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Molecular Endocrinology, doi:10.1210/me.2006-0098
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Molecular Endocrinology 20 (11): 3014-3026
Copyright © 2006 by The Endocrine Society

A Phosphorylation Cluster of Five Serine and Threonine Residues in the C-Terminus of the Follicle-Stimulating Hormone Receptor Is Important for Desensitization But Not for ß-Arrestin-Mediated ERK Activation

Elodie Kara, Pascale Crépieux, Christophe Gauthier, Nadine Martinat, Vincent Piketty, Florian Guillou and Eric Reiter

Institut National de la Recherche Agronomique, Unité Mixte de Recherche 6175 Institut National de la Recherche Agronomique/Centre National de Recherche Scientifique/Université de Tours/Haras Nationaux/Institut Fédératif de Recherche 135, 37380 Nouzilly, France

Address all correspondence and requests for reprints to: Eric Reiter, Unité Mixte de Recherche 6175, 37380 Nouzilly, France. E-mail: reiter{at}tours.inra.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein {alpha} S subunit (G{alpha}s) and activation of the cAMP/protein kinase A (PKA) signaling pathway. ß-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on G{alpha}s/PKA and ß-arrestins. G{alpha}s/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of ß-arrestins. ß-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638–644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced ß-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the ß-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for ß-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a ß-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of ß-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated ß-arrestin-dependent ERK activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-ARRESTINS PLAY A central role in seven membrane-spanning receptor (7TMR) signal transduction. Upon agonist stimulation, 7TMRs recruit ß-arrestins that both uncouple receptors from heterotrimeric guanine nucleotide-binding proteins (G proteins) and target them to clathrin-coated pits for endocytosis (1). In addition, ß-arrestins act as scaffolds: they specifically interact with a growing number of proteins that connect the activated receptors with various signaling pathways (2). So far, the most extensively studied ß-arrestin-dependent signaling system has been the activation of MAPKs such as ERK1 and ERK2. For many 7TMRs like angiotensin type 1a receptor (AT1aR) (3) or protease-activated receptor 2 (4), agonist treatment induces the formation of a signaling module containing the receptor and ß-arrestin together with a MAPK kinase kinase, a MAPK kinase, and a MAPK. Interestingly, for the AT1aR and vasopressin V2 receptor (V2R), it has been recently demonstrated that both G protein and ß-arrestin contribute to the overall ERK signal via two distinct pathways that have different kinetics: a rapid and transient G protein-dependent phosphorylation of ERK, and a slower but sustained ß-arrestin 2-dependent activation (5, 6).

ß-Arrestin binding to activated receptors is regulated by two driving forces: 1) the capacity to interact specifically with receptors in an activated conformation; and 2) the agonist-driven G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor on Ser/Thr residues (7). Moreover, the pattern of phosphorylated serine and threonine residues within the carboxy-terminal domain is presumably key in controlling the avidity of ß-arrestin binding. According to the presence or absence of a Ser/Thr cluster in the carboxy-terminal tail, the receptors are divided into two classes (7, 8). The motif is absent in class A receptors such as ß2 and {alpha}1b adrenergic receptors. These receptors recruit preferentially ß-arrestin 2, and their interaction is not stable because it dissociates at or near the plasma membrane. The cluster of Ser/Thr is present in class B receptors such as V2R or AT1aR. They form complexes with both ß-arrestin 1 and 2 that persist into endocytic vesicles inside the cell after 7TMR internalization.

The FSH-R is a 7TMR specifically expressed in Sertoli cells in male and in granulosa cells in female. This receptor preferentially couples to G{alpha}s and activates the cAMP/PKA signaling pathway. The FSH-R is also known to be phosphorylated by GRKs and to recruit ß-arrestins upon agonist stimulation (9). In cells stably expressing the FSH-R, as well as in cultured primary Sertoli cells, overexpression of various GRKs and ß-arrestins decreases the FSH-induced cAMP generation (10, 11). This receptor also activates ERK 1/2 in its natural target cells (12, 13). It is not currently known whether ß-arrestin contributes to ERK activation via the FSH-R.

Deletion of amino acids 642 to 651 (642SATHNFHARK651) in the carboxy-terminal tail of the rat FSH-R increases internalization and enhances ß-arrestin 2 binding affinity, suggesting that this sequence in the full-length recepetor inhibits the binding of ß-arrestin 2 (14). This result highlights the importance of the carboxy-terminal tail of the FSH-R in ß-arrestin binding. However, previous studies also suggest that the carboxy-terminal tail of the FSH-R is not phosphorylated upon agonist stimulation (15, 16).

Using sequence alignment, we found a putative class B cluster of two Ser and three Thr located in the carboxy-terminal tail of the FSH-R. In the present study, we have investigated the role of this Ser/Thr cluster in ß-arrestin binding and how it impacts on desensitization, internalization, and ERK activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH-R induces ERK 1 and 2 phosphorylation via two distinct pathways
We first investigated whether ERK activation by the FSH-R was due to a dual mechanism involving both G protein and ß-arrestin-dependent pathways. To highlight a potential G protein-dependent component in ERK activation by this receptor, we have inhibited PKA, which reflects G{alpha}s coupling to the FSH-R and cAMP generation, using the chemical inhibitor H89 (6, 17). To determine the proper H89 concentration in our cellular system, we carried out dose-inhibition experiments. The PKA-dependent, forskolin-induced ERK response was profoundly inhibited after 15-min pretreatment with 20 µM H89. In the same conditions, no significant inhibition of PKC-dependent, phorbol myristate acetate-mediated ERK was observed (data not shown). In parallel, we specifically depleted endogenous ß-arrestin 1 or 2 in HEK 293 cells using previously validated small interfering RNA (siRNA) (18). We also used another siRNA that targets a sequence common to ß-arrestin 1 and 2 (19). The efficiency of endogenous ß-arrestin depletion was monitored by Western blotting using an antibody recognizing both ß-arrestins (Fig. 1AGo).


Figure 1
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  		Fig. 1. Effects of PKA Inhibitor and siRNA-Mediated Suppression of ß-Arrestin 1 and/or 2 Expression on the Kinetic Pattern of ERK Activation after Stimulation of the wt FSH-R

HEK 293 cells were transfected simultaneously with the Flag-FSH-R-encoding plasmid and the indicated siRNAs. Three days after transfection, cells were serum-starved for at least 6 h, pretreated for 15 min with vehicle or H89 (20 µM), and then stimulated with 1 nM FSH at 37 C for the indicated periods. After stimulation, cellular extracts were prepared as described in Materials and Methods. Equal amounts of protein (~10 µg) in each sample were used to visualize expression of endogenous ß-arrestins (A), phosphorylated ERK (pERK), total ERK, or GAPDH by immunoblotting (B). C–F, ERK signals were quantified by densitometry and expressed as a ratio of activated over total ERK or GAPDH. The maximal phosphorylation of ERK obtained in control (CTL) siRNA-transfected cells was arbitrarily chosen as 100%. Each data point represents the mean ± SEM from at least four independent experiments. ***, P < 0.001 compared with the entire control curve.

 
As expected, the wild-type FSH-R transiently overexpressed in HEK 293 cells was able to activate ERK 1/2 signaling upon FSH stimulation. Cells were treated with FSH for increasing time periods, and ERK 1/2 activation was revealed using an antibody directed against the phosphorylated ERK (Fig. 1BGo). ERK activation reached the maximum activation level at 5–10 min and appeared to be protracted because approximately 60% of the maximum was still present after 30 min of FSH exposure. When cells were pretreated with H89 (20 µM), FSH-induced ERK activation was dramatically decreased at 5 min (Fig. 1Go, B and C). Interestingly, H89 had only a modest effect at 10 and 30 min of FSH stimulation, suggesting that PKA activation is only required for the early phase of ERK activation by the FSH.

We then measured the impact of ß-arrestin 1 or 2 depletion on FSH-stimulated ERK. Remarkably, ß-arrestin 1 or 2 inhibition had only minor effects at 5 and 10 min on ERK phosphorylation while leading to a substantial decrease of the ERK signal after 30 min of stimulation (Fig. 1Go, B and D). No significant difference was observed between the effect of ß-arrestin 1 and 2 siRNA treatments. Moreover, combined treatment using ß-arrestin 1 or 2 siRNA and H89 led to almost complete inhibition of the ERK response to FSH (Fig. 1Go, B and E). The simultaneous depletion of both ß-arrestins led to a similar pattern of ERK response and sensitivity to H89 as the depletion of each single isoform (Fig. 1Go, B and F).

Taken together, these results demonstrate that the wild-type (wt) FSH-R activates ERK via two different pathways: one that occurs early, is transient and depends on G{alpha}s/PKA activation, whereas the other is slower in onset, sustained, and ß-arrestin 1 and 2 dependent. As a consequence, ß-arrestins are involved in FSH-R signal transduction, not only at the levels of desensitization and internalization but also, as we demonstrate here, because they are able to induce ERK signaling pathways.

Identification of a Ser/Thr cluster in the carboxy terminus of the FSH-R as a major phosphorylation site
To determine which molecular determinants of the FSH-R are important for ß-arrestin recruitment and functions, we have aligned the FSH-R with different class A and B 7TMRs. We found a putative class B cluster of two Ser and three Thr located in the carboxy-terminal tail of the FSH-R (Fig. 2Go).


Figure 2
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  		Fig. 2. Identification of a Putative Class B Cluster in the Carboxy-Terminal End of the Rat FSH-R

An alignment between rat FSH-R and different class A and class B receptor C-terminal sequences was performed. The C-terminal sequences were aligned with respect to the conserved NPxxY motif (boxed residues). The Thr and Ser residues are in bold, whereas experimentally confirmed class B clusters are underlined. Thr 638, Thr 640, Ser 641, Ser 643, and Thr 644 were mutated into alanines by site-directed mutagenesis to generate the 5A FSH-R. NTR-1, Neurotensin receptor 1; OTR, oxytocin receptor; PAR2, protease-activated receptor 2; ß2 adrenergic receptor (ß2AR), {alpha}1b adrenergic receptor ({alpha}1bAR).

 
To evaluate the role of this cluster in regulating ß-arrestin recruitment to the receptor, Thr 638, Thr 640, Ser 641, Ser 643, and Thr 644 were mutated to alanines (5A FSH-R) (Fig. 2Go). The functionality of the 5A FSH-R has been compared with its wt counterpart using binding assays with 125I-porcine FSH (pFSH) on transiently transfected HEK 293 cells. We found no differences in the displacement curves for the two receptors (Fig. 3AGo). The determined IC50 were 3 nM for the wt and 5A FSH-R. To further compare the two receptors, Scatchard plots were calculated (Fig. 3Go, B and C). The dissociation constant (Kd) values were almost identical and in good agreement with the IC50 (2 nM for wt and 5A FSH-R). Together, these results clearly show that the five-point mutations introduced in the FSH-R do not modify its plasma membrane location or its affinity for FSH.


Figure 3
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  		Fig. 3. Pharmacological Characterization of 5A FSH-R

Binding parameters were measured on intact cells transiently transfected with the wt or 5A FSH-R. Incubations were carried out for 1 h at 37 C in the presence of 125I-pFSH and increasing concentrations of unlabeled pFSH (3.10–11 to 10–7 M). Triplicates were done for each concentration of pFSH. A, The displacement curve represents the mean of three different experiments ± SEM. B, C, Equilibrium binding parameters were calculated from the binding data using Scatchard analysis.

 
Next, we compared agonist-induced phosphorylation of the wt vs. 5A FSH-R. All the subsequent experiments reported here have been done with cells expressing equivalent amounts of wt or 5A FSH-R at the plasma membrane. Autoradiographic analysis revealed that, after 5 min of stimulation with 1 nM FSH, the agonist-induced phosphorylation of the 5A FSH-R was diminished by about 60% when compared with the wt FSH-R (Fig. 4Go, A and B). The averaged basal phosphorylation levels were similar in cells expressing wt or 5A FSH-R (data not shown).


Figure 4
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  		Fig. 4. The Ser/Thr Cluster Present in the C terminus of the FSH-R Is a Major Phosphorylation Site

HEK 293 cells were transiently transfected with either wt or 5A FSH-R encoding plasmids. Cells were metabolically labeled with 32Pi and stimulated for 5 min with 1 nM FSH. The wt or 5A receptor was immunoprecipitated and separated on SDS-PAGE. A, A representative autoradiograph is shown. B, Receptor phosphorylation levels were quantified for four individual experiments ± SEM. Signals were expressed as a percentage of wt FSH-R phosphorylation level in FSH-treated cells. ***, P < 0.001 compared with wt FSH-R.

 
The Ser/Thr cluster in the C terminus of the FSH-R is important for ß-arrestin 1 and 2 recruitment
To investigate whether the Ser/Thr-rich motif present in the carboxy-terminal tail of the FSH-R was involved in ß-arrestin recruitment, we have expressed similar amounts of wt or 5A FSH-R at the plasma membrane of HEK 293 cells. By immunoprecipitating the FSH-R with anti-Flag affinity gel, we observed that, upon 5 and 15 min of FSH stimulation, endogenous ß-arrestin 1 and 2 were robustly recruited to the wt FSH-R. A faint nonspecific band that comigrated with ß-arrestin 1 was systematically visible in the nonstimulated conditions as well as in negative controls (i.e. Flag beads alone; data not shown). Interestingly, after a 5-min stimulation, only a minor and not statistically significant difference in ß-arrestin recruitment was found between the wt and 5A FSH-R, whereas after 15-min stimulation, we measured a substantial decrease in ß-arrestin recruitment to the 5A FSH-R ({approx} 80%) when compared with the wt receptor (Fig. 5Go, A and B). This result suggests that the 5A cluster stabilizes ß-arrestin interaction with the receptor.


Figure 5
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  		Fig. 5. The Ser/Thr Cluster Present in the C Terminus of the FSH-R Is Important for ß-Arrestin 1 and 2 Recruitment

HEK 293 cells transiently expressing wt or 5A FSH-R were stimulated with 1 nM pFSH for 5 or 15 min. The wt or 5A FSH-R was immunoprecipitated, and the complexes were separated by SDS-PAGE. A, A representative experiment is shown. Endogenous ß-arrestins were revealed using A1CT antibody that recognizes both ß-arrestin 1 and 2. B, Signals were quantified by densitometry and expressed as a percentage of ß-arrestin 1 and 2 recruitment in wt FSH-R transfected cells upon FSH stimulation. Data correspond to the mean ± SEM from four independent experiments. *, P < 0.05 compared with wt FSH-R.

 
Internalization of the 5A FSH-R is reduced
ß-Arrestins are involved in the targeting of receptors to clathrin-coated pits, and this pathway constitutes the predominant internalization mode of 7TMRs (20, 21). Because the 5A FSH-R has a decreased ability to bind ß-arrestins, it could be anticipated that ß-arrestin-dependent internalization would be altered. We have measured the 125I-FSH internalization at 5, 15, 60, and 120 min of FSH stimulation. We observed a slight but reproducible reduction of 5A FSH-R internalization when compared with the wt FSH-R, and the kinetics remained unchanged (Fig. 6Go).


Figure 6
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  		Fig. 6. The Ser/Thr cluster is involved in the control of FSH-R internalization

Internalizations of wt and 5A FSH-R were compared using 125I-FSH binding and acid washes on whole cells at 37 C according to the procedure described in Materials and Methods. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicates. The percentage of internalized receptor is calculated as the ratio of surface-bound 125I-pFSH to the surface-bound plus intracellular 125I-pFSH. *, P < 0.05 compared with the entire control curve.

 
Desensitization of the 5A FSH-R is impaired
The FSH-R is coupled to G{alpha}s and triggers rapid cAMP accumulation. We have compared FSH-stimulated cAMP production of the wt vs. 5A FSH-R. First, dose/response experiments were carried out. Transfected cells were stimulated for 30 min with increasing FSH concentrations in the presence of isomethylbutylxanthine (IBMX) (100 µM). Similar EC50 values were measured for the two receptors in the 0.5 nM range, which is fairly consistent with our calculated Kd and IC50 (Fig. 3AGo). Interestingly, the maximum cAMP accumulation level was significantly higher (~40% increase) for the 5A FSH-R than for its control counterpart (Fig. 7AGo). To further characterize the cAMP response to the 5A FSH-R, cells were stimulated with 3 nM pFSH in the presence of IBMX for increasing time periods to determine the kinetics of cAMP production. Basal levels were similar and cAMP accumulation reached a maximum level after 5 min of FSH exposure for wt FSH-R. Interestingly, with the 5A FSH-R, cAMP continues to accumulate up to 30 min, indicating that this mutant receptor has a significantly slower desensitization kinetics. Moreover, the 5A FSH-R-stimulated maximum cAMP accumulation level was also significantly enhanced when compared with the wt FSH-R (Fig. 7BGo). Together, these results suggest that the 5A FSH-R is less desensitized than the wt control.


Figure 7
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  		Fig. 7. Comparison of the dose-responses and kinetics of cAMP accumulation upon FSH stimulation of wt vs. 5A FSH-R

HEK 293 cells were transiently transfected with either wt or 5A FSH-R expression plasmids. A, Cells were incubated for 30 min with increasing FSH concentrations (3.10–11 to 10–8 M) in the presence of IBMX (100 µM). B, Cells were stimulated with 3 nM FSH for increasing time periods. Total cAMP levels were measured as described in Materials and Methods, normalized with forskolin stimulation, and expressed as a percentage of the maximum response with wt FSH-R. Results are expressed as mean ± SEM of at least two individual experiments performed in triplicate. *, P < 0.05; ***, P < 0.001% compared with wt FSH-R.

 
The ability of the 5A FSH-R to activate ERK via the ß-arrestin-dependent pathway is enhanced
Because the FSH-R is able to activate MAPK ERK 1 and 2 through a ß-arrestin-dependent mechanism, we sought to compare the ability of the 5A and wt FSH-R to activate ERK phosphorylation. To allow such comparisons, cells expressing similar amounts of either wt or 5A FSH-R at the plasma membrane were simultaneously treated, stimulated, and analyzed on the same blots as described in Materials and Methods. Upon FSH stimulation, we found that the 5A FSH-R induced significantly stronger ERK phosphorylation than the wt receptor (Fig. 8Go, A and B). We expected the 5A receptor-mediated ERK activation to be more G{alpha}s-dependent than its wt counterpart because it produced more cAMP than the wt receptor. In contrast, we postulated that the ß-arrestin-dependent activation of ERK via the 5A FSH-R should be impaired based on its reduced ability to recruit ß-arrestins upon FSH stimulation. Surprisingly, however, siRNA-mediated depletion of either ß-arrestin 1 or 2 in cells expressing the 5A FSH-R induced a significant inhibition of ERK activation upon FSH stimulation (Fig. 8Go, A and C). Both ß-arrestin 1 and 2 siRNA had similar effects on ERK activation by the 5A FSH-R. At 5 min of FSH exposure, a weak reduction in the ERK activation level was observed for ß-arrestin 1 or 2 siRNA-treated cells when compared with control siRNA. After 10 min of FSH treatment, ERK activation level in ß-arrestin 1 or 2-depleted cells was at 62% compared with 5A FSH-R cotransfected with control siRNA (Fig. 8Go, A and C). At 30 min of FSH stimulation, the difference between control and ß-arrestin 1 or 2 siRNA-treated cells was about 35% of the 5A FSH-R control siRNA. In cells pretreated with the PKA inhibitor H89, ERK activation by the 5A FSH-R was clearly decreased at 5 min and more weakly at 10 and 30 min (Fig. 8Go, A and D). In cells depleted in ß-arrestin 1 or 2, treatment with H89 led to a more drastic inhibition than without H89, confirming that the ERK signals that are resistant to ß-arrestin siRNA treatment are actually corresponding to G{alpha}s-mediated ERK activation (Fig. 8Go, A and D).


Figure 8
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  		Fig. 8. 5A FSH-R-Mediated PKA- and ß-Arrestin-Dependent Pathways for ERK Activation Are Enhanced when Compared with the wt FSH-R

HEK 293 cells were transiently transfected with either wt or 5A FSH-R encoding plasmid and the indicated siRNAs simultaneously. Three days after transfection, cells were serum-starved for at least 6 h and then pretreated with vehicle only (vehicle, CTL) or 20 µM H89 for 15 min and stimulated with FSH (1 nM) for the indicated time. After stimulation, cellular extracts were prepared as described in Materials and Methods. Equal amounts of protein (~10 µg) in each sample were used to visualize phosphorylated ERK (pERK) and total ERK by immunoblotting. A, Representative blots are shown for time-course stimulation of cells transfected with CTL and ß-arrestin 1 or 2 siRNAs. B–F, Content of pERK in each lane was quantified by densitometry and normalized according to total ERK signals. The maximal phosphorylation of ERK obtained in control (CTL) siRNA-transfected cells was arbitrarily chosen as 100%. For clarity, some of the wt receptor data presented in Fig. 1Go are compared in Fig. 8Go, B, D, and E, with the values measured with 5A FSH-R. Each data point represents the mean ± SEM from four independent experiments. *, P < 0.05; ***, P < 0.001 compared with the entire control curve.

 
Unexpectedly, when the H89-resistant (i.e. ß-arrestin-dependent) ERK phosphorylation levels were compared side by side, it appeared that the 5A FSH-R was activating significantly more ß-arrestin-dependent ERK than the wt receptor (Fig. 8EGo). The comparison of wt vs. 5A FSH-R-mediated ERK responses generated in ß-arrestin 1- or 2-depleted cells (i.e. G{alpha}s-dependent) revealed that the 5A FSH-R had prolonged ERK phosphorylation (Fig. 8FGo). This latter observation is consistent with the 5A FSH-R-mediated cAMP response being less desensitized over time than the wt control. Overall, the higher activation level of ERK by the 5A FSH-R seems to be due to an increase in both G{alpha}s- and ß-arrestin-dependent activation pathways. Proportionally, the ß-arrestin-dependent mechanism seems to be the main contributor to the enhanced ERK response of the 5A FSH-R.

Modulation of ß-arrestin-dependent ERK activation by different GRK subtypes reveals 5A FSH-R insensitivity to GRK2 depletion
Phosphorylation of agonist-activated 7TMRs by GRKs has been largely accepted as a molecular signature for ß-arrestin recruitment. To investigate what their role in the biphasic ERK response to FSH could be, we specifically depleted endogenous GRK2, -3, -5, and -6 in HEK 293 cells using previously validated siRNA (6, 22). The efficiency and specificity of depletion was monitored for each GRK by Western blotting (Fig. 9AGo).


Figure 9
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  		Fig. 9. GRK5 and -6 Are Required for ß-Arrestin-Dependent ERK Activation by Both wt and 5A FSH-R, whereas GRK2 Depletion Enhances ERK Activation by the wt But Not by the 5A FSH-R

HEK 293 cells were transiently transfected with either wt or 5A FSH-R encoding plasmid and the indicated siRNAs simultaneously. Three days after transfection, cells were serum-starved for at least 6 h and then stimulated with 1 nM FSH at 37 C for the indicated periods. Equal amounts of protein (~10 µg) in each sample were used to monitor siRNA effects on the expression of endogenous GRKs (A), phosphorylated ERK (pERK) and total ERK by immunoblotting (B). C–F, ERK signals were quantified by densitometry and expressed as a ratio of activated over total ERK. The maximal phosphorylation of ERK obtained in control (CTL) siRNA-transfected cells was arbitrarily chosen as 100%. Each data point represents the mean ± SEM from at least four independent experiments. ns, Not significant. **, P < 0.01; ***, P < 0.001, compared with the entire control curve.

 
We compared how depletion of the different GRKs impacted on FSH-stimulated ERK activation by the wt and 5A FSH-R. GRK3 depletion induced no significant change with either receptor (Fig. 9Go, B, C, and E). Interestingly, GRK2 inhibition had a significant stimulatory effect on ERK phosphorylation by the wt FSH-R while inducing no change in 5A FSH-R-mediated ERK signal (Fig. 9Go, B, C, and E). By contrast, GRK5 and GRK6 were both necessary to elicit ß-arrestin-dependent ERK activation by wt as well as 5A FSH-R (Fig. 9Go, B, D, and F). These results are in agreement with the notion that GRK2 and GRK5/6 exert opposite regulation of ß-arrestin-dependent ERK response as recently reported for the AT1aR and V2R (6, 22). Together, these data suggest that the 5A cluster might preferentially be phosphorylated by GRK2, and that GRK5 and GRK6 are required for ß-arrestin-dependent ERK activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
ß-Arrestins have long been known to play crucial roles in the desensitization and internalization of many 7TMRs, including the FSH-R (1, 10, 11). In addition, ß-arrestins have recently emerged as scaffolding proteins with the unique property to bring signaling modules to agonist-occupied receptors (2). The ability of ß-arrestins to assemble and activate ERK signaling modules including MAPK kinase kinase, MAPK kinase, and MAPK has been extensively studied (2, 3). Interestingly, it has been demonstrated for several 7TMRs, including AT1aR (5), V2R (6), and ß2 adrenergic receptor (ß2AR) (5, 17), that the ERK signaling pathway results from the coordinate activation of a G protein-dependent mechanism and a ß-arrestin-dependent mechanism with distinct temporal and spatial characteristics (4, 5, 23, 24). In the present study, we show that ERK activation by the FSH-R involves a similar bimodal activation pattern: a rapid and transient G{alpha}s/PKA-dependent ERK mechanism and a delayed but protracted ß-arrestin 1 and 2-dependent pathway. The involvement of G proteins in ERK activation by the FSH-R had previously been demonstrated (13), but we report here, for the first time, the involvement of ß-arrestins in FSH-mediated ERK activation. Interestingly, ERK activation by the FSH-R depends on both ß-arrestin 1 and 2, indicating that one ß-arrestin cannot replace the other. A similar observation has been recently reported for the ß2AR (17) and PTH-R (18), whereas ß-arrestin 1 and 2 reciprocally regulate ERK activation by the AT1aR (5). One possible explanation is that FSH-R and ß2AR-recruited ß-arrestins might act as heterodimers to activate the ERK pathway. Indeed, Storez et al. (25) recently demonstrated that ß-arrestins can form heterodimers in living cells.

This newly appreciated ability to activate the ERK pathway upon FSH stimulation reinforces the crucial role played by ß-arrestins in FSH-R biology. As a consequence, it is very important to understand the molecular mechanisms controlling FSH-R/ß-arrestin interaction. A short linear sequence (642–651) present in the C-terminal tail of the rat FSH-R has been reported to negatively regulate ß-arrestin 2 recruitment in a phosphorylation-independent fashion (14). However, nothing was known about the motif(s) required for ß-arrestin recruitment to the FSH-R. For other 7TMRs, Oakley et al. (7) have identified a cluster of Ser/Thr in the C-terminal tail that is correlated with a higher binding affinity for ß-arrestins. Class A receptors do not have such a motif; they transiently recruit ß-arrestins and have a marked preference for ß-arrestin 2. In contrast, class B receptors have the cluster, recruit ß-arrestin 1 and 2 equally well, and form stable complexes with these proteins. We identified a putative class B cluster in the C-terminal region (Thr 638, Thr 640, Ser 641, Ser 643, and Thr 644) of rat FSH-R of which the first four residues are highly conserved from human to reptiles (data not shown). In the current study, we provide evidence that this motif might be a functional class B cluster. First, we have observed that the wt FSH-R significantly recruits ß-arrestin 1 in addition to ß-arrestin 2. Second, the 5A FSH-R mutant with five alanines instead of the original Ser and Thr residues within the putative class B motif showed a reduced ability to stably interact with ß-arrestins when compared with the wt FSH-R. The substantial decrease in FSH-mediated phosphorylation of the 5A when compared with the wt receptor is also consistent with data reported by Oakley et al. (7) showing that class B clusters are major phosphorylation sites. To further confirm that the FSH-R belongs to the class B 7TMRs, we tried to visualize ß-arrestin-GFP recruitment to the FSH-R. Unfortunately, although we were able to coimmunoprecipitate endogenous ß-arrestins with the FSH-R, all our attempts to visualize ß-arrestin-GFP recruitment to the activated FSH-R failed. Because there is a competition in the recruitment assays between endogenous ß-arrestins and transfected ß-arrestin-GFP, relatively high receptor expression levels are a prerequisite for this kind of experiments. For unknown reasons, it was impossible to express high levels of FSH-R at the plasma membrane of transfected cells. As a consequence, we cannot formally conclude that the FSH-R is a class B receptor.

The finding that the FSH-R is phosphorylated on its C-terminal region upon FSH stimulation is discordant with Hipkin et al. (15). Those authors reported that the FSH-R was not phosphorylated in the C-terminal tail but rather in the first and third intracellular loops in response to FSH stimulation. They found similar phosphorylation levels between a truncated FSH-R mutant with no C-terminal tail and the wt receptor upon FSH treatment. Truncation of the C-terminal tail is a very drastic modification of the receptor and might have led to a profound conformational change of the receptor. As a consequence, an increased accessibility of phosphorylation sites within the intracellular loops might explain this discrepancy with our results.

Two polymorphisms have recently been described in the C-terminal tail of the human FSH-R that respectively substitute (T665A) or create (N680S) putative phosphorylation sites (26, 27). Interestingly, significant changes in FSH responsiveness are associated with the presence or the absence of a Ser at position 680 in normal or subfertile women (28, 29). In that context, the observation that the FSH-R is phosphorylated in the C-terminal tail upon FSH stimulation is of particular interest. Indeed, it raises the possibility that Thr 665 and/or Ser 680 can be phosphorylated when they are present and provides a potential mechanism to explain the observed phenotypes. Further studies will be required to test this hypothesis.

The Ser/Thr cluster also plays a role in FSH-R internalization. It has been shown that ß-arrestins act as clathrin adaptors during endocytosis (20). Because we show that the ability of the 5A FSH-R to stably associate with ß-arrestins is reduced, we can postulate that the internalization would be altered because the targeting of receptors to clathrin-coated pits is diminished. This hypothesis is supported by previous data showing that a ß-arrestin 319–418 peptide that sequesters clathrin inhibits FSH-R internalization (9). However, the overall inhibitory effects observed in the present study are of modest amplitude, and the kinetics of internalization are the same for both 5A and wt receptors. This is not surprising for several reasons: 1) the 5A FSH-R was still able to recruit significant amounts of ß-arrestins at 5 min of agonist stimulation; 2) FSH-R internalization is not entirely ß-arrestin-dependent and can also be achieved through uncoated vesicles (9); and 3) removal of class B clusters in various 7TMRs has been shown to transform them into class A receptors. Class A receptors transiently interact with ß-arrestins near the cell surface but traffic alone into deeper endosomal compartments (8).

According to our results, the 5A FSH-R constitutes an appropriate model to study the importance of ß-arrestin recruitment in ERK activation. Surprisingly, the decrease of ß-arrestin recruitment to the 5A FSH-R did not diminish but actually enhanced the ß-arrestin-dependent ERK signal compared with the wt FSH-R. This finding suggests that two functionally distinct pools of ß-arrestins are recruited to the FSH-R upon FSH stimulation. We postulate that the predominant ß-arrestin subpopulation interacts with the phosphorylated 638–644 cluster, is involved in both desensitization and internalization, and exerts a negative role on the ERK activated via the ß-arrestin-dependent mechanisms. A distinct ß-arrestin subpopulation is required for ß-arrestin-mediated ERK activation. This second pool of ß-arrestins does not interact with the 638–644 Ser/Thr cluster, but interacts with other, yet to be identified, residues. Moreover, the fact that the 5A FSH-R led to increased ß-arrestin-dependent ERK activation level suggests that the two pools of ß-arrestin are in competition. Several hypotheses can be proposed to explain what the differences between the two ß-arrestin pools are at the molecular level. As already proposed, ß-arrestin interaction with different residues of the receptor might adopt distinct conformations and thereby exert distinct functions (6, 22). Alternatively, the two pools might correspond to ß-arrestins localized in distinct cellular compartments, thereby favoring the assembling of specific ß-arrestin scaffolds upon FSH stimulation.

The notion of two functionally distinct populations of ß-arrestins recruited to the activated receptor and competing with each other is supported by recent data showing that different GRK subtypes exert specialized functions on AT1aR, V2R, ß2AR, and histamine H1 receptor (6, 17, 22, 30). In these studies, GRK2 and -3 both inhibit ß-arrestin-dependent ERK activation (6, 22), whereas GRK5 and -6 are required for ERK activation by the ß-arrestin-dependent mechanism. These studies strongly support our present data obtained with the FSH-R. Interestingly, with other receptors, GRK2 and -3 are responsible for the majority of ligand-induced receptor phosphorylation, ß-arrestin recruitment, desensitization, and internalization. Our observations together with these recently published data raise the hypothesis that the two ß-arrestin subpopulations suggested in this study might result from differential phosphorylation of the FSH-R by specific GRKs. More specifically, our siRNA data support a model where the 638–644 cluster might be preferentially phosphorylated by GRK2. Further studies will be required to confirm this hypothesis.

In conclusion, we have identified a seven-residue region in the C-terminal tail of the FSH-R that serves as a "desensitization motif" for both G{alpha}s-mediated signaling and ß-arrestin-dependent ERK activation. Whether other 7TMRs will display similar functional specialization of their Ser/Thr-rich motifs, in addition to their well-established regulation of receptors, intracellular trafficking is an open and interesting question.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
pFSH was purified by Dr. J. Closset (Université de Liège, Liège, Belgium). Waymouth’s MB752/1 medium, Hank’s balanced salt solution, and trypsin-EDTA were purchased from Invitrogen/GIBCO Life Technologies, Inc. (Gaithersburg, MD). Minimum essential medium, nonessential amino acids, BSA and fetal bovine serum, penicillin, streptomycin, and anti-Flag M2 affinity beads were purchased from Sigma (Uppsala, Sweden). Polyclonal anti-GRK and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ß-arrestin 1 and 2 polyclonal antibody (A1CT) was a kind gift from Dr. R. J. Lefkowitz (Duke University, Durham, NC). Forskolin, phorbol myristate acetate, glutamine, and IBMX were purchased from Sigma-Aldrich Chimie SARL (Uppsala, Sweden). The Fugene 6 transfection reagent was purchased from Roche Diagnostics (Basel, Switzerland), and the sulfo-dithio-bis[succinimidyl propionate] (DSP) was from Pierce (Rockford, IL).

Plasmids and cells
The plasmid encoding the full-length Flag-FSH-R is derived from the pRK-FSH-R/3 provided by Dr. R. Sprengel (Heidelberg, Germany) and has been described previously (13). The 5A FSH-R (T638A, T640A, S641A, S642A, T644A) has been obtained by using the Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA).

HEK 293 N cells were grown in minimum essential medium complemented with 10% heat-inactivated fetal bovine serum, 10 U/ml penicillin, and 10 µg/ml streptomycin. Cells were transiently transfected when 70–80% confluent with 66 ng/cm2 of the plasmid encoding the wt or the 5A rat FSH-R, using the Fugene 6 transfection reagent. Seventy-two hours later, cells were stimulated with 1 nM pFSH or 10 µM forskolin.

siRNA transfection
The siRNA sequences targeting human ß-arrestin 1 and ß-arrestin 2 are 5'-AAAGCCUUCUGCGCGGAGAAU-3' (positions 439–459) and 5'-AAGGACCGCAAAGUGUUUGUG-3' (positions 201–221), respectively. The double-stranded siRNA sequence 5'-AAACCTGCGCCTTCCGCTATG-3' was used to simultaneously target human ß-arrestin 1 (positions 172–190) and ß-arrestin 2 (positions 175–193). The siRNA sequences targeting GRKs are GRK2, 5'-AAGAAGUACGAGAAGCUGGAG-3' (positions 268–288); GRK3, 5'-AAGCAAGCUGUAGAACACGUA-3' (positions 376–396); GRK5, 5'-AAGCCGUGCAAAGAACUCUUU-3' (positions 406–426); and GRK6, 5'-AACAGUAGGUUUGUAGUGAGC-3' (positions 724–744). Indicated position numbers are relative to the start codon. One small RNA duplex, which has no silencing effect, was used as a control (5'-AAGUGGACCCUGUAGAUGGCG-3'). All the siRNAs have been chemically synthesized (Dharmacon Research, Lafayette, CO) and have been previously described and validated (6, 18, 19, 22). Early passage HEK 293 cells at 30% confluency were transfected into 100-mm dishes with siRNA using the GeneSilencer transfection reagent according to manufacturer’s recommendations (Gene Therapy Systems, San Diego, CA). Two micrograms of plasmid encoding the wt or 5A FSH-R were simultaneously transfected with 20 µg of siRNA. After 48 h at 37 C, cells were seeded into 12-well plates. All assays were performed 3 d after transfection.

Hormone labeling
125I-pFSH was labeled with the IODO-GEN (Pierce, Rockford, IL) method as described (31).

Binding assays and Scatchard analysis
Binding parameters for pFSH were measured during a 1-h incubation of intact cells plated in 35-mm wells (typically around 250 µg of total protein per well) with eight concentrations of pFSH ranging from 3.10–11 to 10–8 M at 37 C. Binding reactions were carried out in 300 µl of PBS 20 (1.1 mM KH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl, 0.5 mM MgCl2, 0.9 mM CaCl2, 234 mM saccharose, 0.5 mg/ml BSA; pH 7.4) with 75.10–9 M 125I-pFSH. At the end of the incubation, monolayers were washed three times in 1 ml PBS 20. Cells were then lysed in 1 ml of 0.5 N NaOH, collected, and counted in a {gamma}-counter (Wallac Wizard, Amersham Biosciences, Pittsburgh, PA). Each concentration of pFSH was assayed in triplicate. Equilibrium binding parameters were calculated from the binding data by Scatchard analysis.

Internalization assay
Internalization of the FSH-R was measured using 125I-FSH according to the method described by Krishnamurthy et al. (32). Cells were cultured in 35-mm wells, washed, placed in 1 ml of Waymouth’s MB752/1 medium containing 20 mM HEPES and 1 mg/ml BSA, and incubated with 416 nM 125I-pFSH. pFSH (280 µM) was added to correct the nonspecific binding. After incubation, cells were placed on ice and washed twice with Hank’s balanced salt solution containing 1 mg/ml BSA. The surface-bound hormone was then released by incubating cells in 1 ml of cold 150 mM NaCl, 50 mM glycine (pH 3.0) for 3 min, which was collected in a 3-ml tube. Then, cells were lysed in 1 ml of 1 M NaOH and collected separately. The two series of tubes were counted using a {gamma}-counter.

After each transfection experiment, the expression level of the 5A and wt FSH-R at the plasma membrane was compared. For this purpose, binding of 125I-FSH was performed on intact cells in PBS 20. All experiments were done with cells expressing equivalent amounts of wt or 5A FSH-R at the plasma membrane.

cAMP assay
HEK 293 cells were stimulated with indicated pFSH concentrations in the presence of 100 µM phosphodiesterase inhibitor IBMX. The supernatants were frozen; the cells were stripped in ethanol and then evaporated in a speed-vac concentrator. The pellets were diluted with the supernatant, and the total cAMP content (intra- and extracellular) was measured by RIA, according to the manufacturer’s instructions (Immunotech, Beckman Coulter, Paris, France).

Phosphorylation assay
Before stimulation, cells were incubated in phosphate-free DMEM (Invitrogen, Life Technologies, Inc.) containing 0.1 mCi 32P/ml for 1 h at 37 C. Then, cells were stimulated with 1 nM FSH for 5 min. After stimulation, cells were washed with PBS and harvested in 1 ml glycerol buffer (50 mM HEPES, 0.5% Nonidet P-40, 250 mM NaCl, 2 mM EDTA; pH 8.0; 10% glycerol) with protease and phosphatase inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 10 µg/ml aprotinin). The lysates were tumbled for 3 h at 4 C and then centrifuged at 4 C, 15,000 rpm for 20 min. The FSH-R was immunoprecipitated overnight with M2 agarose beads (Sigma). The beads were washed five times with Tris-buffered saline and eluted with 50 µl 2x Laemmli buffer (0.25 mM Tris HCl, 5% sodium dodecyl sulfate, 50% glycerol, 50 mM ß-mercapto-ethanol, 0.01% bromophenol blue). The immunoprecipitates were loaded on a 10% SDS-PAGE. The gel was dried and autoradiographed. The phosphorylation signal was quantified using a PhosphorImager (Molecular Dynamics, Amersham Biosciences, Pittsburgh, PA).

Coimmunoprecipitation
Endogenous ß-arrestins were coimmunoprecipitated with the activated receptor after DSP cross-linking according to the method described by Luttrell et al. (3). All experiments were done with cells expressing equivalent amounts of wt or 5A FSH-R at the plasma membrane. Cells were stimulated with 1 nM FSH in 100-mm culture dishes for 5 or 15 min in PBS/10 mM HEPES and incubated for 30 min with DSP. The cross-linking reaction was quenched with Tris-HCl (pH 7.3) to a 20-mM final concentration. Cells were washed with PBS/10 mM HEPES three times and lysed in radioimmunoprecipitation assay buffer with protease inhibitors. The lysates were tumbled at 4 C for at least 4 h. Then the pellets were centrifuged at 4 C, 15,000 rpm for 20 min. Ten microliters of total lysate were collected for further analysis. The remaining lysate was incubated with M2-agarose beads overnight at 4 C. The beads were washed three times with radioimmunoprecipitation assay buffer and eluted with 50 µl 2x Laemmli buffer. The samples were incubated at room temperature for 1 h before loading on 10% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane (Roche Diagnostics, Indianapolis, IN), incubated overnight with the polyclonal A1CT antibody (a kind gift of Dr. R. J. Lefkowitz) directed against the ß-arrestins 1 and 2, and diluted to 1:10,000 in Tris-buffered saline, 0.1% Tween 20, and 5% nonfat dry milk. The blots were revealed using an enhanced chemiluminescence reaction ECL (Amersham Pharmacia Biotech, Pittsburgh, PA).

Phospho-ERK 1 and 2 analysis
Cells were stimulated with 1 nM FSH for various time periods, and the supernatants were removed. All experiments were performed with cells expressing equivalent amounts of wt or 5A FSH-R at the plasma membrane. Then, cells were lysed in 2x Laemmli buffer, and equivalent amounts of proteins were loaded for Western blot analysis. The membranes were incubated overnight with a polyclonal anti-phospho-ERK 1/2 antibody (1:2000) (Cell Signaling Technology Inc., Danvers, MA). The same membranes were stripped and incubated with primary antibody against total ERK 1/2 (Santa Cruz Biotechnology) (1:10,000) to monitor loading differences between lanes.

Statistical analysis
Statistical analysis of the data was performed using one-way ANOVA (Bonferroni’s multiple comparison tests) to compare samples or two-way ANOVA to compare entire curves (GraphPad PRISM Software, San Diego, CA).


   FOOTNOTES
 
The authors are grateful to Dr. R. J. Lefkowitz (Howard Hughes Medical Institute, Duke University, Durham, NC) for his invaluable help throughout this work. We also thank Drs. S. Shenoy and S. Ahn (Duke University) for their critical reading of the manuscript and Dr. X. R. Ren (Duke University) for helpful discussions. The wt FSH-R construct was a gift of Dr. R. Sprengel (Heidelberg, Germany).

E.K. is the recipient of doctoral fellowships from the Region Centre, Fond d’aide à la recherche Organon, and Fondation Recherche Médicale. This work was also funded by Institut National de la Recherche Agronomique, Centre National de Recherche Scientifique, and Région Centre.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: ß2AR, ß2 adrenergic receptor; AT1aR, angiotensin type 1a receptor; DSP, sulfo-dithio-bis[succinimidyl propionate]; FSH-R, FSH receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G protein, guanine nucleotide-binding protein; G{alpha}s, G protein {alpha} S subunit; GRK, G protein-coupled receptor kinase; IBMX, isomethylbutylxanthine; pFSH, porcine FSH; PKA, protein kinase A; siRNA, small interfering RNA; 7TMR, seven membrane-spanning receptor; V2R, vasopressin type 2 receptor; wt, wild-type.

Received for publication February 28, 2006. Accepted for publication July 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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