help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2002-0138
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Simon, V.
Right arrow Articles by Cohen-Tannoudji, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simon, V.
Right arrow Articles by Cohen-Tannoudji, J.
Endocrinology Vol. 144, No. 7 3058-3066
Copyright © 2003 by The Endocrine Society

Endogenous G Protein-Coupled Receptor Kinase 6 Triggers Homologous ß-Adrenergic Receptor Desensitization in Primary Uterine Smooth Muscle Cells

Violaine Simon, Marie-Thérèse Robin, Chantal Legrand and Joëlle Cohen-Tannoudji

Laboratoire de Physiologie et Physiopathologie, Unité Mixte de Recherche 7079, Université P & M Curie, 75252 Paris Cedex 05, France

Address all correspondence and requests for reprints to: Dr. Joëlle Cohen-Tannoudji, Laboratoire de Physiologie et Physiopathologie, UMR 7079, Université P & M Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France. E-mail: joelle.cohen-tannoudji{at}snv.jussieu.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that G protein-coupled receptor kinase (GRK) may contribute to ß-adrenergic receptor (ß-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous ß-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the ß-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4–6 families, we demonstrated that only the GRK4–6 family mediated ß-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants (K215RGRK5 and K215RGRK6). Expression of K215RGRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of K215RGRK5 or K220RGRK2 had no effect on ß-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous ß-AR desensitization in myometrial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVEN-TRANSMEMBRANE G protein-coupled receptors (GPCRs) mediate signal transduction of a wide array of molecules, ranging from neurotransmitters, hormones, chemokines, and lipids to light and odorants by activating or inhibiting specific effectors such as adenylyl cyclase, phospholipases C and A2, and ion channels (1). One of the early regulatory mechanisms taking place after GPCR stimulation is receptor homologous desensitization. This is an agonist-dependent adaptive process attenuating cell responsiveness to prolonged or repeated agonist stimulation, without affecting the response to other agonists or activators. Homologous desensitization begins with GPCR uncoupling because of two types of serine/threonine kinases, the second messenger-dependent kinases such as cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) and the more recently discovered G protein-coupled receptor kinases (GRKs) (2). Mechanism of GRK action has been extensively studied for the retinal receptor, rhodopsin, and the ß2-adrenergic receptor (ß2-AR) (3). GRKs specifically phosphorylate the agonist-activated form of GPCR (at serine and threonine residues located in the C-terminal tail of ß2-AR) and promote binding of a cytosolic protein, ß-arrestin, which further uncouples GPCR by preventing receptor-G protein interaction. Seven GRKs were cloned from distinct genes and classified into three families: 1) the GRK1 family, including GRK1 and GRK7, is predominantly localized in the retina and regulates photoreceptor function, 2) the GRK2 family includes GRK2 and GRK3, which are more widely distributed, and 3) the GRK4 family includes GRK4, GRK5, and GRK6. GRK5 and GRK6 are ubiquitously expressed, whereas GRK4 is localized primarily to the testis, suggesting a role in desensitization of gonad-specific GPCRs such as receptors of LH and FSH (3).

In uterine smooth muscle (myometrium), activation of ß-adrenoreceptor/adenylyl cyclase pathway by catecholamines markedly contributes to myorelaxation during pregnancy. In humans as well as in rodents, ß-AR/adenylyl cyclase pathway desensitizes at the end of pregnancy, contributing to initiation of contractions at parturition (4, 5, 6). We previously demonstrated that the sudden decrease of adenylyl cyclase response to ß-agonist, taking place just before parturition in rat, was due to a ß-AR-G{alpha}s protein uncoupling occurring concomitantly with an increased myometrial GRK activity (7). These results suggest that alterations of ß-adrenergic signaling may be triggered by one or several of the myometrial GRK, i.e. GRK2, GRK5, and GRK6. Such a mechanism may be relevant for initiation of uterine contractions and normal delivery in the rat. Although all seven GRKs can phosphorylate ß-AR in vitro (3), little is known about GRK substrate specificity in vivo. Although limited expression of the GRK1 family and GRK4 suggests very specific functions, the targets of widely expressed GRKs are less defined. Several experimental strategies have been recently developed on various cell lines to identify specific roles of endogenous GRK by inhibiting either their synthesis or action.

In this study, we took advantage of two complementary experimental strategies to identify GRK involved in myometrial homologous ß-AR desensitization. We attempted to inhibit the action of endogenous myometrial GRK by introducing monoclonal antibodies known to block GRK2/3 or GRK 4–6 families and overexpressing kinase-dead dominant-negative mutants of GRK5 or GRK6 (K215RGRK5 or K215RGRK6). Experiments were performed on a rat primary cell culture retaining the main phenotypical and functional characteristics of the myometrial tissue. Our results demonstrate, for the first time, that homologous ß-AR desensitization in myometrial cells is triggered by GRK6.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, Hanks’ buffer salt saline, fetal calf serum (FCS), penicillin-streptomycin (PS), collagenase type II, OptiMEM, and lipofectamine Reagent Plus were from Invitrogen (Cergy Pontoise, France). Culture plates, scrynel NYHC nylon 48 µm, 2-well Lab-Tek chamber, slide and aluminum oxide 90 activé neutre were from VWR International (Strasbourg, France). Dulbecco’s PBS, polylysine, mouse monoclonal antimyosin (smooth), cAMP, imidazole, protease cocktail inhibitor, 3-iso-1-methylxanthine (IBMX), cholera toxin, forskolin, heparin, and isoproterenol were from Sigma (L’Isle d’Abeau, France). H89 dihydrochloride was from Coger (Paris, France). Streptavidin fluoresceine reagent and antimouse biotinylated second antibodies were from Amersham Pharmacia Biotech (Les Ulis, France). [3H]CGP-12177 (42.5 Ci/mmol), [2,8-3H]adenine (30 Ci/mmol), and ({gamma}-32P)ATP (6000 Ci/mmol) were from Perkin Elmer Life Sciences (Paris, France). AG50 DOWEX W-X4 was from Bio-Rad Laboratories, Inc. (Marne la Coquette, France). Dual luciferase reporter assay system and pTK-renilla were from Promega Corp. (Charbonnières, France); mouse monoclonal anti-{alpha}-actin (smooth) and rabbit polyclonal anti-GRK6 (C-20) was from TEBU International (Le Perray en Yvelines, France). GRK2 polyclonal antibody and GRK5 polyclonal antibody were generous gifts from Dr. F. Mayor, Jr. (Universitad Autõnoma, Madrid, Spain) and Dr. F. Boulay (CEA, Grenoble, France), respectively.

Monoclonal antibodies (mAbs) directed against GRK 2/3 or GRK4–6 families were kindly donated by Dr. M. Oppermann (University of Göttingen, Göttingen, Germany). Those mAbs were generated against glutathione-S-transferase fusion protein (GST) encompassing the C-terminal GRK domains (8). Urea-treated bovine rod outer segments were obtained from Dr. N. Bennett (CEA, Grenoble, France) (9). {Delta}MMTV-Luc(wtCRE) plasmid was kindly donated by Dr. D. Spengler (Max Planck Institute of Psychiatry, Munich, Germany). Kinase-dead, dominant-negative mutants, pCDNA3-K220RGRK2, PCDM8-K215RGRK5, and pBC12BI-K215RGRK6, were kindly donated by Dr. J. L. Benovic (Philadelphia, PA), Dr. T. Métayé (CHU Poitiers, France), and Dr. F. Boulay (CEA, Grenoble, France), respectively.

Primary culture of myometrial cells
Sprague Dawley pregnant rats (250–300 g) were obtained from Janvier (Le Genest-St-Isle, France). Investigations were conducted in accordance with guidelines for care and use of laboratory animals (NIH Guide). Pregnant rats were killed by cervical dislocation at the end of pregnancy (d 20 of pregnancy). Uterine horns were immediately excised and fetoplacental units were removed. Myometrial tissues were then rapidly trimmed of fat and connective tissues and scraped from adherent endometrium. Myometrial cells were prepared essentially according to Dodge et al. (10). Myometrium was minced into fragments (about 1 mm3) and digested by 375 U/ml collagenase type II in high-glucose (4500 mg/liter) DMEM containing 25 U/ml PS at 37 C in atmosphere of 5% CO2 95% humidified air under agitation. Dispersed cells were separated from tissue fragments by filtration through a 48-µm membrane filter before being centrifuged at 1 500 x g and resuspended in high-glucose DMEM supplemented with 20% FCS and 25 U/ml PS. Suspension of cells obtained after a first 10-min digestion was discarded. Myometrial cells were dispersed by three digestions of 40 min, pooled, and plated in culture plates, at the appropriate density, in high-glucose (4500 mg/liter) DMEM supplemented with 20% FCS and 25 U/ml PS. Cells were maintained at 37 C in humidified conditions under 5% CO2. Medium was replaced by fresh DMEM supplemented with 20% FCS 1 d later and by 10% FCS the following day. Cells were used at confluence 3 d after plating.

Immunocytochemical staining
Cells were plated at a density of 25,000 cells per well on a 2-well Lab-Tek chamber slide coated with 300 µg/ml polylysine. Briefly, confluent cells were washed with cold PBS and fixed in acetone-ethanol (vol/vol) during 3 min at -20 C. After drying for 15 min at room temperature, cells were blocked in PBS containing horse serum during 20 min and then incubated overnight with or without (control) smooth muscle specific {alpha}-actin or myosin mouse antibodies (1:20 and 1:150 dilutions, respectively) at room temperature. Cells were treated with a 1:200 dilution of an antimouse biotinylated second antibody (2 h at room temperature) and then incubated with streptavidin-fluorescein isothiocyanate complex (1:100 dilution) during 45 min at room temperature in darkness. After this period, slides were mounted and observed under fluorescent microscopy. To evaluate the percentage of cells that express these smooth muscle-specific markers, the total number of cells were determined by staining with Evans blue.

Determination of the [3H]cAMP accumulation
The intracellular content of cAMP was determined as described (11). Confluent cells (100,000 cells/well, 6-well plates) were serum deprived in the morning before being incubated overnight in DMEM with 2 µCi [3H]adenine per well (AS: 30 Ci/mmol). Cells were then preincubated (15 min, room temperature) in DMEM with 20 mM HEPES and 250 µM IBMX. After this incubation period, cells were stimulated with a ß-agonist, isoproterenol (10 µM, 5 min); a Gs activator, cholera toxin (3 nM, 2 h); or an adenylyl cyclase activator, forskolin (0.1 mM, 10 min) at 37 C. The incubation medium was removed and the reaction was stopped by addition of 1 ml ice-cold lysis solution containing 2.5% trichloroacetic acid and 3.5% cAMP. Supernatant (0.9 ml) containing cAMP were neutralized with 0.1 ml KOH 4.2 M. After centrifugation (1500 x g, 15 min, 4 C), the cAMP produced was separated by two successive chromatographies on Dowex and Alumina columns as described (12).

Binding assays on intact myometrial cells
Intact cell receptor-binding assays were performed as described (13). Cell monolayers (105 cells) were incubated for 20 min at room temperature with increasing concentrations (0.25–6 nM) of [3H]CGP-12177 in 1 ml binding buffer (10 mM NaHCO3; 20 mM HEPES; 1 mM ascorbic acid, pH 7.35). Nonspecific binding was determined in the presence of 10-5 M propranolol. After binding, cells were washed three times with ice-cold PBS, scraped, and counted. Data from binding experiments were analyzed using a nonlinear curve-fitting GraphPad Prism program (GraphPad Software, Inc., San Diego, CA).

Desensitization experiments
Cells were preincubated in DMEM with 20 mM HEPES (15 min, room temperature) before stimulation with 10 µM isoproterenol for 30 min at 37 C. The medium was then removed and cells were washed extensively with PBS. The cAMP accumulation was measured after a subsequent stimulation of adenylyl cyclase by 10 µM isoproterenol for 5 min in presence of IBMX (250 µM). Desensitization was defined as the percent decrease of cAMP production in response to this subsequent isoproterenol stimulation. In some experiments, effects of isoproterenol pretreatment were evaluated on the cAMP subsequent response to cholera toxin (3 nM, 2 h) or forskolin (0.1 mM, 10 min). For experiments with the GRK inhibitor heparin (14) or GRK antibodies, myometrial cells were permeabilized with saponin (35 µg/ml, 10 min, 37 C). Heparin (1 µM) and GRK antibodies (5 µg/ml) were added or not during saponin treatment. Cells were then washed twice with PBS to remove saponin and incubated with heparin or GRK antibodies 1 h before and during the 30-min desensitization period. We used mouse mAbs directed against either GRK 2/3 or GRK4–6 families. GST mAbs were used as controls.

For experiments with the cell permeant inhibitor of PKA, H89 (15), myometrial cells were preincubated with H89 (20 µM) 1 h before and during the 30-min desensitization period.

Western blot analysis of GRK expression
Plasma membrane fractions were prepared from confluent myometrial cells. Briefly, cells were scraped in solution containing 5 mM Tris, 10 mM EDTA, and a proteases cocktail inhibitor (1:200 dilution). The cell suspension was homogenized, centrifuged at 100,000 x g at 4 C during 1 h, and the plasma membrane pellet resuspended in buffer A (20 mM Tris-HCl, pH 7.4; 5 mM EDTA; 5 mM EGTA; and a 1:200 dilution of proteases cocktail inhibitor). A 100,000 x g plasma membrane fraction was prepared from myometrium as described previously (7). Protein concentrations were determined by the Bradford method (16) using BSA as a standard. Proteins (50 µg) were subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane filters. Blots were probed with antibodies directed against GRK, and the resulting bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). Recombinant GRK overexpressed in Cos-7 cells were used as positive controls (7). The quantification of GRK expression was done by densitometric scanning followed by computer analysis using NIH image 1.62 program.

Measurement of GRK activity
The GRK activity was assessed on cytosolic (100,000 x g supernatant) and plasma membrane fractions of myometrial cells as described previously for myometrium (7). Extraction of membrane-associated proteins was made with 200 mM NaCl before GRK assay. For phosphorylation assay, cytosolic or plasma membrane proteins (50 µg) were incubated with urea-treated rod outer segments (400 pmol rhodopsin) in a 50-µl buffer containing 20 mM Tris-HCl, pH 7.5; 2 mM EDTA; 6 mM MgCl2; and 100 µM [{gamma}-32P]ATP (2500–3000 cpm/pmol). Incubation was carried out for 35 min at 30 C in absence or presence of light. Reaction was stopped by dilution with 20 volumes of ice-cold buffer A and centrifugation (12,000 x g, 15 min). Rhodopsin pellets were electrophoresed on 10% SDS-PAGE. Gels were dried and autoradiographed.

Transfection of myometrial cell culture
After 48 h of culture, myometrial cells (50,000 cells/well, 12-well plate) were cotransfected with {Delta}MMTV-Luc(wtCRE) driven firefly luciferase reporter plasmid (200 ng), used as a cAMP sensor, and pTK-renilla driven renilla luciferase plasmid (40 ng) used to normalize transfection efficiency. Expression vectors for human dominant-negative mutants of GRK2, GRK5, or GRK6 (50 ng) were cotransfected with reporter constructs. These mutants have single-point mutations in their catalytic domain: K220R for GRK2 or K215R for GRK5 and GRK6. Equimolar amounts of respective empty vectors were used as controls. DNA total quantity per well was standardized using PUC19 plasmid DNA.

Cells were transfected at 70–90% of confluence using lipofectamine Reagent Plus according to manufacturer’s instructions. After transfection, cells were serum deprived for 40 h and stimulated or not (basal conditions) by 10 µM isoproterenol during 4 h in presence of 60 µM IBMX. Cells transfected with GRK6 dominant-negative mutants were also stimulated by 0.1 mM forskolin in the same conditions. At the end of isoproterenol or forskolin stimulations, cells were rinsed with PBS and lysed with the lysis buffer. Firefly and renilla luciferase activities were quantified using the dual-luciferase assay system in a scintillation counter (Wallach, Evry, France). The ratio of firefly to renilla luciferase activities served as a measure of normalized luciferase activity.

Each transfection was performed in triplicates or quadruplicates and repeated at least four times with different myometrial cells culture.

Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis were performed using unpaired t test. Values were considered statistically different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of rat myometrial primary cell culture
After 3 d of culture only, confluent rat myometrial cells showed morphological characteristics consistent with cultured smooth muscle cells, including a long and spindle shape as well as a phase dense and homogeneous cytoplasm (Fig. 1AGo), differentiating them from fibroblasts (17). They exhibited an average size of 300 µm as previously reported for pregnant rat uterine smooth muscle cells (18, 19). Myometrial cells retained expression of smooth muscle-specific markers like {alpha}-actin and myosin as detected by immunocytochemical staining (Fig. 1Go, B and C). More than 80% of cultured cells showed a positive labeling.



View larger version (69K):
[in this window]
[in a new window]
  		Figure 1. Phenotypic characterization of rat myometrial primary cells. A, A representative myometrial cell visualized by phase contrast light microscopy in rat subconfluent myometrial primary cell culture. B and C, Immunocytochemical staining of smooth muscle specific {alpha}-actin (B) and myosin (C) in rat confluent myometrial primary cells. Percentage of positive actin or myosin labeling was determined as the number of fluorescent cells (green staining), compared with the total number of cells (red staining by Evans Blue).

 
To demonstrate the functionality of ß-adrenergic signaling pathway, confluent myometrial cells were stimulated by isoproterenol, cholera toxin, or forskolin, and the intact cells cAMP production was measured (Fig. 2Go). Isoproterenol stimulated cAMP production in a dose-dependent manner with an EC50 value of 2.1 x 10-7 M, similar to that reported in late pregnant rat myometrial tissue (Fig. 2AGo) (6). Preincubation with a ß-antagonist, propranolol, completely abolished cAMP production demonstrating the involvement of ß-AR. Maximal stimulation of cAMP production was obtained with 10 µM isoproterenol with an increase of 877 ± 127% over basal levels. Adenylyl cyclase maximal velocity value was of 16.3 pmol/mg protein per 5 min, well within the range of values obtained on rat myometrium (6). The density of ß-AR, evaluated by binding experiments on whole cells using the hydrophilic radioligand [3H]CGP-12177, was approximately 40,000 sites per cell (Fig. 2BGo). Cholera toxin and forskolin increased the cAMP production by 1172 ± 113% and 1825 ± 528% above basal levels, respectively (Fig. 2CGo). Altogether, these data demonstrate that ß-adrenergic signaling pathway is fully functional in rat myometrial primary cell culture.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 2. Functionality of the ß-adrenergic signaling pathway in rat myometrial primary cells. Myometrial cells were grown to confluence in 6-well plates and depleted in serum about 8 h before being incubated overnight with 2 µCi/well [2,8-3H]adenine. A, Myometrial cells were stimulated by increasing doses of a ß-agonist, isoproterenol (ISO) (filled circle, 10-9 to 10-4 M) and 3[H]cAMP produced was separated by chromatography as described in Materials and Methods. The same experiment was performed after pretreatment of myometrial cells with a ß-antagonist, propranolol (open circle, 10-4 M, 5 min). The basal cAMP production was 2.38 ± 0.52 pmol cAMP/mg prot/5 min. B, Saturation plot of [3H]CGP-12177 binding to cultured myometrial cells. The 105 cells were incubated for 20 min at room temperature with increasing doses (0.25–6 nM) of [3H]CGP-12177. Specific binding is expressed as femtomoles per milligram protein, and results are representative of two independent experiments. The dissociation constant (Kd) was 3 nM and the calculated Bmax values corresponded to about 40,000 receptors per cell. C, Myometrial cells were stimulated either by a Gs protein activator, cholera toxin (CTX, 3 nM, 2 h) or an adenylyl cyclase activator, forskolin (FSK, 0.1 mM, 10 min). Results are expressed as percentage of the cAMP production above basal values obtained in nonstimulated cells. The basal cAMP production was 1.72 ± 0.13 pmol cAMP/mg prot/5 min. Data are expressed as means ± SEM of 3–11 independent experiments, each performed in triplicate.

 
Homologous desensitization of ß-adrenergic signaling pathway
We next examined whether ß-adrenergic signaling pathway could be desensitized in cultured myometrial cells. Treatment of cells with 10 µM isoproterenol for 30 min led to a significant reduction of cAMP response to a subsequent isoproterenol challenge (59.3 ± 4.1%, Fig. 3Go). Conversely, pretreatment with isoproterenol had no effect on cholera toxin-stimulated cAMP production and led to only a slight decrease in forskolin-stimulated cAMP production (13.5 ± 3.5%, Fig. 3Go). Hence, short-term isoproterenol stimulation of myometrial cells led to homologous desensitization, predominantly at the ß-AR locus.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Homologous desensitization of ß-adrenergic signaling pathway in rat myometrial primary cells. Confluent myometrial cells were pretreated (filled bar) or not (open bar, control cells) with isoproterenol (10 µM) during 30 min. After extensive washes, myometrial cells were submitted to a subsequent stimulation with isoproterenol (10 µM, 5 min), cholera toxin (3 nM, 2 h), or forskolin (0.1 mM, 10 min). The cAMP produced was measured as described in Fig. 2Go. Results are expressed as percentage of the cAMP produced in control cells in response to the indicated agents. The cAMP produced was 16.1 ± 2.2, 23.3 ± 5.5, and 32 ± 9.6 pmol cAMP/mg prot/5 min in response to isoproterenol, cholera toxin, and forskolin, respectively. Data are means ± SEM of 5–11 independent experiments, each performed in triplicate. *, P < 0.05; **, P < 0.01.

 
Involvement of GRK and PKA in ß-AR desensitization
To explore the potential role of GRK in homologous desensitization of ß-adrenergic signaling pathway, myometrial cells were pretreated with the nonselective GRK inhibitor, heparin (Fig. 4Go). Permeabilization of myometrial cells with saponin (35 µg/ml, 10 min) was performed to permit access of heparin to the cytoplasm. Although decreasing by about 50% the cAMP response to isoproterenol, saponin treatment did not modify the desensitization extent because desensitization values were 59 ± 4% and 62 ± 8% with or without saponin, respectively (Fig. 4AGo). We demonstrated that pretreatment of myometrial cells with heparin inhibited desensitization by 36.2 ± 4.7% (Fig. 4BGo). Albeit being a potent GRK inhibitor (14), heparin also inhibits other protein kinases (20, 21). To confirm the involvement of GRK in homologous desensitization of myometrial ß-AR, we thus evaluated whether agonist stimulation of receptor effectively led to GRK activation. In vitro phosphorylation of rhodopsin by plasma membrane extracts was enhanced after stimulation of myometrial cells with isoproterenol (Fig. 4CGo). This effect was dependent on light activation of rhodopsin because only a faint phosphorylation could be detected in the dark. Altogether these results demonstrate that myometrial ß-AR is the target of GRK in our experimental cell model. Cells were also treated with the PKA inhibitor, H89. Because Penn et al. (22) reported that H89 may act as a ß-AR antagonist, we first checked, in our model, that H89 did not decrease cAMP response to isoproterenol (a similar increase of 7-fold over basal was measured with or without H89). Desensitization of ß-adrenergic signaling pathway was inhibited by 31 ± 2.6% after H89 treatment (Fig. 4DGo), suggesting the involvement of PKA.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Role of GRK and PKA in ß-AR homologous desensitization. A, Effect of saponin on ß-AR desensitization. Cells were permeabilized with saponin (35 µg/ml) during 10 min, and the effect of saponin was evaluated on the extent of ß-AR desensitization. Desensitization was measured as the percent decrease of the cAMP production in response to a subsequent stimulation with 10 µM isoproterenol. Although decreasing by about 50% the cAMP response to isoproterenol (2061 ± 277 vs. 1017 ± 103 cpm/well of cAMP, without and with saponin, respectively), saponin treatment did not modify the extent of desensitization (59 ± 4% vs. 62 ± 8% without and with saponin, respectively). Data are expressed as means ± SEM of four independent experiments, each performed in triplicate. B, Inhibition of ß-AR desensitization by heparin. Cells were permeabilized with saponin (35 µg/ml, 10 min) and were preincubated or not with the nonselective GRK inhibitor (heparin, 1 µM) 1 h before and during the ß-AR desensitization process. Desensitization was measured as the percent decrease of the cAMP production in response to a subsequent stimulation with 10 µM isoproterenol and considered as 100% in absence of heparin. Heparin pretreatment did not affect basal or isoproterenol-stimulated cAMP production but inhibited desensitization by 36.2 ± 4.7% (percent of desensitization: 63.8 ± 4.7%). The isoproterenol-stimulated cAMP production was 9 ± 0.4 pmol cAMP/mg prot/5 min. Data are expressed as means ± SEM of three independent experiments, each performed in triplicate. **, P < 0.01. C, Increased GRK activity after isoproterenol stimulation of myometrial cells. Cells were stimulated with isoproterenol (10 µM, 20 min), lysed, and the GRK activity measured on plasma membrane fractions as described in Materials and Methods. Plasma membrane proteins (50 µg) were incubated in a buffer containing [{gamma}-32P]ATP (2500 cpm/pmol) with 400 pmol rhodopsin for 35 min at 30 C in the light. Samples were separated by SDS-PAGE on a 10% gel and dried for autoradiography. The autoradiogram shown is representative of two independent experiments. D, Inhibition of ß-AR desensitization by H89. Myometrial cells were preincubated or not with PKA inhibitor (H89, 20 µM) 1 h before and during the ß-AR desensitization process. Results are expressed as described in Fig. 4BGo. H89 pretreatment did not inhibit by itself the cAMP response to isoproterenol but inhibited the desensitization extent by 31 ± 2.6% (percent of desensitization: 69 ± 2.6%). Data are expressed as means ± SEM of three independent experiments, each performed in triplicate. **, P < 0.01.

 
GRK expression in rat myometrial cells and myometrium
To characterize GRK expressed in primary cell cultures, we performed Western blotting using specific antibodies directed against GRK. We found proteins, comigrating with recombinant GRK, both in late pregnant myometrium and myometrial cells. These proteins, with the expected molecular size of 80, 68, and 66 kDa, were identified as GRK2, GRK5, and GRK6, respectively (Fig. 5Go). Thus, cultured myometrial cells retained the in vivo pattern of GRK expression.



View larger version (70K):
[in this window]
[in a new window]
  		Figure 5. Identification by Western blot analysis of GRK in rat myometrial primary cells and late pregnant rat myometrium. Proteins (50 µg) from plasma membrane fractions (100,000 x g) prepared from myometrial cells or tissue were resolved on 10% SDS-PAGE and transferred on a nitrocellulose membrane. They were probed with polyclonal antibodies: GRK2 (1:1000 dilution), GRK5 (1:500 dilution), and GRK6 (1:200 dilution) together with respective recombinant proteins obtained by transfection of Cos-7 cells as described previously (7 ). The autoradiogram shown is representative of three independent experiments.

 
Identification of the GRK family mediating myometrial ß-AR desensitization
To identify which GRK family was involved in myometrial ß-AR desensitization, we evaluated the ability of GRK-specific mAbs to inhibit desensitization. Permeabilized cells were incubated or not with GRK2/3- or GRK4–6-specific mAbs or antibodies directed against GST protein (control mAbs) (Fig. 6Go). Treatment with antibodies had no effect by itself on the desensitization extent. Treatment of cells with combination of anti-GRK2/3 and anti-GRK4–6 mAbs inhibited agonist-induced receptor desensitization by 31.8 ± 3%, compared with treatment with control mAbs. The extent of desensitization inhibition was similar to the one obtained with heparin.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Inhibition of isoproterenol-induced ß-AR desensitization by GRK-specific mAbs in permeabilized primary myometrial cells. Permeabilized myometrial cells were incubated or not (no mAbs) either with anti-GRK2/3 mAbs, anti-GRK4–6 mAbs, or the combination of both antibodies 1 h before and during the desensitization process. Anti-GST mAbs were used as control. Desensitization was expressed as the percent decrease of cAMP production in response to 10 µM isoproterenol and was considered as 100% in presence of control mAbs. Treatment with control mAbs had no effect by itself on the cAMP production in response to isoproterenol (1304 ± 257 vs. 1507 ± 374 cpm/well of cAMP in nontreated vs. control mAbs-treated cells), and the desensitization extent in nontreated cells was similar, representing 98.9 ± 4.3% of the GST mAbs. Treatment of cells with both GRK-specific mAbs inhibited desensitization by 31.8 ± 3% (percent of desensitization 68.2 ± 3%). Treatment of cells with anti-GRK4–6 mAb alone inhibited desensitization by 33.2 ± 6%, whereas anti-GRK2/3 mAbs had no effect on ß-AR desensitization (percent of desensitization: 66.8 ± 6% and 97.7 ± 2%, respectively). The isoproterenol-stimulated cAMP production was 7.6 ± 0.8 pmol cAMP/mg prot/5 min. Data are expressed as means ± SEM of three to four independent experiments, each performed in triplicate. **, P < 0.01.

 
To discriminate between both GRK families, mAbs were incubated separately. Treatment of myometrial cells with anti-GRK4–6 mAb alone inhibited agonist-induced receptor desensitization by 33.2 ± 6%, whereas treatment with anti-GRK2/3 mAb alone had no effect. These results clearly demonstrate that GRK5 and/or GRK6 is the endogenous GRK-mediating homologous ß-AR desensitization in primary myometrial cells.

Effects of overexpression of GRK5 and GRK6 dominant-negative mutants on ß-AR desensitization
To discriminate between GRK5 and GRK6 involvement in myometrial ß-AR desensitization, we chose to inhibit either endogenous GRK5 or GRK6 by overexpressing their kinase activity-lacking mutants, K215RGRK5 and K215RGRK6. Cells were also transfected with K220RGRK2.

We first verified that dominant-negative mutants lacked kinase activity by measuring GRK activity using in vitro phosphorylation of rhodopsin in transfected myometrial cells extracts (Fig. 7AGo). As expected, wild-type (wt) GRK2, GRK5, or GRK6 overexpression strongly enhanced rhodopsin phosphorylation, compared with control cells. In contrast, there was only a faint rhodopsin phosphorylation with overexpressed K220RGRK2, K215RGRK5, or K215RGRK6. This result was not due to overexpression deficiency of kinase-dead mutants because we verified by immunoblotting that mutants were indeed overexpressed in myometrial cells (Fig. 7BGo).



View larger version (44K):
[in this window]
[in a new window]
  		Figure 7. Determination of GRK activity and expression in myometrial cells transfected with dominant-negative GRK2 (K220RGRK2), GRK5 (K215RGRK5), or GRK6 (K215RGRK6). Myometrial cells were transfected or not (control) with wt GRK or dominant-negative GRK. After 48 h of culture, cells were lysed. A, GRK activity was measured in cytosolic fractions as described in Fig. 4CGo. The autoradiogram shown is representative of two independent experiments. B, GRK expression was assessed by immunoblotting as described in Fig. 5Go. The autoradiogram shown is representative of three independent experiments.

 
Myometrial cells were cotransfected with GRK mutant constructs, with a cAMP-inducible reporter gene: {Delta}MMTV-Luc (wtCRE) plasmid (23) and pTK-renilla to normalize transfection efficiency. The transfection efficiency of primary myometrial cells was determined using green fluorescent protein expression vector and was of approximately 1% of transfected cells. We determined that a 4-h stimulation with isoproterenol led to a maximal expression of CRELuc gene (data not shown). Isoproterenol increased luciferase activity by approximately 2-fold, compared with nonstimulated cells.

Transfection of each dominant-negative mutant diminished by approximately 25% the basal cAMP production (Fig. 8AGo). Overexpression of dominant-negative mutant K215RGRK5 had no significant effect on isoproterenol-stimulated ß-adrenergic signaling (Fig. 8AGo). Conversely, overexpression of dominant-negative mutant K215RGRK6 resulted in a large increase (~70%) in isoproterenol-stimulated ß-adrenergic signaling (Fig. 8AGo). Furthermore, ß-AR appeared to be the target of K215RGRK6 because overexpression of this kinase did not significantly increase the cAMP production in response to forskolin (Fig. 8BGo). Overexpression of dominant-negative mutant K220RGRK2 had no significant effect on isoproterenol-stimulated ß-adrenergic signaling in agreement with anti-GRK mAbs experimental results. Altogether, these results demonstrate that, among endogenous myometrial GRK, GRK6 is the only GRK-desensitizing ß-AR.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 8. Isoproterenol- and forskolin-stimulated cAMP production in myometrial primary cells transiently transfected with K220RGRK2, K215RGRK5, or K215RGRK6. A, Isoproterenol-stimulated cAMP production in myometrial primary cells transiently transfected with K220RGRK2, K215RGRK5, or K215RGRK6: Myometrial cells were transiently cotransfected with a cAMP-sensitive reporter {Delta}MMTV-Luc(wtCRE) plasmid (200 ng/well) and expression vectors for kinase-dead, dominant-negative K220RGRK2, K215RGRK5, or K215RGRK6 (50 ng/well). Control cells were transfected with respective empty vectors. Renilla luciferase plasmid (40 ng/well) was added to normalize the transfection efficiency. After 48 h of culture, cells were either left unstimulated (basal) or stimulated during 4 h with isoproterenol (10 µM) in the presence of 60 µM IBMX. After stimulation, luciferase activities (Firefly and Renilla) were measured in cell lysates, and normalized luciferase activities were calculated (firefly/renilla activities). Results were expressed as fold over control basal values. Data are representative of four to eight independent experiments, each performed in triplicate or quadruplicate. Isoproterenol-stimulated luciferase values were: 1.56 ± 0.1 and 1.51 ± 0.1 for control and K220RGRK2-transfected cells, 1.99 ± 0.2 and 1.51 ± 0.2 for control and K215RGRK5 transfected cells, 1.46 ± 0.14 and 2.46 ± 0.15 for control, and K215RGRK6-transfected cells; **, P < 0.01 vs. isoproterenol response in control cells. B, Forskolin-stimulated cAMP production in myometrial primary cells transiently transfected with K215RGRK6. Myometrial cells were transiently transfected with dominant-negative K215RGRK6 (50 ng/well) as described in A. After 48 h of culture, cells were either left unstimulated (basal) or stimulated during 4 h with forskolin (0.1 mM) in presence of 60 µM IBMX. Results were expressed as described in A. Data are representative of six independent experiments, each performed in triplicate or quadruplicate. Forskolin-stimulated luciferase values were: 2.94 ± 0.46 and 3.6 ± 0.5 for control and K215RGRK6-transfected cells, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using two complementary experimental strategies, i.e. GRK-specific mAbs and kinase-dead, dominant-negative mutants of GRK on a primary myometrial cell culture, we demonstrated, for the first time, that endogenous GRK6, but not GRK2 or GRK5, triggers ß-AR homologous desensitization.

Our study was conducted on an appropriate cell model that retained most of its in vivo cellular characteristics. Indeed, such cells retained their myogenic origin as indicated by morphological criteria and immunocytochemical studies. Moreover, they expressed a functional ß-AR signaling pathway as evidenced by a cellular cAMP production in response to isoproterenol exhibiting EC50 and maximal velocity values consistent with those previously found on late pregnant rat myometrium (6). Stimulation of adenylyl cyclase was also achieved with agents acting at postreceptor levels like cholera toxin or forskolin. In this primary culture, a significant ß-adrenergic signaling pathway desensitization (60%) could be induced by a short-term treatment (30 min) of cells with isoproterenol. Lastly, Western blot analysis revealed that the cells retained the tissue GRK expression pattern, i.e. GRK2, GRK5, and GRK6 (7). Myometrial primary cell culture thus constituted a suitable model to identify GRK specifically involved in myometrial ß-AR regulation.

We showed that H89 partly inhibited ß-AR desensitization in myometrial cells in agreement with the previously reported regulation of ß-AR by PKA in reconstituted systems or several cell lines (24). Moreover, we demonstrated that GRKs contribute to ß-AR homologous desensitization in myometrial cells. Such involvement was evidenced by an increase of membrane-associated GRK activity after isoproterenol stimulation of cells and a significant inhibition of desensitization after treatment of cells with heparin or anti-GRK mAbs. Permeabilization with saponin, used for those treatments, may constitute a limit to study desensitization mechanisms in some cells because it may be poorly tolerated (25) or it may markedly disturb signaling or desensitization patterns as observed in primary rabbit ventricular myocytes (Oppermann, M., personal communication). We thus established experimental conditions of cell permeabilization that neither led to cellular death nor induced alteration of the ß-AR desensitization extent. Using GRK-specific mAbs, we demonstrated that endogenous GRK5 and/or GRK6 mediates ß-AR homologous desensitization in primary culture of myometrial cells.

The fact that no available GRK antibodies specifically inhibit either GRK5 or GRK6 led us to use an alternative experimental strategy to discriminate between those two GRKs. Rather than overexpressing GRKs, which may generate nonspecific interactions between receptor and GRKs and thus lead to inconclusive data (26), we chose to inhibit endogenous GRKs by introducing into myometrial cells, K215RGRK5 and K215RGRK6, both mutated in their catalytic domain (27, 28). We verified, using in vitro phosphorylation of rhodopsin, that overexpressed mutants did not exhibit GRK activity into myometrial cells. Weak transfection efficiency of primary cultured cells was overcome by cotransfecting cells with a reporter gene (luciferase) containing highly sensitive cAMP responsive elements (23). All three kinase-dead mutants slightly decreased basal luciferase activity in myometrial cells. We previously reported that myometrial ß-AR pathway was submitted to a tonic inhibition mediated via Gi proteins because pertussis toxin pretreatment of myometrial membranes consistently increased isoproterenol-stimulated adenylyl activity at several stages of pregnancy (29). Inhibition of Gi-coupled GPCR desensitization by overexpression of dominant-negative GRKs may explain the observed decrease in basal adenylyl cyclase activity. Results obtained with dominant-negative GRK transfections revealed that myometrial ß-AR is the target of GRK6. In addition to ß-AR identified in this study, several other GPCRs have been recently identified as targets of endogenous GRK6: FSH receptor (30), thromboxane receptor-{alpha} (31), M3 muscarinic receptor (32), calcitonin gene-related peptide receptor (33), and chemokine receptor (34).

Noninvolvement of endogenous GRK5 is rather surprising because GRK5 and GRK6 are closely related kinases. Such a difference was already reported for M3 muscarinic receptor (32). This suggests that recruitment signals, still poorly understood for both kinases, may be different for GRK5 and GRK6. Results obtained with kinase-dead mutant of GRK2 confirmed results obtained with GRK2/3-specific mAbs and demonstrated that uterine smooth muscle ß-AR is not the target of GRK2 in homologous desensitization. This differs from myocardial ß-AR desensitization for which GRK2 involvement was clearly established (35). Discrepancy between cardiac and uterine muscles may be explained by different proportion of ß-AR subtypes expressed in those two muscles. Indeed, rodent cardiac muscle expresses mainly ß1-AR subtype (80%) (36), whereas ß2-AR subtype is largely predominant (80%) in uterine smooth muscle (37). Regulatory mechanisms between these two subtypes may be different, and the fact that GRK phosphorylation sites are distinct in ß1-AR and ß2-AR (38, 39) supports that hypothesis. However, two studies conducted in rat neonatal liver (40) and vascular smooth muscle from hypertensive rats (41) provide convincing evidence that GRK2 may also underlie ß2-AR desensitization, suggesting that GRK-specific substrate may be tissue specific.

Another hypothesis, based on comparison of results obtained in myometrium and primary myometrial cell culture, is that ß2-AR may be the target of different GRKs, depending on the regulatory mechanisms leading to their activation. We previously reported that an increase of myometrial GRK activity at parturition may underlie ß2-AR uncoupling and altered signaling observed at this period. Several experimental data such as an increase of plasma membrane-associated immunoreactive GRK2 and a translocation of GRK activity, strongly suggested that GRK2 mainly contributed to such a desensitization (7). This apparent discrepancy with conclusions derived from the present study can be solved when taking into account the complex regulations of ß-AR signaling occurring in rat uterus during pregnancy. At the end of pregnancy, many contracturant GPCRs, linked to inositol 1,4,5-triphosphate/PKC/Ca2+ pathways, are activated, and it has been reported that stimulation of myometrial cells with contracturant agents such as oxytocin or {alpha}1-adrenergic agonists induces ß-AR heterologous desensitization (42). The fact that GRK2 is activated by PKC and relatively insensitive to calcium-calmodulin inhibition (43, 44) argues for its involvement in heterologous desensitization at parturition. GRK6, which is sensitive to calcium-calmodulin inhibition may not be recruited at parturition and may be rather responsible for ß-AR homologous desensitization occurring during gestation when contractile signaling pathways are poorly expressed or nonfunctional. Different GRKs may thus be activated to desensitize the ß-AR depending on the pregnant or parturient physiological status of the uterine muscle. This hypothesis is under investigation in our laboratory. For this purpose, primary cell cultures represent a suitable model because these cells express important amounts of functional oxytocin receptors, consistent with known receptor density in late pregnant rat (45, 46). Moreover, their activation indeed induces heterologous ß-AR desensitization (Simon, V., unpublished results).

In conclusion, we have assessed GRK involvement in ß-AR homologous desensitization in myometrial primary cells retaining the in vivo myometrial phenotypic characteristics and entities of the ß-AR pathway. Introduction of anti GRK5/6 mAbs or dominant-negative, kinase activity-lacking GRK6 inhibited agonist-induced ß-AR desensitization. In contrast, inhibition of GRK2 or of the closely related kinase GRK5 had no effect on ß-AR desensitization. This study therefore shows, for the first time, that endogenous GRK6 mediates myometrial ß-AR homologous desensitization.


   Acknowledgments
 
The authors thank Drs. N. Bennett (Grenoble, France), J. L. Benovic (Philadelphia, PA), F. Boulay (Grenoble, France), F. Mayor, Jr. (Madrid, Spain), T. Métayé (Poitiers, France), and D. Spengler (Munich, Germany) for their kind gifts of materials. We gratefully acknowledge Dr. M. Oppermann (Göttingen, Germany) for supplying GRK monoclonal antibodies as well as helpful discussion concerning manuscript.


   Footnotes
 
Violaine Simon was recipient of a doctoral fellowship from the "Ministère de la Recherche et de l’enseignement Supérieur."

Abbreviations: ß-AR, ß-Adrenergic receptor; FCS, fetal calf serum; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; GST, glutathione-S-transferase fusion protein; IBMX, 3-iso-1-methylxanthine; mAb, monoclonal antibody; PKA, protein kinase A; PKC, protein kinase C; PS, penicillin-streptomycin; wt, wild-type.

Received December 12, 2002.

Accepted for publication March 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ 1991 Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem 60:653–688[CrossRef][Medline]
  2. Freedman NJ, Lefkowitz RJ 1996 Desensitization of G protein-coupled receptors. Recent Prog Horm Res 51:319–353
  3. Pitcher JA, Freedman NJ, Lefkowitz RJ 1998 G protein-coupled receptor kinases. Annu Rev Biochem 67:653–692[CrossRef][Medline]
  4. Litime MH, Pointis G, Breuiller M, Cabrol D, Ferre F 1989 Disappearance of ß-adrenergic response of human myometrial adenylate cyclase at the end of pregnancy. J Clin Endocrinol Metab 68:1–6
  5. Cruz MA, Sepulveda WH, Rudolph MI 1990 Changes in the response to adrenergic drugs on mouse uterine contractions during pregnancy. Life Sci 46:99–104[CrossRef][Medline]
  6. Cohen-Tannoudji J, Vivat V, Heilmann J, Legrand C, Maltier JP 1991 Regulation by progesterone of the high-affinity state of myometrial ß-adrenergic receptor and of adenylate cyclase activity in the pregnant rat. J Endocrinol 6:137–145
  7. Simon V, Mhaouty-Kodja S, Legrand C, Cohen-Tannoudji J 2001 Concomitant increase of G protein-coupled receptor kinase activity and uncoupling of ß-adrenergic receptors in rat myometrium at parturition. Endocrinology 142:1899–1905[Abstract/Free Full Text]
  8. Oppermann M, Diversé-Pierlussi M, Drazner MH, Dyer SL, Freedman NJ, Peppel KC, Lefkowitz RJ 1996 Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases. Proc Natl Acad Sci USA 93:7649–7654[Abstract/Free Full Text]
  9. Bennett N, Sitaramayya A 1988 Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin). Biochemistry 27:1710–1715[CrossRef][Medline]
  10. Dodge KL, Carr DW, Yue C, Sanborn BM 1999 A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3', 5'-monophosphate inhibitory response in late pregnant rat myometrium. Mol Endocrinol 13:1977–1987[Abstract/Free Full Text]
  11. Diviani D, Lattion AL, Larbi N, Kunapuli P, Pronin A, Benovic JL, Cotecchia S 1996 Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the {alpha}1B-adrenergic receptor. J Biol Chem 271:5049–5058[Abstract/Free Full Text]
  12. Salomon Y, Londos C, Rodbell M 1974 A highly sensitive adenylate cyclase assay. Anal. Biochem 58:541–548
  13. Feve B, Emorine LJ, Briend-Sutren MM, Lasnier F, Strosberg AD, Pairault J 1990 Differential regulation of ß1- and ß2-adrenergic receptor protein and mRNA levels by glucocorticoids during 3T3–F442A adipose differentiation. J Biol Chem 265:16343–16349[Abstract/Free Full Text]
  14. Benovic JL, Stone WC, Caron MG, Lefkowitz RJ 1989 Inhibition of the ß-adrenergic receptor kinase by polyanions. J Biol Chem 264:6707–6710[Abstract/Free Full Text]
  15. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272[Abstract/Free Full Text]
  16. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 72:248–254
  17. Chamley-Campbell J, Campbell GR, Russel R 1979 The smooth muscle cell in culture. Physiol Rev 59:1–61[Free Full Text]
  18. Garfield RE, Somlyo AP 1985 Structure of smooth muscle. Calcium and contractility. Clifton, NJ: Humana Press; 1–36
  19. Kawarabayashi T, Tsukamoto T, Shojo H, Nakamura S, Sugimori H 1997 Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus towards oxytocin during gestation: contractility and calcium signaling. Mol Cell Endocrinol 128:77–84[CrossRef][Medline]
  20. Bretschneider E, Schror K 2001 Cellular effects of factor Xa on vascular smooth muscle cells—inhibition by heparins? Semin Thromb Hemost 27:489–493[CrossRef][Medline]
  21. Mishra-Gorur K, Singer HA, Castellot Jr JJ 2002 Heparin inhibits phosphorylation and autonomous activity of Ca2+/calmodulin-dependent protein kinase II in vascular smooth muscle cells. Am J Pathol 161:1893–1901[Abstract/Free Full Text]
  22. Penn RB, Parent JL, Pronin AN, Panettieri Jr RA, Benovic JL 1999 Pharmacological inhibition of protein kinases in intact cells: antagonism of ß adrenergic receptor ligand binding by H-89 reveals limitations of usefulness. J Pharmacol Exp Ther 288:428–437[Abstract/Free Full Text]
  23. Spengler D, Rupprecht R, Van LP, Holsboer F 1992 Identification and characterization of a 3', 5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter. Mol Endocrinol 6:1931–1941[Abstract/Free Full Text]
  24. Hausdorff WP, Caron MG, Lefkowitz RJ 1990 Turning off the signal: desensitization of ß-adrenergic receptor function. FASEB J 4:2881–2889[Abstract]
  25. Dautzenberg FM, Hauger RL 2001 G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells. Neuropharmacology 40:394–407[CrossRef][Medline]
  26. Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST, Lefkowitz RJ 1997 Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem 272:17734–17743[Abstract/Free Full Text]
  27. Reiter E, Marion S, Robert F, Troispoux C, Boulay F, Guillou F, Crepieux P 2001 Kinase-inactive G-protein-coupled receptor kinases are able to attenuate follicle-stimulating hormone-induced signaling. Biochem Biophys Res Commun 282:71–78[CrossRef][Medline]
  28. Milcent MD, Christophe T, Rabiet MJ, Tardif M, Boulay F 1999 Overexpression of wild-type and catalytically inactive forms of GRK2 and GRK6 fails to alter the agonist-induced phosphorylation of the C5a receptor (CD88): evidence that GRK6 is autophosphorylated in COS-7 cells. Biochem Biophys Res Commun 259:224–229[CrossRef][Medline]
  29. Mhaouty S, Cohen-Tannoudji J, Bouet-Alard R, Limon-Boulez I, Maltier JP, Legrand C 1995 Characteristics of the {alpha}2/ß2-adrenergic receptor-coupled adenylyl cyclase system in rat myometrium during pregnancy. J Biol Chem 270:11012–11016[Abstract/Free Full Text]
  30. Lazari MF, Liu X, Nakamura K, Benovic JL, Ascoli M 1999 Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor. Mol Endocrinol 13:866–878[Abstract/Free Full Text]
  31. Zhou H, Yan F, Tai HH 2001 Phosphorylation and desensitization of the human thromboxane receptor-{alpha} by G protein-coupled receptor kinases. J Pharmacol Exp Ther 298:1243–1251[Abstract/Free Full Text]
  32. Willets JM, Challiss RA, Nahorski SR 2002 Endogenous G protein-coupled receptor kinase 6 regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J Biol Chem 277:15523–15529[Abstract/Free Full Text]
  33. Aiyar N, Disa J, Dang K, Pronin AN, Benovic JL, Nambi P 2000 Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors. Eur J Pharmacol 403:1–7[CrossRef][Medline]
  34. Fong AM, Premont RT, Richardson RM, Yu YR, Lefkowitz RJ, Patel DD 2002 Defective lymphocyte chemotaxis in ß-arrestin2- and GRK6-deficient mice. Proc Natl Acad Sci USA 99:7478–7483[Abstract/Free Full Text]
  35. Lefkowitz RJ 1996 G protein-coupled receptors and receptor kinases: from molecular biology to potential therapeutic applications. Nat Biotechnol 14:283–286[CrossRef][Medline]
  36. Juberg EN, Minneman KP, Abel PW 1985 ß1- and ß2-Adrenoceptor binding and functional response in right and left atria of rat heart. Naunyn Schmiedebergs Arch Pharmacol 330:193–202[CrossRef][Medline]
  37. Vivat V, Cohen-Tannoudji J, Revelli JP, Muzzin P, Giacobino JP, Maltier JP, Legrand C 1992 Progesterone transcriptionally regulates the ß2-adrenergic receptor gene in pregnant rat myometrium. J Biol Chem 267:7975–7978[Abstract/Free Full Text]
  38. Akiyama C, Nagao T, Kurose H 1999 Analysis of domain responsible for desensitization of ß1-adrenergic receptor. Jpn J Pharmacol 81:12–20[CrossRef][Medline]
  39. Seibold A, Williams B, Huang ZF, Friedman J, Moore RH, Knoll BJ, Clark RB 2000 Localization of the sites mediating desensitization of the ß(2)-adrenergic receptor by the GRK pathway. Mol Pharmacol 58:1162–1173[Abstract/Free Full Text]
  40. Garcia-Higuera I, Mayor Jr F 1994 Rapid desensitization of neonatal rat liver ß-adrenergic receptors. A role for ß-adrenergic receptor kinase. J Clin Invest 93:937–943
  41. Gros R, Chorazyczewski J, Meek MD, Benovic JL, Ferguson SS, Feldman R 2000 G-protein-coupled receptor kinase activity in hypertension: increased vascular and lymphocyte G-protein receptor kinase-2 protein expression. Hypertension 35:38–42[Abstract/Free Full Text]
  42. Lindeman KS, Hirshman CA, Kuhl JS, Levitsky HI, Emala CW 1998 Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells. Biol Reprod 59:1108–1115[Abstract/Free Full Text]
  43. Winstel R, Freund S, Krasel C, Hoppe E, Lhose MJ 1996 Protein kinase cross-talk: membrane targeting of the ß-adrenergic receptor kinase by protein kinase C. Proc Natl Acad Sci USA 93:2105–2109[Abstract/Free Full Text]
  44. Chuang TT, Paolucci L, De Blasi A 1996 Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin. J Biol Chem 271:28691–28696[Abstract/Free Full Text]
  45. Engstrom T, Bratholm P, Vilhardt H, Christensen NJ 1999 Effect of oxytocin receptor and ß2-adrenoceptor blockade on myometrial oxytocin receptors in parturient rats. Biol Reprod 60:322–329[Abstract/Free Full Text]
  46. Alexandrova M, Soloff MS 1980 Oxytocin receptors and parturition. II. Concentrations of receptors for oxytocin and estrogen in the gravid and nongravid uterus at term. Endocrinology 106:736–738[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
J. M. Willets, P. J. Brighton, R. Mistry, G. E. Morris, J. C. Konje, and R. A. J. Challiss
Regulation of Oxytocin Receptor Responsiveness by G Protein-Coupled Receptor Kinase 6 in Human Myometrial Smooth Muscle
Mol. Endocrinol., August 1, 2009; 23(8): 1272 - 1280.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Lariviere, G. Garrel-Lazayres, V. Simon, N. Shintani, A. Baba, R. Counis, and J. Cohen-Tannoudji
Gonadotropin-Releasing Hormone Inhibits Pituitary Adenylyl Cyclase-Activating Polypeptide Coupling to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway in L{beta}T2 Gonadotrope Cells through Novel Protein Kinase C Isoforms and Phosphorylation of Pituitary Adenylyl Cyclase-Activating Polypeptide Type I Receptor
Endocrinology, December 1, 2008; 149(12): 6389 - 6398.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Lariviere, G. Garrel, V. Simon, J.-W. Soh, J.-N. Laverriere, R. Counis, and J. Cohen-Tannoudji
Gonadotropin-Releasing Hormone Couples to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway through Novel Protein Kinase C{delta} and -{epsilon} in L{beta}T2 Gonadotrope Cells
Endocrinology, March 1, 2007; 148(3): 1099 - 1107.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
R. Gaspar, E. Ducza, A. Mihalyi, A. Marki, Z. Kolarovszki-Sipiczki, E. Paldy, S. Benyhe, A. Borsodi, I. Foldesi, and G. Falkay
Pregnancy-induced decrease in the relaxant effect of terbutaline in the late-pregnant rat myometrium: role of G-protein activation and progesterone
Reproduction, July 1, 2005; 130(1): 113 - 122.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
X. Luo, L. Ding, J. Xu, R. S. Williams, and N. Chegini
Leiomyoma and Myometrial Gene Expression Profiles and Their Responses to Gonadotropin-Releasing Hormone Analog Therapy
Endocrinology, March 1, 2005; 146(3): 1074 - 1096.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. Teli, D. Markovic, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos
Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization
Mol. Endocrinol., February 1, 2005; 19(2): 474 - 490.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Simon, V.
Right arrow Articles by Cohen-Tannoudji, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Simon, V.
Right arrow Articles by Cohen-Tannoudji, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals