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Molecular Endocrinology, doi:10.1210/me.2008-0122
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Molecular Endocrinology 22 (11): 2520-2530
Copyright © 2008 by The Endocrine Society

A Crucial Role for G{alpha}q/11, But Not G{alpha}i/o or G{alpha}s, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition

Colin D. White, Marla Coetsee, Kevin Morgan, Colleen A. Flanagan, Robert P. Millar and Zhi-Liang Lu

The Medical Research Council Human Reproductive Sciences Unit (C.D.W., M.C., K.M., R.P.M., Z.-L.L.), The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom; The School of Physiology (C.A.F.), The University of Witwatersrand Medical School, Parktown, Johannesburg 2193, South Africa; and Receptor Biology Group (M.C., C.A.F., R.P.M.), Institute for Infectious Diseases and Molecular Medicine, Division of Medical Biochemistry, University of Cape Town, Observatory 7925, South Africa

Address all correspondence and requests for reprints to: Zhi-Liang Lu, The Medical Research Council Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail: z.lu{at}hrsu.mrc.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to G{alpha}q/11, G{alpha}i/o, and G{alpha}s, their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in G{alpha}q/11-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to G{alpha}i or chimeric G{alpha}qi5, transfection of G{alpha}q cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTP{gamma}S binding assays confirmed that the GnRH receptor was unable to directly couple to G{alpha}i but could directly interact with G{alpha}q/11. Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing G{alpha}q, and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to G{alpha}q/11, but not to G{alpha}i/o or G{alpha}s, and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMALIAN HYPOTHALAMIC GnRH (termed GnRH-I) is a decapeptide hormone that plays key roles in the regulation of reproduction. It is synthesized in the hypothalamus and transported in the hypothalamo-hypophyseal portal circulation to the anterior pituitary. Here it binds to its cognate receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) superfamily, and stimulates the biosynthesis and secretion of LH and FSH (1). As well as having a key role in reproductive behavior, evidence suggests that GnRH may act peripherally, via an autocrine/paracrine mechanism, to exert a growth-regulatory effect on certain cell types (2). Indeed, GnRH and the GnRH receptor have been found in extrapituitary tissues such as the ovary (3) and the mammary gland (4). Cancers of the breast (5, 6, 7), ovary (6, 8), endometrium (9), and prostate (10) have also been shown to express both the ligand and the receptor. Additionally, several studies, both in vitro and in vivo, have demonstrated that direct application of GnRH analogs to receptor-expressing cancer cells results in an attenuation of cellular proliferation and activation of proapoptotic signaling mechanisms (11, 12, 13, 14, 15, 16, 17, 18, 19). In many of these cases, activation of the MAPK signaling cascades is thought to play a fundamental role (16, 17, 20, 21).

Several groups have demonstrated that GnRH stimulates phosphorylation of ERK, jun-N-terminal kinase (JNK), and P38, three prominent members of the MAPK superfamily, in {alpha}T3-1 and LβT2 gonadotrope cell lines and a wide variety of GnRH receptor transfected cells (21, 22, 23, 24, 25). How these cascades are initiated upstream by the activated receptor and which of them impinge on cell growth inhibition remains unclear. It has been proposed that whereas the actions of GnRH at the pituitary are mediated by interactions of the receptor with G proteins of the G{alpha}q/11 subfamily and consequent signaling to and activation of, among other molecules, ERK, JNK and P38 (21, 24, 26), the antiproliferative actions of GnRH are best explained via an interaction of the receptor with G{alpha}i/o (17, 27, 28, 29, 30). Resultant induction of apoptosis coincident with phosphorylation of JNK (16) or other members of the MAPK superfamily (17, 20) has also been observed. It is plausible to speculate that interaction of the receptor with different G proteins may explain the published diversity of the mechanisms involved in MAPK activation and the subsequent effects on cellular fate. This hypothesis, however, is not uncontested (26). Indeed, despite circumstantial evidence, direct proof of the activation of multiple G proteins by the agonist-bound GnRH receptor is still missing.

To better understand the pathways involved in GnRH-mediated cell growth inhibition, we set out to study the G protein coupling profile of the GnRH receptor. We provide direct evidence, in multiple cellular backgrounds, that the receptor couples to G proteins of the G{alpha}q/11 subfamily but not to G{alpha}i/o or G{alpha}s as previously suggested. We also demonstrate that ERK, JNK, and P38 activation in response to GnRH treatment may be mediated by the activation of G{alpha}q/11. Furthermore, we show that G{alpha}q/11 may facilitate the induction of proapoptotic signaling by GnRH and that the ERK cascade, but neither the JNK nor the P38 pathway, plays a pivotal role.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH-Stimulated MAPK Phosphorylation May Be Mediated by G{alpha}q/11
To facilitate potential coupling of the GnRH receptor to G proteins other than G{alpha}q/11, we stably transfected the receptor into G{alpha}q/11-negative MEF cells. These cells thus eliminate potential competition from G{alpha}q/11 for binding of the receptor. The MEF S19 cell line expresses more receptors per cell than either the SCL60 or the LβT2 cells, although the binding affinity of GnRH-I is not significantly different (MEF S19, 11.1 ± 2.2 nM; SCL60, 10.1 ± 1.7 nM; and LβT2, 10.1 ± 1.3 nM) (Fig. 1Go). The higher expression of the GnRH receptor in these cells is reflected by their larger size. In addition, the estimated number of GnRH receptor binding sites on {alpha}T3-1 cells is approximately 50% of the number on primary gonadotropes (31). Thus, the MEF S19 cells may better reflect GnRH receptor expression levels encountered in vivo. To elucidate the ability of GnRH to activate ERK, JNK, and P38 in these cells, GnRH-I was applied in both a time- and dose-dependent manner (Fig. 2Go, A and B, respectively). GnRH-I stimulation brought about no significant increase in the levels of pERK1/2, pJNK1, or pP38 at any time or dose tested when compared with vehicle-treated controls. Transient expression of G{alpha}q allowed GnRH-I to elicit an increase in ERK1/2 phosphorylation with a maximal response of approximately 3-fold that of controls after 5 min stimulation. Additionally, JNK1 and P38 phosphorylation became evident, giving maximal responses of approximately 3.5-fold that of controls after 5 min stimulations and 30 min stimulations, respectively. Agonist dose-response analysis yielded EC50 values for the induction of pERK1/2, pJNK1, and pP38 after 10 min stimulation of 0.95, 3.32, and 2.36 nM GnRH-I, respectively. In untransfected cells, the complete absence of G{alpha}q/11 was verified both by Western blotting and lack of phosphoinositide hydrolysis (Fig. 2Go, C and D, respectively). Additionally, when compared with LβT2 cells, the levels of G{alpha}q/11 after transient G protein transfection were within the physiological range.


Figure 1
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  		Fig. 1. GnRH Receptor Expression in the MEF S19, SCL60, and LβT2 Cell Lines as Measured by Radioligand Binding

Intact MEF S19 ({square}), SCL60 ({circ}), and LβT2 ({diamondsuit}) cells were incubated with 125I-labeled [His5,D-Tyr6] GnRH (100,000 cpm/0.5 ml · well) and either vehicle (0.2% propylene glycol) or various concentrations of unlabeled GnRH-I as indicated for 4 h at 4 C. After incubation, cells were rapidly washed twice with cold PBS and solubilized in 0.1 M NaOH. Radioactivity was counted by {gamma}-spectrometry. Data are representative of at least three independent experiments and the mean counts per minute per million cells (CPM/million cells) ± SE is presented.

 

Figure 2
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  		Fig. 2. Immunoblots Depicting the Time and Dose Dependence of GnRH-I-Induced, G{alpha}q-Mediated Phosphorylation of ERK1/2, JNK1, and P38

A, Time course of GnRH-I-stimulated MAPK phosphorylation. MEF S19 cells transiently transfected with vector or G{alpha}q cDNA were serum starved for 16 h before being treated with vehicle (0.2% propylene glycol; V) or 1 µM GnRH-I for the indicated times. Representative blots are shown. Data from at least three independent experiments were quantified (using total ERK1/2 as a loading control), and the mean fold over control ± SE for the activation of ERK1/2 (black bars), JNK1 (white bars), and P38 (gray bars) in cells transfected with vector (middle panel) or G{alpha}q (bottom panel) cDNA is presented below the corresponding blots. *, P < 0.05, representing statistical significance from vehicle-treated controls. B, Dose response of GnRH-I-stimulated MAPK phosphorylation. MEF S19 cells transiently transfected with vector or G{alpha}q cDNA were serum starved for 16 h before being treated with vehicle (0.2% propylene glycol; V) or increasing doses of GnRH-I (0.1, 1, 10, and 100 nM and 1 µM) as indicated for 10 min. Representative blots are shown. Data from at least three independent experiments were quantified (using total ERK1/2 as a loading control), and the mean fold over control ± SE for the activation of ERK1/2 ({circ} and bullet), JNK1 ({diamond} and {diamondsuit}), and P38 ({triangleup} and {blacktriangleup}) in cells transfected with vector (open symbols) or G{alpha}q (filled symbols) cDNA is presented below the corresponding blots. *, P < 0.05, representing statistical significance from vehicle-treated controls. C, Immunoblots depicting the relative expression of G{alpha}q. MEF S19 cells transiently transfected with vector (lane 1) or G{alpha}q (lane 2) cDNA and LβT2 cells (lane 3) were serum starved for 16 h. Unstimulated cell lysates were collected and used to verify transfection efficiency and relative G protein expression level. Representative blots are shown. D, Phosphoinositide hydrolysis assays depicting the functionality of transfected G{alpha}q. MEF S19 cells transiently transfected with vector ({square}) or G{alpha}q ({blacksquare}) cDNA were labeled overnight with 1 µCi/ml myo-D-[3H]inositol before being treated with vehicle (0.2% propylene glycol; V) or increasing doses of GnRH-I (0.1, 1, 10, and 100 nM and 1 µM) as indicated for 30 min. The [3H]inositol phosphates were processed as described in Materials and Methods and quantified by liquid scintillation counting. Data are representative of at least three independent experiments, and the mean counts per minute (CPM) ± SE is presented. ***, P < 0.001, representing statistical significance from vehicle-treated controls.

 
The GnRH Receptor Does Not Directly Interact with G{alpha}i/o or G{alpha}s
Thus far we have confirmed that transfection of G{alpha}q allows the detection of significant activation of ERK, JNK, and P38 in response to GnRH treatment. Nevertheless, several studies have argued that these responses may also be mediated by interactions of the GnRH receptor with other G proteins. Although the above results indicate that in the complete absence of G{alpha}q/11, it was not possible to detect significant increases in MAPK activation when compared with control cells, we attempted to address this theory. Initially, we considered the possibility that transient overexpression of G{alpha}i could facilitate detectable MAPK signaling. In contrast to G{alpha}q, transient transfection of each of the three subtypes of G{alpha}i did not enable GnRH-I to significantly increase ERK1/2, JNK1. or P38 phosphorylation when compared with controls (Fig. 3AGo). Additionally, we made use of a chimeric G protein, G{alpha}qi5. The C terminus of the G protein {alpha}-subunit has been shown to play a pivotal role in defining the specificity of receptor-G protein interactions. Mutation of the last five C-terminal amino acids is sufficient to completely switch receptor coupling selectivity (32). The cDNA of G{alpha}qi5 codes for a G protein {alpha}-subunit consisting of mainly G{alpha}q. The last five C-terminal amino acids have, however, been substituted for the corresponding amino acids from G{alpha}i2. This facilitates G{alpha}i/o-coupled GPCR signaling through phospholipase Cβ (PLCβ), and as such, a classical G{alpha}q-mediated output becomes indicative of receptor coupling to G{alpha}i/o. Transient expression of G{alpha}qi5 in the MEF S19 cell line did not enable the activated receptor to elicit a significant increase in ERK1/2, JNK1, or P38 phosphorylation when compared with control cells. Transfected cells showed significantly increased expression of G{alpha}i1–3 and G{alpha}qi5 (Fig. 3BGo).


Figure 3
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  		Fig. 3. Immunoblots Depicting the Lack of Effect of Transient Overexpression of G{alpha}i1–3 or G{alpha}qi5 on GnRH-I-Induced Phosphorylation of ERK1/2, JNK1, and P38

A, MEF S19 cells transiently transfected with vector, G{alpha}i1, G{alpha}i2, G{alpha}i3, or G{alpha}qi5 cDNA were serum starved for 16 h before being treated with vehicle (0.2% propylene glycol; V) or 1 µM GnRH-I for 10 min. Representative blots are shown. Data from at least three independent experiments were quantified (using total ERK1/2 as a loading control), and the mean fold over control ± SE for the activation of ERK1/2 (black bars), JNK1 (white bars), and P38 (dark gray bars) relative to vehicle-treated controls (light gray bars) is presented below the corresponding blots. B, MEF S19 cells transiently transfected with vector, G{alpha}i1, G{alpha}i2, G{alpha}i3, or G{alpha}qi5 cDNA were serum starved for 16 h. Unstimulated cell lysates were collected and used to verify transfection efficiency and relative G protein expression level. Representative blots are shown.

 
To address the possibility that the GnRH receptor does interact with G{alpha}i/o but that G{alpha}i/o does not activate the MAPK cascades thus making it impossible to identify such interactions using MAPK phosphorylation as an output, we assessed the ability of GnRH to increase GTP{gamma}S binding to G{alpha}i and to inhibit intracellular cAMP accumulation. Furthermore, for the GTP{gamma}S experiments, we used a different cell line to investigate cell-type-specific differences. Stimulation of SCL60 cells with GnRH-I had no effect on GTP{gamma}S binding to G{alpha}i (Fig. 4Go). In contrast, stimulation of CHO-M2 cells with carbachol significantly increased GTP{gamma}S-G{alpha}i binding, thereby providing a positive control for the G{alpha}i-GTP{gamma}S assay system. Stimulation of SCL60 cells with GnRH-I significantly increased GTP{gamma}S binding to G{alpha}q/11 to approximately 1.5-fold that of controls. Moreover, stimulation of MEF S19 cells with isoproterenol increased intracellular cAMP levels to approximately 15-fold that of vehicle-treated controls. Costimulation of the same cells with isoproterenol and GnRH-I did not significantly affect the cAMP response obtained (Fig. 5Go). Significantly, stimulation with GnRH-I alone did not induce any increase in intracellular cAMP. Similar results were observed in COS-7 cells transiently expressing the GnRH receptor (White, C. D., and Z.-L. Lu, unpublished observations).


Figure 4
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  		Fig. 4. Scintillation Proximity Assays Quantifying Receptor-Mediated Activation of G{alpha}q/11 and G{alpha}i

Freshly prepared SCL60 (gray and white bars) and CHO-M2 (black bars) membranes were incubated in a GTP{gamma}S assay buffer in the presence of vehicle (0.2% propylene glycol), 1 µM GnRHI, or 10 µM carbachol and 200 pM [35S]GTP{gamma}S for 1 h at 25 C. Thereafter, antibodies against G{alpha}i1/2 and G{alpha}i3 (gray and black bars) or G{alpha}q/11 (white bars) and anti-IgG-coated SPA beads were added and processed as described in Materials and Methods. Data from at least three independent experiments were quantified, and the mean fold over control ± SE for GTP{gamma}S bound is presented. **, P < 0.01; and ***, P < 0.001, representing statistical significance from vehicle-treated controls.

 

Figure 5
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  		Fig. 5. cAMP Assays Determining the Effects of GnRH-I and Isoproterenol on Intracellular cAMP Accumulation

MEF S19 cells were treated with vehicle (0.2% propylene glycol), 3 µM isoproterenol (Iso), 1 µM GnRH-I, or 1 µM GnRH-I and 3 µM isoproterenol together for 30 min as indicated after a 30-min incubation with 1 mM 3-isobutyl-1-methylxanthine. Intracellular cAMP levels were measured as described in Materials and Methods. Data from at least three independent experiments were quantified, and the mean fold over control ± SE is presented. ***, P < 0.001, representing statistical significance from vehicle-treated controls.

 
Coupling of the GnRH Receptor to G{alpha}q/11 and Consequent Activation of the ERK Pathway Play a Crucial Role in GnRH-Mediated Cell Growth Inhibition
Continuous treatment of MEF S19 cells with GnRH-I resulted in a time- and dose-dependent inhibition of cell growth relative to vehicle-treated controls only in cells transfected with G{alpha}q (Fig. 6AGo). In these cells, significant growth inhibition was evident on d 1 (cell growth was 93.7 ± 1.2% that of controls) and increased in a time-dependent manner to reach a maximal inhibition on d 4 (cell growth was 69.8 ± 2.8% that of controls). SB203580 (a P38 inhibitor) and SP600125 (a JNK inhibitor) were unable to significantly rescue this inhibition, but PD98059 (a MAPK kinase-1/2 inhibitor) significantly decreased the GnRH-I-induced effect (cell growth was 90.4 ± 1.5% that of controls on d 4). Agonist dose-response analysis yielded an IC50 value for the induction of cell growth inhibition on d 4 of 4.54 nM GnRH-I (Fig. 6BGo). Vector-transfected control cells showed no significant inhibition of growth in response to GnRH-I treatment, nor did any of the MAPK inhibitors have a significant effect. Similar results were obtained in the SCL60 cell line (Fig. 6CGo). Continuous treatment with GnRH-I resulted in a time- and dose-dependent inhibition of cell growth relative to vehicle-treated controls. Significant growth inhibition was evident on d 1 (cell growth was 87.6 ± 0.4% that of controls) and increased in a time-dependent manner to reach a maximal inhibition on d 4 (cell growth was 57.3 ± 1.2% that of controls). SB203580 and SP600125 were unable to significantly rescue this inhibition, but PD98059 significantly decreased the GnRH-I-induced effect (cell growth was 81.0 ± 1.5% that of controls on d 4). Agonist dose-response analysis yielded an IC50 value for the induction of cell growth inhibition on d 4 of 6.21 nM GnRH-I (Fig. 6DGo). YM-254890 (a G{alpha}q/11 inhibitor) completely eliminated the GnRH-I-induced inhibition of cell growth. Trypan blue exclusion confirmed that detached cells were dead (as measured by dye uptake when compared with either live cell suspensions or vector-transfected samples), and these dead cells were more numerous than in samples from vector-transfected controls (White, C. D., and Z.-L. Lu, unpublished observations).


Figure 6
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  		Fig. 6. Sulforhodamine B Assays Assessing the Effects of G{alpha}q and the ERK, JNK, and P38 Pathways on GnRH-I-Induced Cell Growth Inhibition

A, Effect of G{alpha}q and the MAPK pathways on GnRH-I-induced cell growth inhibition in MEF S19 cells. MEF S19 cells transiently transfected with vector (open symbols) or G{alpha}q cDNA (filled symbols) were incubated in medium containing 10% serum with or without vehicle (dimethylsulfoxide; {square} and {blacksquare}), 50 µM PD98059 ({circ} and bullet), 50 µM SP600125 ({diamond} and {diamondsuit}), or 20 µM SB203580 ({triangleup} and {blacktriangleup}) before being treated with vehicle (0.2% propylene glycol) or 100 nM GnRH-I for the indicated times. Data from at least three independent experiments were quantified, and the mean percent cell growth relative to control ± SE is presented. **, P < 0.01; ***, P < 0.001, representing statistical significance from vector-transfected vehicle-treated controls (for cells transfected with G{alpha}q and incubated in media containing dimethylsulfoxide) and from G{alpha}q-transfected vehicle-inhibited controls (for cells incubated in medium containing 50 µM PD98059). B, Dose dependence of GnRH-I-induced cell growth inhibition in MEF S19 cells. MEF S19 cells transiently transfected with vector ({square}) or G{alpha}q cDNA ({blacksquare}) were incubated in medium containing 10% serum before being treated with vehicle (0.2% propylene glycol) or increasing doses of GnRH-I (0.1, 1, 10, and 100 nM and 1 µM) as indicated for 4 d. Data from at least three independent experiments were quantified, and the mean percent cell growth relative to control at d 4 ± SE is presented. *, P < 0.05; **, P < 0.01; ***, P < 0.001, representing statistical significance from vehicle-treated controls. C, Effect of G{alpha}q and the MAPK pathways on GnRH-I-induced cell growth inhibition in SCL60 cells. SCL60 cells were incubated in medium containing 10% serum with or without vehicle (dimethylsulfoxide; {blacksquare}), 50 µM PD98059 (bullet), 50 µM SP600125 ({diamondsuit}), 20 µM SB203580 ({blacktriangleup}), or 100 nM YM-254890 ({square}) before being treated with vehicle (0.2% propylene glycol) or 100 nM GnRH-I for the indicated times. Data from at least three independent experiments were quantified, and the mean percent cell growth relative to control ± SE is presented. **, P < 0.01; ***, P < 0.001, representing statistical significance from vehicle-treated controls (for cells incubated in medium containing dimethylsulfoxide) and from vehicle-inhibited controls (for cells incubated in medium containing 50 µM PD98059 or 100 nM YM-254890). D, Dose dependence of GnRH-I-induced cell growth inhibition in SCL60 cells. SCL60 cells were incubated in medium containing 10% serum before being treated with vehicle (0.2% propylene glycol) or increasing doses of GnRH-I (0.1, 1, 10, and 100 nM and 1 µM) as indicated for 4 d. Data from at least three independent experiments were quantified, and the mean percent cell growth relative to control at d 4 ± SE is presented. **, P < 0.01; ***, P < 0.001, representing statistical significance from vehicle-treated controls.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Binding of GnRH to its receptor at the plasma membrane initiates a variety of intracellular signaling events with distinct physiological outcomes. Since the original observations of GnRH-induced cell growth inhibition on receptor-expressing cell lines (33), substantial effort has been directed toward delineating the precise mechanisms involved. Considerable evidence suggests that the signaling cascades involved in GnRH-mediated cell growth inhibition are distinct from those involved in regulating gonadotropin secretion (17, 30, 34), and one possibility to explain this divergent signaling is to hypothesize the inherent ability of the receptor to directly interact with multiple classes of G proteins. Multiplicity of G protein coupling has after all been successfully confirmed for several other GPCRs (35). However, as yet, no group has provided direct evidence of such interactions, and the frequently repeated paradigm of the ability of the GnRH receptor to directly interact with G{alpha}i/o and G{alpha}s has led to confusion regarding the mechanisms by which GnRH can inhibit cell growth. To critically address this issue, we studied the G protein coupling profile of the GnRH receptor in stably transfected HEK293 cells and MEF cells derived from G{alpha}q/11 knockout mice (36, 37). Additionally, we investigated the roles of the various G proteins in the activation of ERK, JNK, and P38 and the downstream cellular effects of activation of these MAPK modules.

GnRH-mediated activation of ERK, JNK, and P38 has been extensively studied over the past two decades (21, 24). We and others have shown that phosphorylation of these proteins readily occurs upon GnRH stimulation of receptor-expressing cell lines and that this activation is dependent on PLCβ and protein kinase C (PKC) (22, 23, 38). These data thus suggest the involvement of G{alpha}q/11-mediated signaling events. The role of G{alpha}i/o in MAPK activation in response to GnRH has also been the subject of much investigation. ERK activation in {alpha}T3-1 cells has been suggested to depend on a dual mechanism involving G{alpha}q/11 and G{alpha}i/o (39). Similarly, in Caov-3 ovarian cancer cells, which have been shown to express GnRH receptor mRNA (although no evidence is given regarding expression at the protein level), ERK activation has been hypothesized as being mediated by a combination of interactions involving G{alpha}i/o and Gβ{gamma} (20). Studies using pertussis toxin have also indicated a role for G{alpha}i/o in the GnRH-induced phosphorylation of JNK and P38 (17). Interestingly, our data contradict these studies because we have found no evidence to support the theory of GnRH-induced activation of these MAPKs by G{alpha}i/o, even when G{alpha}i is artificially overexpressed in our cell systems. We have, however, confirmed that G{alpha}q/11-mediated signaling plays a pivotal role in the GnRH receptor activation of the MAPKs.

Elegant studies have shown that coexpression of G{alpha}qi5 with several other GPCRs successfully enables G{alpha}i/o-coupled receptors to signal through PLCβ and PKC (40). It follows then that the inability of GnRH to induce phosphorylation of ERK, JNK, or P38 when G{alpha}qi5 is expressed suggests that the GnRH receptor does not directly interact with G proteins of the G{alpha}i/o subfamily. This conclusion is strengthened by the observation that GnRH does not inhibit isoproterenol-induced cAMP accumulation. However, by definition, the only direct measure of G protein activation by an activated receptor is receptor-catalyzed GDP/GTP exchange. Other analytical approaches yield only indirect results and the notion of GnRH receptor-G{alpha}i/o interaction has largely been derived from circumstantial evidence using pertussis toxin. In this case, our data, and the observations of Grosse and colleagues (26), which reported exclusive GnRH receptor-mediated labeling of G{alpha}q/11 with [{alpha}-32P]GTP azidoanilide, show definitively that the GnRH receptor does not directly couple to G{alpha}i/o but does interact with G{alpha}q/11. Reports by Shah and colleagues (41) using activated G proteins as an output support this interpretation. Here, stimulation of {alpha}T3-1 cells with GnRH agonists resulted in elevated agonist-induced down-regulation of G{alpha}q/11 reflecting increased G protein turnover. G{alpha}i/o remained unaffected. Interestingly, it has recently been shown that G protein {alpha}-subunits can be activated directly by growth factor receptor tyrosine kinases (42). Thus, it is possible that GnRH receptor-mediated receptor tyrosine kinase transactivation could be responsible for previous interpretations implicating a direct interaction of the receptor with G{alpha}i/o. Furthermore, the demonstration that very high agonist concentrations are required to facilitate GnRH receptor-mediated G{alpha}i/o signaling events (43) suggests that these events may potentially occur distally of the receptor-G protein interface.

In the early 1990s, Janovick and Conn (44) showed that treatment of rat pituitary cultures with cholera toxin increased LH release in response to GnRH treatment and interpreted this finding as being indicative that the GnRH receptor was directly coupled to G{alpha}s. Since then, many other groups have studied this possibility, but the results are inconclusive. The cAMP-protein kinase A pathway is important for gonadotrope function (45, 46); however, the involvement of G{alpha}s in this signaling cascade is still debated. Using palmitoylation as a measure of G protein activation, it was suggested that there was a direct interaction between the GnRH receptor and G{alpha}s in rat pituitary cells (47). This hypothesis was recapitulated in the LβT2 cell line using cell-permeable peptides that uncouple G{alpha}s from the receptor (48). However, based on these and other observations, the Gudermann group (26) examined the possibility of a direct interaction between the activated receptor and G{alpha}s and, in agreement with our findings, failed to detect any G{alpha}s-mediated signal transduction in response to GnRH treatment. More recently, Larivière and colleagues identified a novel signaling pathway involving PKC{delta} and -{epsilon}, which mediate GnRH activation of a cAMP-sensitive promoter (49). These results, viewed collectively with our own data, question the possibility of a direct interaction between the GnRH receptor and G{alpha}s. Interestingly, it has also been shown that a dominant-negative mutant of G{alpha}s blocked not only G{alpha}s-mediated signaling from the calcitonin receptor but also that mediated by G{alpha}q (50) and that specific adenylate cyclase isoforms may be activated directly by Gβ{gamma} (51) or independently of G proteins altogether (52). It would therefore seem possible to conclude that signal transduction involving GnRH induction of the cAMP pathway remains to be fully elucidated but is unlikely to involve a direct interaction between the activated receptor and G{alpha}s. Indeed, given that we cannot detect any cAMP accumulation in response to GnRH in G{alpha}q/11-negative cells, taken together with the fact that the β2-adrenergic receptor agonist isoproterenol can elicit a marked increase in intracellular cAMP, we would suggest that the GnRH receptor-evoked small cAMP responses previously observed in certain cell lines are not mediated by the direct coupling of the receptor to Gs.

The MAPK pathways are evolutionarily conserved kinase cascades that link extracellular signals to the machinery that controls fundamental cellular processes such as growth, proliferation, differentiation, migration, and apoptosis. Historically, ERK signaling is synonymous with cell proliferation (53), although the JNK and P38 pathways are regarded as being stress activated (54). Involvement of the JNK and P38 signaling cascades in GPCR-induced inhibition of cell proliferation has been widely documented (53, 54, 55, 56), and here the GnRH receptor is no exception (16, 17, 21). In contrast to these data, we showed, in agreement with previous studies (20, 57), that P38 and JNK do not influence GnRH-induced cell growth inhibition. Additionally, we find that the ERK signaling pathway plays a critical role; inhibition of ERK activation significantly decreased cell growth inhibition in both cell lines tested.

ERK has been implicated in cell growth inhibition, cell cycle arrest, and the induction of proapoptotic signaling in a number of cell types (58, 59), and a large body of evidence indicates that the strength and duration of the ERK signal is kernel to determining cellular fate (19, 60, 61). Several studies have shown that strong and prolonged activation of ERK by constitutively active Ras or Raf leads to arrest in the G1 phase of the cell cycle by inducing the expression of cyclin-dependent kinase inhibitors such as p53 and p21 (62, 63). As such, it seems plausible to speculate that continuous exposure of our cells to GnRH would result in ERK activation of a similar strength and duration. Thus, by inhibiting this pathway, this activation is abolished and GnRH-induced cell growth inhibition is significantly diminished. Interestingly, Zhang and colleagues (64) demonstrated that in LβT2 cells, p53 is phosphorylated by GnRH in a P38-dependent manner. Although our results do not indicate a role for P38 in GnRH-mediated cell growth inhibition in the cell lines we have studied, it is possible that transformation of the LβT2 cell line with SV40 large T antigen may influence the signaling pathways involved. Additionally, MAPK activation in response to GnRH has also been suggested to be dependent on the cell background in which any studies are carried out (17, 21). Notably, however, some studies that report significant effects of JNK and P38 on GnRH-induced cell growth inhibition also imply the ability of the GnRH receptor to couple to G{alpha}i/o (17, 65). Perhaps, given our current findings, it would be prudent to directly verify these data.

The ability of many hormones and neurotransmitters to evoke diverse physiological and pathological responses, by binding to a single cognate receptor, brought about the hypothesis that one GPCR may have the inherent ability to activate multiple G proteins. As such, the differential effects of GnRH analogs at central and peripheral sites were thought to be mediated via interactions of the activated receptor with G{alpha}q/11, G{alpha}i/o, and G{alpha}s. In the present study, we provide direct evidence, in multiple cellular contexts, that the GnRH receptor does not couple to G proteins of the G{alpha}i/o or G{alpha}s subfamilies, that MAPK activation in response to GnRH treatment is entirely dependent, of the three G protein subtypes tested, on G{alpha}q/11, and that the ERK pathway is significantly involved in GnRH-mediated cell death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The pMEP4 expression vector was kindly provided by Dr. Keith Leppard, University of Warwick, UK. PD98059 (50 µM), SP600125 (50 µM), and SB203580 (20 µM) were all obtained from Calbiochem (Nottingham, UK). YM-254890 was kindly provided by Dr. Masatoshi Taniguchi, Astellas Pharma Inc., Japan. The G{alpha}q/11 and G{alpha}i1–3 protein cDNAs were obtained from the Missouri S&T cDNA Resource Center (Rolla, MO). The G{alpha}qi5 protein cDNA, the G{alpha}q/11-negative MEF cell line, and the G{alpha}i1/2 and G{alpha}i3 antibodies were kindly provided by Professor Graeme Milligan, University of Glasgow, Glasgow, UK. The G{alpha}q/11-negative MEF cell line was originally derived from a combined G{alpha}q/11 double-knockout mouse and has been previously shown to have absolutely no endogenous G{alpha}q/11 (36, 37). The CHO cell line stably expressing the M2-muscarinic receptor (designated CHO-M2) was kindly provided by Professor Noel Buckley, University of Leeds (Leeds, UK). The LβT2 cell line was kindly provided by Dr. Pamela Mellon, University of California. [35S]GTP{gamma}S (1000–1250 Ci/mmol) and anti-IgG-coated scintillation proximity assay (SPA) beads were purchased from GE Healthcare (Buckinghamshire, UK), and the G{alpha}q/11 (C-19) and G{alpha}i/o antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). All other reagents were obtained from Sigma (Dorset, UK) unless otherwise stated.

Cell Culture and Transfection
Full-length GnRH receptor cDNA was subcloned into pMEP4 at Not1 and Xho1 and the construct transfected into G{alpha}q/11-negative MEF cells by electroporation. Cloned cells were selected using hygromycin resistance and screened by radioligand binding (Fig. 1Go) as previously described (66, 67). After appropriate selection, cells stably expressing the GnRH receptor (designated MEF S19) were maintained in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, appropriate antibiotics, and 50 µg/ml hygromycin (Invitrogen Life Technologies, Paisley, UK) at 37 C in a humidified 5% CO2 atmosphere. Cells were seeded at a density of 1.0 x 105 cells/ml and allowed to grow in large culture flasks for 96 h before transfection. Transient transfections were performed by electroporation using a Bio-Rad Gene Pulser XCell (Bio-Rad Laboratories, Hertfordshire, UK) at 320 V/500 µF with 20 µg plasmid DNA/0.4-cm electroporation cuvette. Before stimulation, appropriately transfected or untransfected cells were incubated in serum-free medium (DMEM supplemented with 2 mM glutamine, appropriate antibiotics, 50 µg/ml hygromycin, and 10 mM HEPES) for 16 h for Western blotting for pERK1/2, pJNK1, and pP38 and cAMP assays or medium containing 10% serum for 24 h for sulforhodamine B staining. Serum deprivation for 16 h is common practice in cell signaling studies (16, 17, 68, 69). Nevertheless, the time-dependent effects of starvation on basal MAPK phosphorylation were first evaluated (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Agonist stimulations were performed at 37 C in either serum-free medium or medium containing 10% serum after appropriate incubation with chemical inhibitors as described in the figure legends. HEK293 cells stably expressing the rat GnRH receptor (designated SCL60) generated previously within our laboratory, LβT2 cells, and CHO-M2 cells were maintained in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, appropriate antibiotics, and (for SCL60 and CHO-M2 cells) 500 µg/ml geneticin (G418) (PAA Laboratories, Somerset, UK).

Preparation of Cellular Extract and Immunoblotting
After ligand stimulation, cell monolayers were placed on ice, washed once with ice-cold Dulbecco's PBS, and lysed in a Nonidet P-40 solubilization buffer (250 mM NaCl, 50 mM HEPES, 0.5% Nonidet P-40, 10% glycerol, 2 mM EDTA, pH 8.0) supplemented with 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Solubilized lysates were clarified by centrifugation at 20,000 x g for 15 min, and nuclear contents were sheared by subsequent sonication. Sample protein concentrations were measured using the modified Bradford assay (Bio-Rad) and diluted to a concentration of 1 mg/ml total protein. Clarified whole-cell lysates were mixed with an equal volume of 2x Laemmli sample buffer and resolved by SDS-PAGE. After electrophoretic separation, proteins were electroblotted on to polyvinylidene difluoride membranes (NEN Life Sciences, Buckinghamshire, UK) for protein immunoblotting. Polyvinylidene difluoride membranes were blocked in a 4% BSA (50 mM Tris-HCl, 0.05% Tween 20, 0.05% Nonidet P-40, pH 7.0) blocking solution. Immunoblotting of endogenous pERK1/2, pJNK1, or pP38 was performed using a 1:1000 dilution of rabbit antihuman phosphorylated ERK1/2, JNK1, or P38 antisera (Cell Signaling, Hertfordshire, UK), respectively. A 1:1000 dilution of rabbit antihuman total ERK1/2 antiserum was used to verify protein loading. Visualization of the phosphorylated or unphosphorylated protein was achieved by addition of a 1:10,000 dilution of alkaline phosphatase-conjugated polyclonal antirabbit IgG as a secondary antibody. Each alkaline phosphatase-labeled protein was visualized using an enzyme-linked chemifluorescence reaction (Amersham) and quantified using a Typhoon 9400 PhosphorImager GE Healthcare.

Phosphoinositide Hydrolysis
Assays for ligand stimulation of inositol phosphate production were performed as previously described (66, 67). Briefly, appropriately transfected MEF S19 cells were labeled overnight with 1 µCi/ml myo-D-[3H]inositol (Amersham) in inositol-free DMEM containing 1% dialyzed fetal calf serum before being incubated in 0.5 ml buffer A (140 mM NaCl, 20 mM HEPES, 8 mM glucose, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml BSA) containing 10 mM LiCl at 37 C for 30 min. Thereafter, appropriate ligand stimulation was carried out for an additional 30 min. The reaction was terminated by removal of the medium and addition of 0.5 ml of 10 mM formic acid. The [3H]inositol phosphates were isolated from the formic acid extracts using Dowex AG 1-X8 ion exchange resin (Bio-Rad), collected with 1 M ammonium formate containing 0.1 M formic acid and quantified by liquid scintillation counting.

Membrane Preparation
Cells were collected in a harvesting buffer (20 mM HEPES, 100 mM EDTA, pH 7.5) and ruptured with 20 strokes of a glass dounce homogenizer. Nuclei and unbroken cells were separated by centrifugation at 200 x g for 15 min. The resultant supernatant was then subjected to a high-speed spin at 40,000 x g for 45 min and resuspended in a GTP{gamma}S assay buffer (5 mM MgCl2, 100 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol). To ensure optimal assay conditions, membranes were prepared fresh rather than stored. Membrane concentrations were determined as previously described (70).

Scintillation Proximity Assay
The SPA was performed as previously described (71). Briefly, cell membranes expressing the receptor of interest (approximately 75 µg protein/well) were incubated in the presence or absence of ligand and 200 pM [35S]GTP{gamma}S for 1 h at 25 C. After incubation, membranes were solubilized in a 0.3% Nonidet P-40 solution for 30 min. Thereafter, antibodies (using dilutions of 1:440 for G{alpha}i1/2 and G{alpha}i3 and 1:1000 for G{alpha}q/11) and anti-IgG-coated SPA beads were added and incubated for an additional 3 h. Plates were centrifuged at 3000 x g for 10 min and counted on a Wallac MicroBeta Trilux β-counter [PerkinElmer Life and Analytical Sciences, (UK) Ltd., Buckinghamshire, UK].

Measurement of Intracellular cAMP
After a 30-min incubation with 1 mM 3-isobutyl-1-methylxanthine and appropriate ligand stimulation, cell monolayers were placed on ice, washed twice with ice-cold PBS, and lysed in 0.1 M HCl. Intracellular cAMP concentrations were determined using an ELISA kit (Biomol, Exeter, UK) as per the manufacturer’s instructions.

Measurement of Cell Growth
After continuous agonist stimulation, cell monolayers were placed on ice, and an equal volume of cold 25% trichloroacetic acid was added directly to the culture medium. Cells were left at 4 C for 1 h after which cell growth was determined using the sulforhodamine B assay as previously described (72).

Statistical Analysis
All experiments were repeated independently at least three times. In addition, all assays were performed in triplicate. Statistical significance was set at P < 0.05, indicated by asterisks in figures, and analyses were performed using the Student’s t test. For agonist dose-response analyses, data representing the mean ± SE from at least three independent experiments were plotted and analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Sigmoidal dose-response curves were fitted to the relevant data sets and the EC50 value determined.


   ACKNOWLEDGMENTS
 
We acknowledge Ryan Gallagher, Vicki Warrender, Robin Sellar, Laura Melville, and Donald Wilson for their expert technical assistance. We also thank Axel Thomson and Rachel Forfar for critically reviewing the manuscript before submission.


   FOOTNOTES
 
This work was funded by the United Kingdom and South African Medical Research Councils.

This work was presented at the 89th Annual Meeting of The Endocrine Society, Toronto, Canada, June 2007 (Abstract P1-399).

Disclosure Summary: C.D.W., M.C., K.M., C.A.F. and Z.-L.L. have nothing to disclose. R.P.M. consults for Ardana Biosciences.

First Published Online September 18, 2008

Abbreviations: GPCR, G protein-coupled receptor; JNK, jun-N-terminal kinase; PKC, protein kinase C; PLCβ, phospholipase Cβ; SPA, scintillation proximity assay.

Received for publication April 15, 2008. Accepted for publication September 9, 2008.


   REFERENCES
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 DISCUSSION
 MATERIALS AND METHODS
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