Involvement of G Protein-Coupled Receptor Kinases and Arrestins in Desensitization to Follicle-Stimulating Hormone Action

Carine Troispoux, Florian Guillou, Jean-Marc Elalouf, Dmitri Firsov, Luisa Iacovelli, Antonio De Blasi, Yves Combarnous and Eric Reiter

INRA/CNRS URA 1291 (C.T., F.G., Y.C., E.R.) Station de Physiologie de la Reproduction des Mammifères Domestiques 37380 Nouzilly, France
Département de Biologie Cellulaire et Moléculaire (J.-M.E., D.F.) Service de Biologie Cellulaire CEA Saclay 91191 Gif-sur-Yvette Cedex, France
Consorzio Mario Negri Sud (L.I., A.D.B.) Istituto di Ricerche Farmacologiche "Mario Negri" 66030 Santa Maria Imbaro, Italy


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as ß-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or ß-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that ß-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative ß-arrestin 2 [319–418] potentiates it 8-fold (P < 0.001); 3) that ß-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH plays a central role in mammalian reproduction. This hormone is necessary for gonadal development and maturation at puberty and for gamete production during the fertile phase of life. FSH acts by binding to specific receptors localized exclusively in the male and female gonads (reviewed in Ref. 1). The FSH receptor (FSH-R) is a complex protein characterized by seven hydrophobic helices spanning the membrane. The intracellular domain of the receptor is functionally coupled to Gs (2). The binding of FSH to the receptor extracellular domain initiates a cascade of events that ultimately leads to the specific biological effects of the hormone (1).

As in many other hormonal systems, prolonged exposure of target cells to FSH leads to a decreased response over time, a process called desensitization (3, 4, 5, 6). The early desensitization step after FSH binding and stimulation is the reduction of FSH-R function due to its uncoupling from Gs (7, 8). Later, other mechanisms, such as the increase of nucleotide-phosphodiesterase activities (9) or the reduction of receptor number (down-regulation) (10, 11, 12), become involved in the decreased cell response to prolonged FSH stimulation.

The mechanisms governing receptor uncoupling have been extensively studied at molecular level for rhodopsin and ß2-adrenergic receptors (reviewed in Ref. 13). For these receptors, uncoupling occurs through phosphorylation of the C-terminal intracellular domain. Two classes of protein kinases may be involved in this process: 1) second messenger-dependent kinases such as protein kinase A (PKA) or protein kinase C (PKC), which are responsible for agonist-independent, or heterologous, desensitization, and 2) G protein-coupled receptor kinases (GRKs), which trigger the agonist-specific, or homologous desensitization (13).

GRKs are serine/threonine kinases showing the unique property of interacting only with agonist-occupied receptors while leaving unaltered nonactivated receptors. To date, six distinct mammalian GRK genes have been identified and classified into three subfamilies on the basis of sequence and functional similarities (14). The first subfamily includes GRK1 only (rhodopsin kinase). The second subfamily is composed of GRK2 and GRK3 (ß-adrenergic receptor kinases 1 and 2), while GRKs 4, 5, and 6 represent the third subfamily. Expression of GRK1 is preferentially confined to retinal photoreceptor cells where it phosphorylates retinal opsins (13). Likewise, GRK4 is predominantly expressed in testicular germ cells, but its receptor substrate is still unknown (15, 16). In contrast, the other GRKs (i.e. GRKs 2, 3, and 5 and the two isoforms of rat GRK6) are ubiquitously expressed (13, 17) and display broad and possibly overlapping substrate specificities (13).

After phosphorylation of agonist-occupied receptors by GRKs, arrestins bind to the intracellular domain of the receptors, thereby preventing further signal transduction (reviewed in Ref. 18). Arrestins are also known to interact with clathrin, providing a mechanism for internalization or sequestration of the activated receptors (19). The arrestin family includes visual arrestin, cone arrestin, ß-arrestin 1 (arrestin 2), and ß-arrestin 2 (arrestin 3), in addition to arrestin D and arrestin E, which are only partially characterized. Visual and cone arrestins are predominantly expressed in retinal tissues where they regulate photoreceptor signaling. In contrast, the other arrestins are ubiquitously expressed and possibly interact with a wide variety of G protein-coupled receptors (18, 19).

The molecular mechanisms involved in the FSH-R uncoupling are still poorly understood. Studies on embryonic kidney cell line (HEK 293) permanently transfected with the rat FSH-R have shown that phosphorylation of the receptor is induced within a few minutes either by FSH or phorbol esters and does not involve PKA. This phosphorylation occurs on both serine and threonine residues, which are probably located upstream of amino acid 635 (20, 21). In addition, recent studies by Nakamura et al. (22) have established that 1) FSH-induced phosphorylation of the FSH-R maps to the first and third intracellular loops and is involved in the processes of coupling with Gs and internalization; 2) FSH-dependent phophorylation of the FSH-R is increased by GRK2 overexpression (23); and 3) FSH-R internalization is enhanced when ß-arrestin 2 and/or GRK2 are overexpressed (23). In the natural FSH-R-bearing Sertoli cells, it has been demonstrated that both PKA and PKC participate in desensitization of the FSH-induced response, but that an additional PKA/PKC-independent uncoupling mechanism also exists (24). Moreover, in rat granulosa cells overexpressing the FSH-R, a specific staurosporine-sensitive receptor kinase may be responsible for hormone-induced phosphorylation and uncoupling of the FSH-R (25, 26).

Taken together, the available data suggest that, although the partial involvement of PKA and/or PKC in the phosphorylation of the FSH-R is possible, the early modulation of FSH action seems to be largely due to specific receptor kinase(s). The experiments presented herein demonstrate that the GRK/arrestin system is involved in homologous desensitization of the FSH-R signaling in Ltk cells stably expressing the rat FSH-R and producing cAMP in response to FSH action (27).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Demonstration of FSH-Induced Homologous Desensitization in Ltk 7/12 Cells
To evaluate FSH-induced desensitization in Ltk 7/12 cells that stably overexpress the rat FSH-R, we first investigated the time course of FSH-stimulated cAMP production in this cell line. First, we observed that FSH-induced cAMP response reached a plateau within 30 min (Fig. 1aGo). In addition, when the cells were preincubated for 2 h with increasing concentrations of FSH and then rechallenged for 30 min with a maximal concentration of FSH (100 ng/ml), cAMP production was significantly reduced at preincubation concentrations >10 ng/ml (Fig. 1bGo). These results suggest that FSH-R undergoes desensitization in Ltk 7/12 cells. The time delay and the FSH doses required to induce desensitization in Ltk 7/12 cells were higher than those previously described for rat Sertoli cells (28). This is probably a consequence of the higher density of FSH-R expressed in the Ltk 7/12 cells [~10,000 receptors per Ltk 7/12 cell (27)] than in primary rat Sertoli cells [~1,000 receptors per Sertoli cell (1)]. Moreover, this high level of overexpression in a cell line that normally does not express the FSH-R, could determine a stoichiometry between the molecules involved in the coupling and in the desensitization, which is different from a physiological system.



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Figure 1. Homologous Desensitization of the FSH-R in Ltk 7/12 Cells

a, Time course of FSH-induced cAMP production. Ltk 7/12 cells were stimulated with 100 ng/ml of FSH for indicated times. Intracellular cAMP content was measured as described in Materials and Methods. b, FSH-induced desensitization of the cAMP production. Cells were preincubated for 2 h with increasing concentrations of FSH. Cells were then washed with culture medium and stimulated with 100 ng/ml of FSH for 30 min. Intracellular cAMP content was measured. ***, P < 0.001, significant statistical difference from cells preincubated without FSH. These data represent the mean ± SE of two independent experiments, each with four replicates.

 
Expression of the GRKs and ß-Arrestins in the Ltk 7/12 Cells
We first investigated the expression of four GRKs (2, 3, 5, 6) and of ß-arrestins 1 and 2, using RT-PCR analyses of total RNA in Ltk 7/12 cells (Fig. 2aGo). GRKs 1 and 4 were excluded from this study due to their tissue-specific expression (i.e. retina for GRK1, male germ cells for GRK4). Specific fragments of the four GRKs and of the two ß-arrestins were amplified, as demonstrated by direct sequencing of the different PCR products. These data indicate that the corresponding genes are transcribed in Ltk 7/12 cells. The efficiencies of the different RT-PCR reactions were not compared in this study principally because all the primers were designed from rat cDNA sequences that were the closest sequences available for the GRKs and ß-arrestins when these experiments were performed. As a consequence, this set of RT-PCR did not allow us to compare the relative mRNA abundances for the different GRKs and ß-arrestins in the Ltk 7/12 cells.



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Figure 2. Expression of GRKs and ß-Arrestins in Ltk 7/12 Cells

a, RT-PCR analysis. Total RNAs (80 ng) extracted from Ltk 7/12 cells were submitted to RT-PCR using primer pairs designed to amplify fragments from GRK 2, 3, 5, 6 or from ß-arrestin (ß-Arr) 1 and 2 transcripts. Reactions were performed in the presence of [{alpha}-32P]dCTP and were stopped after 25, 30, and 35 cycles. The expected sizes of the different amplified products are indicated on the right. Positive controls (+) were obtained by direct amplification of the appropriate expression vector. Negative control reactions (-) were carried out in the absence of reverse transcriptase (RT). Amplified products were resolved on a 2% agarose gel and detected by autoradiography. b, Specificities of the anti-GRK antibodies. Cytosolic or membrane proteins from HEK293 cells transfected with one of the different GRKs or ß-arrestins were prepared, electrophoresed on SDS-polyacrylamide gels, and transferred to PVDF membranes. A monoclonal antibody recognizing both GRK2 and GRK3 and three rabbit polyclonal antibodies raised against GRK2, GRK5, and GRK6 were used for GRK immunodetections. The blots were developed using the Renaissance chemiluminescence detection system (NEN Life Science Products). The signals corresponding to GRKs are indicated by arrows. Data are representative of three separate experiments. c, Western blot analysis. Cytosolic or membrane proteins from Ltk 7/12 cells and transfected positive controls were electrophoresed on SDS-polyacrylamide gels, transferred to PVDF membranes, and analyzed with the antibodies described above. ß-Arrestins 1 and 2 were detected with a monoclonal antibody raised against a epitope common to both isoforms. Colored standards allowed determination of mol wt marker positions. The signals corresponding to GRKs and ß-arrestins are indicated by arrows. Data are representative of three separate experiments.

 
Western blot analyses of these different proteins were also carried out on Ltk 7/12 preparations. The specificity of the antibodies used was confirmed using different transfected preparations as positive controls (Fig. 2bGo). In addition, no signal was obtained for GRK2 using the polyclonal antibody after preincubation with the corresponding control peptide (data not shown). Immunoreactive bands of the appropriate molecular weights, which comigrated with the positive controls, were observed in Ltk 7/12 cytosolic extracts for GRK2, GRK3, GRK5, and GRK6. GRK3 was more abundant than GRK2 in cytosolic extracts (Fig. 2cGo). However, in whole-cell extracts, GRK2 was clearly predominant (Fig. 5cGo). The monoclonal antibody F4C1 raised against an epitope that is conserved in both ß-arrestins 1 and 2, was also used. In Ltk 7/12 cytosolic preparations, two bands were detected with apparent Mr of 45,000 and 50,000, corresponding to ß-arrestin 2 and ß-arrestin 1 from transfected positive control, respectively. The ß-arrestin 2 was more abundant than ß-arrestin 1 in these cytosolic extracts, but the ratio was found inverted in whole cell extracts (data not shown). These results confirm our RT-PCR experiments and definitely establish that Ltk 7/12 cells are equipped with a potentially functional GRK/arrestin system. Thus, this cellular model is suitable to study the involvement of these regulatory proteins in desensitization of FSH-R.



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Figure 5. Transient Overexpression of GRKs Antisense (AS) Constructs in Ltk 7/12 Cells

a, Ltk 7/12 cells were cotransfected with pSOMLuc and with various GRK AS constructs (1 µg GRK2 AS, 1 µg GRK3 AS, 2 µg GRK5 AS, 2 µg GRK6 AS per well). Cells were stimulated with FSH (100 ng/ml) ({square}) or were not stimulated (). Luciferase activity was determined in the cell lysate. Values were expressed in percentage; the mean activity obtained for unstimulated cells transfected with pSOMLuc and pCMV5 was taken as 100%. *, P < 0.05, significant statistical differences from the FSH-stimulated cells transfected with pSOMLuc and pCMV5. These data are representative of two independent experiments, each with four replicates. b, Cross-hybridization assay: unlabeled sense cRNAs corresponding to the full-length sequences of GRK2, -3, -5, and -6A were applied in 2-fold serial dilutions on nitrocellulose filters using a slot blot apparatus. Four replicates were done and each of them was hybridized with 15 x 106 cpm of a 32P-labeled antisense cRNA corresponding to GRK2, -3, -5, or -6. c, Antisense-mediated inhibition of endogenous GRKs: Ltk 7/12 cells were transiently cotransfected with both an expression vector for GFP and one of the antisense contruct or pCMV5 as control. GFP-positive living cells were then sorted using fluorescence-activated cell sorting. Twenty micrograms of whole-cell extracts from each sorted population were analyzed by Western blotting as described above. The transferred proteins were stained with Coomassie blue and scanned for subsequent densitometric analysis. Antibodies specific for GRK2, GRK2/3, GRK5, and GRK6 were used. The inhibition levels (values presented between brackets) were determined by densitometric analysis of the different immunoblots, corrected according to gel loading and transfer efficiency as determined by Coomassie blue staining of the PVDF membranes (data not shown). d, Control of relative transfection efficiencies: Ltk 7/12 cells were transfected by lipofection as described in Materials and Methods. Cells were stimulated with FSH (100 ng/ml) for 4 h. Luciferase activities and the amounts of plasmid transfected were determined in parallel for each condition, luciferase activities being corrected according to the relative transfection efficiencies. The results were arbitrary expressed in percent, the pCMV5 control being taken as 100%. *, P < 0.05; **, P < 0.01; significant statistical differences from pCMV5 control cells.

 
A cAMP-Inducible Gene Reporter System to Measure the FSH Response in Transfected Ltk 7/12 Cells
To examine the role of regulatory proteins in the FSH-R desensitization mechanism, a variety of expression vectors for the GRKs and ß-arrestins were transfected in Ltk 7/12 cells. As in a standard transfection procedure, only a limited number of cells incorporate DNA, we decided to use the cAMP-sensitive pSOMLuc construct as a reporter system [i.e. the luciferase reporter gene driven by a part of the somatostatin gene 5'-end region containing cAMP-responsive elements (39)]. In Ltk 7/12 cells transiently transfected with pSOMLuc, luciferase activity was induced within 10 min of FSH treatment (Fig. 3aGo), and a clear saturation of cell response was observed within 20–60 min. In this experiment, cells were stimulated for the indicated time, washed, and then maintained in serum-free medium for a total of 4 h after the beginning of the stimulation to allow luciferase gene expression. These results, which are similar to those of Fig. 1aGo, demonstrate that the pSOMLuc reporter gene system used here is suitable to measure the effects of potential regulatory proteins on the FSH-R coupling. Figure 3bGo shows that for stimulation longer than 2 h, luciferase activity was systematically higher in FSH-treated conditions than in untreated controls. The maximum response intensity was observed after 4 h and, on the basis of this experiment, 4-h treatments were used in further experiments.



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Figure 3. FSH-Induction of pSOMLuc Expression in Transiently Transfected Ltk 7/12 Cells

a, Short-term induction of pSOMLuc expression by FSH. Ltk 7/12 cells were transiently transfected with 1 µg per well of pSOMLuc. After 24 h of culture, cells were stimulated for the indicated times with FSH (100 ng/ml). Then, cells were washed and maintained in serum free medium to allow time for luciferase gene expression. Four hours after the beginning of the stimulation, luciferase activity was measured in the cell lysates. Values were expressed in percentage, with the activity of the unstimulated control being taken as 100%. b, Long-term induction of pSOMLuc expression by FSH. Ltk 7/12 cells were transiently transfected with 1 µg per well of pSOMLuc. After 24 h of culture, cells were maintained for the indicated times under control conditions ({square}) or exposed to 100 ng/ml FSH ({circ}). At the end of the incubation period, luciferase activity was measured in the cell lysates. These data represent the mean ± SE of two independent experiments, each with four replicates.

 
First, we used pCMVß-Gal as constitutively expressed standard to monitor transfection efficiencies between plates. Early on, however, we suspected that the expression of this construct was influenced by FSH and indeed, when pCMVß-Gal was transfected alone and the cells treated with either FSH or (Bu)2cAMP, ß-galactosidase activity was significantly stimulated by both treatments (data not shown). This suggests that cAMP response element-like sequences are present in the pCMVß-Gal vector.

Transient Overexpression of Various GRKs: Attenuation of the FSH-Stimulated Response
To investigate the possible role of different GRK subtypes in the attenuation of FSH-R-induced response, rat GRKs 2, 3, 5, 6a, and 6b were transiently overexpressed along with the pSOMLuc reporter gene in Ltk 7/12 cells. We measured the effects of GRK overexpression on basal and FSH-induced luciferase production (Fig. 4Go, a–e). Each of the tested rat GRKs was shown to attenuate the luciferase response to FSH. The inhibition levels were directly related to the amount of expression vector transfected (P < 0.001). The maximum inhibition levels recorded in these experiments were between 71.2 ± 0.9% (GRK5) and 96.6 ± 0.2% (GRK3) in the presence of FSH (Table 1Go). The relative transfection efficiencies were determined for each plate by hybridization of DNA extracts prepared from half of the transfected cells, with a luciferase cDNA probe. The CaPO4 precipitate method was not used in this control experiment as the transfection levels are too low to allow direct detection of the transfected plasmids. This control experiment confirmed that all the GRKs decreased the FSH response (Fig. 4fGo). The fact that similar results were obtained using two different transfection methods demonstrate that our results are independent from possible Ca++- or liposome-induced alterations of cell fate and/or signaling. The observation that the five GRKs analyzed were all capable of reducing the FSH response is not surprising. Indeed, numerous reports in the literature have shown that when overexpressed, these kinases often lose their substrate specificities (reviewed in Ref. 13). These results provide evidence that GRKs are able to interact with FSH-R. However, these data are not sufficient to definitely demonstrate the involvement of the GRKs in the desensitization to FSH action. It is interesting, however, to note that the basal levels of luciferase activity were also inhibited after GRK overexpression. This finding is unexpected as GRKs are known to interact only with agonist-occupied receptors. It can be hypothesized that when overexpressed, FSH-R may display constitutive activity in the absence of agonist as already described for the ß2-adrenergic receptor (30). GRKs could then be able to desensitize the constitutively active FSH-R. Another explanation could be that the overexpressed GRKs interact with other Gs-coupled receptors activated by autocrine effectors or by residual serum factors (Ltk 7/12 cells are cultured with 10% serum before transfection and a 48-h starvation period).



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Figure 4. FSH-R Uncoupling in Ltk 7/12 Cells by Transiently Overexpressed GRKs

Ltk 7/12 cells were cotransfected with pSOMLuc and various quantities of the following: panel a, pCMV5-GRK2 (0.5, 1, and 2 µg); panel b, pCMV5-GRK3 (0.25, 1, and 2 µg); panel c, pCR3-GRK5 (0.5, 1, and 2 µg); panel d, pCB6-GRK6A (1, 2, and 3 µg); or panel e, pCB6-GRK6B (0.5, 1, and 2 µg). Calcium phosphate precipitations were used for transfections. Cells were stimulated ({square})or not () with FSH (100 ng/ml) for 4 h. Luciferase activity was measured in the cell lysate. Values were expressed in percentage, the activity of the unstimulated control was taken as 100%. Panel f, 1 µg of each GRK expression vector was transfected by lipofection as described in Materials and Methods. Cells were stimulated with FSH (100 ng/ml) for 4 h. Luciferase activities and the amounts of plasmid transfected were determined in parallel for each condition, luciferase activities being corrected according to the relative transfection efficiencies. The results were arbitrary expressed in percent, the control being 100%. These data are representative of two independent experiments, each with four replicates. *, P < 0.05, significant statistical differences from pCMV5 control cells.

 

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Table 1. Maximum Inhibition of the FSH Response in Ltk 7/12 Cells Overexpressing GRKs

 
Effects on FSH-Stimulated Response of the Specific Inhibition of Endogenously Expressed GRKs Using Antisense cDNAs
To test whether endogenous GRKs in Ltk 7/12 cells were able to regulate FSH-R function, we investigated the effects of specific inhibition of GRK subtypes. Vectors containing GRK cDNAs in antisense orientation were used as specific inhibitors. The cotransfection scheme was as follows: one antisense construct of either rat GRK2, -3, -5, or -6 was transfected along with the pSOMLuc reporter gene in Ltk 7/12 cells, and its effect on the FSH-induced luciferase production was measured. As shown in Fig. 5aGo, strong increases in the cell response to FSH treatment were recorded in cells transfected with antisense for GRK2, -5, and -6 [2- to 3-fold over the FSH-treated control, P < 0.05; see (Table 2Go)]. The antisense for GRK3 did not affect the FSH-induced response of the cells. However, the ability of this GRK3 antisense construct to induce increased cell response to an agonist was observed in another cellular model (C. Troispoux, F. Guillou, Y. Combarnous, and E. Reiter, unpublished data). Parallel to the attenuation of basal luciferase activity after the overexpression of sense GRKs (Fig. 4Go), the four antisense constructs tested (Fig. 5aGo) significantly increased the basal levels by 2- to 4-fold (P < 0.05, Table 2Go). The antisense for GRK3 also increased the basal level and did not affect FSH-stimulated response, suggesting that basal luciferase activity is due to the activation of receptor(s) other than the FSH-R by residual serum factor(s) or by an autocrine mechanism. In addition, this confirms that the GRK3 antisense construct effectively inhibits GRK3 action.


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Table 2. Stimulation of the FSH Response in Ltk 7/12 Cells Transfected with Antisense cDNAs for the Different GRKs

 
To assess the specificity of the antisense experiments above, slot blot hybridizations were carried out using both sense and antisense cDNAs corresponding to each GRK subtype (Fig. 5bGo). The only significant cross-hybridizations were obtained between GRK2 and GRK3. These results were expected as GRK2 and GRK3 display the closest homologies among GRKs. GRK2/3 cross-hybridizations were less than 10% and, more importantly, GRK2 antisense triggered a significant increase in FSH-induced response while GRK3 antisense did not. To check the specificity and the efficiency of antisense activities on GRK protein synthesis, cells were transiently cotransfected with both an expression vector for green fluorescent protein (GFP) and one of the antisense constructs. Doubly-transfected cells were selected by fluorescence-activated cell sorting, using the GFP fluorescence and expression of the different GRK proteins was assessed by Western blotting (Fig. 5cGo). The antisense-specific decreases in protein levels were 22% for GRK2, 17% for the GRK2/3 ratio, 51% for GRK5, and 11% for GRK6. No cross-inhibition between different GRKs was observed in the different populations of sorted GFP-positive cells (data not shown). Not surprisingly, using our antisense constructs, only partial inhibition were observed, as it is well known that the use of full-length cDNA in antisense orientation is not the most efficient antisense strategy. In our model, we showed that pronounced biological effects can be obtained at low inhibition rates for GRK2 and GRK6. The relative efficiencies of the antisense treatments depend probably also on the abundance of each kinase in the Ltk 7/12 cells. When pSOMLuc responses were corrected with the relative transfection efficiencies, pronounced potentializations of the FSH response were demonstrated for GRK2AS, GRK6AS, and, to a lesser extent, for GRK5AS (Fig. 5dGo).

Transient Overexpression of ß-Arrestins 1 and -2 and Dominant Negative ß-Arrestin 2 [319–418] in Ltk 7/12 Cells
To investigate the possible role of arrestins in the homologous desensitization of FSH-R-stimulated-response, Ltk 7/12 cells were transiently cotransfected with the pSOMLuc reporter gene along with either control or ß-arrestin 1 or ß-arrestin 2 expression vectors. Significant signal blunting was achieved by ß-arrestin 1 or ß-arrestin 2 overexpression (Fig. 6aGo), this effect being proportional to the quantity of plasmid DNA transfected. Similarly, the overexpression of ß-arrestin 2 [319–418], a potent dominant negative inhibitor of ß-arrestins (19), strongly potentiates FSH action (Fig. 6bGo). These results were not due to differential transfection efficiencies as shown in Fig. 6cGo. It is well established that arrestins bind to the intracellular domain of the agonist-occupied receptors after their phosphorylation by GRKs, thereby desensitizing signal transduction to heterotrimeric G proteins (18). Thus, this experiment strongly suggests that endogenously expressed GRKs, probably GRK2, GRK5, and GRK6 as indicated by the results with the antisense constructs, might phosphorylate agonist-occupied FSH-R in Ltk 7/12 cells. To ascertain whether the overexpressed arrestins were indeed able to potentiate the effects of GRKs, we assessed the ability of the different GRK/arrestin combinations to desensitize FSH-R. Transient cotransfections were carried out using a submaximal plasmid concentration to detect whether the effects of GRKs and arrestins were additive (Fig. 7Go). The trend of the results was quite similar for both arrestins, but stronger inhibition was consistently observed with ß-arrestin 2. GRK3, GRK6b, and, to a lesser extent, GRK2 generated additional signaling inhibition with the two arrestins. In contrast, the combination of GRK6a with ß-arrestin 1 or ß-arrestin 2 displayed the same signal dampening as arrestins alone. GRK5 raised the inhibitory effects of ß-arrestin 2 but not ß-arrestin 1. These data suggest that some GRK/ß-arrestin combinations are more efficient than others in the inhibition of FSH-R coupling.



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Figure 6. Transient Overexpression of ß-Arrestins 1, 2, and Dominant Negative ß-Arrestins 2 [319–418] in Ltk 7/12 Cells

a, Ltk 7/12 cells were transfected with pSOMLuc and either ß-arrestin 1 (ß-arr1) or ß-arrestin 2 (ß-arr2) expression vectors. Two quantities (0.25 and 0.5 µg per well) of the ß-arrestin plasmids were tested. All the cells were stimulated by FSH. *, P < 0.05; **, P < 0.01; significant statistical differences from control cells. b, Ltk 7/12 cells were transfected with pSOMLuc and ß-arrestin 2 [319–418] expression vectors. Two quantities (1 and 2 µg per well) of the ß-arrestin 2 [319–418] were tested, the quantities of DNA being normalized using the pCMV5 empty vector. All the cells were stimulated by FSH. *, P < 0.05; ***, P < 0.001; significant statistical differences from control cells. c, Control of relative transfection efficiencies: Ltk 7/12 cells were transfected using lipofection as described in Materials and Methods. Cells were stimulated with FSH (100 ng/ml) for 4 h. Luciferase activities and the amounts of plasmid transfected were determined in parallel for each condition, luciferase activities being corrected according to the relative transfection efficiencies. The results were arbitrary expressed in percent; the pCMV5 control was taken as 100%. *, P < 0.05; **, P < 0.01; significant statistical differences from pCMV5 control cells.

 


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Figure 7. FSH-R Uncoupling in Ltk 7/12 cells by Transient Overexpression of Combinations of ß-Arrestins and GRKs

a, ß-Arrestin 1 plus GRKs. Ltk 7/12 cells were cotransfected with pSOMLuc and 0.5 µg of each GRK construct alone, or together with 0.25 µg per well of pCMV5-ß-arr1. b, ß-Arrestin 2 plus GRKs. Ltk 7/12 cells were cotransfected with pSOMLuc and 0.5 µg of each GRK construct alone, or together with 0.25 µg per well of pCMV5-ß-arr2. Values were expressed in percentage. The activity measured for cells transfected with pSOMLuc and pCMV5 in nonstimulated conditions () was taken as 100%. Significantly different from cells transfected with the ß-arrestin 1 or 2 construct alone: *, P < 0.05; **, P < 0.01; ***, P < 0.001. These data are representative of two independent experiments, each with four replicates.

 
The GRK/ß-Arrestin System Is Involved in the Modulation of the FSH-Induced cAMP Response
The luciferase gene reporter did not allow measurement of short-term stimulation by FSH. For this reason, we measured the rapid (15 min) FSH-induced cAMP generation in Cos-7 cells (which do not express the FSH-R) transiently cotransfected with both rat FSH-R expression vector and GRK2, ß-arrestin 2, or GRK2AS constructs. Both GRK2 and ß-arrestin 2 severely blunted the FSH-stimulated cAMP production when compared with controls (Fig. 8Go). On the contrary, the antisense construct for GRK2 significantly enhanced cAMP accumulation after short-term (15 min) FSH stimulation. This experiment suggests that the GRK/ß-arrestin system is involved in the control of the acute FSH-promoted cAMP accumulation.



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Figure 8. Effect of GRK2, ß-arrestin 2, and GRK2AS on the Acute FSH-Promoted cAMP Accumulation in Cos-7 Cells

Cos-7 cells were transiently cotransfected with the rat FSH-R and with either pCMV5, GRK2, ß-arrestin 2, or GRK2AS. After 48 h transfection, cells were stimulated with 100 ng/ml of FSH for 15 min. Intracellular cAMP content was measured as described in Materials and Methods. Values were expressed in percentage. The FSH-dependent cAMP induction measured for cells transfected with the FSH-R and pCMV5 was taken as 100%. These data represent the mean ± SE of two independent experiments, each with four replicates. **, P < 0.01; ***, P < 0.001; significant statistical difference from control conditions.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The gonadotropin receptors (LH and FSH-R) are members of the rhodopsin-like subfamily of G protein-coupled receptors (GPCRs) and bind their ligands with high affinities. Both LH and FSH-Rs as well as the TSH-receptor (TSH-R), are characterized by very large ectodomains (31). Like other GPCRs, LH-R and FSH-R undergo homologous desensitization (reviewed in Ref. 1). They become rapidly phosphorylated on serine and threonine residues in response to agonist stimulation, and this phosphorylation facilitates agonist-induced functional uncoupling and internalization (21, 22). However, despite the key role of gonadotropin receptors in the control of reproductive functions, very little is known concerning the regulatory proteins involved in their agonist-induced desensitization process. Premont et al. (32) have shown that transient cotransfection of HEK 293 cells, with rat LH-R and either GRK2 or one of the four human GRK4 isoforms, dampened the cAMP response to a subsequent LH treatment. Recent studies have also demonstrated the involvement of the GRK/arrestin regulatory proteins in the homologous desensitization of the structurally related TSH-R (33, 34). Nakamura et al. (23) have reported that the overexpression of GRK2 enhances agonist-dependent FSH-R phosphorylation. These authors have also shown that ß-arrestin 2 overexpression stimulates FSH-R internalization. However, so far, it has not been proven that GRKs and arrestins promote the functional uncoupling of the FSH-R in a way similar to that of the ß2-adrenergic receptor and rhodopsin models (13). In contrast, since gonadotropin hormones dissociate very slowly from their receptors, it has been proposed that LH-R and FSH-R may utilize unusual mechanisms for deactivation (22).

In this study, we have used a cAMP-sensitive reporter gene to indirectly monitor the coupling between G{alpha}s and the FSH-R. The results obtained using this reporter system and many data of the literature suggest that GRKs and ß-arrestins participate to the functional uncoupling of the FSH-R. However, direct measurement of early signaling events (i.e. receptor phosphorylation, G{alpha}s activation, and adenylate cyclase activity) will be necessary to confirm that the mechanisms described for the ß2-adrenergic receptor are applicable to the homologous desensitization of the FSH-R (13). This study describes three distinct aspects of the relationships between GRKs, ß-arrestins, and the rat FSH-R in Ltk 7/12 cells: 1) individual GRKs inhibit FSH-R-mediated signaling as demonstrated by the diminished response to FSH in cells transfected with specific GRK subtypes; 2) ß-arrestins quench FSH-R signal transduction when transiently overexpressed, and this effect is potentiated by certain GRKs; and 3) FSH-R desensitization depends on endogenous GRKs and ß-arrestins; in fact, transient overexpression of antisense constructs for the different GRKs and of a dominant negative construct for ß-arrestins enhances FSH stimulation. All the tested GRKs were able to dampen the FSH-induced response. Since high levels of overexpressed proteins were reached in Ltk 7/12 cells for both receptor and GRKs, a problem of specificity was raised. In fact, it is possible that, by mass action, even GRK subtypes with low affinity for the agonist-bound receptor may phosphorylate it in heterologous system but not in physiological conditions.

To investigate further the interaction between the FSH-R and the different GRKs, we used GRK antisense constructs to selectively inhibit the different kinases. Increased signaling responses were recorded with the antisense cDNA constructs for GRK2, GRK5, and GRK6, but not GRK3. These four kinases are detected in Ltk 7/12 cells using RT-PCR and Western blotting. We were confident about the specificity of the antisense approach as we could show that GRK3 antisense had no effect in a cell line expressing this kinase. On the basis of our control experiments, the possibility of cross-reactivities between GRK2, GRK5, and/or GRK6 could be excluded. As assessed by the increase in signal stimulation in the presence of antisense GRK constructs, the FSH-R appears to interact with three of the four GRKs tested. The regulation of FSH-R by one or more GRKs in vivo is further validated by our previous finding that GRK2, -3, -5, and -6 are expressed in rat Sertoli cells (C. Troispoux, F. Guillou, Y. Combarnous, and E. Reiter, unpublished data). It could be anticipated, however, that in a physiological model, the specificity of the FSH-R/GRK interactions may be different and probably more stringent. Until now, only GRK5 in thyroid cells (33) and GRK2 in both CHO and A-431 human epidermoid carcinoma cells (35) have been selectively inhibited using antisense cDNA or oligodeoxynucleotides. Here, we demonstrate that the use of antisense cDNAs is sufficient to selectively inhibit virtually any GRK allowing us to study the substrates of these kinases.

Previous work has demonstrated that overexpression of ß-arrestin augments the desensitization of various GPCRs (34, 36). In agreement with these reports, we demonstate that FSH-R signaling is blunted by transiently overexpressed ß-arrestins and is strongly potentiated by the dominant negative form of this molecule. These data demonstrate that endogenous ß-arrestins could play a role in the desensitization of FSH-R. Both ß-arrestins potentiate the GRK-induced uncoupling of the FSH-occupied receptors, and the degree of inhibition obtained varies according to the types of the cotransfected GRK/ß-arrestin couples. In spite of the limits of this transfection approach (see discussion above), these data suggest that preferential GRK/ß-arrestin associations might exist in vivo for the interaction with the FSH-R. Interestingly, GRK6a completely fails to enhance the effects of ß-arrestin 1 or 2, while the GRK6b isoform displays marked additive action with both ß-arrestins. This is the first direct evidence of a functional difference between the two isoforms of GRK6, whose expression patterns in the rat are different: the GRK6b is the predominant subtype in most tissues, including the testis, while GRK6a is the main isoform expressed in the brain (17).

By analogy with other receptors shown to be GRK substrates, serine and threonine residues present in the intracellular parts of the FSH-R would most likely be sites for GRK phosphorylation (13). In a recent work, Nakamura et al. (22) have shown that the agonist-induced phosphorylation of the rat FSH-R maps on residues located in the first (T369, T370, S371, and T376) and third (T536, T541, S544, S545, S546, S547, and T549) intracellular loops. When three of the four putative phosphorylation sites present in the first loop were mutated, the FSH-induced, but not PMA-induced, phosphorylation of the receptor was almost completely abolished. Moreover, this mutated receptor was found significantly less uncoupled after a 15-min pretreatment with FSH than the wild-type or a third loop mutant of FSH-R (the phosphorylation sites of the third loop were substituted into alanine). The first loop mutant was also significantly less internalized than the wild-type FSH-R or the third loop mutant. These data suggest that the first intracellular loop of the agonist-occupied FSH-R could be important for the interaction with GRKs.

In summary, the present study demonstrates that overexpressed GRKs or ß-arrestins, probably by interacting with agonist-occupied FSH-R, can inhibit receptor signaling. Antisense cDNAs, selectively inhibiting the synthesis of each endogenous GRK, were found to potentiate FSH action.This demonstrates the involvement of GRK2/6 and, to a lesser extent, of GRK5 in the homologous desensitization of the rat FSH-R expressed in Ltk 7/12 cells. To appreciate the specificity of GRK/arrestin interactions with the FSH-R in physiological conditions (i.e. very low levels of both receptor and GRK), primary cultures of Sertoli or granulosa cells should be studied in future works.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell Culture
MEM, geneticin, trypsin-EDTA, and FCS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Penicillin, streptomycin, 4-methyl-umbelliferyl-ß-D-galactopyranoside, and (Bu)2 cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). Porcine FSH (pFSH) was purified in our laboratory (CY 1737 III: 41x NIH FSH P1 in homologous porcine RRA).

RT-PCR
MMLV reverse transcriptase was from Life Technologies, Inc. Taq DNA polymerase was from Pharmacia Biotech (Uppsala, Sweden). Radiolabeled [{alpha}-32P]dCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL).

Plasmid Constructs
pCMVßgal was from CLONTECH Laboratories, Inc. (Palo Alto, CA). The cAMP-sensitive reporter construct pSOMLuc was kindly donated by Dr. B. Peers (Liège, Belgium). pCMV5-rat GRK2, pCMV5-rat GRK3, pCMV5-rat ß-arrestin 1, and pCMV5-rat ß-arrestin 2 were generous gifts from Dr. R. J. Lefkowitz (Durham, NC) (37, 38). pCR-ratGRK5 and pCR-ratGRK5 antisense were kindly donated by Dr. Y. Nagayama (Nagasaki, Japan) (33), while pCB6-rat GRK6a and pCB6-rat GRK6b were previously described (17). The pCMV5-GRK2 antisense construct was generated by digesting pCMV5-GRK2 vector with BglII/HindIII, blunting the ends, inserting the GRK2 full-length cDNA into SmaI-linearized pCMV5 vector, and selecting a clone with the appropriate antisense orientation. The pCMV5-GRK3 antisense was constructed by cutting pCMV5-GRK3 with EcoRI. The 2.4-kb fragment was then ligated with EcoRI-linearized pCMV5. A clone displaying the appropriate antisense orientation was selected. The pCMV5-GRK6 antisense construct was obtained by cutting pBSSK-GRK6B with HindIII and EcoRI enzymes and inserting the fragment into the HindIII/EcoRI-digested pCMV5. The pRK-FSHR/3 plasmid was a kind gift from Dr. R. Sprengel (Heidelberg, Germany). The ß-arrestin 2 [319–418] expression vector was kindly donated by Dr. J. L. Benovic (Philadelphia, PA).

Cell Culture and Transfection
Mouse Ltk cell line permanently transfected with the rat FSH-R (Ltk 7/12 FSHR) was a kind gift from Dr. E. Nieschlag (Münster, Germany) (27). These cells were cultured in MEM supplemented with 10% heat-inactivated FCS and 200 mg Geneticin per 500 ml culture medium. Cos-7 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, glutamine, and antibiotics. Cells were maintained at 37 C in a humidified atmosphere of 5% CO2.

Cells, plated at a density of 2.5 x 105 per well in 12-well culture plates (Corning, Inc., Corning, NY), were transfected 24 h later with various quantities of the appropriate plasmid mixtures. The calcium phosphate precipitation method was used: 62 µl CaCl2 (2 M) were mixed with the constructs and the volume adjusted to 500 µl with water. A DNA precipitate was formed by adding this mixture dropwise to 500 µl HEPES-buffered saline (HBS), after which 120 µl of precipitate were added per well followed by incubation for 4 h. The transfection medium was then aspirated and 300 µl glycerol (15% in HBS) was added for 1 min. Cells were rinsed with MEM and were incubated without serum for 40 h. Alternatively, in some experiments, transfections were carried out using Transfast liposomes (Promega Corp., Madison, WI) according to the manufacturer’s instructions. Cells were stimulated with either 100 ng/ml FSH or saline for 4 h (without phosphodiesterase inhibitor), and then cells were then collected to determine luciferase activity. In all the experiments, the empty vector was added to keep the transfected DNA quantities constant. Cotransfections of the pSOMLuc reporter gene alone with the empty vector were used as control. Each transfection was repeated at least four times, with at least two different DNA preparations for each construct. Results were expressed in percentage as relative light unit values are directly related to cell density at the moment of transfection. Relative variations were highly reproducible among all experiments. Repeated transfections were performed to measure efficiencies of transfection between precipitates. The variabilities between precipitates were always lower than 25% (data not shown).

In some experiments, cells were trypsinized and divided in two equivalent pools: the first to measure luciferase activities and the second to determine the relative amounts of plasmid transfected. Briefly, frozen cell pellets were digested overnight at 37 C under gentle agitation in 100 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5 mM EDTA, 0.2% SDS, and 7.5 mg/ml of proteinase K. The digested mixtures were extracted by phenol-chloroform and were then digested with RNAse A for 1 h at 37 C. The different DNA samples were immobilized on Hybond-N+ (Amersham Pharmacia Biotech) membranes using a slot blot apparatus and standard denaturation/neutralization protocols. The membranes were hybridized with a random priming labeled cDNA probe corresponding to Photinus pyralis luciferase coding sequence. Prehybridizations and hybridizations were performed for 1 and 16 h, respectively, at 65 C in mixtures recommended by the manufacturer. Quantifications were done using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) after autoradiography using a phosphorscreen (Phosphorimager, Molecular Dynamics, Inc.).

RT-PCR Analysis of GRKs and ß-Arrestins
Total RNA was extracted from Ltk 7/12 cells by the single-step guanidium-phenol-chloroform method described by Chomczynski and Sacchi (29). The expression of the GRK2, -3, -5, and -6 and ß-arrestin 1 and 2 genes in Ltk 7/12 cells was studied using RT-PCR assays.

One microgram of total RNA was reverse-transcribed into single-strand cDNA by MMLV reverse transcriptase (Gibco BRL Europe, Ghent, Belgium). Reverse transcription was carried out in a 50 µl reaction volume 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 Biotech), and 200 U of reverse transcriptase. After completion of RT (45 min at 42 C), the temperature was raised to 70 C for 15 min to inactivate the enzyme.

The reverse transcribed first-strand cDNAs were amplified with a set of primer pairs designed to specifically amplify parts of the different GRK and ß-arrestin transcripts: GRK2 sense primer (5'-TCCAGTCGGTGGAAGAGACACA-3') and antisense primer (5'-GCTGAATCAGTGGCACCTTGCT-3') corresponded to bases 1960–1981 and 2169–2190 of the rat GRK2 cDNA, respectively. GRK3 sense primer (5'-CATGTCTGTGGAGGAGACCCAA-3') and antisense primer (5'-CAGATGAATATTCAATTCCAC-3') corresponded to bases 1821–1842 and 2050–2072 of the rat GRK3 cDNA, respectively. GRK5 sense primer (5'-CAAGGAGCTGAATGTGTTCGGAC-3') and antisense primer (5'-GCTGCTTCCAGTGGAGTTTGAAT-3') corresponded to bases 1746–1768 and 1931–1953 of the rat GRK5 cDNA, respectively. GRK6 primers have been already described and they generated a 252-bp insert (15). ß-Arrestin1 sense primer (5'-GTCAAAGTGAAGCTGGTGGTGTC-3') and antisense primer (5'-CCATCATCCTCTTCGTCCTTGTC-3') corresponded to bases 1002–1024 and 1239–1262 of the rat ß-arrestin 1 cDNA, respectively. ß-Arrestin 2 sense primer (5'-TACAGGGTCAAGGTGAAGCTGGT-3') and antisense primer (5'-GGTCATCACAGTCGTCATCCTTC-3') corresponded to bases 1158–1180 and 1391–1413 of the rat ß-arrestin 2 cDNA, respectively.

PCRs were carried out on 4 µl of RT in 100 µl reaction volumes 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 [{alpha}-32P]dCTP, and 1.25 U of Taq polymerase (Pharmacia Biotech). 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); the last extension was for 10 min at 72 C. The DNA fragments were separated by electrophoresis through 2% agarose gels. The gels were fixed in 10% acetic acid, dried, and submitted to x-ray film autoradiography.

Immunoblotting
Ltk 7/12 cells were washed twice in PBS and homogenized at 0 C in lysis buffer (10 mM Tris-Cl, pH 7.4, 2 mM EDTA with the following protease inhibitors: 0.1 mM phenylmethylsulfonyl fluoride; 10 µg/ml benzamidine, leupeptin, and soybean trypsin inhibitor; 5 µg/ml aprotinin; 1 µg/ml pepstatin A). The supernatants of a 1000 x g spin were subsequently centrifuged for 1 h at 150,000 x g. The supernatants of this second centrifugation were designated as the cytosolic fractions, while the membrane fractions were the 150,000 x g pellets resuspended in lysis buffer. Cytosolic and/or membrane fractions were applied to 10% SDS-polyacrylamide gels. Colored mol wt markers (Amersham Pharmacia Biotech) were simultaneously loaded on the gels. Membrane or cytosol extracts from HEK 293 cells transfected with a single GRK or arrestin were used as positive controls. Western blots were prepared by electrophoretically transferring the proteins (2 h at 250 mA using 25 mM Tris, 192 mM glycine, pH 8.3, and 20% methanol) onto polyvinylidene fluoride (PVDF) transfer membranes (NEN Life Science Products, Boston, MA). The membranes were blocked for 2 h at room temperature with buffer A (Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20, 5% instant nonfat dry milk). Next, they were incubated for 2 h at room temperature in buffer A containing immune serum. Rabbit polyclonal antibody raised against GRK2 (epitope: amino acids 675–689 of human GRK2) and the corresponding control peptide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The GRK2/GRK3 monoclonal antibody C5/1, raised against the C terminus of GRK2, was kindly donated by Dr. R. J. Lefkowitz. Rabbit polyclonal antibodies raised against GRK5 (N-terminal peptide) and GRK6 (C-terminal peptide) were kind gifts from Dr. F. Boulay. The monoclonal antibody F4C1, raised against the highly conserved epitope DGVVLVD, identical in ß-arrestins, was kindly provided by Dr. L. A. Donoso. After this, each membrane was washed three times for 5 min with buffer B (Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). The blots were incubated for 1 h in buffer A containing a peroxidase-conjugated second antibody. Each was then washed three times for 5 min with buffer B and once for 5 min with buffer C (Tris-HCl, pH 8.0, 150 mM NaCl). Finally, the blots were developed using the ECL chemiluminescence detection system (Amersham Pharmacia Biotech).

Cross-Hybridization Assay
The full-length GRK2, -3, -5, and -6A cDNAs were subcloned in pBluescript SK, and each was linearized at both ends in the polylinker. Complementary RNAs (cRNAs) corresponding to the different GRKs were then synthesized in both sense and antisense orientations using T3 and T7 RNA polymerases (Promega Corp.). The antisense cRNAs were synthesized in the presence of [{alpha}-32P]CTP (NEN Life Science Products) while the sense cRNAs were unlabeled.

The sense cRNAs were denatured at 50 C for 15 min in 10x SSC (1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) and 5% formaldehyde (vol/vol). The material was applied to nitrocellulose filters (Schleicher & Schuell, Inc., Dassel, Germany) in serial 2-fold dilutions using a slot blot apparatus. The filters were baked 2 h at 80 C and were then hybridized with one of the purified antisense probes (15 106 cpm). Prehybridizations and hybridizations were performed for 2 and 16 h, respectively, at 60 C in the manufacturer’s recommended mixtures. Quantifications were done using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) after autoradiography using a phosphorscreen.

Sorting and Analysis of Antisense Transfected Cells
Ltk 7/12 cells were transiently cotransfected with both an expression vector for GFP (CLONTECH Laboratories, Inc. Palo Alto, CA) and one of the antisense constructs described above. Transfast liposomes were used in a 1:1 ratio; 2.5 µg of GFP expression vector and 40 µg of either pCMV5 or antisense construct were used for each 75-cm2 culture flask. After 48 h, GFP positive living cells were than selected using a FACStar Plus cell sorter (Beckton Dickinson and Co., Franklin Lakes, NJ). To avoid contaminations with untransfected cells, only 15–20% of cells presenting the more intense GFP signal (~50% of the cells were GFP positive) were sorted for subsequent analysis. About 3 x 105 cells were obtained for each condition. Whole-cell extracts were prepared using the lysis buffer described above plus 0.5% NP40. Solubilization was carried out for 1 h on ice. The extracts were then cleared by centrifugation, and the amount of protein was determined. Twenty micrograms of the different extracts were loaded on gels and blotted as described above. The blots were stained with Coomassie blue and scanned to allow quantifications. The densitometric measurement of the blots was achieved using NIH Image software.

cAMP Assay
For cAMP assay, 2-day cultured cells were stimulated by FSH for various times without phosphodiesterase inhibitor. Cells were frozen and lysed and the intracellular cAMP content was determined. The intracellular cAMP content was assayed using a RIA kit (Sanofi Pharmaceuticals, Inc., Pasteur, France).

Luciferase Activity Measurement
The luciferase activity was measured using the luciferase assay system supplied by Promega Corp. Briefly, cells were scraped in 100 µl of reporter lysis buffer. The cell lysate was centrifuged (12,000 x g, 2 min, 4 C), and the supernatant was collected. Each sample (40 µl) was mixed with 100 µl of luciferase assay reagent, containing the substrate. The light produced was measured in a luminometer (Lumat LB 9507, EG&G Berthold, Turku, Finland) and expressed in relative light units.

Statistics
Statistical analysis of the data was performed using a single mean Student’s t test (Statview, Abacus Concepts, Berkeley, CA).


    ACKNOWLEDGMENTS
 
We thank Drs. R. J. Lefkowitz (Durham, NC), J. L. Benovic (Philadelphia, PA), E. Nieschlag (Münster, Germany), Y. Nagayama (Nagasaski, Japan), B. Peers (Liège, Belgium), R. Sprengel (Heidelberg, Germany), and F. Boulay (Grenoble, France) for their kind gifts of materials. We are grateful to Drs D. Kerboeuf and Y. Le Vern (Nouzilly, France) for cell sorting analysis. Thanks are also due to Mrs. C. Barc and Mr. S. Marion for technical assistance with the Western blot analyses and to Mr. A. Beguey for the photography.


    FOOTNOTES
 
Address requests for reprints to: Eric Reiter, INRA/CNRS URA 1291, Station de Physiologie de la Reproduction des Mammifères Domestiques, 37380 Nouzilly, France.

Carine Troispoux was recipient of a doctoral fellowship from the Ministère de la Recherche et de l’Education Nationale.

Received for publication June 24, 1998. Revision received May 27, 1999. Accepted for publication June 2, 1999.


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 DISCUSSION
 MATERIALS AND METHODS
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