
N17Cdc42, and N19RhoA cloned into LZRS-IRES-zeo (26) were introduced
into B103-LPA1 cells by retroviral transduction and
selected on Zeocin (0.25 mg/ml). HA-tagged Tiam1 constructs have been
described (28). COS7 cells were transfected using the DEAE-dextran method.
Cell Migration--
Cell migration was measured in Transwell
chambers (Costar Corp., pore size 8 µm). Filters were coated with 10 µg/ml laminin-1 (in PBS overnight at 4 °C), rinsed once with PBS,
and placed in the lower chamber containing serum-free DMEM supplemented
with agonists. Cells suspended in serum-free DMEM containing 0.1%
fatty acid-free bovine serum albumin were added to the upper chamber (1 × 105 cells/well). Cells were allowed to migrate
for 4 h at 37 °C. Non-migratory cells were removed from the top
filter surface with a cotton swab. Migrated cells, attached to the
bottom surface, were fixed in 3% formaldehyde/PBS, permeabilized in
methanol, stained with crystal violet, and counted.
Rho GTPase Activation Assays and Western
Blotting--
Activation of Rac/Cdc42 was measured using GST-Pak
pull-down assays (26). Briefly, 2 × 106 cells were
seeded in poly-L-lysine-coated 10-cm dishes. Clarified lysates from serum-starved cells were incubated with the Rac/Cdc42 binding domain of GST-Pak produced in Escherichia coli and
immobilized on glutathione-coupled Sepharose beads (45 min at 4 °C).
RhoA activation was measured using a GST fusion protein containing the
RhoA binding domain of Rhotekin, as described previously (29). Beads
were washed, eluted in sample buffer, and analyzed by Western blotting
using monoclonal anti-Rac antibody (Transduction Laboratories; 1:2000),
rabbit anti-Cdc42 (SC-87; Santa Cruz Biotechnology; 1:500), or rabbit
anti-RhoA (SC-418; Santa Cruz Biotechnology; 1:250). Activated ERK1/2
and Akt/protein kinase B were detected by
anti-phosphomitogen-activated protein kinase (New England Biolabs;
1:1000) and anti-phospho-AKT (Ser-473; New England Biolabs; 1:500), respectively.
Fluorescence Microscopy--
Cells were grown on
laminin-1-coated glass coverslips and fixed in 3.7% formaldehyde/PBS
for 10 min. Cells were permeabilized in 0.1% Triton X-100 and blocked
with 2% bovine serum albumin in PBS. HA-tagged LPA1 was
detected by antibody 3F10 (Roche Molecular Biochemicals) and
fluorescein isothiocyanate-labeled secondary antibodies
(Zymed Laboratories Inc.). Cells were stained
simultaneously with rhodamine-conjugated phalloidin (Molecular Probes).
Images were collected by confocal microscopy (Leica).
LPA1/Edg2 Receptor Signaling and Motility
Responses in B103 Cells--
Most cell types co-express at least two
distinct LPA receptors, which hampers the dissection of
receptor-specific signaling pathways. One exception is the B103
neuroblastoma cell line, which lacks detectable expression of LPA
receptors (30). Through retroviral transduction, we obtained B103 cells
that stably express the epitope-tagged LPA1/Edg2 receptor
at relatively modest levels (B103-LPA1 cells), as shown by
immunoprecipitation and immunoblot analysis (results not shown). In
serum/LPA-containing medium, the parental B103 cells have a rounded,
refractile appearance and tend to grow in small islands. In contrast,
B103-LPA1 cells display a flattened morphology
characterized by prominent lamellipodia and membrane ruffles, whereas
they are more "scattered" throughout the dish (Fig.
1A), suggesting that
LPA1 receptor expression promotes random cell motility.
Proper membrane localization of LPA1 receptors was examined
by confocal microscopy, which reveals that LPA1 receptors localize preferentially to lamellipodia (Fig. 1B). We found
that LPA1 receptors undergo rapid internalization from the
plasma membrane after LPA addition (Fig. 1B) and mediate
pertussis toxin (PTX)-sensitive activation of ERK1,2; the latter
response was not observed in the parental B103 cells (Fig.
1C). Thus, LPA1 receptors expressed in B103
cells are functional and couple to Gi/o-mediated activation of ERK1,2.
When maintained in serum-free medium, B103-LPA1
cells underwent rapid cell rounding and process retraction following
addition of LPA (Fig. 2A),
very similar to the contractile responses observed in LPA-treated
N1E-115 neuroblastoma cells (12). LPA-induced cell contraction was
complete after about 3 min and was observed in at least 80% of the
cells in a randomly selected microscopic field (Fig. 2A). In
the continuous presence of LPA, rounded cells began to re-spread after
15-30 min. Cell rounding and re-spreading were fully prevented by
retrovirally introduced dominant-negative N19RhoA and the Rho kinase
inhibitor Y-27632 but not by
PTX2 (see also Ref. 30).
LPA-induced cell rounding was associated with a rapid increase in
RhoA-GTP levels, as measured by the GST-Rhotekin pull-down assay (Fig.
2B). In most experiments, however, RhoA activation was hard
to detect and always very transient (peaking at 2-3 min and lasting
<10 min). From these results, together with previous findings (13,
22-24, 31), we conclude that LPA1 receptors couple to a
G
Because LPA1 receptor expression seems to confer a motile
phenotype on B103 cells, we measured LPA-induced cell migration using a
Transwell system, in which a laminin-1-coated membrane filter separated
the upper cell-containing chamber from the lower LPA-containing
chambers. As shown in Fig. 3A,
LPA strongly stimulates cell migration in B103-LPA1 cells
but not in the parental B103 cells. In the absence of added LPA,
B103-LPA1 cells are slightly more motile than the parental
cells (Fig. 3A); one plausible explanation for this finding
is that some "autocrine" LPA may accumulate in the cellular
microenvironment during the course of the experiment (3-4 h). Very
similar migratory responses were observed when LPA was present in both
chambers, indicating that LPA1 receptors mediate both
random and directed cell migration (chemokinesis and chemotaxis, respectively). LPA-induced cell migration was markedly, although not
completely, inhibited by PTX but not by the mitogen-activated protein
kinase/ERK kinase inhibitor PD98059 at doses that block ERK1,2
activation (Figs. 3A and 1C). Migration in
control cells was also reduced by PTX treatment, indicating that
Gi is required to sustain basal cell motility. Thus,
LPA1 receptor-driven cell migration is mediated by
Gi but independent of the Gi-ERK1,2 activation pathway.
Cell migration during wound healing of fibroblast monolayers (in
serum-containing medium) depends on the activity of RhoA, Rac, and
Cdc42, with active Rac inducing forward cell movement (20). We examined
the requirement of Rac, RhoA, and Cdc42 for LPA-induced cell migration
by expressing their dominant-negative versions in B103-LPA1
cells via retroviral transduction. As shown in Fig. 3B,
expression of dominant-negative N17Rac led to a significant inhibition
of LPA-induced migration. Expression of dominant-negative N19RhoA and
N17Cdc42 also inhibited cell migration, albeit to a lesser degree.
These results indicate that LPA-induced cell migration requires the
activity of all three Rho GTPases.
We next measured the activation state of Rac and Cdc42 in
LPA-stimulated B103-LPA1 cells, using GST-PAK pull-down
assays. As shown in Fig. 3C, LPA1 receptor
stimulation leads to a rapid increase in Rac-GTP levels. Unlike the
relatively weak and transient RhoA activation response (Fig.
2B), LPA-induced Rac activation was robust and prolonged,
decreasing to above basal levels after about 30 min (Fig.
3C). The GST-PAK fusion protein can also be used to detect
Cdc42 activity. However, we did not detect increased Cdc42 activity
above basal levels in LPA-stimulated B103-LPA1 cells
(n = 6; results not shown).
Activation of Rac by cell-surface receptors in general, and GPCRs in
particular, occurs through incompletely characterized effector routes.
In many cases, however, Rac activation is critically dependent on PI3K
activity. We found that LPA-induced Rac activation in
B103-LPA1 cells is inhibited by PTX and the PI3K inhibitor wortmannin (Fig. 3D), consistent with Rac being activated
via Gi-mediated stimulation of PI3K activity. In keeping
with this, LPA activates the PI3K downstream target Akt/protein kinase
B in a PTX- and wortmannin-sensitive manner (Fig.
3D). PI3K isozymes can be activated by binding to receptor
protein tyrosine kinases, activated Ras or G protein The GDP/GTP Exchange Factor Tiam1 Mediates LPA-induced
Rac Activation--
The available evidence indicates that activation
of a given Rho GTPase occurs through stimulation of a GDP/GTP exchange
factor (GEF), rather than by inhibition of a GTPase-activating protein (for review see Ref. 36). The above results therefore prompt the
question of which GEF(s) may link LPA receptors to Rac activation in a
PI3K-dependent manner. Little is still known about the
identity of Rac-GEFs that act downstream of GPCRs. However, one
attractive candidate is Tiam1, a Rac-specific GEF that was originally
isolated as a lymphoma invasion-inducing gene product (37). Tiam1 is widely expressed and has been implicated not only in tumor cell invasion and metastasis but also in neurite outgrowth and cell-cell adhesion (38, 39). Tiam1 function is PI3K-dependent by
virtue of the presence of an N-terminal pleckstrin homology domain that binds preferentially to PtdIns(3,4,5)P3 (40), but otherwise the upstream signaling pathways that lead to Tiam1 activation remain unknown.
Whereas Tiam1 is highly expressed in B103 cells,2
interference approaches were not feasible since dominant-negative
versions of Tiam1 are not available, and our experiments using
RNAi-expressing vectors met with little success so far. To examine the
possible role of Tiam1 in LPA1 receptor signaling, we
therefore took advantage of LPA-responsive cells that lack endogenous
Tiam1, notably COS7 cells (38), Ras-transformed MDCK cells (41), and
Tiam1-null fibroblasts (see below). These cell types predominantly
express LPA1 receptors, with little LPA2 and/or
LPA3 mRNA
detectable.3 In COS7 cells,
LPA activates the Gi-mediated Ras-ERK1/2 pathway (42) yet
fails to stimulate Rac activity (Fig.
4A). In Tiam1-transfected COS7
cells, however, a significant increase in Rac-GTP levels was observed
in response to LPA.
We then turned to MDCK epithelial cells, which express endogenous Tiam1
(39). When transformed by activated V12Ras, MDCK cells (MDCK-f3) revert
from an epithelial to a mesenchymal phenotype, which is associated with
transcriptional down-regulation of Tiam1 and reduced Rac-GTP levels
(41). As shown in Fig. 4B, LPA activates Rac in wild-type
MDCK cells but not in the Tiam1-deficient MDCK-f3 cells.
Re-introduction of Tiam1 restores Rac activation by LPA (Fig.
4B). Taken together, these results show that LPA-induced Rac
activation requires Tiam1.
Next, we compared Rac activation in MEFs derived from Tiam1 homozygous
knockout mice with that in wild-type MEFs. Tiam1-deficient mice are
phenotypically normal, but they are resistant to skin carcinogenesis
(25). Wild-type and Tiam1 knockout (
Assessment of the migratory behavior of Tiam(
In conclusion, our findings indicate that LPA1 receptors
couple to a Gi-mediated PI3K-Rac activation pathway that is
essential for the stimulation of cell motility. We have identified
Tiam1 as the GDP/GTP exchange factor that is necessary and sufficient for LPA to activate Rac in three different cell types. This newly established Gi-Tiam1-Rac pathway counteracts the
G12/13-linked RhoGEF-RhoA activation pathway, as
schematically depicted in Fig. 5. In the
simplest signaling scheme that is compatible with the current evidence,
Tiam1 is activated by direct binding of PI3K lipid products
(particularly PtdIns(3,4,5)P3; Ref. 40) to its N-terminal
pleckstrin homology domain. However, it could well be that additional
signals emanating from Gi are required for full activation
of Tiam1, perhaps similar to the situation in P-Rex1, a Rac-specific
GEF that is synergistically activated by PtdIns(3,4,5)P3
and G12/13-linked Rho-specific guanine nucleotide
exchange factors, it is unknown how LPA receptors may signal to Rac.
Here we report that the prototypic LPA1 receptor
(previously named Edg2), when expressed in B103 neuroblastoma cells,
mediates transient activation of RhoA and robust, prolonged activation
of Rac leading to cell spreading, lamellipodia formation, and
stimulation of cell migration. LPA-induced Rac activation is inhibited
by pertussis toxin and requires phosphoinositide 3-kinase
activity. Strikingly, LPA fails to activate Rac in cell types that lack
the Rac-specific exchange factor Tiam1; however, enforced expression of
Tiam1 restores LPA-induced Rac activation in those cells.
Tiam1-deficient cells show enhanced RhoA activation, stress fiber
formation, and cell rounding in response to LPA, consistent with
Tiam1/Rac counteracting RhoA. We conclude that LPA1
receptors couple to a Gi-phosphoinositide 3-kinase-Tiam1
pathway to activate Rac, with consequent suppression of RhoA activity,
and thereby stimulate cell spreading and motility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
12/13-mediated activation of the small
GTPase RhoA (12, 13); however, opposite morphological responses to LPA
have also been observed, i.e. cell spreading and
pseudopodia formation (14, 15).
12/13
subunits; furthermore, they have led to the identification of a family
of Rho-specific guanine nucleotide exchange factors (RhoGEFs) that
provide a direct link between G
12/13 and RhoA-GTP accumulation (22-24). In contrast, very little is still known about how LPA receptors may modulate the activity of Rac and/or Cdc42. In
particular, the identity of the Rac-GEF(s) (and/or Cdc42-GEFs) that may
act downstream of LPA receptors remains unknown. In the present study,
we set out to analyze the activation of the three Rho GTPases
downstream of the prototypic LPA1/Edg2 receptor and to
determine how their activity relates to LPA-induced cell migration. We
show that the LPA1 receptor, in addition to
transiently activating RhoA, mediates prolonged activation of Rac via a
Gi-PI3K pathway leading to lamellipodia formation, cell
spreading, and migration. Furthermore, we show that the Tiam1 exchange
factor provides the link between LPA1/Edg2 receptors and
Rac activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Functional expression of
LPA1 receptors in B103 neuroblastoma cells.
A, morphology of B103 and B103-LPA1 cells in
serum/LPA-containing medium. B, cell-surface expression of
HA-tagged LPA1 receptors (green) and F-actin
(red) in B103-LPA1 cells. Receptors are only
detectable in a subpopulation of cells with sufficiently high
expression levels. Right panel, note HA-LPA1
receptor internalization after addition of LPA (1-oleoyl; 1 µM; 20 min). Bar, 20 µm. C,
detection of activated ERK1/2 (pERK(1,2)) in
serum-starved B103 and B103-LPA1 cells, determined by
Western blotting using anti-pERK1,2 antibodies. Sphingosine 1-phosphate
(S1P), which acts on its own G protein-coupled receptors,
was used as a positive control. LPA (1 µM) and S1P (100 nM) were added 5 min prior to cell lysis. PTX,
pertussis toxin (200 ng/ml, overnight preincubation); PD,
MEK inhibitor PD98059 (20 µM; 10 min
preincubation).
12/13-linked RhoGEF-RhoA-Rho kinase pathway to mediate
rapid but transient actomyosin-driven cytoskeletal contraction.
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Fig. 2.
LPA-induced cell rounding and RhoA
activation. A, morphological responses of serum-starved
B103 and B103-LPA1 cells to LPA (1 µM; 3 min). Note rapid LPA-induced cell rounding and process retraction
within 3 min in virtually all B103-LPA1 cells (in the same
microscopic field), with no response observed in the parental B103
cells. Bar, 40 µm. B, rapid activation of RhoA
by LPA and S1P (1 µM; 2 min), as determined by the
GST-Rhotekin pull-down assay and Western blotting.
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Fig. 3.
LPA1 receptor-mediated cell
migration and Rac activation. A, Transwell cell
migration and effects of inhibitors. Cell migration was measured after
4 h. Concentrations: LPA, 1 µM; PTX, 200 ng/ml;
PD98059, 20 µM. Each data point is the average number of
cells in four randomly selected fields. Numbers represent
the mean ± S.D. of duplicate wells (n = 4).
B, effects of dominant-negative Rho GTPases (introduced
by retroviral transduction) on LPA-induced cell migration.
Inset, expression of Myc-tagged N17Rac, N17Cdc42,
and N19RhoA, as determined by Western blotting. C,
LPA-induced Rac activation and its kinetics, as determined by GST-Pak
pull-down assays and Western blotting in B103-LPA1 cells.
LPA concentration, 1 µM. D, activation of Rac
and Akt/protein kinase B in B103-LPA1 cells
stimulated for 3 min with LPA (1 µM) or insulin
(Ins, 1 µg/ml), and the inhibitory effects of PTX (200 nM, overnight) and wortmannin (Wm, 100 nM, 20 min).
subunits.
Previous antibody-blocking experiments have implicated the PI3K
isoform in LPA signaling (32), whereas more recent findings indicate
that this ubiquitously expressed
-isoform is activated by G
dimers both in vitro and in vivo (33-35).
Therefore, LPA-induced Rac activation is most likely mediated by the
G
-regulated PI3K
isoform, although this remains to be
established experimentally.
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Fig. 4.
Effects of Tiam1 expression on LPA-induced
Rac/Rho activation. A, LPA activates Rac in COS-7 cells
transfected with Tiam1 (HA-tagged) but not in the parental cells. Total
Rac levels and expression of Tiam1 were determined by Western blotting.
B, upper panel, LPA-induced Rac activation in
MDCK epithelial cells and their Ras-transformed counterparts (MDCK-f3
cells) in which endogenous Tiam1 is down-regulated (41).
Re-introduction of Tiam1 (HA-tagged) in MDCK-f3 cells by retroviral
transduction restores LPA-induced Rac activation. Lower
panel, expression controls of endogenous Tiam1 and transfected
HA-Tiam1. C, activation of Rac and ERK1,2 by LPA (1 µM; 3 min) in wild-type (wt) and Tiam1( /
)
MEFs (derived from homozygous knockout mice). D, upper
panel, confocal images of serum-starved wild-type and Tiam1
/
MEFs, before or after LPA stimulation (1 µM, 5 min) and
stained for F-actin. Bar, 20 µm. Lower panel,
RhoA activation by LPA (2 min, 1 µM) detected in
Tiam1(
/
) but not wild-type MEFs.
/
) MEFs express
LPA1 and little LPA2 but not LPA3
mRNA.3 In serum-starved MEFs, LPA induced a significant
increase in Rac-GTP levels (Fig. 4C). In contrast, no
LPA-induced increase in Rac-GTP levels was detected in the Tiam1(
/
)
cells, whereas ERK1,2 activation was not impaired (Fig. 4C).
Thus, three independent lines of evidence obtained in different cell
systems (COS7, MDCK, and Tiam1-knockout MEFs) demonstrate that Tiam1 is
necessary and sufficient for LPA to activate Rac. In other words,
LPA-induced Rac activation is mediated by
Tiam1.4
/
) MEFs was hindered
by the fact that Tiam1 deficiency led to marked cell contraction, enhanced stress fiber formation, and reduced cell adhesion, when compared with wild-type MEFs (Fig. 4D and results not
shown). LPA stimulation led to further cytoskeletal contraction in the Tiam1(
/
) cells but not in the wild-type MEFs. These observations strongly suggest that RhoA signaling is enhanced following Tiam1 deletion. Indeed, although no RhoA activation was detectable in wild-type MEFs, the Tiam(
/
) cells showed a significant RhoA activation response to LPA (Fig. 4D). It thus appears that
the balance between Rac and RhoA activity depends on Tiam1 expression, a notion consistent with previous observations (29, 38) showing that
Tiam1/Rac activation inhibits RhoA. Whereas the mechanism underlying
this negative cross-talk remains to be elucidated, these results show
that the Rac-RhoA activity balance in a given cellular context is
critical in determining whether LPA receptor stimulation leads to cell
spreading and migration or to cell rounding and reduced adhesion.
subunits (44). In addition to the identification of Tiam1
as a key player in LPA-induced Rac activation, our findings highlight
the importance of LPA1/Edg2 as a cell motility-stimulating GPCR. Whether the other LPA receptor members, LPA2/Edg4 and
LPA3/Edg7, can play a similar physiological role is
currently under investigation.
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Fig. 5.
LPA1 receptor signaling pathways
leading to activation of Rac and RhoA. In this scheme, Rac
activation is mediated by Gi( ) and involves
PI3K-dependent activation of the Tiam1 RacGEF; PI3K
is
the most likely PI3K isoform involved (see text). RhoA activation
occurs in parallel via one or more G
12/13-linked
RhoGEF(s) (22-24). Through an inhibitory cross-talk mechanism that
remains to be elucidated, Tiam1/Rac signaling suppresses both basal and
LPA-induced RhoA activation. Coordinate regulation of Rac and RhoA
activity thus controls LPA-induced cytoskeletal changes (cell spreading
and rounding, respectively) and cell motility. See text for further
details.
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ACKNOWLEDGEMENTS |
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We thank Laurens van Meeteren, Ingrid Verlaan, Trudi Hengeveld, and Gerben Zondag for their excellent assistance, and Angeliki Malliri for providing the MEFs.
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FOOTNOTES |
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* This work was supported by the Dutch Cancer Society and a European Commission fellowship (to C. O.).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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Division of
Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-512-1971; Fax:
31-20-512-1989; E-mail: w.moolenaar@nki.nl.
Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M210151200
2 F. van Leeuwen, unpublished results.
3 L. van Meeteren, B. Giepmans, and W. H. Moolenaar, unpublished results.
4 A previous study (43) reported that LPA induces threonine phosphorylation of Tiam1, but not Rac activation, in 3T3 cells. Although we could readily detect basal threonine phosphorylation of Tiam1 using anti-phosphothreonine antibodies, we failed to detect an increase in Tiam1 phosphorylation following LPA stimulation in any of the cell types used here.2 We therefore conclude that enhanced threonine phosphorylation is not involved in Tiam1 activation.
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ABBREVIATIONS |
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The abbreviations used are: LPA, lysophosphatidic acid; ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; MEFs, mouse embryo fibroblasts; PI3K, phosphoinositide 3-kinase; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PTX, pertussis toxin; S1P, sphingosine 1-phosphate; HA, hemagglutinin; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; MDCK, Madin-Darby canine kidney cells.
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