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Regular Article |
a UMR 6073, INRA/CNRS/Université de Tours, Station de Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle-stimulating hormone receptor, kinases, Sertoli cells, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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s subunit of heterotrimeric G proteins (Gs) coupling, but recent observations have indicated that PKA does not account for all of the intracellular targets of cAMP [5, 6]. Like many other GPCRs, the FSH-R becomes functionally uncoupled from Gs upon continuous stimulation [5, 7]. This process, desensitization, is initiated by phosphorylation of intracellular loops i1 and i3 of the agonist-activated receptor [8]. Second messenger-stimulated kinases PKA and protein kinase C (PKC) have been reported to participate in the desensitization of the FSH response [9–11]. More recently, it has been established that G protein-coupled receptor kinases (GRKs) play a key role in the homologous desensitization of the FSH-R [12, 13].
GRKs specifically recognize and phosphorylate a wide range of agonist-occupied GPCRs [14]. Additionally, alternative homologous desensitization mechanisms have been described for other receptors such as ß3-adrenergic [15, 16], 2C-adrenergic [17], and 5-HT2A-serotonin [18] receptors. Receptor phosphorylation by GRKs is required for subsequent stoechiometric binding of an adaptor protein, arrestin, which prevents further interaction with Gs by sterical obstruction [19]. This receptor-arrestin complex associates with clathrin and is then internalized via clathrin-coated pits [20]. Sequestered GPCRs are then either recycled back to the plasma membrane or degraded in the lysosomes. Interaction of phosphorylated FSH-R with non-visual arrestins is essential for its functional uncoupling and internalization [13, 21]. The interactions between GRKs/arrestins and the activated GPCRs necessitate recruiting these regulatory proteins from diverse subcellular compartments to the plasma membrane. Several mechanisms involved in the relocalization of these proteins have already been proposed such as protein-protein interactions, recognition of specific lipid species, and covalent modification such as acylation or prenylation [14]. Besides their role in terminating or attenuating GPCR signaling, there is increasing evidence that the ß-arrestins also play novel roles as scaffolds, which recruit additional signaling molecules to ligand-activated GPCRs [22]. The expanding role of ß-arrestins in the signaling of many GPCRs enhances the importance of delineating their roles in the control of the FSH response.
To date, the data concerning the role of GRKs and arrestins in the desensitization and internalization of the FSH-R have been obtained using non-gonadal cell lines with enforced expression of the receptor. This approach could alter the stoechiometry within the signaling complexes and ultimately modify cell responses to the agonist [23]. In this context, because some GPCRs are also desensitized through GRK/ß-arrestin independent mechanisms [15–18], it is important to explore the involvement of GRKs and ß-arrestins in modulating the FSH-induced response, in natural FSH-R-bearing gonadal cells.
Using primary rat Sertoli cells that model in vivo conditions more precisely, we investigated various aspects of GRK2, -3, -5, and -6 and ß-arrestins 1 and 2: 1) their transcript and protein levels in freshly isolated and cultured Sertoli cells, 2) the functional consequences for FSH response of their transient overexpression, and 3) the FSH-induced relocalization of endogenous GRKs/ß-arrestins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leibovitz (L 15) medium and Dulbecco minimum essential medium (DMEM) were purchased from Gibco-BRL Life Technologies (Gaithersburg, MD). Penicillin, streptomycin, trypsin, trypsin inhibitor (soybean), deoxyribonuclease type-I, human transferrin, and all the factors that supplemented the DMEM were obtained from Sigma Chemical Co (St. Louis, MO). Porcine FSH (pFSH) was kindly donated by Y. Combarnous (Université de Tours, Nouzilly, France). Moloney murine leukemia virus reverse transcriptase was provided by Gibco-BRL, Taq DNA polymerase was from Pharmacia (Uppsala, Sweden), and radiolabeled [-32P]dCTP (3000 Ci/mmol) was from Amersham (Arlington Heights, IL).
Porcine cytomegalovirus Renilla was obtained from Promega (Madison, WI). The constructs for the different GRKs and ß-arrestins and the cAMP-sensitive reporter construct pSOMLuc have been previously described [13].
The GRK2/3 monoclonal antibody C5/1 and GRK4–6 monoclonal antibody A16/17 were purchased from Upstate Biotechnology (Waltham, MA). The monoclonal antibody F4C1, raised against the DGVVLVD epitope highly conserved among arrestins, was kindly provided by Dr. L.A. Donoso. The anti-E-cadherin monoclonal antibody was supplied by Transduction Laboratories (Lexington, KY).
Isolation and Culture of Rat Sertoli Cells
Sertoli cells were prepared from testes of 11- to 12-day-old rats (Wistar, Janvier, France), according to the method described by Dorrington et al. [24]. Collagenase digestion was replaced by one additional mechanical dispersion performed after trypsin treatment. Our current Sertoli cell preparations were contaminated by less than 10% germ cells and 2% myoid cells as previously described [25].
Cells were plated at a density of 106 cells/well in 24-well culture plates (Falcon; Becton Dickinson, Franklin Lakes, NJ) for transfection studies and a density of 30 x 106 cells in 100-mm dishes for subcellular fractionation. Culture was performed in DMEM supplemented with 100 U/ml penicillin, 2.5 µg/ml amphotericin B, 100 µg/ml streptomycin, 5 µg/ml human transferrin, 2 mM glutamine, 200 ng/ml vitamin E (-tocopherol), and 50 ng/ml vitamin A (retinol) at 34°C in a humidified atmosphere of 5% CO2.
Transfection of Rat Sertoli Cells
After 24 h of culture, Sertoli cells were cotransfected with 500 ng/well of pSOMLuc driven firefly luciferase reporter plasmid used as cAMP sensor and 25 ng of renilla luciferase plasmid added to normalize transfection efficiency. Expression vectors for the different GRKs and ß-arrestins were cotransfected with these reporter constructs. The vector without insert was systematically used as control. A calcium phosphate precipitation method was used as follows. Calcium chloride (62 µl, 2 M) was mixed with the constructs, and the volume was adjusted to 500 µl with water. A DNA precipitate was formed by adding this mixture by drops to 500 µl of Hepes-buffered saline. The precipitate (60 µl) was added to each well and incubated for 4 h. The transfection medium was removed, and cells were incubated in supplemented DMEM. Less than 1% of primary Sertoli cells were transfected using this method (determined by transfection of a green fluorescent protein expression vector), but this treatment had virtually no effect on Sertoli cell viability.
Forty-eight hours after transfection, cells were stimulated with FSH (100 ng/ml) for 6 h in fresh medium and then rinsed in PBS and lysed according to the manufacturer's instructions (Dual luciferase assay, Promega). Both firefly and renilla luciferase activities were quantified on a luminometer (Lumat LB 9507-EG & G Berthold, Turku, Finland).
Reverse Transcription Polymerase Chain Reaction Analysis of GRKs and ß-Arrestins
Total RNA was extracted from cells by the single-step guanidium-phenol-chloroform method described by Chomczynski and Sacchi [26]. Reverse transcription (RT) polymerase chain reaction (PCR) was carried out to assay expression of GRK2, -3, -5 and -6, ß-arrestin 1, and ß-arrestin 2 genes in Sertoli cells.
Reverse transcription of total RNA (1 µg) was carried out in a 50-µl reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 µM of each deoxynucleotide triphosphate, 10 mM dithiothreitol, 100 pmol of oligo(dT)12–18 (Pharmacia), and 200 U of reverse transcriptase. After completion of RT for 45 min at 42°C, the enzyme was heat inactivated at 70°C for 15 min.
Single-strand cDNAs were amplified with specific sets of primer pairs designed to amplify parts of the different GRKs and ß-arrestins as previously described [13].
PCRs were carried out with 4 µl of reverse transcriptase in 100-µl reaction mixtures containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each deoxynucleotide triphosphate, 10 pmol of each primer, 0.5 µCi/nmol [-32P]dCTP, and 1.25 U of Taq polymerase (Pharmacia). The samples were overlaid with mineral oil and processed for 25, 30, or 35 PCR cycles (95°C, 1 min; 60°C, 1 min; 72°C, 1 min), with a final extension step at 72°C for 10 min. We have determined that 30 PCR cycles were within the linear range of amplification for each of the primer pairs tested. The DNA fragments were separated by electrophoresis using 2% agarose gels. The gels were fixed in 10% acetic acid, dried, and exposed to x-ray film for autoradiography. The specificity of the amplified fragments was assessed by direct sequencing [13]. To evaluate the risk of amplification from genomic DNA potentially present in our total RNA preparations, all samples were amplified by PCR with each primer pair in the absence of reverse transcriptase. In the absence of the enzyme, no amplification product was detected after 35 cycles.
In separate experiments, PCR was carried out with known amounts of plasmids corresponding to each GRK or ß-arrestin. We were then able to compare the relative efficiencies of the different PCRs. The ranking of transcript levels was then determined on the basis of relative signal intensities recorded for each GRK or ß-arrestin amplified from a single RT and normalized with relative efficiencies of the different PCRs.
RT-PCR Detection of GRK6 Isoforms
The expression of both rat variants of GRK6 was determined in primary rat Sertoli cells according to the method of Firsov and Elalouf [27]. The sense primer used for PCR amplification was end-labeled with 
-33P. The RT samples were subjected to 29 cycles (95°C, 30 sec; 62°C, 30 sec; 72°C, 30 sec), with a final elongation time of 6 min at 72°C. The amplification products were then digested with PstI, cutting 19 base pairs (bp) downstream from the 2-bp insert to avoid heterogeneity due to incomplete elongation or to incorporation of extra adenosine by the Taq polymerase. The samples were subsequently denatured, electrophoresed through a 6% polyacrylamide 7 M urea sequencing gel, and processed for autoradiography.
Membrane and Cytosol Preparation
Membranes Twenty-four hours after isolation, Sertoli cells were stimulated or not for 4 min with FSH (100 ng/ml). The medium was then removed on ice, and the flasks were rinsed with ice-cold PBS. Cells were scraped into ice-cold buffer A (20 mM Hepes pH 7.5, 0.15 M NaCl, 250 mM sucrose) containing protease inhibitors (1 mM PMSF, 1 µM pepstatin A, 1 µM leupeptine, 5 mM EDTA, 3 mM EGTA, 50 mM ß-glycerophosphate, 10 mM sodium fluoride, 0.1 mM orthovanadate, and 100 nM okadaic acid) and 10 mg/ml of cycloheximide as previously described [28]. Cells were collected and subjected to 15 strokes with a teflon-glass homogenizer. The extracts were centrifuged at 4°C for 10 min at 700 x g to pellet nuclei and cellular fragments, and the postnuclear supernatants were layered over prechilled 10%–40% continuous sucrose gradients (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM PMSF, 10 mg/ml cycloheximide) prepared with a peristaltic pump and subjected to isopicnic centrifugation at 100 000 x g for 16 h at 4°C in a SW 50.1 rotor (Beckman Instruments, Palo Alto, CA). The 200-µl fractions were collected on ice from the bottom and stored at -20°C until analysis. Fractions were analyzed for their E-cadherin content, a classical marker for plasma membranes [29]. E-cadherin-positive fractions were pooled and subsequently analyzed for GRK/arrestin detection.
Cytosols Postnuclear supernatants prepared from Sertoli cells were centrifuged at 100 000 x g for 16 h at 4°C in a SW 50.1 rotor. Supernatants were collected and considered as soluble cytosolic fractions.
Immunoblotting
Sertoli cells were homogenized on ice in lysis buffer (20 mM Hepes, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 3 mM EGTA, 1 mM PMSF, 1 µM leupeptin, 1 µM pepstatin A, 50 mM ß-glycerophosphate, 10 mM NaF, 0.1 mM orthovanadate, 100 nM okadaïc acid, and 0.5% NP40) with 10 strokes of a teflon-glass homogenizer. The extracts were incubated on ice for 1 h and were subsequently centrifuged (15 000 x g for 15 min at 4°C). Samples (20 µg of proteins of the resulting supernatants or of subcellular fractions) were prepared in reducing conditions (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% ß-mercaptoethanol, 2% SDS, and 0.02% bromophenol blue) followed by immersion in a boiling water bath for 5 min and were then loaded in 10% SDS-polyacrylamide gels. Western blots were carried out as already described [13]. The blots were developed using chemiluminescence detection (NEN).
Statistics
Statistical analysis of the data was performed by one-way analysis of variance. Simultaneous confidence intervals were determined to investigate possible differences between groups using the Scheffé test. Results were considered significant at the 5% level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first used RT-PCR analyses to investigate the existence of transcripts corresponding to 4 GRKs (2, 3, 5, and 6) and to the 2 ß-arrestins in freshly isolated and cultured primary Sertoli cells (Fig. 1a). Because of their tissue specificity (i.e., retina and male germ cells, respectively) GRK1 and GKR4 were not studied [14, 30, 31]. Specific fragments were amplified for the 4 GRKs and the 2 ß-arrestins in all the samples tested. The specificity of the amplified products was assessed by direct sequencing (data not shown). The efficiencies of the different RT-PCRs were compared using known amounts of the homologous plasmids. Using this approach, the relative transcript levels in Sertoli cells were as follows: GRK2 >> GRK6 > GRK3 = GRK5 = ß-arrestin 1 = ß-arrestin 2. To discriminate the transcripts coding for the 2 GRK6 isoforms, we used a previously described high-resolution PCR-based method to detect the 2-bp deletion, which induces a frame shift in the C-terminal part of GRK6 [29]. Both a and b isoform transcripts were detected in Sertoli cells (Fig. 1b). In all the cases, our culture conditions did not modify the expression pattern.
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Western blot analyses of the corresponding proteins were also carried out on Sertoli cells (Fig. 2). Immunoreactive bands of the appropriate molecular weights were observed for GRK2, GRK3, GRK5, and GRK6. Discrepancies between mRNA steady-state levels, as determined by RT-PCR, and protein relative abundances were observed, which was not surprising because our RT-PCR assay was qualitative rather than quantitative. These results demonstrated that Sertoli cells are equipped with a potentially functional GRK/ß-arrestin system. Moreover, the relative abundances of these different factors were quite similar in cultured Sertoli cells and in freshly isolated Sertoli cells, suggesting that plating and in vitro serum-free culture conditions did not alter the expression of the GRK/ß-arrestin system.
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Transient Overexpression of Various GRKs in Primary Sertoli Cells Attenuates FSH-Stimulated cAMP Response
To investigate whether some GRKs were able to attenuate the FSH-R-induced response in primary Sertoli cells, GRK2, -3, -5, -6a, and -6b were transiently overexpressed. We measured the effects of GRK overexpression on basal and FSH-induced CRE-dependent luciferase production (Fig. 3). Each of the rat GRKs tested attenuated the luciferase response to FSH. Several amounts of GRK constructs were transfected (data not shown), and the conditions providing the highest inhibition levels are shown in Figure 3. The maximum inhibition levels recorded in these experiments range between 70% (GRK5) and 42% (GRK2) in the presence of FSH. Basal levels of luciferase activity were also inhibited after GRK overexpression. To determine whether GRK-mediated loss of responsiveness was due to events occurring upstream of adenylyl cyclase, primary Sertoli cells cotransfected with pSOMLuc and with 1 of the GRK expression vectors were stimulated with the adenylyl cyclase-specific activator forskolin. GRK-transfected cells remained fully responsive to forskolin (Fig. 3).
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Transient Overexpression of ß-Arrestins 1 and 2 Also Dampens FSH-Induced cAMP Response in Primary Sertoli Cells
To investigate the possible role of ß-arrestins in the homologous desensitization of FSH response in cells naturally bearing FSH-R, Sertoli cells were transiently cotransfected with the pSOMLuc reporter gene along with either empty or ß-arrestin expression vectors. Significant signal blunting was achieved by ß-arrestin 1 or ß-arrestin 2 overexpression (Fig. 4). Again, both basal and stimulated cells were affected by ß-arrestin transfection (30–40% inhibition in the presence of FSH), and ß-arrestin-transfected cells remained fully responsive to forskolin treatment.
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FSH Rapidly Triggers the Relocalization of Endogenous GRK2, -3, -5, and -6 and ß-Arrestins 1 and 2 in Primary Sertoli Cells
To gain better insight into the physiological relevance of GRK and ß-arrestin actions in FSH signaling, their subcellular localization (membranes vs. cytosol) was examined by Western blotting (Fig. 5). The endogenous GRK2, -3, -5, and -6 and ß-arrestins 1 and 2 accumulated in membrane fractions (E-cadherin positive) after 4 min of agonist exposure, when compared with control membranes. Symmetrically, significant decreases in these protein amounts were observed in cytosolic extracts when compared with untreated controls. After exposure to FSH (Table 1), normalized levels of GRK and ß-arrestin increased 1.4- to 2.4-fold in the membrane fraction in contrast to a 1.2- to 2.0-fold decrease in the cytosolic fraction of primary Sertoli cells.
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DISCUSSION |
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Various GRKs and ß-arrestins (i.e., GRK2, -3, -5, and -6 and ß-arrestins 1 and 2) are endogenously expressed in rat primary Sertoli cells. Their respective transcripts and proteins were detected both in freshly isolated and cultured Sertoli cells. In accordance with these results, Lazari et al. [12] previously reported the presence of GRK2 and GRK6 in MSC-1 cells, a cell line derived from Sertoli cells. An attempt was made to determine the relative steady-state mRNA levels of these GRKs and ß-arrestins in Sertoli cells. GRK2 is mostly expressed in this model. However, because relative affinities of the antibodies raised against the different GRK or ß-arrestin isoforms are not known, it was impossible to confirm GRK2 predominance at the protein level. When total Sertoli cell extracts were analyzed (Fig. 2), the signal corresponding to GRK3 was very faint, raising some doubts about the existence of this kinase in our model. However, in subcellular preparations (i.e., cytosol vs. E-cadherin-positive plasma membranes; Fig. 5) the GRK3:GRK2 ratio was systematically increased and the GRK3 band was very obvious, clearly showing that significant levels of this protein are present in Sertoli cells.
When GRKs and ß-arrestins were transfected into primary Sertoli cells with a cAMP-sensitive gene reporter system, significant blunting of FSH response was achieved. These results are similar to those previously described in Ltk cells overexpressing rat FSH-R [13]. However, the degrees of inhibition observed here are much lower than those measured in previous studies. Basal levels were also inhibited after GRK or ß-arrestin overexpression. We obtained similar results in Ltk cells overexpressing rat FSH-R [13]. We hypothesized that when overexpressed, FSH-R displays constitutive activity in the absence of agonist, as already described for the ß2-adrenergic receptor [32]. Moreover, ß-arrestins bind to the intracellular domain of the agonist-occupied receptors once phosphorylated by GRKs, thereby desensitizing signal transduction to heterotrimeric G proteins [19]. Thus, the results of this experiment clearly suggest that endogenously expressed GRKs phosphorylate the agonist-occupied FSH-R in Sertoli cells.
All the GRKs and ß-arrestins tested were able to dampen the FSH-induced response. Because high levels of overexpressed proteins were reached in transfected cells, it is unlikely that the effectiveness of GRKs and ß-arrestins implies that they are all physiologically active in regulating FSH-R activation of adenylate cyclase in vivo. By mass action, even GRK or ß-arrestin subtypes with low affinity for the agonist-bound receptor may interact with it in overexpressing cells but not under physiological conditions. These results highlight the limits of overexpression methods such as transient transfection when addressing physiologic issues. To further investigate the interaction between FSH-R and the different GRKs and ß-arrestins, we tried to inhibit endogenous GRKs and ß-arrestins using various antisense and dominant negative (i.e., ß-arrestin 2 [319–418]) constructs, respectively. These approaches, which were previously successful in Ltk cells overexpressing the rat FSH-R [13], were inefficient in rat primary Sertoli cells (data not shown). A possible explanation for this result is that in natural FSH-R-bearing cells the ratio between the receptor and GRKs/ß-arrestins could largely be in favor of the desensitizing factors because of the low expression level of the receptor (±1000 receptors/cell [5]). However, nongonadal immortalized cell lines overexpress a large number of FSH-R (10 000–100 000 receptors/cell), probably resulting in an inverted ratio. One assumption based on this hypothesis is that the inhibition of a given GRK or ß-arrestin in primary Sertoli cells must be very strong to potentiate FSH signaling, whereas in reconstituted cells, partial inhibition is sufficient to enhance cAMP response. Null mouse lines for the different GRKs and ß-arrestins have recently been developed and offer new possibilities for working with primary Sertoli cells where the expression of a single desensitization factor is completely abolished [22, 33, 34].
At this point of our study, the overexpressions required to set up our functional assays combined with our inability to achieve efficient inhibition of the endogenously expressed GRKs and ß-arrestins cast some doubts over the specificity of the observed effects on FSH signaling. However, we were able to show significant changes in subcellular localization of endogenous GRKs and ß-arrestins in untreated vs. FSH-stimulated primary Sertoli cells. FSH clearly triggered a rapid (i.e., 4 min) cytosol-to-membrane recruitment of the different GRKs and ß-arrestins endogenously expressed in the Sertoli cells. FSH-induced relocalization was also observed with GRK5, which was previously thought to be constitutively associated with membranes through interaction with a C-terminal polybasic region [14]. In accordance with this view, we observed that the ratio of GRK5:GRK6 was dramatically lower in cytosol than in membrane. However, a slight GRK5 signal was detected in cytosol but systematically decreased after FSH stimulation. The polybasic region of GRK5 could be transiently masked by a mechanism yet to be determined. The very low FSH-R expression in primary Sertoli cells interferes with the ability of classical methods to demonstrate direct interactions between the receptor and the different desensitizing factors. In a very recent study, we coimmunoprecipitated GRK2 with a tagged FSH-R after transient cotransfection in Cos 7 cells. This interaction was enhanced in FSH-stimulated cells [35]. Moreover, Lazari et al. [12] showed that GRKs phosphorylate the FSH-R in an agonist-dependent fashion in HEK 293 cells. These data obtained in nongonadal cell lines suggest that physical interaction between GRKs and the FSH-R occurs in vivo.
Because neither overexpression nor pharmacological treatments were used in these experiments, our results support the existence of a functional link between FSH signaling and the GRK/ß-arrestin system in a physiological system. These data and those from our functional assays clearly suggest that FSH-R transduction is modulated by GRKs and ß-arrestins, most likely by several desensitizing factors rather than a single GRK/ß-arrestin pair.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 S.M. is the recipient of a doctoral fellowship from the Région Centre-INRA. P.C. is funded by a fellowship from the Fondation d'Aide à la Recherche ORGANON. This work was also funded by the Ligue contre le cancer. 
2 Correspondence. FAX: 33 2 47427743; reiter{at}tours.inra.fr
Accepted: August 14, 2001.
Received: April 3, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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