From the Department of Biochemistry, Institute for
Developmental Research, Aichi Human Service Center, Kasugai, Aichi
480-0392 and the § Faculty of Bioscience and Biotechnology,
Tokyo Institute of Technology, Midori-ku,
Yokohama 226-8501, Japan
Received for publication, August 18, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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The pertussis toxin-sensitive G protein,
Gi, has been implicated in lysophosphatidic
acid-induced cell mitogenesis and migration, but the mechanisms remain
to be detailed. In the present study, we found that pertussis toxin
blocks lysophosphatidic acid-induced cell spreading of NIH 3T3
fibroblasts on fibronectin. This prevention of cell spreading was
eliminated by the expression of constitutively active mutants of Rho
family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In
addition, activation of the endogenous forms was suppressed by
pertussis toxin, indicating that Gi-induced cell spreading
is mediated through the Rac and Cdc42 pathway. Transfection of
constitutively active mutants of G Most cell types respond to extracellular matrix proteins by
adhering and then spreading out to acquire a flattened morphology. The
cell spreading is also regulated by soluble factors from serum such as
lysophosphatidic acid (LPA)1
and platelet-derived growth factor, which then induce cell growth and
migration (1, 2). The process is accomplished by complex dynamic
rearrangements of the actin cytoskeleton, and the dynamics appear to be
coordinated in space and time by intracellular signaling pathways
involving Rho family small GTP-binding proteins (3, 4), although the
precise mechanisms are poorly understood.
Rho family members such as Rho, Rac, and Cdc42 are part of the Ras
superfamily of small GTP-binding proteins that act as guanine nucleotide-regulated switches (3). Their activity is regulated primarily by two groups of proteins: guanine nucleotide exchange factors (GEF), which catalyze exchange of GDP for GTP; and
GTPase-activating proteins, which stimulate hydrolysis of GTP to GDP.
Upon binding to GTP, Rho family GTP-binding proteins interact with and
activate various downstream effector proteins such as Rho kinases and
p21-activated kinases (PAK). Rho stimulates the formation of actin
stress fibers and focal adhesions, whereas Cdc42 activation triggers
the extension of filopodia and Rac controls growth factor-stimulated
membrane ruffling and formation of lamellipodia (3). It has been
defined how soluble extracellular factors induce the assembly of focal adhesions and actin filaments in serum-starved adherent fibroblasts through activation of Rho. However, there are several reports that Rho
family small GTP-binding proteins are also involved in the generation
of intracellular morphological structures during cell spreading on an
extracellular matrix (5-7). Activation of Rac and Cdc42 and consequent
stimulation of PAK have been observed in NIH 3T3 cells in response to
plating cells on fibronectin (5). With Rat1 cells, cell spreading was
significantly reduced by expression of dominant negative mutants of
Rac, Cdc42, and Rho (6). More recently, it was shown that plating Swiss
3T3 cells on fibronectin-coated dishes elicited a transient inhibition
of Rho activation, suggesting the existence of an
adhesion-dependent negative feedback loop (7).
LPA induces multiple cellular responses through a G protein-coupled
receptor and activates Ras and Rho family GTP-binding proteins via
different pathways (1). In fibroblasts, LPA stimulates cell growth and
lowers cAMP levels in a pertussis toxin (PTX)-sensitive manner,
suggesting coupling of the LPA receptor to Gi family G proteins (8). LPA-induced activation of the Ras/mitogen-activated protein kinase cascade also occurs in a PTX-sensitive fashion, and
G It is well known that LPA induces cell migration, which is blocked by
PTX, suggesting the involvement of Gi. However, regulation of Rho family GTP-binding proteins by Gi has not been
clearly shown to date. In the present study, we found that PTX blocks LPA-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. We
show here Gi stimulation via the LPA receptor leads to Rac and Cdc42 activation during cell spreading.
Materials--
pCMV5-G Production of GST-CRIB--
PAK1 cDNA was a generous gift
from L. Lim (National University of Singapore). The Cdc42/Rac
interactive binding domain (CRIB) of PAK1 (amino acids 67-150) (25)
was amplified by polymerase chain reaction using PAK1 cDNA as a
template, ligated into pCMV-Myc, and confirmed by DNA sequencing (20).
The cDNA of Myc-CRIB was cloned into a pGEX-2T vector (Amersham
Pharmacia Biotech). Expression and purification of GST fusion protein
were performed in accordance with the manufacturer's instructions and
published protocols (26). Escherichia coli BL21 cells
transformed with the GST-Myc-CRIB construct were grown at 37 °C, and
expression of recombinant protein was induced by addition of 0.1 mM isopropyl-1-thio- Transfection and the Cell Spreading Assay--
NIH 3T3 cells
were grown in Dulbecco's modified essential medium supplemented with
10% calf serum. For transient transfections, cells were plated at a
density of 6 × 105 cells/10-cm dish 24 h before
transfection with LipofectAMINE Plus reagent according to the
manufacturer's instructions (Life Technologies, Inc.). The final
amount of the transfected DNA for a 10-cm dish was adjusted to 4 µg
containing 3.6 µg of plasmid of interest or empty vector pCMV5 and
0.4 µg of pEGFP-C3 (CLONTECH). Medium replacement
with serum-free medium was performed after 24 h, and the cells
were incubated for another 24 h, in the presence or absence of 20 ng/ml PTX for the last 16 h. They were then washed with
phosphate-buffered saline, detached with trypsin-EDTA, and washed with
Dulbecco's modified essential medium containing 0.3 mg/ml trypsin
inhibitor and 1 mg/ml fatty acid-free bovine serum albumin. Cells were
then resuspended in Dulbecco's modified essential medium, replated on
fibronectin-coated glass coverslips or dishes, and incubated for 1 h, unless otherwise specified, in the absence or presence of 10 µM LPA, 10 ng/ml platelet-derived growth factor, 10 ng/ml
epidermal growth factor, and 5 µM BIM. After incubation, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline
for immunocytochemistry or lysed with 1% SDS in 20 mM
Tris-HCl, pH 8.0, 1 mM EDTA for immunoblot analyses.
Untransfected cells were stained for F-actin with tetramethylrhodamine
isothiocyanate-phalloidin. To quantitate cell spreading, images of
cells were obtained using a laser scanning microscope (Fluoview,
Olympus), and the areas of fluorescence-positive or GFP-positive cells
were measured using Fluoview image analysis software. For each
experiment, the areas of 50 cells were quantitated. All results shown
represent the mean ± S.D. of at least three independent
experiments. Data were analyzed by performing unpaired Student's
t test. For immunoblotting, cell lysates were subjected to
SDS-polyacrylamide gel electrophoresis (27) or
Tricine/SDS-polyacrylamide gel electrophoresis (28) for immunoblotting
with various antibodies.
GTPase Pull-down Assay--
This was performed essentially as
described previously (26). After replating on fibronectin-coated
dishes, cells were washed with phosphate-buffered saline, incubated for
5 min at 0 °C in buffer B (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10% glycerol,
0.2 mM phenylmethylsulfonyl fluoride, and 2 µg/ml trypsin inhibitor) containing 1% Nonidet P-40, and centrifuged at 21,000 × g for 5 min at 4 °C. Aliquots were taken from the
supernatant to compare protein amounts. For this purpose, incubation
with bacterially produced GST-Myc-CRIB fusion proteins bound to
glutathione-coupled Sepharose 4B beads at 4 °C for 60 min was
followed by washing three times in an excess of buffer B containing
0.5% Nonidet P-40. Proteins bound to the beads were eluted in Laemmli
sample buffer (27) and then analyzed for bound Rac and Cdc42 by
immunoblotting using monoclonal mouse antibodies against human Rac1 and
Cdc42, respectively. Detection was with a chemiluminescence reagent
(PerkinElmer Life Sciences) and densitometry analysis was
performed using the LAS-1000 system (Fujifilm).
To investigate the role of Gi signaling during cell
spreading, serum-starved NIH 3T3 fibroblasts were incubated for 16 h in the absence or presence of PTX, replated on fibronectin-coated plastic dishes, then exposed to 10 µM LPA for various
times, and stained with rhodamine-phalloidin to label F-actin (Fig.
1). Cells incubated in the absence of PTX
spread over a period of 60 min. This was markedly diminished with PTX
treatment. To confirm that PTX abolishes only the function of
Gi, we examined the effects of PTX with induction by other
stimulants known to enhance cell spreading or cell migration.
Fibronectin alone (none), platelet-derived growth factor, and epidermal
growth factor induced cell spreading, although the staining of F-actin
in cell cortex was much weaker than that observed in LPA-stimulated
cells, and this was not affected by PTX (Fig.
2). These results suggest that PTX
specifically blocks Gi activation via the LPA receptor. The
apparently paradoxical result that spreading occurs equally on
fibronectin in the presence or absence of LPA, but is inhibited by PTX
only in the presence of LPA will be explained later (see
"Discussion").
i and
G
11 and G
subunits enhanced spreading of pertussis
toxin-treated cells. G
1 with G
12, a major
G
form in fibroblasts, was more effective for increasing cell
spreading than G
1
2 or G
1
plus G
12S2A, a mutant in which Ser-2, a phosphorylation
site for protein kinase C, is replaced with alanine. In addition, a
protein kinase C inhibitor diminished
G
1
12-induced cell spreading, suggesting a
role for phosphorylation of the protein. These findings indicate that
both G
i and G
stimulate Rac and Cdc42 pathways
with lysophosphatidic acid-induced cell spreading on fibronectin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of G proteins transduce this effect (9, 10). In
contrast, in a variety of cells, stimulation of phospholipase C by LPA
was shown to be PTX-insensitive, so that LPA receptor coupling to
Gq family G proteins may also be important (11). LPA
induces the Rho-dependent formation of actin stress fibers and focal adhesions in quiescent Swiss 3T3 fibroblasts (12, 13).
G
12 and G
13 probably mediate this action,
because microinjection of constitutively active mutants of
G
12 and G
13 into fibroblasts triggers
actin polymerization in a Rho-dependent manner (12, 13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i2Q205L,
pCMV5-G
11Q209L, pCMV5-G
sQ227L,
pCMV5-G
12Q229L, pCMV5-G
2,
pCMV5-G
12, pCMV5-G
12S2A,
pCMV5-G
1, the pCMV5 carboxyl terminus of
-adrenergic
receptor kinase 1 (
ARKct), pCMV5-FLAG-RhoAT19N, pCMV5-FLAG-Rac1T17N,
pCMV5-FLAG-Cdc42HsT17N, pCMV5-FLAG-RhoAG14V, pCMV5-FLAG-Rac1G12V, and
pCMV5-FLAG-Cdc42HsG12V were constructed as detailed previously
(14-20). Antibodies against G
i2, G
7,
G
12, phospho-G
12, and G
subunits,
generated by ourselves, have been described previously (21-24). Rabbit
polyclonal antibodies against phosphotyrosine, G
q/11,
G
s, and G
12 were purchased from Santa
Cruz Biotechnology and mouse monoclonal antibodies against human Rac1
and Cdc42 from Transduction Laboratories. Mouse monoclonal antibodies
against the FLAG and Myc epitopes were purchased from Sigma and Roche
Molecular Biochemicals, respectively. Tetramethylrhodamine isothiocyanate-phalloidin was obtained from Molecular Probe, PTX from
Seikagaku Kogyo, bisindolylmaleimide I (BIM) from
Calbiochem-Novabiochem Co., LPA from Avanti Polar Lipids Inc.,
platelet-derived growth factor and epidermal growth factor from
Immunobiological Laboratories, glutathione-coupled Sepharose 4B beads
from Amersham Pharmacia Biotech, and
isopropyl-1-thio-
-D-galactopyranoside and fibronectin from Wako Pure Chemical Industries.
-D-galactopyranoside for
4 h. Cells were harvested, resuspended in buffer A (50 mM Tris-HCl, pH 8.2, 2 mM MgCl2,
0.2 mM Na2S2O3, 10%
glycerol, 20% sucrose, 1 µg/ml trypsin inhibitor, 0.2 mM
phenylmethylsulfonyl fluoride), then sonicated on ice, and mixed gently
for 30 min after addition of 10% Triton X-100 to a final concentration
of 1%. Cell lysates were centrifuged at 4 °C for 10 min at
12,000 × g, and the supernatant was incubated with
glutathione-coupled Sepharose 4B beads for 30 min at room temperature
followed by further incubation at 4 °C for 30 min. The beads were
washed three times in lysis buffer A and once with phosphate-buffered
saline. GST-Myc-CRIB fusion protein-bound beads were suspended in
phosphate-buffered saline, and glycerol was added to a
final concentration of 50% and stored at
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Effects of PTX on LPA-induced spreading of
NIH 3T3 fibroblasts on fibronectin. NIH 3T3 cells were cultured in
the absence (left) or presence (right) of 20 ng/ml PTX for 16 h, trypsinized, and replated on
fibronectin-coated coverslips in the presence of 10 µM
LPA. A, cells were fixed at the indicated times after
plating and stained for F-actin with tetramethylrhodamine
isothiocyanate-phalloidin. Scale bar, 50 µm. B,
quantification of spreading by measuring of cell areas. Open
circles, PTX-untreated; solid circles, PTX-treated.
Values are means ± S.D. from three experiments. *,
p < 0.01 compared with PTX-untreated cells.
View larger version (52K):
[in a new window]
Fig. 2.
Effects of PTX on cell spreading induced by
LPA, platelet-derived growth factor, and epidermal growth factor.
NIH 3T3 cells were cultured in the absence (left) or
presence (right) of 20 ng/ml PTX for 16 h, trypsinized,
replated on fibronectin-coated coverslips in the absence
(none) or presence of 10 µM LPA, 10 ng/ml
platelet-derived growth factor (PDGF) or 10 ng/ml epidermal
growth factor (EGF) and stained for F-actin after 60 min.
Scale bar, 50 µm.
To examine which subtypes of Rho family GTP-binding proteins contribute
to cell spreading induced by LPA, we transfected plasmids with various
Rho family mutants into NIH 3T3 cells. Cells expressing dominant
negative mutants of Rac (RacT17N) and Cdc42 (Cdc42T17N) were rounded,
whereas dominant negative Rho (RhoT19N) was without effect (Fig.
3, A and B). On the
other hand, constitutively active mutants of Rac (RacG12V) and Cdc42
(Cdc42G12V) enhanced spreading of PTX-treated cells (Fig. 3,
A and C). In contrast, the expression of the
constitutively active form of Rho (RhoG14V) rather caused rounding of
both PTX-treated and untreated cells (Fig. 3). The LPA receptor
activates not only Gi, but also other G proteins such as
Gq/11 and G12/13, which are PTX-insensitive,
and G12/13 is involved in Rho-dependent stress
fiber formation (12). Therefore, it is possible that Rho is also
activated during spreading. In fact, transfection of a dominant
negative mutant of Rho restored cell spreading when the cells were
treated by PTX (Fig. 3C). These observations suggest that
the LPA receptor induces activation of Rac/Cdc42 and Rho through
Gi and probably G12/13, respectively. Rho-mediated cell rounding appears with inhibition of Gi
pathway by PTX.
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To examine whether PTX indeed affected the activation of endogenous Rac
and Cdc42 during cell spreading, we performed pull-down assays of Rac
and Cdc42 using GST-Myc-CRIB-bound Sepharose beads. First, to confirm
that GST-Myc-CRIB specifically binds to active forms of Rac and Cdc42
(29), GST-Myc-CRIB Sepharose beads were incubated with lysates from NIH
3T3 cells expressing FLAG epitope-tagged mutants of Rho family
GTP-binding proteins. GST-Myc-CRIB beads retained constitutively active
forms of Rac and Cdc42 but not dominant negative forms of Rac and
Cdc42, indicating a high specificity for the GTP-bound state. Neither
constitutively active nor dominant negative mutants of Rho were
retained by GST-Myc-CRIB beads, consistent with published data (29)
(Fig. 4A).
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To determine whether Rac and Cdc42 were activated during LPA-stimulated cell spreading on fibronectin, cell lysates were incubated with GST-Myc-CRIB beads and the retained proteins were analyzed by immunoblotting with antibodies against Rac and Cdc42 (Fig. 4, B and C). In the control cells, Rac activity was increased 1.9-fold (Fig. 4B), and Cdc42 activity 1.7-fold (Fig. 4C). In contrast, Rac and Cdc42 activities did not significantly increase within 120 min in PTX-treated cells (Fig. 4, B and C). These results also indicate that Rac and Cdc42 activation is mediated by the Gi pathway during LPA-induced cell spreading.
To examine further the involvement of Rac and Cdc42 activation in LPA-induced cell spreading, we transfected Myc-CRIB into NIH 3T3 cells and replated on fibronectin-coated coverslips in the presence of LPA. Expression of Myc-CRIB strongly inhibited cell spreading (control, 1592 ± 156 µm2; Myc-CRIB, 461 ± 43 µm2).
Next we determined which G protein subunit, G or G
, is
responsible for cell spreading. First we transfected plasmids with several constitutively active mutants of
subunits of G proteins and
tested the restoration of spreading of PTX-treated cells in the
presence of LPA (Fig. 5B).
Constitutively active mutants of G
i2
(G
i2Q205L) and unexpectedly G
11
(G
11Q209L) restored cell spreading to the levels
observed in Mock cells in the absence of PTX. Other active mutants,
G
12 and G
s (G
12Q229L and
G
sQ227L), were not effective in PTX-treated cells (Fig.
5B). These active mutants, however, decreased cell spreading
in the absence of PTX (Fig. 5A), suggesting that
G
12 and G
s cause cell rounding in these
conditions. Then, we cotransfected G
1 with various G
forms into NIH 3T3 cells. G
expression also restored spreading of PTX-treated cells (Fig. 6). To further
test its involvement, we transfected
ARKct, which binds and
sequesters free G
, and showed this to reduce the LPA-induced cell
spreading (Fig. 6).
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We previously reported that the Ser-2 of the G12
subunit, a major G
in fibroblasts, is specifically phosphorylated by
PKC and that this is blocked by PTX (23, 24), as shown in Fig. 6. We
have also demonstrated that phosphorylation of G
12
enhances migration of NIH 3T3 cells (16). Expression of
G
1
12 was more effective at inducing cell
spreading than that of G
1
2 or
G
1 with the mutant G
12S2A, in which a
phosphorylation site is replaced with alanine (Fig. 6). To examine
whether phosphorylation of G
12 by PKC is involved in
cell spreading, we tested the effect of the PKC inhibitor BIM (30)
(Fig. 7). It significantly decreased cell
spreading induced by G
1
12 as well as
phosphorylation of G
12, while being essentially without
effect on G
1
2-induced cell spreading. It
should be note that G
1
12-induced cell
spreading in the presence of BIM is comparable to
G
1
2-induced cell spreading. In
PTX-untreated Mock cells, BIM suppressed LPA-induced cell spreading and
phosphorylation of G
12. Gq/11 is known to
stimulate phospholipase C, which produces diacylglycerol, then
activates PKC. Expression of G
11Q209L increased
phosphorylation of G
12 in PTX-treated cells (Fig. 7),
and the treatment with BIM diminished cell spreading induced by
G
11Q209L, but not by G
i2Q205L (Fig. 7).
Thus PKC-dependent phosphorylated G
12 may
partially contribute to G
11Q209L-induced cell
spreading.
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To examine whether cell spreading with Gi2Q205L,
G
11Q209L, and G
is mediated through Rac and Cdc42
pathways, we cotransfected dominant negative mutants of Rac and Cdc42
with G
i2Q205L, G
11Q209L, and G
into
cells (Fig. 8). The dominant negative
mutant of Rac inhibited cell spreading induced by
G
i2Q205L, G
11Q209L, and G
. However,
the dominant negative form of Cdc42 appeared to be less effective,
especially with G
11Q209L-induced cell spreading.
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DISCUSSION |
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It has been reported that PTX blocks cell growth and migration
induced by LPA (1, 31). In the present study, we demonstrated that
Gi mediates LPA-induced cell spreading, one of important steps of growth and migration processes. LPA receptor is coupled not
only with Gi but also with Gq and
G12 family G proteins. G12 and
G
13, which activate Rho, induce the formation of actin
stress fibers and focal adhesions (12, 13). The present investigation indicated that Gi activates Rac and Cdc42 during cell
spreading stimulated by LPA. The evidence that the dominant negative
mutant of Rho enhanced spreading of PTX-treated cells (Fig.
3C) suggests that this signal protein is also activated with
LPA stimulation. Previous reports indicated that Rac down-regulates Rho
activity in NIH 3T3 cells (32), or Rac may counteract Rho (33). In the
latter case, PAK, which is activated by Rac and Cdc42, was found to
block the phosphorylation of myosin light chain induced by Rho. Ren
et al. (7) have described that plating Swiss 3T3 cells on
fibronectin-coated dishes elicits a transient inhibition of Rho,
followed by an activation phase, suggesting the existence of an
adhesion-dependent negative feedback loop. In addition, cell rounding observed by expression of G
12Q229L (Fig.
5A) suggests that G
12-mediated Rho activation
caused cell rounding in these conditions. Taken together, the available
information indicates that activation of Rac and Cdc42 by
Gi promotes cell spreading and probably interferes with
Rho-mediated cell rounding in LPA-stimulated cells (Fig.
9). On the other hand, it has been shown
that Rac, Cdc42, and Rho were activated during cell spreading on
fibronectin (5-7), and cells also tend to spread probably due to the
down-regulation of Rho by Rac activation (Fig. 9). The degree of
spreading may be determined by a balance between Rac/Cdc42 activation
and Rho activation. The evidence that blockage of Gi by PTX
leads to cell rounding on fibronectin in the presence of LPA suggests
that activation of Rho by G12/13 and fibronectin is much
larger than activation of Rac/Cdc42 by fibronectin (Fig. 9). This
scheme explains the apparently paradoxical result (Fig. 2) that
spreading occurs equally on fibronectin in the presence or absence of
LPA, but is inhibited by PTX only in the presence of LPA.
|
It has been shown that Gi and G
released from
Gi by receptor stimulation regulate adenylyl cyclase and
potassium channels (34). The present study demonstrated that they both
contribute to LPA-induced cell spreading. The fact that expression of
ARKct partially inhibited cell spreading (Fig. 6) supported an
involvement of G
i in addition to G
. We have
demonstrated previously that cotransfection of G
1 with
various G
forms into NIH 3T3 cells increases cell migration (16).
Cell motility of G
12-expressing cells was much greater
than those of G
2-, G
5-, and
G
7-expressed cells. Because the effect of
G
12 was decreased by replacement of Ser2 by alanine, we
concluded that protein phosphorylation enhances cell migration (16).
The present study demonstrated G
1
12 to be
more efficient at stimulating cell spreading than G
1
2 or
G
1
12S2A, consistent with results obtained
for G
-induced motility.
In addition to Gi2Q205L, the expression of
G
11Q209L restored cell spreading in PTX-treated cells
(Fig. 5, 7, and 8). Since this was partially blocked by an PKC
inhibitor, the existence of PKC-dependent and -independent
pathways in G
11-induced cell spreading can be
speculated. Phosphorylation of G
12 in
G
11Q209L-expressing cells points to an involvement in a
PKC-dependent pathway. Although PTX-insensitive
Gq family G proteins would be expected to be activated by
LPA in PTX-treated cells, cell spreading did not occur. The physiological significance of G
11-induced cell spreading
remains uncertain.
A previous study demonstrated that increase of intracellular cAMP
contents by forskolin or a cAMP phosphodiesterase inhibitor blocked
LPA-induced cell spreading and migration of carcinoma cells (35). High
levels of intracellular cAMP induced by forskolin also block chemotaxis
of human embryonic kidney cells expressing the interleukin-8 receptor
(36), and Gi and G
inhibit several types of
adenylyl cyclase (34). It has, furthermore, been reported that LPA
decreases cAMP levels in Swiss 3T3 cells in a PTX-sensitive manner (8).
Therefore, suppression of cAMP levels by Gi may be
important in LPA-induced cell spreading. In fact, the expression of the
constitutively active mutant G
sQ227L decreased cell
spreading (Fig. 5A).
Small GTP-binding proteins are regulated by GEF and
GTPase-activating proteins (3, 37), and recent reports have
indicated that G12 and G
13 are able to
bind directly to p115-RhoGEF (38, 39) or PDZ-RhoGEF (40).
G
13 but not G
12 stimulates the GDP-GTP exchange reaction of p115-RhoGEF. G
may also be associated with the NH2-terminal of Dbl, a kind of RhoGEF (41), but its GEF activity toward Rho is not influenced by G
binding. With respect to GTPase-activating proteins, G
i specifically binds to
that for Rap1, a member of the Ras family. Stimulation of the
Gi-coupled m2-muscarinic receptor translocates rap1GAPII
from cytosol to the membrane and decreases the amount of GTP-bound Rap1
(42). Our finding that G
i and G
induce cell
spreading in a Rac/Cdc42-dependent manner suggests the
existence of GEF or GTPase-activating proteins, which directly interact
with G
i or G
subunits.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. I. Simon, T. Nukada, R. A. Cerione, K. Kaibuchi, and L. Lim for supplying the plasmids. We are also grateful to Dr. Y. Kaziro for encouragement.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by a grant from CREST of Japan Science and Technology.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.
¶ Present address: Dept. of Molecular Cell Pharmacology, National Children's Medical Research Center, Tokyo 154-8509, Japan.
Present address: Graduate School of Agriculture and Life
Science, University of Tokyo, Tokyo 113-8657, Japan.
** To whom correspondence should be addressed: Dept. of Biochemistry, Inst. for Developmental Research, Aichi Human Service Center, Kamiya-cho, Kasugai, Aichi 480-0392, Japan. Tel.: 81-568-88-0811; Fax: 81-568-88-0829; E-mail: toasano@inst-hsc.pref.aichi.jp.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M007541200
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ABBREVIATIONS |
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The abbreviations used are: LPA, lysophosphatidic acid; G protein, heterotrimeric guanine nucleotide-binding regulatory protein; PTX, pertussis toxin; BIM, bisindolylmaleimide I; GST, glutathione S-transferase; GEF, guanine nucleotide exchange factor; PAK, p21-activated kinase; CRIB, Cdc42/Rac interactive binding domain; PKC, protein kinase C; Tricine, N-tris(hydroxymethyl)methylglycine.
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