From the Institut für Pharmakologie, Freie Universität
Berlin, Thielallee 67-73, D-14195 Berlin, Germany
Lysophosphatidic acid (LPA) utilizes a
G-protein-coupled receptor to activate the small GTP-binding protein
Rho and to induce rapid remodeling of the actin cytoskeleton. We
studied the signal transduction from LPA receptors to Rho activation.
Analysis of the G-protein-coupling pattern of LPA receptors by labeling
activated G-proteins with [
-32P]GTP azidoanilide
revealed interaction with proteins of the Gq, Gi, and G12 subfamilies. We could show that in
COS-7 cells, expression of GTPase-deficient mutants of
G
12 and G
13 triggered Rho activation as
measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of G
12 and G
13 induced formation of actin
stress fibers and assembly of focal adhesions in a
Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization was suppressed by
microinjected antibodies directed against G
13, whereas
G
12-specific antibodies showed no inhibition. The
tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth
factor (EGF) receptor-specific tyrphostin AG 1478 completely blocked
actin stress fiber formation caused by LPA or activated
G
13 but not the effects of activated G
12. Also, expression of the dominant negative EGF receptor mutant EGFR-CD533 markedly prevented the LPA- and G
13-induced
actin polymerization. Coexpression of EGFR-CD533 and activated
G
13 in COS-7 cells resulted in decreased Rho-GTP levels
compared with expression of activated G
13 alone. These
data indicate that in Swiss 3T3 cells, G13 but not
G12 is involved in the LPA-induced activation of Rho.
Moreover, our results suggest an involvement of the EGF receptor in
this pathway.
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INTRODUCTION |
Upon stimulation, a number of heterotrimeric G-protein-coupled
receptors initiate cellular responses involving small GTP-binding proteins. For instance, the water-soluble phospholipid lysophosphatidic acid (LPA)1 binds to a
G-protein-coupled heptahelical receptor (1, 2) and stimulates the
activation of Ras and Rho proteins via different pathways (3, 4). In
fibroblasts, LPA lowers cAMP levels in a pertussis toxin
(PTX)-sensitive manner, suggesting the coupling of the LPA receptor to
G-proteins of the Gi subfamily (5). LPA-induced activation
of the Ras/Raf/mitogen-activated protein kinase cascade also occurs in
a PTX-sensitive fashion (3, 6), and G
dimers of heterotrimeric
G-proteins are thought to transduce this effect via intermediary
protein-tyrosine kinases (7, 8). In contrast, in a large variety of
cells, the stimulation of phospholipase C induced by LPA was shown to
be PTX-insensitive (9), suggesting a coupling of the LPA receptor to
members of the Gq subfamily. In addition to these pathways,
LPA induces the Rho-dependent formation of focal adhesions
and actin stress fibers in quiescent Swiss 3T3 fibroblasts (4, 10). A
tyrosine kinase has been implicated in the LPA-induced Rho stimulation,
since the tyrosine kinase inhibitor tyrphostin A 25 blocks the effects
of LPA but not of microinjected Rho on actin polymerization (11).
However, the G-proteins coupling the LPA receptor to Rho activation
have not been specified. LPA-induced cytoskeletal reorganizations are
PTX-insensitive and therefore presumably not transmitted via
Gi subfamily members (12). Gq proteins are
apparently not involved in this pathway, because neither the activation
of protein kinase C nor mobilization of intracellular calcium or
expression of a GTPase-deficient mutant of G
q induces
the formation of actin stress fibers or focal adhesions (12-14).
Interestingly, microinjection of constitutively active mutants of
G
12 and G
13 into Swiss 3T3 cells triggers
actin polymerization in a Rho-dependent manner (13).
In the present study, we determined the G-protein coupling pattern of
the LPA receptor in membrane preparations and characterized the pathway
leading from LPA receptor activation to stress fiber formation in
intact cells. We report direct coupling of the LPA receptor to proteins
of the G12, Gq, and Gi subfamilies.
Furthermore, our data suggest that in intact cells G13 but
not G12 mediates LPA-induced Rho activation involving the
epidermal growth factor (EGF) receptor tyrosine kinase activity.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Swiss 3T3 cells, kindly provided by Dr. Alan
Hall (London), and COS-7 cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 10% fetal bovine serum. To obtain
quiescent and serum-starved Swiss 3T3 cells, cultures were rinsed three times in serum-free DMEM and incubated in DMEM supplemented with 25%
Ham's F-12, 0.2% NaHCO3, 10 mM Na-Hepes, and
0.1% fetal bovine serum (modified DMEM) for 24 h followed by an
incubation in modified DMEM without fetal bovine serum for 18 h.
Membrane Preparations--
Membranes were prepared from
subconfluent Swiss 3T3 cells. Cells were rinsed and scraped in a buffer
containing 20 mM Tris/HCl, pH 8, 1 mM EDTA, 140 mM NaCl, 20 mM
-mercaptoethanol, and 10 mM phenylmethylsulfonylfluoride. The pelleted cells were
resuspended in the buffer described above devoid of NaCl but containing
20 µg/ml leupeptin. Cells were disrupted by forcing the suspension 10 times through a 26-gauge needle. Undisrupted cells were pelleted (750 × g, 2 min), and the supernatant was centrifuged
at 50,000 × g for 30 min at 4 °C. Membranes were
resuspended and stored at
70 °C until use. Protein concentrations
were determined according to Lowry et al. (15).
SDS-PAGE and Immunoblotting--
For immunological detection of
G-proteins, SDS-PAGE using 10% (w/v) polyacrylamide separating gels
was employed. Proteins were transferred to nitrocellulose as described
(16). The subtype specific antisera AS 232 and AS 233 (anti-
12); AS 343, AS 342, and AS 272 (anti-
13); AS 369 (anti-
q/11), and AS 266 (anti-
i) were characterized previously (17). Antisera
against G
12 were incubated with the peptides (10 µg/ml) used for their generation, as published elsewhere (17, 18).
Detection of filter-bound proteins was carried out using the enhanced
chemiluminescence (ECL) Western blotting system purchased from Amersham
(19).
Photolabeling of G
Subunits--
Synthesis of
[
-32P]GTP azidoanilide from [
-32P]GTP
(NEN Life Science Products) was performed according to published
protocols (20). For photolabeling of G
subunits, sedimented Swiss
3T3 membranes (200 µg of protein/tube) were preincubated in the
absence or presence of 10 µM LPA at 30 °C, followed by
the addition of [
-32P]GTP azidoanilide (3 µCi/tube;
specific activity 3,000 Ci/mmol) at 30 °C for different times: 3 min
for G
i, 10 min for G
q/11, and 30 min for
G
12 and G
13 (18). Samples were irradiated
for 15 s at 4 °C with a 254-nm UV lamp. Photolabeled membranes
were pelleted and solubilized in 40 µl of 4% SDS (w/v) at room
temperature, followed by the addition of precipitating buffer (280 µl) containing 10 mM Tris/HCl, pH 7.5, 1 mM
EDTA, 150 mM NaCl, 1% deoxycholate (w/v), 1% Nonidet P-40
(w/v), 1 mM dithiothreitol, 0.2 µM
phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin. Samples were
centrifuged (13,000 × g) for 5 min at 4 °C to
remove insoluble material. The antisera (20 µl) AS 369 for
G
q/11, AS 233 for G
12, AS 343 for
G
13, and AS 266 for G
i were added to the
supernatants followed by constant rotation of the samples for 2 h
at 4 °C. Protein A-Sepharose beads (7 µg) were added, and samples
were incubated at 4 °C overnight. The immunoprecipitates were washed
three times, boiled in SDS-sample buffer, and subjected to SDS-PAGE.
Gels were subsequently dried and analyzed using autoradiography.
Transient Transfection of COS-7 Cells and Rho Activation
Assay--
For transfection experiments, 1.2 × 105
COS-7 cells were plated in 10-cm diameter cell culture dishes, grown
overnight, and subsequently serum-starved for 18 h. Cells were
then washed with phosphate-buffered saline, and 12 µg of DNA mixed
with 60 µl of LipofectAMINE (Life Technologies, Inc.) in 6 ml of
Opti-MEM (Life Technologies, Inc.) were added. In cotransfection
experiments with two different plasmids, 6 µg of each plasmid were
added. The total amount of DNA was kept constant by supplementing with DNA from a vector encoding
-galactosidase. Twelve h after
transfection, COS-7 cell transfectants were labeled with
[32P]orthophosphate (0.4 mCi/ml for 4 h) in
phosphate-free Eagle's minimum essential medium. Cells were lysed on
ice in 1 ml of 50 mM Hepes buffer, pH 7.4, containing 1%
Triton X-100, 0.5% deoxycholate, 0.05% SDS, 150 mM NaCl,
5 mM MgCl2, 1 mM EGTA, 1 mg/ml of
bovine serum albumin, 10 mM benzamidine,
leupeptin-aprotinin-soybean trypsin inhibitor (10 µg/ml), and 1 mM phenylmethylsulfonylfluoride. Nuclei and cellular debris
were sedimented, and NaCl was added to lysates at a final concentration
of 500 mM. After preclearing for 30 min with 8 µg of
protein A-Sepharose, samples were incubated with 4 µg of rabbit
polyclonal anti-Rho A (Santa Cruz Biotechnology) for 60 min at 4 °C.
Immunocomplexes were collected with 15 µg of protein A-Sepharose for
90 min. The beads were washed eight times in 50 mM Hepes
buffer, pH 7.4, 500 mM NaCl, 0.1% Triton X-100, and
0.005% SDS. Nucleotides were eluted in 5 mM EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and
0.5 mM GDP for 20 min at 68 °C and separated by
thin-layer chromatography on polyethyleneiminecellulose plates (Merck)
run in 1 M KH2PO4, pH 3.4. The
positions of unlabeled GDP and GTP standards on the plates were
visualized under 254-nm UV light. Quantitations of radiolabeled
nucleotides were performed with phosphoimaging (Fuji BAS 1500).
Microinjection--
For microinjection studies, quiescent,
serum-starved Swiss 3T3 cells (approximately 103
cells/mm2) plated on glass coverslips marked with squares
to facilitate the localization of injected cells were used. The
cDNAs of G
12 (Q229L; G
12QL),
G
13 (Q226L; G
13QL), and G
q
(R183C; G
qRC), carried by the cytomegalovirus
promotor-containing vector pCis, G
11 (Q209L;
G
11QL) cloned in pZeo, and empty vector controls, were
applied for microinjection. Plasmids were microinjected into the
nucleus alone or with Texas red-coupled dextran (5 mg/ml, Molecular
Probes) to visualize injected cells. Clostridium botulinum C3 toxin was co-microinjected with the cDNAs described above at a
final concentration of 100 µg/ml. After microinjection, the cells
were returned to the incubator for a further 90 min until fixation. As
indicated, cells were treated with tyrphostin A 25 (150 µM), tyrphostin A 1 (150 µM), or genistein
(30 µg/ml) for 60 min. Tyrphostin AG 1478 (1 µM) or
tyrphostin AG 1296 (10 µM) were added 120 min before
fixation. To study cytoskeletal effects of extracellular factors, cells
were incubated with LPA (0.3 µM), thrombin (40 ng/ml),
EGF (10 ng/ml), or bombesin (10 nM) for 10 min. The
phosphatase inhibitor orthovanadate (100 µM) was added to
serum-starved cells 25 min before fixation. Antibodies against G
subunits were diluted and microinjected into the cytosol of cells
followed by an incubation period of 60 min. The dominant negative EGF
receptor mutant EGFR-CD533 carried by the pRK5 vector was expressed in
Swiss 3T3 cells by nuclear microinjection. About 100 cells/field were
injected in each case using a manual injection system (Eppendorf).
Needles were pulled from capillaries with a horizontal micropipette
puller (Sutter).
Fluorescence Microscopy--
Microinjected or growth
factor-treated cells were fixed in 4% paraformaldehyde for 20 min and
permeabilized in 0.2% Triton X-100 for 5 min. For localization of
actin filaments, cells were stained with 0.5 µg/ml fluorescein
isothiocyanate (FITC)-phalloidin (Sigma) for 40 min. Vinculin was
detected after blocking with phosphate-buffered saline supplemented
with 5% fetal bovine serum for 30 min, followed by incubation with a
monoclonal anti-vinculin antibody (VIN-11-5; Sigma) for 60 min.
Subsequently, cells were labeled with a FITC-conjugated goat anti-mouse
antibody (Sigma) for 45 min. Antibody incubations were performed at
room temperature. The coverslips were mounted on glass slides and
examined on an inverted microscope (Zeiss Axiovert 100).
 |
RESULTS |
To characterize the G-protein coupling pattern of the LPA receptor
in Swiss 3T3 fibroblasts, we first determined the expression of
different PTX-insensitive G
subunits in these cells by performing immunoblot experiments with subtype-specific antibodies. Immunoblotting of membrane proteins with an antibody specific for G
q/11
(AS 369) identified the expression of the corresponding 42-kDa proteins (Fig. 1, lane 1). Antisera
raised against a C-terminal peptide of G
12 (AS 232, AS
233) recognized a protein with an apparent molecular mass of 43 kDa
(Fig. 1, lanes 2 and 3). In addition, these
antisera cross-reacted with several unknown proteins of higher and
lower molecular masses that had not been detected in rodent and
nonrodent brain membranes (17). To assure the specificity of AS 232 and
AS 233, which have been extensively characterized (17, 18), we
performed peptide competition experiments. Upon preincubation with the
peptide used for their generation, the detection of G
12
was abolished (data not shown). Immunoblotting of membranes with an
antiserum specific for G
13 (AS 343) recognized a protein
of approximately 43 kDa (Fig. 1, lane 4). These experiments indicate the expression of both G
12 and
G
13 in Swiss 3T3 cell membranes.

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Fig. 1.
Immunological detection of G-protein subunits subunits expressed in Swiss 3T3 cells. Membrane proteins
prepared from Swiss 3T3 cells (150 µg of protein/lane)
were acetone-precipitated and separated by SDS-PAGE using separating
gels containing 10% acrylamide. After blotting, nitrocellulose strips
were incubated with anti- q/11 (AS 369, lane
1), anti- 12 (AS 232, lane 2; AS 233, lane 3), and anti- 13 (AS 343, lane
4) antisera. All antisera were diluted 1:150. The ECL system was
used for detection of filter-bound antibodies. The position of a 43-kDa
marker protein is indicated.
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In previous studies, interactions of the LPA receptor with G-proteins
of the Gi and Gq subfamilies were postulated
mainly from experiments showing the existence of PTX-sensitive as well as PTX-insensitive cellular effects of LPA (9). To directly show the
LPA-mediated activation of G-proteins, membranes of subconfluent Swiss
3T3 cells were photolabeled with [
-32P]GTP
azidoanilide in the presence or absence of LPA. G-protein G
subunits
were subsequently immunoprecipitated with anti-
q/11 (AS
369), anti-
12 (AS 233), anti-
13 (AS 343),
or anti-
i (AS 266) antisera and visualized by
autoradiography. Fig. 2 shows that
stimulation of membranes with 10 µM LPA induced an
incorporation of [
-32P]GTP azidoanilide into G
subunits of Gq/11, G12, G13, and
Gi proteins. For studying [
-32P]GTP
azidoanilide incorporation into G
12 and
G
13, the experiments were performed with prolonged
incubation times of 30 min, whereas an agonist-induced activation of
G
i was only observed with short incubation times,
reflecting the substantial basal guanine nucleotide turnover of
Gi proteins. No increased incorporation of
[
-32P]GTP azidoanilide into G
12 or
G
13 was found using lower concentrations of LPA
(e.g. 1 µM).

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Fig. 2.
Coupling of the LPA receptor to G-proteins in
Swiss 3T3 cell membranes. Cell membranes (200 µg of
protein/tube) were photolabeled with [ -32P]GTP
azidoanilide (3 µCi/tube) in the absence ( ) or presence (+) of LPA
(10 µM) as described under "Experimental Procedures." Membranes were solubilized and immunoprecipitated with
anti- q/11 (AS 369), anti- 12 (AS 233),
anti- 13 (AS 343), and anti- i (AS 266)
antisera and protein A-Sepharose beads. Precipitated proteins were
subjected to SDS-PAGE (separating gels with 10% acrylamide). Gels were
dried, and labeled proteins were visualized by autoradiography. The
position of a 43-kDa marker protein is indicated. Results are
representative of three independent experiments.
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Expression of constitutively active mutants of G12
subfamily members has been reported to induce a
Rho-dependent formation of actin stress fibers in Swiss 3T3
cells (13). To further characterize the functional role of
G12 and G13 in LPA-induced and Rho-mediated regulation of the actin cytoskeleton, we first expressed constitutively active mutants of G
12 (G
12 Q229L;
G
12QL) and G
13 (G
13 Q226L; G
13QL) in Swiss 3T3 fibroblasts by microinjecting
expression plasmids into quiescent cells. Both G
subunits stimulated
the formation of actin stress fibers, whereas GTPase-deficient forms of
G
q (G
q R183C; G
q RC) and
G
11 (G
11 Q209L; G
11 QL)
were unable to induce stress fiber formation (Fig.
3). The expression of
G
12QL and G
13QL was also accompanied by
the formation of vinculin-containing spots with a characteristically
elongated, arrowhead shape typical of Rho-induced assembly of focal
adhesions (22) that were not observed upon microinjection of plasmids encoding G
qRC and G
11QL (Fig. 3). The
dependence of G
12QL- and G
13QL-mediated
cytoskeletal effects on stimulation of Rho activity could be
demonstrated employing purified recombinant C3 exoenzyme from C. botulinum (13). C3 exoenzyme has been shown to inhibit the
isoforms of Rho but no other members of the Rho family (21, 22).
Co-microinjection of C3 exoenzyme (100 µg/ml) abolished the actin
stress fiber formation induced by G
12QL and G
13QL (Fig. 4). Likewise,
focal adhesion assembly caused by both activated G
subunits was
inhibited by C3 exoenzyme (data not shown). These results confirm that
G12 and G13 are able to trigger Rho-dependent effects in Swiss 3T3 cells, whereas
cytoskeletal remodeling typical of an activation of Cdc42 or Rac,
i.e. formation of filopodia or membrane ruffles (23,
24), was not observed. Interestingly, the phosphotyrosine phosphatase
inhibitor orthovanadate (100 µM), which has previously
been shown to stimulate Rho in Swiss 3T3 fibroblasts (11), also induced
a selective formation of actin stress fibers without membrane ruffling
or formation of filopodia (not shown). To test G12
subfamily-induced Rho activation biochemically, we transiently
overexpressed G
12QL and G
13QL in COS-7
cells and analyzed the incorporation of radioactive nucleotides in Rho
A. Expression of activated G
12 and G
13,
but not of the
-galactosidase control, caused an accumulation of
Rho-bound radioactive GTP. The ratio of bound GTP to total nucleotides
bound to Rho [(GTP/GTP + GDP) × 100] was 22 ± 0.57% for the
lacZ control, 28.75 ± 0.92% for G
12QL, and
35.7 ± 4.1% for G
13QL (Fig.
5). These data demonstrate that
expression of G
12QL and G
13QL leads to an
activation of Rho.

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Fig. 3.
Stimulation of actin stress fiber formation
and focal adhesion assembly by constitutively active G
subunits. Expression plasmids (100 ng/µl) containing the
cDNAs of G 12 (Q229L), G 13 (Q226L),
G q (R183C) or G 11 (Q209L), or the empty
vector (control) were microinjected into the nucleus of serum-starved,
subconfluent Swiss 3T3 fibroblasts. Cells were fixed after 90 min with
paraformaldehyde and permeabilized with Triton X-100. Actin stress
fibers were revealed using FITC-labeled phalloidin (left
panels), and focal adhesions were localized with a mouse
anti-vinculin antibody followed by a FITC-conjugated goat anti-mouse
antibody (right panels). Shown are cells from one of four
independent experiments.
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Fig. 4.
Botulinum C3 exoenzyme abolishes the
cytoskeletal effects induced by constitutively active
G 12, G 13, and LPA. Serum-starved Swiss 3T3 cells were stimulated with LPA (200 ng/ml) or microinjected with expression plasmids (100 ng/µl) encoding G 12
(Q229L) or G 13 (Q226L). Cells in the right
panel were co-microinjected with botulinum C3 exoenzyme (100 µg/ml). Actin stress fibers were visualized by indirect
immunofluorescence as described under "Experimental Procedures."
Cells from one of three independent experiments are shown.
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Fig. 5.
Rho-activation due to overexpression of
constitutively active G 12 and G 13.
COS-7 cells were transiently transfected with cDNAs encoding
activated G 12 and G 13. After metabolic
labeling with [32P]orthophosphate, cells were lysed and
RhoA was immunoprecipitated. A, radioactive nucleotides
bound to Rho were eluted and resolved by TLC. The position of GDP and
GTP standards is indicated. B, ratio of GTP to total labeled
nucleotides complexed to Rho [(GTP/GTP + GDP) × 100]. Data represent
means ± S.D. (n = 2). Error bars not shown are
masked by the symbols.
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To characterize the function of individual G12 subfamily
members for LPA-induced activation of Rho in intact cells, we
microinjected antibodies directed against the carboxyl termini of
G
12 and G
13. Actin stress fiber formation
caused by LPA at a concentration of 0.3 µM was suppressed
by application of G
13-specific antiserum AS 343 (dilution of 1:50), whereas microinjection of the corresponding preimmune serum failed to influence LPA-induced cytoskeletal effects. Inhibition of LPA-induced stress fiber formation was also obtained using other G
13-specific antisera, i.e. AS
342 and AS 272 (not shown). Surprisingly, the
G
12-specific antisera (AS 232, AS 233) did not inhibit
the LPA-induced actin polymerization using dilutions of 1:1 to 1:50
(Fig. 6). The application of antibodies
against G
q/11 (AS 369) showed no effects on cytoskeletal
reorganisation caused by LPA. These results suggest that in intact
Swiss 3T3 cells, LPA-induced Rho stimulation may be mediated by
G13 but not by G12.

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Fig. 6.
Suppression of LPA-induced cytoskeletal
effects by antibodies against G 13. Formation of
actin stress fibers is shown in serum-starved Swiss 3T3 cells
stimulated with 0.3 µM LPA (control). Cells were
microinjected with anti- 13-antiserum AS 343, corresponding preimmune serum (AS 343-P),
anti- 12-antiserum AS 232, anti- 12-antiserum AS 233, or
anti- q/11-antiserum AS 369, each diluted 1:50. Actin stress fibers were visualized by fluorescently tagged phalloidin, as
described under "Experimental Procedures." Cells from one of three
independent experiments are shown.
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Since the tyrosine kinase inhibitor tyrphostin A 25 has been shown to
block stress fiber formation evoked by LPA, but not by microinjected
Rho protein, an involvement of a tyrosine kinase acting upstream of Rho
in the pathway of LPA-induced Rho activation appears to be likely (11).
To further study the mechanism whereby G13 mediates the
LPA-induced activation of Rho, we investigated effects of tyrosine
kinase inhibitors on cytoskeletal changes induced by different stimuli.
As demonstrated in Fig. 7A,
tyrphostin A 25 (150 µM) completely inhibited stress
fiber formation induced by LPA and G
13QL (100 ng/µl of
cDNA) but not the cytoskeletal effects of G
12QL (100 ng/µl of cDNA), further confirming the notion that LPA signals
via G13 to Rho. Focal adhesion assembly caused by LPA or
G
13QL was also blocked by incubation with tyrphostin A
25, whereas focal adhesions induced by G
12QL were not
influenced by this tyrosine kinase inhibitor (Fig. 7B). To
exclude that these differences in tyrphostin sensitivity were results
of different expression levels of G
12QL and
G
13QL, we studied cytoskeletal effects caused by various
concentrations of expression plasmids. Cytoskeletal effects of
G
13QL induced by injection of up to 1 µg/µl of
cDNA were abolished by tyrphostin A 25. However,
G
12QL-induced submaximal stress fiber formation after
microinjection of 10 ng/µl of cDNA was tyrphostin A
25-insensitive. Unlike tyrphostin A 25, the tyrosine kinase inhibitor
genistein (30 µg/ml), which has previously been shown to act
downstream of Rho (12), blocked the formation of actin stress fibers
induced by LPA, G
13QL, and G
12QL (Fig.
7A). The biologically inactive tyrphostin A 1 (150 µM) showed no effects on cytoskeletal changes caused by
LPA or the activated G
subunits (data not shown). Using more
specific inhibitors of tyrosine kinases, we found that 1 µM EGF receptor-specific tyrosine kinase inhibitor
tyrphostin AG 1478 (25) completely blocked both the LPA- and
G
13QL-induced formation of actin stress fibers (Fig.
8). Lower concentrations of tyrphostin AG
1478 (e.g. 250 or 500 nM) partially suppressed
actin polymerization caused by G
13QL. Cytoskeletal
effects caused by G
12QL were not influenced by this
tyrphostin at concentrations of up to 20 µM. Tyrphostin AG 1296 (10 µM), a selective platelet-derived growth
factor receptor tyrosine kinase inhibitor, failed to affect stress
fiber formation induced by LPA and G
13QL or by
G
12QL (Fig. 8). Actin stress fiber formation caused by
orthovanadate was also inhibited by tyrphostin A 25 and tyrphostin AG
1478 but not by tyrphostin AG 1296 (data not shown).

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Fig. 7.
Effects of tyrosine kinase inhibitors on
cytoskeletal reorganization caused by LPA, active G 13,
or G 12. Serum-starved Swiss 3T3 cells were treated
with 0.3 µM LPA for 10 min or microinjected with plasmids
(100 ng/µl) encoding G 12 (Q229L) or G 13
(Q226L). Cells were incubated with control buffer ( ), tyrphostin A 25 (150 µM), or genistein (30 µg/ml) for 60 min as
indicated, fixed in paraformaldehyde, and permeabilized using Triton
X-100. Actin filaments (A) or focal adhesions (B)
were visualized with FITC-phalloidin and an anti-vinculin antibody,
respectively, as described under "Experimental Procedures." Shown
are cells from one of four independent experiments.
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Fig. 8.
Inhibition of G13-mediated Rho
activation by tyrphostin AG 1478. Actin stress fiber formation is
shown in Swiss 3T3 cells treated with LPA (0.3 µM) or
injected with plasmids encoding constitutively active
G 12 or G 13. As indicated, cells were
incubated with tyrphostin AG 1478 (1 µM), tyrphostin AG
1296 (10 µM), or buffer (control). Actin cytoskeleton was
visualized by staining of fixed cells with FITC-phalloidin. Cells from
one of three independent experiments are shown.
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The inhibitory effects observed with tyrphostins suggested a
participation of the EGF receptor in G
13QL-induced Rho
activation. Therefore, we expressed an EGF receptor mutant lacking 533 C-terminal amino acids (EGFR-CD533) in Swiss 3T3 cells by
microinjection. It has been shown that this mutant exerts a dominant
negative function on EGF receptor signaling by formation of
signaling-incompetent heterodimers with the wild type receptor (26).
Fig. 9 shows that expression of the
truncated receptor markedly suppressed actin stress fiber formation
caused by LPA or by G
13QL. In agreement with the
tyrphostin data shown above, G
12QL-induced actin
polymerization was not inhibited by expression of EGFR-CD533.
Furthermore, expression of the dominant negative EGF receptor mutant
also abolished the cytoskeletal effects caused by orthovanadate (Fig.
9). To assess a role for the EGF receptor in
G
13-mediated Rho activation, we transiently coexpressed
G
13QL and EGFR-CD533 in COS-7 cells and determined the
activation state of Rho by analyzing Rho-bound nucleotides. Fig.
10 demonstrates that the
G
13QL-induced Rho-GTP accumulation was attenuated by
co-expression of the mutated EGF receptor. The ratio of bound GTP to
total nucleotides bound to Rho [(GTP/GTP + GDP) × 100] was 17.8 ± 1.9% for the lacZ control, 25.8 ± 0.2% for the coexpression
of G
13QL with lacZ, and 22.5 ± 0.1% for the
coexpression of G
13QL with EGFR-CD533. This demonstrates that the dominant negative EGF receptor (EGFR-CD533) was able to
inhibit G
13-induced activation of Rho. The expression of
EGFR-CD533 with lacZ did not affect basal Rho-GTP levels compared with
lacZ (not shown). These data clearly suggest the EGF receptor as a critical component in the pathway leading from G
13 to
Rho activation.

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Fig. 9.
Suppression of cytoskeletal effects by
expressing the dominant negative EGF receptor mutant EGFR-CD533.
Cells were incubated with LPA (0.3 µM) or orthovanadate
(100 µM) or microinjected with plasmids encoding
G 12QL or G 13QL. In addition, fibroblasts were co-microinjected with plasmids encoding the dominant negative EGF
receptor mutant (EGFR-CD533) or the empty vector (control). After
fixation, cells were stained with FITC-phalloidin. Shown are cells from
one of three independent experiments.
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Fig. 10.
Reduction of G 13-induced Rho
activation after expression of the dominant negative EGF receptor
mutant EGFR-CD533. COS-7 cells were transfected with either lacZ
alone or G 13QL/lacZ or G 13QL/EGFR-CD533.
Cells were labeled with [32P]orthophosphate for
4 h, and RhoA was immunoprecipitated from the lysates.
A, migration of Rho-associated radioactive nucleotides after
separation by TLC in comparison to cold GDP/GTP standards. B, ratio of GTP to total labeled nucleotides complexed to
Rho [(GTP/GTP + GDP) × 100]. Data represent means ± S.D.
(n = 2). Error bars not shown are contained within the
symbols.
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DISCUSSION |
The phospholipid LPA has been characterized as a multifunctional
messenger acting on adenylyl cyclases, phospholipases,
mitogen-activated protein kinases, and the actin cytoskeleton via
different pathways (9). Previous studies demonstrated the existence of
high affinity, guanine nucleotide-sensitive binding sites for LPA
in membranes prepared from Swiss 3T3 cells and rat brains (27).
Recently, the cloning of LPA receptors from neuronal cells and
Xenopus oocytes has been described (1, 2). It has been
suggested from experiments using PTX that some cellular effects of LPA
are mediated by G-proteins belonging to Gi or
Gq subfamilies (9). The signaling cascade coupling the LPA
receptor to an activation of Rho and subsequent remodeling of the actin
cytoskeleton leading to stress fiber formation has, however, remained
unclear. Results from previous studies indicated that LPA-induced Rho
signaling is PTX-insensitive and that constitutively activated
G
q subunits are not able to stimulate Rho, excluding
Gi and Gq proteins from this pathway (9, 13). On the other hand, expression of constitutively active mutants of
G
12 and G
13 has been shown to trigger
Rho-mediated stress fiber formation in Swiss 3T3 cells (13). Therefore,
we studied the involvement of G12 subfamily proteins in the
pathway from LPA receptor to stimulation of Rho/stress fiber formation.
Activated G12 and G13 induced Rho-mediated
stress fiber formation (see Fig. 3; Ref. 13). Furthermore, we were able
to demonstrate activation of Rho by constitutively active mutants of
G
12 and G
13 (see Fig. 5). In
photolabeling experiments, both members of the G12 subfamily appeared to interact with the LPA receptor; however, in
intact cells, only G13, but not G12, mediated
signaling from LPA receptor to Rho. This was suggested by
discriminating effects of microinjected subtype-specific antibodies, by
differences in tyrphostin sensitivity, and by the different requirement
of EGF receptor function in LPA and G
13QL-
versus G
12QL-induced Rho activation (see
below). These data led us to identify G13 as a mediator
from LPA receptors to Rho. A likely explanation for the observed
discrepancy between the photoaffinity assay and microinjection/Rho activation studies is that photolabeling studies had to be performed employing a high concentration of LPA (10 µM), whereas a
significantly lower concentration of 0.3 µM LPA was
sufficient to induce maximum Rho activation in live Swiss 3T3 cells.
Moreover, our data suggest that selectivity in receptor signaling is
often found to be higher in intact complex systems than in in
vitro assays. Similar conclusions may be deducible from the
specificity of G
dimers in signaling pathways, since studies with
antisense oligonucleotides performed in intact cells revealed a higher
selectivity than biochemical assays using purified components (28-31).
The identification of G13 as signal transducer from LPA
receptor to Rho may suggest that other agonists of G-protein-coupled
receptors that induce effects on the actin cytoskeleton may also act
via G13 and/or G12.
Previously, Nobes et al. (11) have indicated the existence
of a protein-tyrosine kinase acting in the LPA pathway upstream of Rho.
One major finding of the present study is that LPA- as well as
G
13QL-induced effects on the actin cytoskeleton can be blocked with tyrphostins, suggesting that a tyrosine kinase activity is
required for the signaling from G13 to Rho. Although an
inhibition of GTPase activity of transducin by tyrphostin A 25 has been
reported (32), it appears unlikely that the suppression of
G
13QL-induced Rho activation by this inhibitor might be
a result of nonspecific interactions between tyrphostin A 25 and the
G-protein, since GTPase-deficient mutants of G
subunits were used.
Furthermore, also the specific EGF receptor inhibitor tyrphostin AG
1478 blocked the effects. The implication of EGF receptor kinase
activity in the LPA/G
13QL-induced,
Rho-dependent stress fiber formation was substantiated by
the expression of a dominant negative EGF receptor mutant in Swiss 3T3
cells. In addition, G
13-induced Rho-GTP accumulation was
at least partially reversed after overexpression of the truncated EGF
receptor mutant. Recently, several G-protein-coupled receptors have
been reported to induce a ligand-independent activation of the EGF or
platelet-derived growth factor receptor with subsequent activation of
mitogen-activated protein kinase cascades, and an involvement of
Gi-type G-proteins in this transactivation pathway has been
suggested (33-35). However, neither active G
i nor
G
dimers lead to a reorganization of the actin cytoskeleton (13). Our data suggest an involvement of the receptor tyrosine kinase EGF
receptor in the pathway of G13-mediated actin stress fiber formation. The presumably complex mechanism whereby the EGF receptor influences signaling mediated by activated G
13 has yet
to be resolved. Using serum-starved Swiss 3T3 cells stimulated with LPA, we found neither a coimmunoprecipitation of G
13
with the EGF receptor or with a nonreceptor tyrosine kinase of the Src family nor a tyrosine phosphorylation of G
13, arguing
against the possibility of a direct interaction between
G
13 and a tyrosine kinase.2 On the other hand,
strong similarities were observed between cytoskeletal events induced
by LPA or G
13QL and those caused by orthovanadate,
pointing to the possibility that activated G
13 may cause
a ligand-independent stimulation of the EGF receptor by interacting
with a phosphotyrosine phosphatase. The identification of a putative
receptor tyrosine kinase/phosphatase cycle controlling the
communication between activated G13 and Rho is under
current investigation. Taken together, our data indicate that
G13 couples the LPA receptor to Rho activation in Swiss 3T3
cells. In addition, we provide evidence for an involvement of the EGF
receptor in the pathway leading from G13 to Rho
activation/stress fiber formation.
We thank Nadine Albrecht for expert technical
assistance. We are grateful to Dr. Klaus Aktories (Freiburg) for
providing botulinum C3 exoenzyme, Dr. Alan Hall (London) for the
donation of Swiss 3T3 fibroblasts, Dr. Melvin I. Simon (Pasadena) for
providing cDNAs of wild type and constitutively active forms of
G
q, G
11, G
12, and
G
13, Dr. Christoph Sachsenmaier (München/Seattle) and Andreas Herrlich (Berlin/Karlsruhe) for the construct encoding the
EGF receptor mutant EGFR-CD533, and Dr. Karsten Spicher (Berlin/Los Angeles) for providing antisera against G
subunits. Furthermore, we thank Dr. Thomas Gudermann and Dr. Christian Harteneck for helpful
suggestions and critical reading of the manuscript and Dr. Frank
Kalkbrenner for help with the microinjection technique.