Gbeta gamma -independent Coupling of alpha 2-Adrenergic Receptor to p21rhoA in Preadipocytes*

Sandrine Bétuing, Danièle Daviaud, Céline Pagès, Elisabeth Bonnard, Philippe Valet, Max Lafontan, and Jean Sébastien Saulnier-BlacheDagger

From the INSERM U317, Institut Louis Bugnard, Université Paul Sabatier, CHU Rangueil, Batiment L3, 31403, Toulouse Cedex 4, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In preadipocytes, alpha 2-adrenergic receptor (alpha 2-AR) stimulation leads to a Gi/Go-dependent rearrangement of actin cytoskeleton. This is characterized by a rapid cell spreading, the formation of actin stress fibers, and the increase in tyrosyl phosphorylation of the focal adhesion kinase (pp125FAK). These cellular events being tightly controlled by the small GTPase p21rhoA, the existence of a Gi/Go-dependent coupling of alpha 2-AR to p21rhoA in preadipocytes was proposed.

In alpha 2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line and which stably expresses the human alpha 2C10-adrenergic receptor) alpha 2-adrenergic-dependent induction of cell spreading, formation of actin stress fibers, and increase in tyrosyl phosphorylation of pp125FAK were abolished by pretreatment of the preadipocytes with the C3 exoenzyme, a toxin which impairs p21rhoA activity by ADP-ribosylation. Conversely, C3 exoenzyme had no effect on the alpha 2-adrenergic-dependent increase in tyrosyl phosphorylation and shift of ERK2 mitogen-activated protein kinase. alpha 2-Adrenergic stimulation also led to an increase in GDP/GTP exchange on p21rhoA, as well as to an increase in the amount of p21rhoA in the particulate fraction of alpha 2AF2 preadipocytes. Stable transfection of alpha 2AF2 preadipocytes with the COOH-terminal domain of beta ARK1 (beta ARK-CT) (a blocker of Gbeta gamma -action), strongly inhibited the alpha 2-adrenergic-dependent increase in tyrosyl phos- phorylation and shift of ERK2, without modification of the tyrosyl phosphorylation of pp125FAK and spreading of preadipocytes. These results show that alpha 2-adrenergic-dependent reorganization of actin cytoskeleton requires the activation of p21rhoA in preadipocytes. Conversely to the activation of the p21ras/mitogen-activated protein kinase pathway, the alpha 2-adrenergic activation of p21rhoA-dependent pathways are independent of the beta gamma -subunits of heterotrimeric G proteins.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha 2-Adrenergic receptors (alpha 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, alpha 2-ARs belong to the alpha 2A-subtype and are expressed in both adipocytes and preadipocytes. In adipocytes alpha 2-ARs mediate inhibition of lipolysis through a Gi-dependent coupling to adenylyl cyclase (2). In preadipocytes, stimulation of alpha 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 alpha 2-ARs to the small GTPase p21ras (4). alpha 2-Adrenergic activation of the Ras/MAPK pathway is mediated by the beta gamma -subunits of the heterotrimeric G proteins as demonstrated by the ability of the COOH-terminal domain of beta ARK1 (a beta gamma -binding protein) to block this activation (5, 6).

In preadipocytes, stimulation of alpha 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 alpha 2-ARs in preadipocytes, we analyzed (i) the involvement of p21rhoA in alpha 2-adrenergic-dependent reorganization of actin cytoskeleton and tyrosyl phosphorylation of pp125FAK; (ii) the existence of a functional coupling between alpha 2-ARs and p21rhoA; (iii) the putative involvement of beta gamma -subunits of G proteins in this coupling. In the present study we demonstrate that, in alpha 2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line stably expressing the human alpha 2C10-adrenergic receptor), alpha 2-adrenergic-dependent reorganization of actin cytoskeleton involves the activation of p21rhoA independently of a Gbeta gamma -mediated transduction pathway.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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). alpha 2AF2 (alpha 2AF2) preadipocytes were previously obtained by permanent transfection of the human alpha 2C10-adrenergic receptor gene in the 3T3F442A preadipose cell line followed by G418 selection (3). As determined by radioligand binding analysis, alpha 2-adrenergic receptor density in alpha 2AF2 preadipocytes was 2050 ± 90 fmol/mg protein. Stable expression of the carboxyl-terminal domain of beta ARK1 (beta ARK-CT) was obtained by transfection of alpha 2AF2 preadipocytes with a pZeo/beta ARKCT vector, followed by the double selection G418/zeocin. pZeo/beta ARKCT vector was obtained by subcloning an EcoRI-SalI fragment from pRK-beta 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 alpha 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 beta -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 alpha 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-beta -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-alpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Influence of the C3 Exoenzyme on alpha 2-Adrenergic-dependent Regulation of Preadipocyte Morphology-- In a previous study, we demonstrated that in preadipocytes, alpha 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 alpha 2-adrenergic-dependent regulation of preadipocyte morphology, the effect of C3 exoenzyme, a toxin that impairs the function of p21rho (12), on alpha 2-adrenergic-mediated spreading, stress fibers formation, and pp125FAK phosphorylation in alpha 2AF2 preadipocytes was tested.

In total alpha 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 alpha 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 alpha 2AF2 preadipocytes and ADP-ribosylate p21rho proteins.


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Fig. 1.   C3 exoenzyme catalyzed ADP-ribosylation in alpha 2AF2 preadipocytes. alpha 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.

After 15-h serum starvation, alpha 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 alpha 2-adrenergic agonist UK14304 led the spreading of almost 80% of retracted alpha 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 alpha 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 alpha 2-adrenergic-induced spreading in alpha 2AF2 preadipocytes. alpha 2AF2 preadipocytes were treated (C3) or not (Control) with C3 exoenzyme (72 h, 10 µg/ml). After serum starvation, control and C3 exoenzyme-treated alpha 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 alpha 2adrenergic-dependent stress fiber formation in alpha 2AF2 preadipocytes. alpha 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 alpha 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.

Five-min treatment of 24-h serum-starved alpha 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 alpha 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 alpha 2-adrenergic-stimulated tyrosyl phosphorylations. alpha 2AF2 preadipocytes were treated (C3) or not (Control) with C3 exoenzyme (72 h, 10 µg/ml). After serum starvation, control and C3 exoenzyme-treated alpha 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.

These results revealed that, in alpha 2AF2 preadipocytes, active p21rho proteins are required to mediate alpha 2-adrenergic-dependent (i) spreading; (ii) actin stress fibers formation; and (iii) tyrosyl phosphorylation of the focal adhesion kinase.

alpha 2-Adrenergic Stimulation Activates RhoA Protein-- The above results suggested the existence of a functional coupling between alpha 2-adrenergic receptors and p21rhoA. To test this hypothesis, we analyzed the influence of the alpha 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 alpha 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 Galpha i2, which is exclusively localized in plasma membrane, was not modified by UK14304 treatment (Fig. 5B).


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Fig. 5.   alpha 2-Adrenergic-dependent translocation of RhoA in alpha 2AF2 preadipocytes. A, cytosolic (CF) and particulate (PF) fractions of nonstimulated alpha 2AF2 preadipocytes were separated, and the amount of RhoA was measured by Western blot analysis as described under "Materials and Methods." B, alpha 2AF2 preadipocytes were exposed (UK) or not (Control) to 1 µM UK14304 for various time. Particulate fractions of control and stimulated alpha 2AF2 preadipocytes were rapidly separated from the cytosolic fraction, and the amount of RhoA as well as alpha 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.

The influence of alpha 2-adrenergic stimulation on GDP/GTP exchange on p21rhoA was tested by a GTP-loading assay. Stimulation of 24-h serum-starved alpha 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 alpha 2-adrenoreceptors and p21rhoA proteins.


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Fig. 6.   alpha 2-Adrenergic stimulation of GDP/GTP exchange on RhoA. 32P-Labeled alpha 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.

Gbeta gamma -independent Induction of Spreading and Tyrosyl Phosphorylation of the Focal Adhesion Kinase-- We have shown previously that alpha 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 Gbeta gamma -subunits of activated G proteins in this regulation, alpha 2AF2 preadipocytes were stably transfected with the COOH-terminal domain of beta ARK1 (beta ARK-CT), a truncated protein known to block the action of the Gbeta gamma -subunits of G proteins when overexpressed in a cell (6). The selection of the cell clones exhibiting a blockade of Gbeta gamma -subunits was based on the inhibition of the alpha 2-adrenergic-dependent tyrosyl phosphorylation and shift of ERK2. The activation of ERK proteins resulting from a Gbeta gamma -dependent activation of p21ras (5) should be blocked by transfection of beta ARK-CT. Conversely Gbeta gamma -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 alpha 2-adrenergic receptor density was not significantly different between clone 50 and nontransfected alpha 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 alpha 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 beta ARK-CT transfection (Fig. 7).


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Fig. 7.   Influence of stable transfection of the COOH-terminal domain of beta ARK1 (beta ARK-CT) on alpha 2-adrenergic-stimulated tyrosyl phosphorylation of ERK2 in alpha 2AF2 preadipocytes. alpha 2AF2 preadipocytes (A) and clone 50 (alpha 2AF2 preadipocytes stably transfected with beta 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.

We then tested the influence of Gbeta gamma blockade on the alpha 2-adrenergic-dependent activation of p21rhoA-mediated pathways. For that, we studied, in clone 50, the alpha 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 alpha 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 alpha 2AF2 preadipocytes (Fig. 9).


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Fig. 8.   Influence of the stable transfection of the COOH-terminal domain of beta ARK1 (beta ARK-CT) on alpha 2-adrenergic-induced spreading in alpha 2AF2 preadipocytes. alpha 2AF2 preadipocytes (white bars) and clone 50 (alpha 2AF2 preadipocytes stably transfected with beta 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 beta ARK1 (beta ARK-CT) on alpha 2-adrenergic-stimulated tyrosyl phosphorylation of pp125FAK in alpha 2AF2 preadipocytes. alpha 2AF2 preadipocytes (A) and clone 50 (alpha 2AF2 preadipocytes stably transfected with beta 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.

These results show that, in alpha 2AF2 preadipocytes, beta ARK-CT-mediated blockade of Gbeta gamma -subunits did not alter the alpha 2-adrenergic-dependent regulation of cell spreading and focal adhesion kinase phosphorylation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrates that (i) the coupling between alpha 2-adrenergic receptors and the small GTPase p21rhoA is involved in the alpha 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 beta gamma -subunits of heterotrimeric G proteins.

As described with other agents such as lysophosphatidic acid, sphingosine 1-phosphate, or thrombin (8, 10, 11), alpha 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 alpha 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 alpha 2-adrenergic receptors and p21rhoA.

This hypothesis was confirmed, since we demonstrated that alpha 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 alpha 2-adrenergic receptor to activate p21rhoA. alpha 2-Adrenergic stimulation also leads to an increase in the amount of p21rhoA in the particulate fraction of alpha 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 alpha 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 alpha 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 alpha 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 alpha 2-adrenergic-dependent activation of p21ras, via the Gbeta gamma -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 Gbeta gamma -subunits in the alpha 2-adrenergic-dependent activation of p21rhoA could reasonably be suspected. Transient or permanent expression of beta ARK-CT, a Gbeta gamma -binding protein, have been demonstrated as being a useful strategy to discriminate between alpha - and beta gamma -mediated pathways (6). Our results demonstrate that permanent transfection of beta ARK-CT in alpha 2AF2 preadipocytes almost completely blocks the Gbeta gamma -dependent activation of p21ras/MAPK pathway generated by alpha 2-adrenergic receptor stimulation. This blockade appears to be specific of Gbeta gamma , since the Gbeta gamma -independent activation of p21ras/MAPK generated by growth factor-containing serum was not modified. Conversely to the Ras/MAPK pathway, the alpha 2-adrenergic-dependent regulation of spreading and increase of tyrosyl phosphorylation of pp125FAK were not altered by Gbeta gamma blockade. These data demonstrate that beta gamma -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 Gbeta gamma -independent coupling between alpha 2-adrenergic receptors and p21rhoA in preadipocytes involves the alpha i/alpha o-subunits of the heterotrimeric G proteins. We showed previously that alpha 2AF2 preadipocytes express the three pertussis toxin-sensitive alpha -subunits, alpha i2, alpha i3 and alpha o, but not alpha i1 (24). Further investigations will be necessary to determine which of these subunits are involved in alpha 2-adrenergic-dependent activation of p21rhoA.

The existence of a Gbeta gamma -independent coupling between alpha 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 alpha 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 alpha 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 alpha 2AF2 preadipocytes.2 Therefore, it is reasonable to think that p21rhoA could be involved in the alpha 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 alpha 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 alpha 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 Gbeta gamma -dependent p21ras/MAPK pathway, alpha 2-adrenergic receptors can also activate the p21rhoA/cytoskeleton pathway in a Gbeta gamma -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 (beta gamma or alpha i/alpha o) the combined Gi/Go-mediated stimulation of p21ras and p21rhoA can cooperate in the mediation of the alpha 2-adrenergic receptor-dependent regulation of preadipocyte proliferation and/or differentiation.

    FOOTNOTES

* 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.

Dagger To whom correspondence should be addressed. Tel.: 33-5-6217-2956; Fax: 33-5-6133-1721; E-mail: saulnier{at}rangueil.inserm.fr.

1 The abbreviations used are: alpha 2-AR, alpha 2-adrenergic receptor; FAK, focal adhesion kinase; ERK, extracellular signal-regulated kinase; beta ARK-CT, COOH-terminal domain of beta -adrenergic receptor kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline.

2 S. Bétuing, personal communication.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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