From the INSERM U317, Institut Louis Bugnard, Université Paul
Sabatier, CHU Rangueil, Batiment L3, 31403, Toulouse Cedex
4, France
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INTRODUCTION |
2-Adrenergic receptors
(
2-AR)1 are
G-protein-coupled receptors that mediate the action of catecholamines
(epinephrine, norepinephrine) in a wide range of tissues (1). In white
adipose tissue,
2-ARs belong to the
2A-subtype and
are expressed in both adipocytes and preadipocytes. In adipocytes
2-ARs mediate inhibition of lipolysis through a
Gi-dependent coupling to adenylyl cyclase (2).
In preadipocytes, stimulation of
2-ARs generates a
Gi/Go-dependent increase in cell
proliferation. This effect was associated with rapid and transient
tyrosine phosphorylation of the p44 and p42 mitogen-activated protein
kinases (MAPK) ERK1 and ERK2 (3), reflecting the
Gi/Go-mediated coupling of the
2-ARs to the small GTPase p21ras (4).
2-Adrenergic activation of the Ras/MAPK pathway is
mediated by the 
-subunits of the heterotrimeric G proteins as
demonstrated by the ability of the COOH-terminal domain of
ARK1 (a

-binding protein) to block this activation (5, 6).
In preadipocytes, stimulation of
2-ARs is also
associated with striking
Gi/Go-dependent rearrangement of
actin cytoskeleton. This is characterized by a rapid spreading of the
cells on their growing substratum, the formation of actin stress
fibers, and the increase in the tyrosyl phosphorylation of the pp125
focal adhesion kinase (pp125FAK) (7). These cellular events
are known to be tightly controlled by another GTPase belonging to the
Ras superfamily, p21rhoA (8, 9). It has particularly been
demonstrated that the C3 exoenzyme from Clostridium
botulinum (a toxin which specifically block the action of
p21rhoA by catalyzing its ADP-ribosylation) is able to block
the activation of cell motility, cell morphology, and cell growth,
generated by several agonists acting through G protein-coupled
receptors such as lysophosphatidic acid, sphingosine 1-phosphate, and
thrombin (10, 11).
Considering the morphological changes generated by stimulation of the
2-ARs in preadipocytes, we analyzed (i) the involvement of p21rhoA in
2-adrenergic-dependent
reorganization of actin cytoskeleton and tyrosyl phosphorylation of
pp125FAK; (ii) the existence of a functional coupling
between
2-ARs and p21rhoA; (iii) the putative
involvement of 
-subunits of G proteins in this coupling. In the
present study we demonstrate that, in
2AF2 preadipocytes (a cell
clone derived from the 3T3F442A preadipose cell line stably expressing
the human
2C10-adrenergic receptor),
2-adrenergic-dependent reorganization
of actin cytoskeleton involves the activation of p21rhoA
independently of a G
-mediated transduction pathway.
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MATERIALS AND METHODS |
Cell Culture and Transfection--
The cells were grown at
37 °C in Dulbecco's modified Eagle's medium supplemented with 10%
donor calf serum (Life Technologies, Inc.) as described previously (7).
2AF2 (
2AF2) preadipocytes were previously obtained by permanent
transfection of the human
2C10-adrenergic receptor gene in the
3T3F442A preadipose cell line followed by G418 selection (3). As
determined by radioligand binding analysis,
2-adrenergic receptor density in
2AF2
preadipocytes was 2050 ± 90 fmol/mg protein. Stable expression of
the carboxyl-terminal domain of
ARK1 (
ARK-CT) was obtained by
transfection of
2AF2 preadipocytes with a pZeo/
ARKCT vector,
followed by the double selection G418/zeocin. pZeo/
ARKCT vector was
obtained by subcloning an EcoRI-SalI fragment
from pRK-
ARK1 vector (6) (generous gift from Dr. Lohse) into
pcDNA3.1/Zeo vector (Invitrogen) linearized by
EcoRI-XhoI. The validity of the construct was
verified by sequencing.
Expression and Purification of C3 Exoenzyme--
Recombinant C3
exoenzyme were expressed as glutathione S-transferase fusion
proteins in Escherichia coli (generous gift from Dr. Alan
Hall) and purified on glutathione-Sepharose beads as described
previously (12). C3 exoenzyme was released from the beads by thrombin
(Calbiochem) cleavage and concentrated by centricon-3 (Amicon Inc.,
Beverly, MA).
C3 Exoenzyme-catalyzed ADP-ribosylation of Rho
Proteins--
Cells were pretreated or not with C3 exoenzyme for
various period of time, washed twice in PBS, and homogenized in
extraction buffer (0.25 M sucrose, 20 mM
Tris-HCl, 3 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 2 mM benzamidine, pH 7.5). ADP-ribosylation was
carried out in a total volume of 100 µl containing 20 mM
Hepes, pH 8.0, 2 mM MgCl2, 10 mM
thymidine, 0.1% deoxycholate, 10 mM NAD, 1 µCi of
[32P]NAD, 100 µg of cell homogenate, and 0.5 µg of C3
exoenzyme. The reaction was carried out at 30 °C for 37 min and
terminated by addition of 11 µl of 70% trichloroacetic acid for 15 min at 4 °C. The proteins in the pellet were dissolved in 50 µl of
Laemmli buffer (200 mM Tris-HCl, pH 6.8, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol) and
separated on 12.5% SDS-polyacrylamide gel, stained with Coomassie
Brilliant Blue R-250, dried, and autoradiographed. The intensity of the
spots was analyzed using an Image'quant software.
Morphological Analysis--
Cell spreading activity was
determined as described previously (7). Briefly, cells were previously
retracted by serum starvation (which was characterized by an increased
cell refringency) before addition of an
2-adrenergic agonist. The proportion of retracted cells was determined as the ratio between the number of refringent cells and the total number of cells present in a microscope field. Each
value correspond to the mean of at least five separate fields.
Actin filaments were visualized as described previously (13). Cells
grown on plastic culture plates were washed twice with PBS and fixed
for 15 min in 3.7% (v/v) formaldehyde in PBS. Fixed cells were washed
twice with PBS and permeabilized with 0.1% Triton X-100 in PBS for 2 min. Permeabilized cells were washed twice with PBS and incubated with
150 nM of fluorescein isothiocyanate-conjugated phalloidin
PBS for 20 min. The cells were then extensively washed with PBS and
examined using an epifluorescence microscope.
Detection of Tyrosyl-phosphorylated
Proteins--
Immunoprecipitation of tyrosyl-phosphorylated FAK, ERK2,
and Western blot was carried out as described previously (7). Briefly,
tyrosyl-phosphorylated proteins were immunoprecipitated with 15 µl of
protein G-Sepharose beads (Sigma) conjugated with an antiphosphorylated
antibody (PY20, Transduction Laboratories) and resuspended in 50 µl
of Laemmli buffer. Tyrosyl-phosphorylated proteins were then separated
on 8% SDS-polyacrylamide gel. After electrophoresis, proteins were
transferred to nitrocellulose and incubated with an anti-focal adhesion
kinase antibody (FAK C-20; 0.5 µg/ml, Santa Cruz Biotechnology, Inc.)
followed by horseradish peroxidase-labeled secondary anti-rabbit
(Immunotech). Immunoreactive bands were visualized by enhanced
chemiluminescence detection (ECL, Amersham Pharmacia Biotech). After
FAK detection, the blot was stripped for 30 min at 50 °C in 100 mM
-mercaptoethanol, 2% SDS, 62.5 mM
Tris-HCl, pH 6.5, followed by extensive washes in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20). The blot was then incubated with anti-ERK2 (C-14, 1 µg/ml, Santa
Cruz Biotechnology, Inc.) to detect tyrosyl-phosphorylated ERK2. The
intensity of the bands was measured using Image'quant software.
Detection of RhoA and
i2 Proteins in Particulate
Subcellular Fraction--
Cells were lysed in extraction buffer (25 mM Tris-HCl, pH 7.5, 5 mM EGTA, pH 7.5, 15 mM NaCl, 1%
n-octyl-
-D-glucopyranoside, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin). Lysates were
centrifuged at 120,000 × g for 45 min to separate
cytosolic and particulate fractions. Particulate fraction were
resuspended in RIPA buffer (0.01 M Tris-HCl, pH 7.0, 150 mM Nacl, 2 mM EDTA, 1 mM sodium
orthovanadate, 0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM phenylmethylsulfonyl fluoride) and centrifuged at
15,000 × g to eliminate nonsolubilized material.
Protein concentrations were determined according to the method of Lowry
et al. (14) and separated on 12.5% SDS-polyacrylamide gel
for Western blot analysis using anti-RhoA antibody (26C4, 1 µg/ml,
Santa Cruz Biotechnology, Inc.), and anti-
i2 antibody
(gift from Dr. Rouot).
Determination of GTP/GDP Ratio--
The determination of the
GTP/GDP ratio of p21rhoA was essentially as described (15).
Cells were serum-starved for 18 h and subsequently labeled with
0.25 mCi/ml [32P]orthophosphate for 3 h. Cells
were stimulated with 1 mM UK14304 for various times and
lysed in 0.5% Nonidet P-40 buffer containing 20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 250 mM sucrose, 2 mM EGTA, 1 mM Na4P2O7, 1 mM NaF, 1% Triton X-100, 1 mM ATP, 0.1 mM GTP, 0.1 mM GDP, 1 mM
phenylmethylsulfonyl fluoride. The soluble cell extract was incubated
for 3 h at 4 °C with 15 µl of protein G-Sepharose beads
(Sigma) conjugated with a mouse anti-RhoA antibody (119, Santa Cruz
Biotechnology, Inc.). 32P-Labeled GDP and GTP were eluted
from the immunocomplexes with a buffer containing 5 mM
EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM
GDP, and 0.5 mM GTP for 20 min at 68 °C. After extensive washes GTP/GDP were separated by thin layer chromatography (Bakerflex PEI-F cellulose TLC plates, Bakerflex) in 1 M
KH2PO4, pH 3.4, and autoradiographed. The
intensity of the spots was analyzed using Image'quant software.
 |
RESULTS |
Influence of the C3 Exoenzyme on
2-Adrenergic-dependent Regulation of
Preadipocyte Morphology--
In a previous study, we demonstrated that
in preadipocytes,
2-adrenergic stimulation promotes
rapid spreading, pp125FAK tyrosyl phosphorylation and actin
stress fiber formation (7). These cellular events are known to be
tightly controlled by the small GTPases of the Rho family particularly
p21rhoA (8). To determine the involvement of RhoA in the
2-adrenergic-dependent regulation of
preadipocyte morphology, the effect of C3 exoenzyme, a toxin that
impairs the function of p21rho (12), on
2-adrenergic-mediated spreading, stress fibers
formation, and pp125FAK phosphorylation in
2AF2
preadipocytes was tested.
In total
2AF2 preadipocyte lysate C3 exoenzyme catalyzed the
[32P]ADP-ribosylation of a unique band exhibiting a
molecular mass of 21 kDa corresponding to p21rho proteins (Fig.
1A) (12, 16). Seventy-two hour
pretreatment of intact
2AF2 preadipocytes with 10 µg/ml C3
exoenzyme led to about 80% reduction in the amount of the 21-kDa band
(Fig. 1B), demonstrating the ability of C3 exoenzyme to
penetrate into intact
2AF2 preadipocytes and ADP-ribosylate
p21rho proteins.

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Fig. 1.
C3 exoenzyme catalyzed ADP-ribosylation in
2AF2 preadipocytes. 2AF2 preadipocytes were exposed
(C3) or not (Control) to 10 µg/ml C3 exoenzyme
for 24, 48, or 72 h. Cell lysates were prepared and subjected to
in vitro C3 exoenzyme-catalyzed ADP-ribosylation as
described under "Materials and Methods." A,
representative experiment. B, quantification from three
separate experiments. Values correspond to the mean ± S.E.
Comparison with the control was performed using Student's t
test: *, p < 0.05.
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After 15-h serum starvation,
2AF2 preadipocytes exhibited a
retracted morphology (Fig. 2)
characterized by the absence of actin stress fibers (visualized with
fluorescein isothiocyanate-labeled phalloidin) (Fig.
3A). Fifteen-minute treatment
with a 1 µM amount of the specific
2-adrenergic agonist UK14304 led the spreading of almost
80% of retracted
2AF2 (Fig. 2) and by the formation of actin stress
fibers (Fig. 3B). UK14304-induced cell spreading and
reorganization of actin cytoskeleton were abolished by 72-h pretreatment of
2AF2 preadipocytes with 10 µg/ml C3 exoenzyme (Figs. 2 and 3, C and D).

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Fig. 2.
Influence of C3 exoenzyme on
2-adrenergic-induced spreading in 2AF2
preadipocytes. 2AF2 preadipocytes were treated (C3)
or not (Control) with C3 exoenzyme (72 h, 10 µg/ml). After
serum starvation, control and C3 exoenzyme-treated 2AF2
preadipocytes were exposed (+) or not ( ) to 1 µM
UK14304 for 15 min. Cell spreading was measured by quantifying the
proportion of refringent cells present in a field (mean of five
separate fields). Values represent the mean ± S.E. of three
separate experiments. Comparison with the control was performed using
Student's t test: *, p < 0.05.
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Fig. 3.
Influence of C3 exoenzyme on
2adrenergic-dependent stress fiber formation
in 2AF2 preadipocytes. 2AF2 preadipocytes were treated
(C, D) or not (A, B) with
C3 exoenzyme (72 h, 10 µg/ml). After serum starvation control and C3
exoenzyme-treated 2AF2 preadipocytes were exposed (B,
D) or not (A, C) to 1 µM
UK14304 for 15 min. Actin filaments were visualized using fluorescein
isothiocyanate-labeled phalloidin as described under "Materials and
Methods." The pictures are representative of at least three separate
experiments.
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Five-min treatment of 24-h serum-starved
2AF2 preadipocytes with 1 µM UK14304 led to an increase in the tyrosyl
phosphorylation of the focal adhesion kinase (pp125FAK)
(Fig. 4A, Control).
The same stimulation also led to an increase in the tyrosyl
phosphorylation of the mitogen-activated protein kinase ERK2 associated
with a shift corresponding to the biphosphorylated (on tyrosine and
threonine residues) and active form of ERK2 (Fig. 4B,
Control). Seventy-two-h pretreatment of
2AF2
preadipocytes with 10 µg/ml C3 exoenzyme led to a 75-80% reduction
of UK14304-induced tyrosyl phosphorylation of pp125FAK
(Fig. 4A, C3) without alteration of
UK14304-induced shift and tyrosyl phosphorylation of ERK2 (Fig.
4B, C3).

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Fig. 4.
Influence of C3 exoenzyme on
2-adrenergic-stimulated tyrosyl phosphorylations.
2AF2 preadipocytes were treated (C3) or not
(Control) with C3 exoenzyme (72 h, 10 µg/ml). After serum
starvation, control and C3 exoenzyme-treated 2AF2 preadipocytes were
exposed (+) or not ( ) to 1 µM UK14304 for 2 min. The
level of tyrosyl phosphorylation of pp125FAK (A)
and ERK2 (B) was determined and quantified as described
under "Materials and Methods." Notice that ERK2 phosphorylation is
also associated with a shift (ERK2*). Values correspond to
the mean ± S.E. of three separate experiments. Comparison with
the control was performed using Student's t test: *,
p < 0.05.
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These results revealed that, in
2AF2 preadipocytes, active
p21rho proteins are required to mediate
2-adrenergic-dependent (i) spreading; (ii)
actin stress fibers formation; and (iii) tyrosyl phosphorylation of the
focal adhesion kinase.
2-Adrenergic Stimulation Activates RhoA
Protein--
The above results suggested the existence of a functional
coupling between
2-adrenergic receptors and
p21rhoA. To test this hypothesis, we analyzed the influence of
the
2-adrenergic stimulation on the translocation of
p21rhoA from cytosol to plasma membrane and the capacity of
GDP/GTP exchange on p21rhoA.
In control cells, the amount of p21rhoA in the particulate
fraction was lower as compared with the cytosoluble fraction (Fig. 5A). Stimulation of 24-h
serum-starved
2AF2 preadipocytes with 1 µM UK14304 led
to a increase in the amount of RhoA in the particulate fraction. This
effect was not significant (1.6-fold increase over control) before
15-min treatment. (Fig. 5B). Conversely, the amount of
G
i2, which is exclusively localized in plasma membrane,
was not modified by UK14304 treatment (Fig. 5B).

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Fig. 5.
2-Adrenergic-dependent translocation
of RhoA in 2AF2 preadipocytes. A, cytosolic
(CF) and particulate (PF) fractions of
nonstimulated 2AF2 preadipocytes were separated, and the amount of
RhoA was measured by Western blot analysis as described under
"Materials and Methods." B, 2AF2 preadipocytes were
exposed (UK) or not (Control) to 1 µM UK14304 for various time. Particulate fractions of
control and stimulated 2AF2 preadipocytes were rapidly separated
from the cytosolic fraction, and the amount of RhoA as well as
i2 content (after stripping the blot) was measured by
Western blot analysis as described under "Materials and Methods."
Values correspond to the mean ± S.E. of three separate
experiments. Comparison with the control was performed using Student's
t test: *, p < 0.05.
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The influence of
2-adrenergic stimulation on GDP/GTP
exchange on p21rhoA was tested by a GTP-loading assay.
Stimulation of 24-h serum-starved
2AF2 preadipocytes with 1 µM UK14304 led to a rapid and transient increase in the
proportion of p21rhoA-GTP versus p21rhoA-GDP
with a maximum increase of 6-fold after 2 min (Fig.
6). These results revealed the existence
of a functional coupling between
2-adrenoreceptors and
p21rhoA proteins.

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Fig. 6.
2-Adrenergic stimulation of
GDP/GTP exchange on RhoA. 32P-Labeled 2AF2
preadipocytes were exposed (UK) or not (Control)
to 1 µM UK14304 for various time. The relative proportion
of [32P]GTP and -GDP present in RhoA immunoprecipitate
was determined as described under "Materials and Methods."
A, representative experiment. B, quantification
from three separate experiments. Values correspond to the mean ± S.E. Comparison with the control was performed using Student's
t test: *, p < 0.05.
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G
-independent Induction of Spreading and Tyrosyl
Phosphorylation of the Focal Adhesion Kinase--
We have shown
previously that
2-adrenergic-dependent
activation of actin cytoskeleton in preadipocytes was pertussis
toxin-sensitive, a result demonstrating the involvement of
heterotrimeric G proteins of the Gi/Go family
(7). To determine the putative influence of the G
-subunits of
activated G proteins in this regulation,
2AF2 preadipocytes were
stably transfected with the COOH-terminal domain of
ARK1
(
ARK-CT), a truncated protein known to block the action of the
G
-subunits of G proteins when overexpressed in a cell (6). The
selection of the cell clones exhibiting a blockade of G
-subunits
was based on the inhibition of the
2-adrenergic-dependent tyrosyl
phosphorylation and shift of ERK2. The activation of ERK proteins
resulting from a G
-dependent activation of
p21ras (5) should be blocked by transfection of
ARK-CT.
Conversely G
-independent activation of the p21ras/ERK
pathway, such as that induced by growth factors contained in the serum,
should not be altered.
Based on these criteria, two cell clones (clone 5 and clone 50)
exhibiting similar behavior were selected. Only results obtained with
clone 50 are presented. As determined by radioligand binding assay
2-adrenergic receptor density was not significantly
different between clone 50 and nontransfected
2AF2 preadipocytes
(1750 ± 110 versus 2050 ± 90 fmol/mg protein).
In clone 50 the tyrosyl phosphorylation of ERK2 induced by 1 µM UK14304 treatment was reduced about 80% as compared
with nontransfected
2AF2 preadipocytes (Fig.
7). This was accompanied by an almost
complete disappearance of the shifted form of ERK2 (Fig. 7).
Conversely, the tyrosyl phosphorylation and shift of ERK2 promoted via
treatment with 10% fetal calf serum was unaffected by
ARK-CT
transfection (Fig. 7).

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Fig. 7.
Influence of stable transfection of the
COOH-terminal domain of ARK1 ( ARK-CT) on
2-adrenergic-stimulated tyrosyl phosphorylation of ERK2
in 2AF2 preadipocytes. 2AF2 preadipocytes (A) and
clone 50 ( 2AF2 preadipocytes stably transfected with ARK-CT
polypeptide) (B) were serum-starved and exposed to 1 µM UK14304 or 10% FCS treatment for time indicated. The
shift (ERK2*) and the level of tyrosyl phosphorylation of
ERK2 were determined as described under "Materials and Methods."
Values correspond to the mean ± S.E. of three separate
experiments. Comparison with the control was performed using Student's
t test: *, p < 0.05.
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We then tested the influence of G
blockade on the
2-adrenergic-dependent activation of
p21rhoA-mediated pathways. For that, we studied, in clone 50, the
2-adrenergic-dependent regulation of
spreading and tyrosyl phosphorylation of pp125FAK, cellular
events demonstrated above to be dependent on p21rhoA
activation. In clone 50, the induction of preadipocyte spreading generated by 15-min exposure to 1 µM UK14304 was not
significantly different as compared with nontransfected
2AF2
preadipocytes (Fig. 8). Similarly, in
clone 50, the increase of pp125FAK tyrosyl phosphorylation
induced by treatment with 1 µM UK14304 was not
significantly altered as compared with
2AF2 preadipocytes (Fig.
9).

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Fig. 8.
Influence of the stable transfection of the
COOH-terminal domain of ARK1 ( ARK-CT) on
2-adrenergic-induced spreading in 2AF2
preadipocytes. 2AF2 preadipocytes (white bars) and
clone 50 ( 2AF2 preadipocytes stably transfected with ARK-CT
polypeptide) (black bars) were serum-starved and exposed
(UK) or not (Control) to 1 µM
UK14304 for 15 min. Cell spreading was measured by quantification the
proportion of retracted cells present in a field (mean of five separate
fields). Values represent the mean ± S.E. of three separate
experiments. Comparison with the control was performed using Student's
t test: *, p < 0.05.
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Fig. 9.
Influence of the stable transfection of the
COOH-terminal domain of ARK1 ( ARK-CT) on
2-adrenergic-stimulated tyrosyl phosphorylation of
pp125FAK in 2AF2 preadipocytes. 2AF2
preadipocytes (A) and clone 50 ( 2AF2 preadipocytes stably
transfected with ARK-CT polypeptide) (B) were
serum-starved and exposed to 1 µM UK14304 or 10% FCS
treatment for time indicated. The level of tyrosyl phosphorylation of
pp125FAK was determined as described under "Materials and
Methods." Values correspond to the mean ± S.E. of three
separate experiments. Comparison with the control was performed using
Student's t test: *, p < 0.05.
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These results show that, in
2AF2 preadipocytes,
ARK-CT-mediated blockade of G
-subunits did not alter the
2-adrenergic-dependent regulation of cell
spreading and focal adhesion kinase phosphorylation.
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DISCUSSION |
The present study demonstrates that (i) the coupling between
2-adrenergic receptors and the small GTPase
p21rhoA is involved in the
2-adrenergic-dependent regulation of
preadipocyte actin cytoskeleton and pp125FAK and that (ii)
conversely to the Ras/MAPK pathway, the
Gi/Go-dependent activation of
p21rhoA/cytoskeleton pathway does not involve the

-subunits of heterotrimeric G proteins.
As described with other agents such as lysophosphatidic acid,
sphingosine 1-phosphate, or thrombin (8, 10, 11),
2-adrenergic-dependent activation of actin
cytoskeleton was blocked by C3 exoenzyme pretreatment, a toxin known to
specifically suppress the activity p21rhoA (17, 18). Under its
active form (bound to GTP), p21rhoA plays a key role in the
reorganization of the actin network, particularly in the formation of
actin stress fibers, but also in the formation of focal adhesion
plaques and the tyrosyl phosphorylation of the focal adhesion kinase
(8, 9, 19). Therefore, our results obtained with the C3 exoenzyme
demonstrated the implication of p21rhoA in the
2-adrenergic-dependent regulation of the
actin cytoskeleton and in the control of adhesion of the preadipocyte.
This observation suggested the existence of a possible coupling between
2-adrenergic receptors and p21rhoA.
This hypothesis was confirmed, since we demonstrated that
2-adrenergic receptor stimulation leads to the
activation of GDP/GTP exchange on p21rhoA characterized by a
rapid and transient increase in the p21rhoA-GTP form as
previously demonstrated for p21ras (4). This observation
directly demonstrates the ability of the
2-adrenergic
receptor to activate p21rhoA.
2-Adrenergic
stimulation also leads to an increase in the amount of p21rhoA
in the particulate fraction of
2AF2 preadipocytes. This observation can be interpreted as the result of the translocation of a fraction of
RhoA from the cytosol to the plasma membrane as classically described
for the GTPases of the Rho family (20-22). It is noticeable that
whereas
2-adrenergic receptor-stimulated RhoA GTP
loading was maximal within 2 min of stimulation, no significant
translocation was observed until 15 min. This suggests that, in our
model, RhoA translocation is not required for RhoA activation. Knowing
the close relationship between cytoskeleton activity and protein
translocation, it is possible that RhoA translocation is a consequence,
rather than a cause, of RhoA-mediated reorganization of actin
cytoskeleton. Another possibility is that RhoA translocation is
independent of RhoA activation as proposed previously (33). Further
investigations will be necessary to test these hypotheses.
In a previous study, we have demonstrated that the
2-adrenergic-dependent reorganization of
actin cytoskeleton in preadipocytes was suppressed by pertussis toxin
pretreatment of preadipocytes (7). Therefore, it is reasonable to
propose that the coupling between
2-adrenergic receptors
and p21rhoA involves an heterotrimeric G protein of the
Gi/Go family. Gi proteins have been
demonstrated to be involved in the
2-adrenergic-dependent activation of
p21ras, via the G
-subunits of heterotrimeric G proteins
(5, 23). Since p21rhoA and p21ras belong to the same
superfamily they potentially exhibit very similar mode of regulation.
Therefore, the involvement of the G
-subunits in the
2-adrenergic-dependent activation of
p21rhoA could reasonably be suspected. Transient or permanent
expression of
ARK-CT, a G
-binding protein, have been
demonstrated as being a useful strategy to discriminate between
-
and 
-mediated pathways (6). Our results demonstrate that
permanent transfection of
ARK-CT in
2AF2 preadipocytes almost
completely blocks the G
-dependent activation of
p21ras/MAPK pathway generated by
2-adrenergic
receptor stimulation. This blockade appears to be specific of G
,
since the G
-independent activation of p21ras/MAPK
generated by growth factor-containing serum was not modified. Conversely to the Ras/MAPK pathway, the
2-adrenergic-dependent regulation of
spreading and increase of tyrosyl phosphorylation of
pp125FAK were not altered by G
blockade. These data
demonstrate that 
-subunits are not involved in the regulation of
actin cytoskeleton nor in the activation of the focal adhesion kinase,
two p21rhoA-dependent controlled cellular events.
Therefore, based on the action of pertussis toxin, it is likely that
the G
-independent coupling between
2-adrenergic
receptors and p21rhoA in preadipocytes involves the
i/
o-subunits of the heterotrimeric G proteins. We showed
previously that
2AF2 preadipocytes express the three pertussis
toxin-sensitive
-subunits,
i2,
i3 and
o, but not
i1 (24). Further
investigations will be necessary to determine which of these subunits
are involved in
2-adrenergic-dependent activation of p21rhoA.
The existence of a G
-independent coupling between
2-adrenergic receptors and p21rhoA asks the
question of its functional consequences in preadipocytes. Modifications
of actin cytoskeleton are associated with numerous cellular events such
as proliferation, differentiation, and motility (25, 26). We have
demonstrated previously that
2-adrenergic receptor
stimulation increases preadipocyte proliferation, an effect that is
associated with the tyrosyl phosphorylation of ERK1 and ERK2 MAPK (3),
kinases which are dependent upon p21ras activation (27) and are
implicated in cell cycle regulation (28). The results of the present
study clearly demonstrate that p21rhoA is not involved in the
2-adrenergic-dependent activation of p21/MAPK pathway, since C3 exoenzyme was without effect on ERK2 activation. However, even though the precise mechanism has not been
completely elucidated, it has clearly been demonstrated by several
groups that Rho proteins are regulators of cell cycle regulation (29,
30). It has indeed been shown that C3 exoenzyme strongly inhibits the
growth of several cell types (29), including
2AF2
preadipocytes.2 Therefore, it
is reasonable to think that p21rhoA could be involved in the
2-adrenergic-dependent regulation of preadipocyte proliferation via an ERK-independent transduction pathway.
Recently, Jinsi-Parimoo et al. (31) proposed that
p21rhoA is involved in the
2-adrenergic
stimulation of phospholipase D in PC12 cells. Phospholipase D is an
enzyme involved in a broad spectrum of cellular events, including
mitogenesis (32). Therefore, it could be proposed that
2-adrenergic-dependent regulation of preadipocyte proliferation could involve a
p21rhoA-dependent activation of phospholipase
D.
In conclusion, this study emphasizes that in preadipocytes, in addition
to their involvement in the activation of the
G
-dependent p21ras/MAPK pathway,
2-adrenergic receptors can also activate the
p21rhoA/cytoskeleton pathway in a G
-independent manner.
Control of p21rhoA activity and actin cytoskeleton not only
plays an important role in cell morphological changes but is also
crucial for cell cycle regulation. Therefore, depending on each kind of
subunit (
or
i/
o) the combined
Gi/Go-mediated stimulation of p21ras
and p21rhoA can cooperate in the mediation of the
2-adrenergic receptor-dependent regulation
of preadipocyte proliferation and/or differentiation.