From the Institut für Pharmakologie, Freie
Universität Berlin, Thielallee 67-73, D-14195 Berlin, Germany and
§ Pharmacology Department, University of Texas Southwestern
Medical Center, Dallas, Texas 75235-9041
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ABSTRACT |
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The ubiquitously expressed heterotrimeric guanine
nucleotide-binding proteins (G-proteins) G12 and
G13 have been shown to activate the small GTPase Rho. Rho
stimulation leads to a rapid remodeling of the actin cytoskeleton and
subsequent stress fiber formation. We investigated the involvement of
G12 or G13 in stress fiber formation induced
through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and
G Heterotrimeric guanine nucleotide-binding proteins
(G-proteins)1 act as
molecular switches that couple receptors for hormones, neurotransmitters, and other extracellular stimuli to effector systems
such as enzymes or ion channels (1-4). G-proteins are composed of an
The G Studies conducted in plasma membrane preparations have shown that
G12 and G13 are activated by receptors that
also couple to Gq/G11 (7-9). The
Gq family is constituted by four members (Gq,
G11, G14, and G15/16) that couple
receptors to To analyze the role of G Materials--
Angiotensin II, bradykinin, carbachol,
endothelin-1, isoproterenol, lysophosphatidic acid, thrombin, and
[Arg8]vasopressin were from Sigma. Endothelin-1(8-21),
N-Suc-[Glu9,Ala11,15](IRL-1620),
(±)-2,5-dimethoxy-4-iodoamphetamine (DOI) hydrochloride, trans-azetidine-2,4-dicarboxylic acid, BQ-123, and BQ-788
were purchased from RBI.
Cell Culture--
Wild-type fibroblasts and fibroblasts lacking
both G Expression Vectors--
Expression vectors carrying cDNAs of
the human muscarinic M1 and M2 receptors, the
murine serotonin 5-HT2C receptor, the human vasopressin
V1A receptor, the human Microinjection--
For microinjection studies, cells were
seeded at a density of approximately 103
cells/mm2 on glass coverslips imprinted with squares to
facilitate the localization of injected cells and grown overnight. To
obtain quiescent and serum-starved fibroblasts, cultures were rinsed in
serum-free DMEM and incubated in DMEM supplemented with 25% Ham's F-12
medium, 0.2% NaHCO3, 10 mM Hepes, and 0.1%
fetal bovine serum (modified DMEM) for 24 h, followed by a 48-h
incubation in modified DMEM devoid of fetal bovine serum. Plasmids were
injected into the nucleus together with Texas Red dextran (5 mg/ml;
Molecular Probes) to visualize injected cells. Clostridium
botulinum C3 exoenzyme, which was kindly provided by K. Aktories
(Albert-Ludwigs-Universität, Freiburg, Germany), was
co-microinjected with the cDNAs at a concentration of 100 µg/ml.
To test the effects of dominant negative mutants of G Intracellular Calcium Measurements--
For dye loading, cells
were incubated with 2 µM fura-2 acetoxymethyl ester at
37 °C for 30 min in DMEM. Cells were then washed in 138 mM NaCl, 6 mM KCl, 1 mM
MgCl2, 5.5 mM glucose, 1 mM
CaCl2, and 20 mM Hepes (pH 7.4). Determinations
of [Ca2+]i in single microinjected
cells were performed in the same buffer, applying a digital imaging
system and FUCAL 5.12 software (T.I.L.L. Photonics, München,
Germany). Maximum and minimum fluorescence values were determined after
the addition of 10 µM ionomycin (Calbiochem) and 10 mM CaCl2 or 5 mM EGTA.
Fluorescence Microscopy--
Microinjected cells were stimulated
with various agonists for 15 min at 37 °C, fixed in 4%
paraformaldehyde for 20 min, and permeabilized in 0.2% Triton X-100
for 5 min. To visualize the cytoskeleton, cells were stained for
polymerized actin by incubation with 0.5 µg/ml fluorescein
isothiocyanate-phalloidin (Sigma) for 40 min. The coverslips were
mounted on glass slides and examined using an inverted microscope
(Zeiss Axiovert 100).
Quantification of Stress Fiber Formation--
For studies on
endogenous receptors, the percentage of stress fiber-positive cells
relates to the total number of investigated cells in randomly chosen
visual fields. In the case of heterologously expressed receptors,
microinjected cells were first identified by their Texas Red
fluorescence, and then the fraction of stress fiber-positive cells
among these cells was counted. Typically, the average
receptor-dependent induction of actin stress fiber formation resulted in an increase of approximately 40% in fluorescence intensity compared with control cells when the average fluorescence intensity values of fluorescein isothiocyanate-labeled polymerized actin/cell were calculated using a digital imaging system and FUCAL
5.12 software. A minimum of 100 cells was investigated in each case.
Experiments were performed in duplicate and repeated at least twice. In
most cases, quantifications were performed by two independent observers
in a blind manner, i.e. observers were not informed of the
conditions used in the indicated experiment.
Assessment of cAMP Accumulation--
For the determination of
cAMP levels, HEK 293 cells were seeded in 12-well plates at a density
of 1 × 105 cells/well and grown overnight. Cells were
transfected with cDNAs encoding the Stress fiber formation can be specifically induced by
constitutively active mutants of G To assure a functional expression of the investigated receptors, we
first measured the receptor-dependent calcium mobilization in wild-type fibroblasts. Fig.
1A (left part)
shows that thrombin and LPA, acting through endogenous receptors,
initiate a calcium mobilization in wild-type cells. The receptors for
thrombin and LPA have been shown to couple to Gq/11,
G12, G13, and Gi (8, 9). To test a
possible involvement of Gi-type G-proteins in calcium
mobilization induced through these receptors, Gi family members were inactivated by pretreating cells with PTX. The abrogation of Gi-mediated PLC activation slightly reduced the
thrombin- and LPA-induced calcium mobilization (Fig. 1A,
left part). Similarly, the agonist-dependent
activation of ETA, ETB, AT1A, and
B2 receptors that were heterologously expressed by
intranuclear microinjection of respective cDNAs induced partially
PTX-sensitive calcium responses. In contrast, elevation of
[Ca2+]i mediated by the activation
of heterologously expressed V1A, 5-HT2C,
M1, and mGluR1q/G
11-deficient mice, we show that
agonist-dependent activation of the endogenous receptors
for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A,
endothelin ETA, and serotonin 5-HT2C receptors
induced stress fiber formation in either the presence or absence of
G
q/G
11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate
subtype 1
receptors was dependent on Gq/G11
proteins. The activation of the Gq/G11-coupled
endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber
formation was dependent on G
13 and involved epidermal
growth factor (EGF) receptors, whereas thrombin, ETA, and
V1A receptors induced stress fiber accumulation via
G
12 in an EGF receptor-independent manner. Our data
demonstrate that many Gq/G11-coupled receptors
induce stress fiber assembly in the absence of G
q and
G
11 and that this involves either a G
12
or a G
13/EGF receptor-mediated pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-subunit and are characterized by the identity of
the
-subunit that binds and hydrolyzes GTP. On the basis of sequence
and functional homologies, G-protein
-subunits can be classified
into four families: (a) G
s, (b)
G
i/o, (c) G
q, and
(d) G
12 (5).
12 subfamily consists of the ubiquitously expressed
members G
12 and G
13, whose functions are
still incompletely understood. They share a 67% sequence identity and
are less than 45% homologous to other G-protein
-subunits (6).
G12 and G13 are activated through various
receptors, including receptors for thrombin, thromboxane A2, and lysophosphatidic acid (LPA) (7-9). Because no
receptors selectively coupling to G
12 or
G
13 have been found thus far, G12/G13-mediated signaling pathways have been
studied using constitutively active mutants of G
12 and
G
13. Both G
12 and G
13 can
regulate the Na+/H+ antiporter (10-12), the
Jun kinase/stress-activated protein kinase pathway (13), and the
Rho-dependent formation of actin stress fibers (14).
Whereas there are clear similarities in the effects induced by
constitutively active forms of G
12 and
G
13, differences in the involved signal transduction
mechanisms have been reported (9, 11, 15). Functional differences
between G
12 and G
13 are also suggested
from studies on G
13-deficient mice that die at
midgestation due to an angiogenetic defect. Fibroblasts derived from
G
13-deficient mice express G
12 but show
severely impaired chemokinetic responses to thrombin (16). Effectors
directly regulated by G
12 and G
13 remain
to be discovered. Recently, it was shown that a guanine nucleotide
exchange factor (GEF) for Rho, p115 RhoGEF, can be directly regulated
by G
13 (17, 18). Whereas G
13 activates
the p115 RhoGEF-catalyzed nucleotide exchange on Rho,
G
12 counteracts the G
13-mediated
stimulation. In addition, p115 RhoGEF functions as a GTPase-activating
protein for both G
12 and G
13.
-isoforms of phospholipase C (PLC) in a pertussis toxin
(PTX)-insensitive manner, resulting in the generation of inositol
phosphates and the mobilization of intracellular calcium (19-21).
Whereas G
q and G
11 are widely expressed
and are primarily responsible for PTX-insensitive PLC-
activation,
the expression of G
14 and G
15/16 is
restricted to a few tissues (22, 23). Studies on
G
q/G
11-double deficient mice and cells
have confirmed that G
q and G
11 are the
primary mediators of PTX-insensitive PLC-
activation and that their
cellular functions are highly redundant (24, 25).
q/G
11,
G
12, and G
13 in receptor-mediated
rearrangements of the actin cytoskeleton, we studied the signaling
processes induced through endogenous and heterologously expressed
receptors in wild-type, G
q/G
11-deficient
and G
13-deficient cells. Our data show that most, but
not all, receptors tested mediate stress fiber formation in the absence
of G
q and G
11. Unexpectedly, we found
that G
q/G
11-independent stress fiber formation involved either G
12 or G
13.
Moreover, the EGF receptor represents an essential intermediate in the
G
13-induced stress fiber formation but not the
G
12-induced stress fiber formation in fibroblasts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q and G
11 were derived from
embryonic day 10.5 mouse embryos originating from intercrosses of
G
q(
/+) and G
11(
/+) mice. The
generation of G
q and G
11 mutant mice has
been described previously (25). Fibroblasts lacking
G
q/G
11 or G
13 were
prepared and cultured as described previously (16, 26).
2 adrenergic
receptor, and
-galactosidase have been described previously (27).
G
12G228A and G
13G225A were generated by
overlap extension polymerase chain reaction and cloned into pCIS (28),
and mutations were verified by sequencing. The rat metabotropic
glutamate receptor subtype 1
(mGluR1
) cDNA was
inserted in pcDNA-Amp (29), and the human angiotensin
AT1A receptor cDNA was inserted in pcDNA3
(Invitrogen). Human endothelin ETA and ETB
receptor cDNAs were cloned in pMEsf
(30), and the human
bradykinin B2 receptor was carried by pcDNA1 (Invitrogen). RhoA G14V was in pcEXV (31). The dominant negative EGF
receptor mutant EGFR-CD533 was carried by the pRK5 vector (32).
12
(G
12G228A; G
12GA) and G
13
(G
13G225A; G
13GA), plasmids encoding for
receptors (0.1 µg/µl) were co-microinjected with G
12GA or G
13GA (0.5 µg/µl). As a
control, receptor plasmids (0.1 µg/µl) supplemented with a vector
encoding
-galactosidase (0.5 µg/µl) to maintain the total amount
of injected DNA constant were microinjected in an adjacent field on the
same coverslip. The dominant negative EGF receptor mutant EGFR-CD533
(0.5 µg/µl) was co-expressed with various receptors (0.1 µg/µl)
by nuclear microinjection. To test the specificity of dominant negative
mutants of G
12 and G
13, RhoA G14V (0.1 µg/µl) was co-microinjected with G
12GA or
G
13GA (0.5 µg/µl). About 150 cells/field were
injected in each case, using a manual injection system (Eppendorf,
Hamburg, Germany). To inhibit the EGF receptor tyrosine kinase activity pharmacologically, cells were incubated with the EGF receptor-selective tyrphostin AG1478 (1 µM) for 120 min before fixation. A
25-min treatment with the tyrosine phosphatase inhibitor vanadate (100 µM) was performed before fixation.
2 adrenergic
receptor and the M2 muscarinic receptor and either dominant
negative G
12 or G
13 or the
-galactosidase control, as described previously (27). The cells were
subsequently labeled with [3H]adenine, and
[3H]cAMP was isolated by double chromatography over Dowex
and Alumina columns as described previously (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 and
G
13 in a Rho-dependent manner (14). The
G12- and G13-coupling receptors identified to
date appear to activate Gq family members as well (7-9).
Stress fiber formation induced through these receptors is accordingly
accompanied by a PTX-insensitive stimulation of PLC-
and a
subsequent elevation of cytosolic [Ca2+]. To investigate
whether Gq/G11-coupled receptors can, in
general, activate G12/G13-mediated stress fiber
formation and to test whether these cytoskeletal rearrangements are
dependent on Gq/G11 signaling, we studied the
agonist-induced stress fiber assembly through various Gq/11-coupled endogenous or heterologously expressed
receptors in G
q/G
11-deficient fibroblasts.
receptors was not affected by pretreatment of cells with PTX (Fig. 1A, right
part). These data demonstrate that the tested receptors were
functionally expressed at densities comparable to each other and to
those of endogenous thrombin and LPA receptors.
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Fig. 1.
Intracellular calcium mobilization in
response to activation of endogenous or heterologously expressed
receptors. Wild-type fibroblasts (A) or
G q(
/
);G
11(
/
) fibroblasts
(B) were activated with 1 µM LPA or 1 unit/ml
thrombin (left parts of A and B). For
studies on heterologously expressed receptors, cells were first
microinjected with expression plasmids carrying cDNAs of the
indicated receptors (right parts of A and
B) and then challenged with receptor agonists. To test the
effect of PTX, cells were preincubated overnight in the absence (
)
or presence of 100 ng/ml PTX (
). The increases in free cytosolic
[Ca2+]i over basal
[Ca2+]i are shown (in
nM; ordinate). Note the different scales in A
and B. The ETA receptor was specifically
activated with endothelin-1 (0.1 µM) in the presence of
the ETB receptor antagonist BQ-788 (1 µM).
The ETB receptor was specifically activated with the
ETB receptor-specific agonist IRL-1620 (1 µM)
in the presence the ETA receptor antagonist BQ-123 (10 µM). The V1A receptor was stimulated with AVP
(1 µM), the AT1A receptor was activated with
angiotensin II (1 µM), and bradykinin (1 µM) was used to activate the B2 receptor. The
5-HT2C receptor was stimulated with the serotonin receptor
subtype-specific agonist DOI (1 µM), the M1
receptor was activated with carbachol (10 µM), and the
mGluR1
receptor was activated with the metabotropic glutamate receptor agonist
trans-azetidine-2,4-dicarboxylic acid (10 µM).
Bars represent the mean values ± S.D. of calcium
measurements in 30-50 individual cells in two independent experiments.
C, typical calcium transients obtained after activation of
heterologously expressed 5-HT2C and mGluR1
receptors in wild-type fibroblasts (upper panels) or
Gq/11-deficient fibroblasts (lower panels).
Cells were stimulated with 1 µM DOI to activate
5-HT2C receptors or with 10 µM
trans-azetidine-2,4-dicarboxylic acid to activate
mGluR1
receptors. The calcium responses of five
individual cells each are shown. Arrows indicate the time of
agonist addition.
To selectively study the agonist-dependent calcium
mobilization induced by activation of the tested receptors in the
absence of Gq- and G11-proteins, we used
Gq/G
11-double deficient embryonic fibroblasts. Activation of the heterologously expressed
V1A, 5-HT2C, M1, and
mGluR1
receptors did not elevate basal intracellular calcium levels, whereas stimulation of the endogenous receptors for
thrombin and LPA and of the heterologous ETA,
ETB, AT1A, and B2 receptors in
G
q/G
11-deficient cells induced a slight
increase in the intracellular calcium concentration that was completely PTX-sensitive (Fig. 1B). Thus, the
G
q/G
11-deficient, PTX-treated fibroblasts
did not respond to various receptor agonists with measurable,
PLC-mediated elevation of
[Ca2+]i.
The receptors for thrombin and LPA have previously been shown to
mediate Rho-dependent stress fiber formation in Swiss 3T3 cells (34). To investigate whether the fibroblast cell lines derived
from wild-type or Gq/G
11-deficient mice
provide a suitable assay system to study receptor-induced,
Rho-dependent stress fiber formation, we first assessed the
ability of thrombin and LPA to trigger stress fiber assembly in
serum-starved, wild-type fibroblasts. Fig.
2A shows that both agonists
clearly stimulated stress fiber formation in about 90% of the cells.
Stress fiber assembly was insensitive to pretreatment with PTX but was
completely blocked by C3 transferase, which specifically inactivates
Rho by ADP-ribosylation (35). Similarly, agonist-dependent
activation of the heterologously expressed ETA,
V1A, B2, 5-HT2C, M1,
and mGluR1
receptors resulted in a pronounced
PTX-insensitive and C3 exoenzyme-sensitive stress fiber formation. We
could not detect any actin polymerization in fibroblasts activated
through the AT1A or ETB receptors, which clearly mediated calcium responses (Fig. 1A).
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We then investigated whether stress fiber formation can be triggered in
the absence of PLC-mediated calcium mobilization. Stress fiber assembly
in response to the activation of M1 or
mGluR1 receptors in
G
q/G
11-deficient fibroblasts was
drastically reduced by 90-95% compared with wild-type cells,
indicating that it largely depended on the presence of
Gq/11-proteins (Fig. 2B). In contrast, stress
fiber assembly triggered through the activation of the endogenous
receptors for thrombin and LPA and heterologously expressed ETA, V1A, B2, and
5-HT2C receptors was induced as efficiently in
G
q(
/
);G
11(
/
) cells as in
wild-type fibroblasts. In all cases,
G
q/G
11-independent stress fiber assembly
was insensitive to PTX but was sensitive to C3 exoenzyme. We conclude
that Gq/11- and Gi-proteins are dispensable for
Rho-mediated actin reorganization induced through many, but not all,
Gq/11-coupled receptors. Because activated forms of
G
12 and G
13 are able to induce stress
fiber assembly (14), these data suggest that
G
q/G
11- and Gi-independent, Rho-mediated stress fiber formation involves exclusively
G12 family proteins.
By labeling activated G-proteins with [-32P]GTP
azidoanilide in plasma membrane preparations, we have previously
demonstrated that LPA receptors activate G
12 and
G
13, whereas LPA-induced stress fiber formation is
selectively mediated via G
13 and does not involve
G
12 (9). To test specific roles for G
12
and G
13 in the same cell, we have used dominant negative
forms of G
12 and G
13 and
G
13-deficient fibroblasts. Receptors that were shown to
mediate stress fiber formation in a
G
q/G
11-independent way (see Fig.
2B) were co-expressed with point mutants of
G
12 (G
12GA) and G
13
(G
13GA). The analogous substitution of glycine for
alanine in the nucleotide binding pocket of G
s has been
shown to block G
s activation induced by
receptor-mediated guanine nucleotide exchange. Therefore, this mutation
is believed to create a dominant negative-acting protein (36, 37).
Expression of G
12GA and G
13GA did not
interfere with stress fiber formation induced by the constitutively
active RhoA mutant RhoA G14V or by the incubation of the cells with
orthovanadate (9), indicating that dominant negative forms of
G
12 or G
13 did not unspecifically
inhibit stress fiber formation (data not shown). In addition, the
expression of dominant negative G
12 or
G
13 did not interfere with the
Gq/11-dependent calcium release (Table
I), nor did dominant negative
G
12 or G
13 interfere with signal
transduction pathways dependent on Gi-type G-proteins. The
latter is evident from the fact that G
12GA and
G
13GA did not influence the carbachol-mediated
inhibition of cAMP accumulation compared with control cells expressing
-galactosidase (Table I). These data clearly demonstrate that
dominant negative mutants of G
12 and G
13
do not interfere with Gq/11- and
Gi-dependent signal transduction pathways.
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Interestingly, G12GA blocked thrombin-induced stress
fiber formation by about 80% in
G
q(
/
);G
11(
/
) cells compared with the control vector, whereas G
13GA had no inhibitory
effect. In contrast, LPA-dependent actin reorganization was
not affected by G
12GA, whereas G
13GA
reduced the LPA-stimulated stress fiber assembly by about 70% (Fig.
3A). To further explore a
specific role for G
12 and G
13 in stress
fiber assembly, we co-expressed G
12GA and
G
13GA with receptors shown to mediate stress fiber formation in a Gq/G11-independent way (see Fig.
2B). In G
q/G
11-deficient cells, stress fiber formation induced through the stimulation of
ETA and V1A receptors was strongly reduced by
G
12GA, whereas G
13GA had no effect. Using
the same experimental design, G
13GA but not
G
12GA blocked stress fiber assembly induced through the B2 and 5-HT2C receptors. Thus,
G
12 and G
13 appear to differentially couple receptors to Rho-dependent stress fiber formation.
Stress fiber assembly induced by the activation of the M1
and mGluR1
receptors was dependent on
Gq/11-mediated PLC-
stimulation and calcium mobilization
(see Fig. 2B). However, stress fiber formation induced
through the M1 and mGluR1
receptors in
wild-type fibroblasts was inhibited by 60-90% after expression of
G
13GA, whereas the expression of G
12GA
was without effect (data not shown).
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To further prove a selective involvement of either G12
or G
13 in receptor-mediated stress fiber formation, we
used G
13(
/
) fibroblasts. Fig.
4A demonstrates that the
activation of the LPA, B2, and 5-HT2C
receptors, which mediate stress fiber formation in a
G
13GA-sensitive manner, failed to induce stress fibers
in G
13(
/
) cells. In contrast, stimulation of the
thrombin, ETA, and V1A receptors, which trigger
stress fiber assembly in a G
12GA-sensitive manner,
occurred in almost all of the G
13-deficient cells. This clearly confirms that both G
12 and G
13
are capable of mediating receptor-induced stress fiber accumulation.
However, different receptors selectively utilize G
12 or
G
13 for the stimulation of Rho activation and the
subsequent formation of actin stress fibers.
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We have previously demonstrated that the EGF receptor is an essential
signaling intermediate in LPA-induced, G13-mediated stress
fiber formation but not in the G12-induced stress fiber formation in Swiss 3T3 fibroblasts (9). To investigate a general involvement of the EGF receptor in
G13-dependent rearrangements of the actin
cytoskeleton, we first determined the effects of the EGF
receptor-specific tyrosine kinase inhibitor tyrphostin AG1478 on the
receptor-induced stimulation of stress fiber formation in wild-type
fibroblasts. Fig. 5A shows
that tyrphostin AG1478 blocked the G13-mediated stress
fiber formation induced through activation of the LPA, B2,
and 5-HT2C receptors. In contrast, the
G12-mediated stress fiber formation triggered by
stimulation of the thrombin, ETA, or V1A
receptors was not reduced by inactivation of the EGF receptor with
tyrphostin AG1478. Furthermore, a cytosolically truncated EGF receptor
(EGFR-CD533) was expressed in fibroblasts. This EGF receptor mutant
exerts a dominant negative function on EGF receptor signaling due to
the formation of signaling-incompetent heterodimers with endogenous EGF
receptors (32). In agreement with the effects observed after inhibition
of the EGF receptor tyrosine kinase activity with tyrphostin AG1478,
expression of EGFR-CD533 abolished the G13-mediated stress
fiber formation induced through LPA, B2, and
5-HT2C receptors, whereas the
G12-dependent stress fiber formation was not
sensitive to an inhibition of EGF receptor function (Fig.
5B).
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DISCUSSION |
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The aim of the present study was to investigate the involvement of
Gq/G11- and
G12/G13-proteins in
receptor-dependent stress fiber formation. Whereas
constitutively active mutants of G12 and
G
13 are known to induce stress fiber assembly (14), it is not clear whether physiological activation of
G12/G13 through receptors is alone sufficient
to mediate actin reorganization. Our data demonstrate that activation
of LPA, thrombin, ETA, V1A, B2, and
5-HT2C receptors efficiently triggers PTX-insensitive stress fiber formation in the absence of
Gq/11-dependent calcium mobilization. We
conclude that Gq/11- and Gi-proteins are
dispensable for stress fiber formation evoked through many
Gq/11-coupled receptors.
Stress fiber assembly induced by activation of M1 and
mGluR1 receptors was dependent on
Gq/11-mediated PLC-
stimulation and calcium
mobilization. However, using dominant negative mutants of
G
12 and G
13, we could show that stress
fiber formation induced through M1 and
mGluR1
receptors in wild-type fibroblasts was inhibited
in 60-90% of the cells after the expression of G
13GA, whereas the expression of G
12GA was without effect. In
addition, the activation of M1 and mGluR1
receptors in G
13-deficient fibroblasts failed to promote
stress fiber assembly (data not shown). These results suggest that
Gq/G11-dependent signaling can
critically contribute to G13-mediated stress fiber
formation induced through some receptors, although the expression of
constitutively active Gq-proteins alone is not sufficient
to form stress fibers (9, 14). Experiments in cardiomyocytes have
indicated an involvement of G
q in receptor-induced and
Rho-mediated actin reorganization (38).
It has been reported that stimulation of a voltage-dependent calcium channel in rat portal vein myocytes through AT1A receptors involves G13 (39). In contrast to these studies, we could not detect any G12/G13-dependent actin polymerization in fibroblasts activated through AT1A receptors. This may suggest that G13 is indirectly involved in calcium-current regulation via AT1A receptors.
Stimulation of ETA receptors effectively triggered
G12-dependent stress fiber formation. In
contrast, the activation of ETB receptors failed to induce
stress fiber assembly. These results suggest that ETA
receptors activate G12, whereas ETB receptors do not couple to G12 family members, which evoke actin reorganization.
We have previously demonstrated that the LPA receptor induces stress
fiber formation selectively via G13. By labeling
activated G-proteins with [
-32P]GTP azidoanilide in
plasma membrane preparations, it was shown that LPA activates both
G12 and G13, but that this activation of
G12 was apparently not linked to stress fiber assembly (9). To test a possible selective involvement of G
12 and
G
13 in stress fiber formation through other receptors,
we have used dominant negative forms of G
12 and
G
13 and G
13-deficient cells. Our data
show that stress fiber formation induced through the thrombin, ETA, and V1A receptors was mediated by
G
12 and occurred in the absence of G
13.
In contrast, stress fiber accumulation induced through the LPA,
B2, and 5-HT2C receptors was completely
dependent on the presence of G
13 and was not reduced by
a dominant negative form of G
12. This clearly shows that
both G
12 and G
13 are capable of mediating
receptor-induced stress fiber formation, although different receptors
selectively recruit G
12 or G
13 for the
regulation of actin stress fiber accumulation.
The EGF receptor has been identified as a critical component in
LPA/G13-mediated stress fiber formation in Swiss 3T3
fibroblasts (9). Using the EGF receptor-selective tyrphostin AG1478 and the dominant negative EGF receptor mutant EGFR-CD533, we now show that
receptors utilizing either G12 or G13 for
stress fiber formation can be further distinguished by the involvement
of EGF receptors in the process of actin polymerization. Whereas EGF
receptors contributed to the LPA, B2, and
5-HT2C receptor-mediated,
G13-dependent stress fiber formation in
wild-type fibroblasts, the G
12-dependent stress fiber assembly induced through thrombin, ETA, and
V1A receptors occurred via a different, EGF
receptor-independent pathway. These results implicate the EGF receptor
as a signaling intermediate of general importance in
G
13-dependent stress fiber formation in fibroblasts.
Whereas no difference in the coupling of e.g. thrombin or
LPA receptors to G12 and G13 has been observed
in plasma membrane fractions, it is conceivable that receptors
preferentially activate G12 or G13 under
physiological conditions in intact cells. This could be caused by
different affinities of receptors for G12 and G13 or by the subcellular localization of the involved
downstream signaling components like EGF receptors. It is also possible
that in the intact cell, G12 and G13 are
specifically preassembled with individual receptors. Finally,
regulators of G-protein signaling (RGS) proteins may selectively
influence the G12- or G
13-mediated signaling in a receptor-specific way. The recently described GEF for
Rho, p115 RhoGEF, functions as an RGS protein for G
12
and G
13 in vitro, but its GEF-activity is
only regulated by G
13 (17, 18). However, it is not clear
how p115 RhoGEF behaves in intact cells. In addition, other RGS
proteins specific for G
12 or G
13 may exist.
In summary, we have taken advantage of a
Gq/G
11-deficient cell line and dominant
negative mutants of G
12 and G
13 and
G
13-deficient cells to demonstrate that many
Gq/G11-coupled receptors induce stress fiber
formation in the absence of G
q and G
11
and that this involves either a G
12 or a
G
13/EGF receptor-dependent pathway.
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ACKNOWLEDGEMENTS |
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We thank U. Brandt and B. Klages for excellent technical assistance and A. Hall, F. Hess, and T. Masaki for expression plasmids.
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FOOTNOTES |
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* This work was supported by funds from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie and by National Institutes of Health Grant DK47890 (to T. M. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 49-30-8445-1812; Fax: 49-30-8445-1818; E-mail: gschultz{at}zedat.fu-berlin.de.
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ABBREVIATIONS |
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The abbreviations used are: G-protein, heterotrimeric guanine nucleotide-binding protein; LPA, lysophosphatidic acid; PTX, pertussis toxin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GEF, guanine nucleotide exchange factor; PLC, phospholipase C; DOI, 2,5-dimethoxy-4-iodoamphetamine; DMEM, Dulbecco's modified Eagle's medium.
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REFERENCES |
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