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INTRODUCTION

Study analyzes p21RhoA involvement in a2-adrenergic preadipocyte changes and functional coupling. Treatment with C3 exoenzyme affects cell morphology and tyrosine phosphorylation.

ruthmeyer
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INTRODUCTION

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  1. INTRODUCTION Catecholamines act through a set of specific a2 and b-adrenoceptors. Whereas the role of catecholamines in the control of white adipocyte metabolism (lipolysis)has been well established, their invlvement in preadipocyte proliferation and differentiation remains poorly understood. We previously demonstrated that a2A-adrenoceptor-stimulation increases in a Gi/Go-dependent pathway preadipocytes proliferation (Bouloumie et al./J.B.C/269/30254-30259/1994). This effect was associated with rapid and transient tyrosine phosphorylation of the p44 and p42 mitogen-activated protein kinases (MAPK). a2-adrenergic activation of the ras/MAPK pathway is mediated by the bg-subunits of the heterotrimeric Gi/Go proteins. In preadipcytes, stimulation of a2-adrenoceptors 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). These cellular events are known to be tightly controlled by a GTPase belonging to the Ras superfamily, p21RhoA.

  2. AIM OF THE STUDY Considering the morphological changes generated by stimulation of the a2-adrenoceptors stimulation in preadipocytes, we analysed (1) the involvement of p21RhoA in a2-adrenergic-dependent reorganization of actin cytoskeleton and tyrosyl phosphorylation of pp125FAK, (2) the existence of a functional coupling between a2-adrenoceptors and p21RhoA and (3) the putative involvement of bg-subunits of G proteins in this coupling. This study was realized in a2AF2 preadipocytes, a cell clone derived from the 3T3F442A preadipose cell line stably expressing the human a2C10-adrenergic receptor.

  3. 80 60 % of retracted cells 40 * 20 0 - + - + UK C3 Control INFLUENCE OF C3 EXOENZYME ON a2-ADRENERGIC-DEPENDENT REGULATION OF a2AF2 PREADIPOCYTE MORPHOLOGY Influence of C3 exoenzyme on a2-adrenergic-induced spreading and stress fibers formation. a2AF2 preadipocytes were treated (C3) or not (Control) with C3 exoenzyme (72h, 10mg/ml). After serum starvation, control and C3 exoenzyme-treated a2AF2 preadipocytes were exposed (+) or not (-) to 1 mM UK14304 for 15 min. (A) Cell spreading was measured by quantifying the proportion of refringent cells present in a field. Values represent the mean +/- S.E of three separate experiments. (B) Actin filaments were visualized using fluorescein isothiocyanate-labeled phallooidin.

  4. * * * 100 100 80 80 % of maximum phosphorylation 60 % of maximum phosphorylation 60 40 40 20 20 0 0 - + - + UK - + - + Control C3 Control C3 INFLUENCE OF C3 EXOENZYME ON a2-ADRENERGIC-DEPENDENT TYROSYL PHOSPHORYLATION OF PP125FAK AND ERK2 REGULATION IN a2AF2 PREADIPOCYTES A B ERK2* FAK ERK2 - + - + - + - + UK UK Control C3 Control C3 a2AF2 were treated (C3) or not (Control) with C3 exoenzyme (72h, 10mg/ml). After serum starvation, control and C3 exoenzyme-treated a2AF2 preadipocytes were exposed (+) or not (-) to 1 mM UK14304 for 2 min. The level of tyrosyl phosphorylation of pp125FAK (A) and ERK2 (B) was determined after immunoprecipitation of tyrosyl-phosphorylated proteins andwestern-blot western-blot and quantified. ERK2 phosphorylation is associated with a shift (ERK2*). Values correspond to the mean +/- S.E.

  5. * 60 GDP * 50 40 * GTP 30 % GTP/(GTP+GDP) 20 10 0 Origin 2' 5' 15' Control UK 2’ 5’ 15’ Control UK ALPHA2-ADRENERGIC STIMULATION PROMOTES GDP/GTP EXCHANGE ON RHOA IN a2AF2 PREADIPOCYTES A B [32P] labelled a2AF2 preadipocytes were exposed (UK) or not (control) to 1mM UK14304 for various time. The relative proportion of [32P] GTP and GDP present in rhoA immunoprecipitate was determined as described in Materials and Methods. (A) Representative experiment. (B) Quantification from three separate experiments. Values correspond to the mean +/- SEM. Comparison with the control was performed using Student’s t test : *, P<0.05.

  6. 80 ERK2* B Clone 50 ERK2 60 2’ 5’ 2’ 5’ Control UK FCS % of retracted cells 40 * * 20 C Clone 50 FAK 0 Control UK (15MIN) 2’ 5’ 2’ 5’ Control UK FCS INFLUENCE OF THE STABLE TRANSFECTION OF THE COOH-TERMINAL DOMAIN OF b-ARK1 ON a2-ADRENERGIC-STIMULATED CELL SPREADING AND TYROSYL PHOSPHORYLATION OF PP125FAK, ERK2 A Clone 50 (a2AF2 preadipocytes stably transfected with bARK-CT polypeptide) were serum starved and exposed to 1mM UK14304 or FCS10% (foetal Calf Serum) treatment for time indicated. (A) Cell spreading was measured by quantifying the proportion of refringent cells present in a field. The level of tyrosyl phosphorylation of ERK2 (B) and pp125FAK (C) was determined after immunoprecipitation of tyrosyl-phosphorylated proteins andwestern-blot. Values correspond to the mean +/- SEM of three separate experiments.

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