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Endocrinology Vol. 141, No. 9 3087-3097
Copyright © 2000 by The Endocrine Society

ARTICLES

Gonadotropin-Releasing Hormone Receptor Activation of Extracellular Signal-Regulated Kinase and Tyrosine Kinases in Transfected GH3 Cells and in {alpha}T3–1 Cells1

Melanie S. Johnson, Eve M. Lutz2, Christopher J. MacKenzie3, W. Bart Wolbers, Derek N. Robertson, Pamela J. Holland and Rory Mitchell

Medical Research Council Membrane and Adapter Proteins Cooperative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom EH8 9XD; and Medical Research Council Brain Metabolism Unit, Edinburgh, United Kingdom EH8 9JZ

Address all correspondence and requests for reprints to: Dr. Melanie S. Johnson, Medical Research Council Membrane and Adapter Proteins Cooperative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, United Kingdom EH8 9XD.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion.

We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and {alpha}T3–1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in {alpha}T3–1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in {alpha}T3–1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in {alpha}T3–1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in {alpha}T3–1 cells.

In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in {alpha}T3–1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GnRH RECEPTOR belongs to the seven-transmembrane spanning, G protein-coupled receptor (G7) superfamily, which includes ß-adrenergic receptors, muscarinic receptors, and odorant receptors. Sequences of the mouse (1), rat (2), and human (3) GnRH receptors show a high degree of homology. The GnRH receptor is atypical compared with other receptors in the G7 receptor superfamily; there is a lack of a C-terminal, intracellular tail; a change of a conserved Asp87-Asn318 motif in the second and seventh transmembrane regions, respectively, for the inverse amino acid motif Asn87-Asp318, and a change in a conserved DRY140xxV motif in the second intracellular loop for DRS140xxV. These characteristics in the GnRH receptor sequence are probably involved in some of the unusual properties displayed when the GnRH receptor binds and is activated by GnRH. There is little rapid desensitization of the receptor on exposure to GnRH, and internalization/down-regulation of the receptor is a relatively long term event (4, 5, 6). Although the major route of signal transduction leading to hormone secretion appears to be through Gq/11{alpha} (7, 8) [activating phospholipase C (PLC) and hence eliciting inositol phosphate ([3H]InsP) production and raised intracellular Ca2+] (6), there is evidence that this receptor may interact with other heterotrimeric G proteins (9, 10, 11). Indeed, other workers, who produced a GH3-derived cell line similar to that used in this study, have shown that the GnRH receptor can couple through a cholera toxin-sensitive Gs{alpha} to affect PRL synthesis and (long-term) secretion (12). Although GnRH receptors in both transiently transfected COS-7 cells and stably transfected GH3 cells can elevate cAMP levels, there is no good evidence that cAMP is a direct mediator of acute LH secretion in gonadotropes (9, 12). The receptor can also activate tyrosine kinases in {alpha}T3–1 cells and pituitary tissue by an unknown pathway (13) and possibly tyrosine phosphatases in ovarian-carcinoma tissue (14). Activation of the GnRH receptor in {alpha}T3–1 cells can result in activation of the mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase 1/2 (ERK1/2) (11, 15, 16), p38 MAPK (17), and Jun N-terminal kinase (18). Finally, the GnRH receptor displays the phenomenon of GnRH self-priming; that is, after an appropriate preexposure to steroid hormones, an initial challenge of GnRH greatly augments GnRH-induced LH release on a subsequent exposure (19).

To study the signaling capability of the GnRH receptor from native tissue such as the mixed cell population in anterior pituitary or from {alpha}T3–1 cells where the link between receptor and secretion has been affected by the transformation to an immortal cell line (20) is problematic. To overcome these difficulties we created a model of GnRH receptor function in a single cell type that retains a functional stimulus-secretion mechanism. The GnRH receptor complementary DNA (cDNA) was transfected into GH3 cells to produce stably expressing cell lines that could secrete both PRL and GH in response to GnRH and agonists of native receptors. In this study we produced such a clonal cell line, wt28, and showed that GnRH-induced hormone secretion was mediated through activation of PLC and concomitantly [3H]InsP production. However, some aspects of intracellular signaling that have been recorded for native tissue, i.e. pituitary gonadotropes or {alpha}T3–1 cells, are different in these cells, namely GnRH-induced tyrosine and ERK1/2 phosphorylation. A role for tyrosine kinase action in the sustained ERK1/2 phosphorylation seen in {alpha}T3–1 cells is suggested by the ability to reproduce it in wt28 cells with the addition of pervanadate. Thus, even though {alpha}T3–1 and wt28 cells are both derived from pituitary tissue, the endogenous signaling pathways converging on ERK1/2 activation are different. These data highlight the variety of responses that can result from the same receptor depending on the host tissue in which it is expressed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transfection of GH3 cells with cDNA encoding the murine GnRH receptor
GH3 cells were transfected with the GnRH receptor encoding cDNA (1.31-kb insert in pcDNA1 containing the neomycin resistance gene, named SCS56) (1) by electroporation of 5 x 106 cells with 2 µg cDNA/cuvette. Two days after transfection, cells were selected by incubation in DMEM with sodium pyruvate containing 5% FCS and 100 U each of penicillin and streptomycin supplemented with 500 µg/ml geneticin (selection medium). After 1 month of growing cells in geneticin-containing medium, clones were selected, replated into 24-well plates, and maintained in the selection medium. Once cell numbers had reached a sufficient level, clonal lines were assayed for [3H]InsP production in response to GnRH and selected accordingly. Clonal cell lines were maintained in flasks in the same medium, but with the geneticin concentration reduced to 200 µg/ml.

Northern blot analysis
Total RNA was isolated from cells using the Catrimox-14 surfactant solution (VH Bio Ltd., Newcastle-upon-Tyne, UK). Approximately 15 µg total RNA from each cell line were separated by electrophoresis on denaturing 1.2% agarose/formaldehyde gels, transferred to a nitrocellulose membrane, and baked for 2 h at 80 C. The membrane was then hybridized with the 1.3-kb EcoRI/XhoI/HindIII fragments of the mouse GnRH receptor cDNA, SCS56 that had been labeled with [32P]deoxy-CTP [DuPont (UK) Ltd., Stevenage, UK] using random hexanucleotide primers (Pharmacia Biotech, St. Albans, UK) and the Klenow fragment of Escherichia coli DNA polymerase (21). Hybridization was performed for 1 h in QuikHyb solution (Stratagene, Cambridge, UK) with 100 µg/ml denatured herring sperm DNA at 68 C. The filter was washed twice for 15–30 min each time in 2 x SSC buffer (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at room temperature, then for 15 min at 60 C with 0.1 x SSC-0.1% SDS, and exposed to Fuji Photo Film Co., Ltd. RX film (Tokyo, Japan).

[3H]Inositol phosphate production
Cells cultured in 12-well tissue culture plates (maintained at 37 C in a 5% CO2-95% O2 environment) were labeled with 0.5 µCi/ml myo-[2-3H]inositol (Amersham Pharmacia Biotech, Aylesbury, UK) for 16–18 h in Earle’s balanced salt solution (EBSS) with 10 mM glucose and 10 mM HEPES, pH 7.4 (NaOH). The cells were then washed twice in EBSS with 10 mM glucose, 10 mM HEPES, 0.2% BSA (fraction V; Sigma-Aldrich Corp., Poole, UK), pH 7.4 (NaOH), and preincubated for 10 min with 10 mM LiCl before agonist (GnRH and TRH, both from Sigma) stimulation. During stimulation the cells were maintained at 37 C in a 5% CO2-95% O2 environment. Reactions were stopped by aspiration of medium and addition of 700 µl ice-cold 1.34 M trichloroacetic acid to each well. The wells were scraped, and the solution was centrifuged to pellet the precipitated protein (5 min, 12,000 x g); a 500-µl sample of the supernatant was then added to 50 µl 0.1 M EDTA and 500 µl of a 1:1 mixture of 1,1,2-trichlorotrifluoroethane and tri-n-octylamine. The sample was vortexed and centrifuged (5 min, 12,000 x g), and a 300-µl volume of the aqueous phase was added to 200 µl 1 M NaHCO3 containing universal indicator. The sample was applied to a 1-ml column of Dowex anion exchange resin (1 x 8 resin, formate form, 200–400 mesh; Bio-Rad Laboratories, Inc., Hemel Hempstead, UK), and a stepwise gradient of ammonium formate was used to elute the [3H]inositol phosphates; a method previously described by Berridge et al. (22).

Whole cell binding
The GnRH agonist, [D-Ala6,des-Gly10]GnRH-ethylamide (GnRH-A; Peninsula Laboratories, Inc., Belmont, CA) was iodinated by the chloramine-T procedure and purified by the method of Sandow and Konig (23). Binding of [125I]GnRH-A to transfected GH3 cells was carried out in 12-well plates according to the method of Slice et al. (24). Briefly, cells were washed twice with medium 199 containing 0.2% BSA before being incubated on ice for 90 min in the same medium with [125I]GnRH-A and in the absence or presence of increasing concentrations of unlabeled GnRH-A. In experiments investigating the time course of association of ligand to receptor, the cells were incubated for different lengths of time at 37 C, and nonspecific binding was determined using unlabeled GnRH-A (3 µM). At the end of the incubation the cells were washed three times with EBSS-0.1% BSA at 4 C and then incubated for 5 min with ice-cold acid-strip solution (0.2 M acetic acid and 0.5 M NaCl). This solution was then removed, and the 125I content was determined. If measurement of internalized label was also required, cells were dissolved in lysis buffer (2% NaHCO3, 1% SDS, and 0.1 M NaOH), and this was also removed and counted for 125I. Protein estimations were performed on cells using the Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL), and estimations of receptor number from the displacement curve that was constructed were performed using the method of Swillens (25) after determination of the IC50 with the nonlinear, error-weighted program P.FIT (Biosoft, Cambridge, UK).

Secretion of PRL and GH
Cells were seeded into 12-well plates, and the medium was changed every day for 3 days before secretion experiments were undertaken. On the day of the experiment cells were washed twice in MEM (Earle’s salts with HEPES) before MEM containing GnRH (Sigma), vasoactive intestinal polypeptide [VIP; Calbiochem-Novabiochem (UK) Ltd., Nottingham, UK], or no peptide was added to the cells. In experiments with U73122 (20 µM; Affiniti Research Products Ltd., Exeter, UK), this compound was added to the appropriate wells in ethanol (to a final concentration of 1%, which was also added to all wells not treated with U73122) 2 h before the cells went through the wash procedure. At the end of a specified length of time (usually 10 min) of incubation at 37 C under an atmosphere of 95% O2-5% CO2, medium was changed for fresh medium with the appropriate drugs added, and the incubation was repeated. All of the medium samples removed from the wells were stored frozen (-20 C) until they were assayed for PRL or GH by RIA (26). Separation of bound radiolabel was achieved using the double antibody technique (secondary antibody a gift from the Scottish Antibody Production Unit, Carluke, UK). The standards used were NIDDK RP-3 for PRL and RP-1 for GH.

Immunoblotting with phosphotyrosine and phospho-ERK1/2 antibodies
Cells were seeded in equal numbers in 12-well plates and grown to confluence. Twenty-four hours before treatment with GnRH, medium was exchanged for serum-free medium to quiesce the cells. Both {alpha}T3–1 cells and wt28 cells were treated with GnRH concentrations in the range 1 nM to 1 µM, and other drugs [phorbol 12,13-dibutyrate (PDBu; Sigma), forskolin (FSK; Calbiochem), and Bay K 8644 (RBI, Sigma)] were prepared as stock solutions (10 mM) in dimethylformamide and kept at -20 C for times ranging between 2–60 min before washing in ice-cold HBSS (Ca2+ and Mg2+ free) and scraping into SDS sample buffer (2% SDS, 20 mM Tris, and 5% mercaptoethanol). The immunoblots were carried out as described previously (26) using a mouse monoclonal antibody to phosphotyrosine, 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), and rabbit polyclonal antibodies to ERK1/2 and phospho-ERK1/2 (New England Biolabs, Inc., Hitchin, UK).

Before immunoblotting was carried out, blots were stained with PhastGel Blue R (Amersham Pharmacia Biotech) to check that protein loading of the lanes was even. In many cases the blots were scanned at this point so that an arbitrary measure of protein could be made to use for correction when blots were scanned and bands were analyzed using densitometry (Scan Analysis, Biosoft, Cambridge, UK), and the data were displayed as a graph. For the phospho-ERK blots, some blots were stripped (Re-Blot reagent, Chemicon International Ltd., Harrow, UK) and reprobed with the ERK antibody to detect variations in protein loading (the values obtained were very similar in relative terms to those seen using the PhastGel Blue R). This also allowed us to check specifically that there was no effect of time or drugs on ERK1/2 protein levels; none was found. All blots were repeated at least twice and usually three or more times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production and characterization of a stable cell line containing the murine GnRH receptor
Transfection of the GnRH receptor into GH3 cells resulted in the creation of 36 clonal cell lines, and an initial screen of these lines was carried out by assaying GnRH-induced [3H]InsP production compared with responses of the native TRH receptor. From this screen, 5 cell lines were chosen (wt5, wt6, wt25, wt26, and wt28). GnRH (1 µM)-induced [3H]InsP responses in these cell lines were approximately 2-, 2-, 9-, 11-, and 12-fold increases over basal, respectively, compared with approximately 8-fold increases evoked by 1 µM TRH (response measured over 30 min). Expression of the 1.3-kb construct in these clonal cell lines, but not in the parental GH3 cell, was confirmed by Northern analysis (Fig. 1AGo). Secretion of PRL in response to acute (10-min) exposure to GnRH and VIP, agonist for the native VPAC2 receptor, was also measured in the 5 cell lines. All 5 displayed significant increases in PRL secretion in response to these peptides (data not shown). Responses to GnRH were greatest in the 3 cell lines that expressed the higher levels of GnRH receptor, and the line selected for further study (wt28) demonstrated a PRL secretion response to 100 nM GnRH of 6.6 ± 0.3-fold the basal value (n = 4). Figure 1BGo shows a homologous displacement curve for [125I]GnRH-A binding to whole wt28 cells. The receptor number was calculated as 202 ± 32 fmol GnRH receptor/mg protein, which when corrected for protein per cell gave a value of 34,600 ± 7,400 receptors/cell. These estimates were repeated several times and on each occasion gave closely similar results. The time course of association of [125I]GnRH-A to the cell surface GnRH receptor and internalization of this complex into wt28 cells is shown in Fig. 1CGo. Maximum specific binding of label occurred by 2 min, was considerably reduced at 5 min, and decreased slowly thereafter. Internalization of [125I]GnRH-A into a compartment resistant to acid washing of the cells was detectable by 5 min, rose to a plateau at 10 min, and was around 6 ± 1% of maximum binding for the duration of the experiment. This time course of association and internalization of GnRH-A to the GnRH receptor corresponds closely to observations in both transiently transfected COS-7 cells and {alpha}T3–1 cells (data not shown).



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  		Figure 1. A, Northern blot of GnRH receptor messenger RNA from five clonal cell lines (wt5, wt6, wt25, wt26, and wt28) with accompanying control blots, including {alpha}T3–1 cell RNA. Each lane was loaded with approximately 15 µg RNA. Lanes are numbered 1–11, where 1 is GH3 cell RNA only, 2 is wt28, 3 is wt26, 4 is wt25, 5 is wt6, 6 is wt5, 7 is wt2, 8 is SH-SY5Y cells, 9 is {alpha}T3–1 cells, 10 is AtT20 cells, and 11 is RNA from COS-7 cells. wt2 was a clonal cell line that showed no significant [3H]InsP production or hormone secretion. B and C, GnRH receptor binding in clonal cell line wt28. B, The homologous displacement of [125I]GnRH-A binding by GnRH-A in clonal cell line wt28 gave an IC50 value of 0.64 ± 0.01 nM. The ratio of maximum counts bound to total counts added was approximately 1:25. At each point the value is the mean ± SEM (n = 4). C, Association of [125I]GnRH-A to wt28 cells at 37 C. Values for specifically bound ligand that was removed by acid wash (external; •) and that which was resistant (internal; {blacksquare}) are shown. At each point the value is the mean ± SEM (n = 4).

 
GnRH-induced [3H]InsP production in wt28 cells displayed an EC50 of 2.8 ± 0.8 nM, and maximal responses were achieved between 30–100 nM (Fig. 2AGo). The time course of [3H]InsP production in response to 100 nM GnRH in wt28 cells showed that the initial rate (0.41 ± 0.01-fold of basal/min) was maintained over the first 20 min, but then showed a small decline (to 0.19 ± 0.01-fold of basal/min) over the next 40 min (Fig. 2BGo). Similarly, the GnRH response in {alpha}T3–1 cells maintained a steady rate for at least 20 min, whereas the initial high rate of [3H]InsP production in response to TRH in wt28 cells decreased after 5 min, as reported previously (27), but this was not investigated in greater detail here.



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  		Figure 2. Concentration-response curve at 20 min (A) and time course to 100 nM GnRH ({blacksquare}; B) for GnRH-induced [3H]InsP production in wt28 cells. A, When the concentration-response curve was fitted, an EC50 value of 2.8 ± 0.8 nM GnRH was obtained. Basal [3H]InsP production was 1398 ± 130 dpm over the 20-min assay period. At each point the value is the mean ± SEM (n = 4). B, Data are depicted as the fold increase over basal [3H]InsP levels, where the basal value was 3588 ± 244 dpm. Basal levels did not significantly change over the 60-min period investigated. Data for TRH (100 nM)-induced [3H]InsP production in wt28 cells (•) and GnRH (100 nM)-induced [3H]InsP production in {alpha}T3–1 cells ({square}) are included for comparison. At each point, the value is the mean ± SEM (n = 4–6).

 
Acute secretion of PRL in response to both GnRH and VIP was statistically significant by 5 min, and output was maintained for 30–40 min (Fig. 3AGo). GnRH-induced GH secretion was detectable by 5 min, continued for the next 30–40 min, and then returned to a slow rate similar to basal. VIP-induced GH reached significant levels only after 15 min and then continued for up to 1 h (Fig. 3BGo). The EC50 value for GnRH-induced PRL secretion (measured at 10 min) was 5.6 ± 3.3 nM (Fig. 3CGo). As the secretagogue effects of GnRH and VIP on anterior pituitary cells are considered to be mediated largely through PLC and adenylyl cyclase, respectively, the effects of the selective PLC inhibitor U73122 (20 µM) on GnRH- and VIP-induced PRL secretion from wt28 cells were compared (Fig. 3DGo). U73122 reduced GnRH-induced PRL release to levels not significantly different from those seen with U73122 alone, whereas there was no detectable effect of U73122 on VIP-induced PRL release.



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  		Figure 3. Secretory responses of wt28 cells. The time course for 100 nM GnRH (•)- and 100 nM VIP ({blacksquare})-induced PRL (A) and GH (B) release and concentration-response curve for GnRH-induced PRL release from wt28 cells (C). Data from stimulated groups were stripped of basal release at each time point. Basal release of PRL started at 8.1 ± 0.3 at 5 min and maintained a rate of approximately 1.8 ng/ml·min; that for GH started at 18.9 ± 0.4 and maintained a rate of 4.9 ng/ml·min. At each point, n = 4 for basal and stimulated values. The concentration-response curve (C) for GnRH-induced PRL release was measured over a 10-min assay period. PRL release was significantly greater than basal at 10 and 100 nM GnRH (*, P < 0.05, by Student’s t test). At each point, n = 6, and the value is the mean ± SEM. The EC50 value (5.6 ± 3.3 nM GnRH) obtained assumed a maximum response at 100 nM GnRH. This value was assessed from other experiments of concentration-response data using higher concentrations of GnRH (data not shown). D, Effect of the PLC inhibitor U73122 (20 µM) on GnRH (100 nM)- and VIP (100 nM)-induced PRL release from wt28 cells. Stimulus-induced release in the presence of GnRH/VIP alone (compared with basal release) is depicted in the open bars. GnRH- and VIP-induced release in the presence of U73122, compared with the effect of U73122 alone, is indicated by the hatched bars. Whereas VIP-induced release was still increased by 2-fold, GnRH-induced PRL release was significantly (*, P < 0.05) reduced by U73122. For each group, n >= 4. Values are the mean ± SEM.

 
Effect of GnRH receptor activation on ERK1/2 and tyrosine phosphorylation in wt28 compared with {alpha}T3–1 cells
An involvement of ERK1/2 and tyrosine kinase activation has been noted in GnRH action on {alpha}T3–1 cells and pituitary gonadotrophs (11, 13, 15, 16). To determine whether the GnRH receptor expressed in wt28 cells could affect these kinases, immunoblots with antiphospho-ERK1/2 and antiphosphotyrosine antibodies were carried out using {alpha}T3–1 cells as a comparison. Figure 4AGo shows the effect of a 10-min incubation with GnRH (0.1 nM to 1 µM) on phospho-ERK1/2 immunoreactivity in wt28 and {alpha}T3–1 cells as well as the effect of TRH on its native receptor in wt28 cells. The GnRH response in {alpha}T3–1 cells showed marked phosphorylation of both p44ERK1 and p42ERK2, whereas in wt28 cells both agonists predominantly caused phosphorylation of p42ERK2. Correspondingly, a difference in the relative amounts of the two ERKs could be seen in the pan (total)-ERK1/2 blot between the two cell types, with wt28 cells being relatively deficient in ERK1 (clearly shown in Figs. 7CGo and 9AGo). The time courses of ERK1/2 activation showed some differences between the two cell types (Fig. 4BGo), with a sustained response (up to 1 h) in {alpha}T3–1 cells, but a more transient one (declining after 15 min) for both GnRH and TRH in wt28 cells (see also Fig. 8AGo). GnRH in a range of concentrations caused no detectable phosphotyrosine staining in wt28 cells, whereas the marked GnRH-induced appearance of phosphotyrosine-containing proteins in {alpha}T3–1 cells was concentration dependent (Fig. 5AGo). Even when the enhanced chemiluminescence film from immunoblots of GnRH-stimulated wt28 cells was exposed for a much longer period, to reveal basal phosphotyrosine levels (Fig. 5BGo), no GnRH-dependent action was apparent. Similarly, no TRH-induced tyrosine phosphorylation was detectable in wt28 cells (Fig. 5CGo), even though the intensity of phospho-ERK1/2 immunoreactivity seen with TRH in wt28 cells approached that seen with GnRH in {alpha}T3–1 cells (Fig. 4Go).



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  		Figure 4. Immunoblots of {alpha}T3–1 and wt28 cells showing the concentration response (A) and time course (B) of peptide agonist-induced phospho-ERK1/2 immunoreactivity. The concentration-response data were obtained at 10 min, and GnRH or TRH at 100 nM was used for the time-course data. A direct comparison of the efficacies of ERK1/2 phosphorylation by GnRH and TRH in the wt28 cells is shown in A, where 1 µM of each agonist was used in the same experiment (TRH row). When measured using densitometry, the GnRH-induced phospho-ERK1/2 immunoreactivity was 59% of that induced by TRH.

 


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  		Figure 7. Effects of GnRH combined with the activators shown in Fig. 6Go on ERK1/2 phosphorylation. A, An immunoblot of {alpha}T3–1 samples, where the effects of FSK (1 µM) on GnRH (100 nM)-induced phospho-ERK1/2 immunoreactivity at an early and a later time point are shown. B, The effects of PDBu (1 µM) and Bay K 8644 (1 µM) on GnRH (100 nM)-induced phospho-ERK1/2 immunoreactivity in {alpha}T3–1 and wt28 cells at 10 min. The numbers represent incubation with the following agents: 1) vehicle alone, 2) GnRH, 3) PDBu, 4) Bay K 8644, 5) GnRH plus PDBu, and 6) GnRH plus Bay K 8644. Blots were stripped and reprobed for total ERK1/2 immunoreactivity present in the lanes. C, The effect of PV (100 µM) on GnRH (100 nM)-induced phospho-ERK1/2 immunoreactivity in {alpha}T3–1 and wt28 cells at 10 min. Again, reprobed blots for pan-ERK1/2 immunoreactivity are shown for comparison.

 


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  		Figure 9. The effects of different concentrations of PV and GnRH on ERK1/2 phosphorylation in {alpha}T3–1 and wt28 cells. Immunoreactivity for both ERK1/2 and phospho-ERK1/2 are shown (A) in the two cell types at 10 min. In these data it can be seen that there was less total ERK1/2 in the wt28 GnRH (3 nM)/PV (1 mM) sample. This was corrected for when the data were transformed using scanning densitometry to produce histograms B and C. B, Data for {alpha}T3–1 cells; C, data for wt28 cells. Open bars show data for PV alone, lightly hatched bars show the addition of 3 nM GnRH, and heavily hatched bars show the addition of 30 nM GnRH.

 


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  		Figure 8. Effects of PV and GnRH on the time course of phospho-ERK1/2 (A) and phosphotyrosine (B) immunoreactivities. A, Phospho-ERK1/2 immunoblots from {alpha}T3–1 and wt28 cells that were exposed to PV (100 µM), GnRH (100 nM), or both together over time points up to 1 h were compared. The blots were stripped and reprobed with pan-ERK1/2 antibody, thus demonstrating no effect of any treatment on total ERK1/2 over the time period investigated. B, The same samples probed for phosphotyrosine.

 


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  		Figure 5. Antiphosphotyrosine immunoblots of wt28 and {alpha}T3–1 cells treated with GnRH. A, Concentration-response data to GnRH of {alpha}T3–1 and wt28 cells at 15 min. B and C, Effects of GnRH (100 nM; B) and TRH (100 nM; C) on wt28 cells. In B and C, the enhanced chemiluminescence film was extensively exposed to visualize basal levels of phosphotyrosine-containing proteins. Any slight differences between control and treated samples were not consistent across experiments (n = 3).

 
In view of such differences in GnRH-induced ERK1/2 and tyrosine phosphorylation between {alpha}T3–1 and wt28 cells, we decided to examine responses to activation of various intracellular signaling pathways that may lead from the GnRH receptor to ERK and tyrosine kinase (Fig. 6Go). Concentrations of activators were used that cause near-maximal effects, thus PDBu (1 µM) to activate protein kinase C (PKC), FSK (1 µM) to activate adenylyl cyclase, Bay K 8644 (1 µM) to activate L-type voltage-gated calcium channels, and pervanadate (PV; 1 mM) to inhibit tyrosine phosphatases and thus unmask tyrosine kinase activity. Figure 6AGo shows the effects of these activators on phospho-ERK1/2 immunoreactivity. Only PDBu, and to much lesser extent Bay K 8644, activated ERK1/2 in {alpha}T3–1 cells. However, in wt28 cells the most effective activator was FSK, followed by PDBu, PV, and then Bay K 8644. Figure 6BGo shows the effects of the various agents on phosphotyrosine levels. The major difference between the two types was that PDBu caused phosphorylation of tyrosine-containing proteins in {alpha}T3–1 cells, but had no such effect in wt28 cells. The cell lines were similar in showing increased tyrosine phosphorylation in response to PV, but not Bay K 8644 or FSK. Figure 6Go, C and D, depicts densitometric data from typical phospho-ERK1/2 and phosphotyrosine immunoblots when both {alpha}T3–1 cells and wt28 cells were stimulated via a variety of individual or combined conditions. The robust response to PDBu in {alpha}T3–1 cells was only marginally increased by adding either Bay K 8644 or PV, whereas in wt28 cells both of these drugs greatly increased ERK1/2 phosphorylation. However, the maximal ERK1/2 phosphorylation in wt28 cells was seen with either PV plus FSK or PDBu plus FSK. In contrast, adding FSK to PDBu in {alpha}T3–1 cells caused a marked reduction in ERK1/2 phosphorylation compared with PDBu alone. Most other combinations appeared to show approximately additive or moderately greater than additive responses. Phosphotyrosine immunoreactivity was greatest for both cell types with either PV plus Bay K 8644 or PV plus FSK, but only with {alpha}T3–1 cells was there a large response to PDBu plus PV (Fig. 6DGo). FSK had no inhibitory effect on PDBu-induced phosphotyrosine in {alpha}T3–1 cells.



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  		Figure 6. Effects of signaling pathway activators on phospho-ERK1/2 and phosphotyrosine immunoreactivity in {alpha}T3–1 and wt28 cells. A, The effects of vehicle (0.1% dimethylformamide), PDBu (1 µM), Bay K 8644 (1 µM), FSK (1 µM), and PV (1 mM) on phosphorylation of ERK1/2. Cells were incubated for 10 min with the drugs before the experiment was terminated by aspiration of the medium and addition of 2 x Laemmli buffer. B, The same samples were immunoblotted for phosphotyrosine. Interactions between the drugs were also investigated. C and D, Densitometric analysis of phospho-ERK1/2 (C) and phosphotyrosine (D) data in histogram form. {alpha}T3–1 cells are represented by the lightly hatched bars, and wt28 cells are shown with the heavily hatched bars. The numbers on the horizontal axis represent incubations with the following agents: 1) vehicle alone, 2) Bay K 8644, 3) PV, 4) PV plus Bay K 8644, 5) FSK, 6) FSK plus Bay K 8644, 7) FSK plus PV, 8) PDBu, 9) PDBu plus Bay K 8644, 10) PDBu plus PV, and 11) PDBu plus FSK. Concentrations were the same as in A. The blots were scanned, and densitometry was used to convert the data into an arbitrary gray scale, presented here as a percentage of the maximum immunoreactivity detected within the dataset analyzed. Phospho-ERK1/2 data were prenormalized for protein abundance using pan-ERK1/2 blot data from the same blots that had been stripped and reprobed. Phosphotyrosine data were corrected in the same way using measurements from PhastGel Blue R staining of the blot.

 
Figure 7Go shows the effect of the signaling pathway activators on GnRH-induced phospho-ERK1/2 immunoreactivity. In {alpha}T3–1 cells FSK inhibited the GnRH-induced responses in 5-min incubations, but at 30 min there was much less effect of the drug (Fig. 7AGo). PDBu and Bay K 8644 had an additive effect with GnRH-induced phospho-ERK1/2 immunoreactivity in wt28 cells at 10 min (Fig. 7BGo), whereas in {alpha}T3–1 cells GnRH alone produced the maximal response. PV increased GnRH-induced ERK1/2 phosphorylation in wt28 cells more than in {alpha}T3–1 cells, and on its own it had a greater effect in wt28 cells (Fig. 7CGo).

Involvement of tyrosine kinase activation in phospho-ERK1/2 production by GnRH
We conjectured that the sustained time course of ERK1/2 phosphorylation in {alpha}T3–1 cells may result from tyrosine phosphorylation, and thus the transient nature of the phospho-ERK1/2 response in wt28 cells may be caused by the absence of this tyrosine phosphorylation. Thus, we investigated the interaction of PV with GnRH in the two cell types over time (Figs. 7CGo and 8AGo). Again there was little effect of PV on {alpha}T3–1 cells up to 60 min, GnRH produced a sustained ERK1/2 phosphorylation over this period, whereas PV and GnRH together produced an additive effect, as determined using densitometry. With wt28 cells, an increasing effect of PV with time could be seen, the transient response with GnRH alone was recapitulated, and together these stimuli produced a synergistic phosphorylation of ERK1/2 (2–3 times the sum of the components), which was now maintained up to 60 min. Equivalent data for tyrosine phosphorylation are shown in Fig. 8BGo; no similar synergy was seen in the wt28 data here, although an additive effect of PV and GnRH can be seen in {alpha}T3–1 cells.

These data support the idea that tyrosine kinase activity is necessary for the sustained response of phospho-ERK1/2 to GnRH in {alpha}T3–1 cells. However, to check that effects were not being overlooked because of the responses being maximal, concentration-response data were obtained for PV and GnRH at 10 min. Figure 9AGo shows the greater effect of PV on phospho-ERK1/2 in wt28 cells compared with {alpha}T3–1 cells. When used with increasing concentrations of GnRH, the synergism of PV with GnRH was evident in wt28 cells. In contrast, there was little effect of PV on {alpha}T3–1 cells with or without GnRH. Figure 9BGo shows densitometry quantification of these data and emphasizes that GnRH-evoked activation of ERK1/2 in wt28 cells was facilitated at PV concentrations (e.g. 100 µM) that have little effect alone. In contrast, PV has no marked effect on the phosphorylation of ERK1/2 in {alpha}T3–1 cells caused by submaximal concentrations of GnRH.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have created a stable cell line containing the GnRH receptor with some of the characteristics of native tissue. The measurements of GnRH-induced [3H]InsP production and PRL/GH secretion were similar to those recorded after activation of native GH3 cell receptors (i.e. TRH and VIP receptors). The concentration of receptors expressed per mg protein in the wt28 line was in a similar range as that recorded in {alpha}T3–1 cells [{alpha}T3–1 cells, 1.3–1.9 pmol/mg protein (28) compared with 0.202 pmol/mg in wt28]. Overexpression of receptors in clonal cell lines can lead to the anomalous activation of signal transduction pathways (29); however, the levels of expression recorded for wt28 are on par with the native receptors in GH3 cells. For example, VIP receptor number has been estimated at 9,000/cell and TRH receptors at 135,000/cell (30) compared with 34,600 GnRH receptors/cell in wt28 cells. The suppression of GnRH-induced PRL release by the PLC inhibitor U73122, which had no effect on VIP-induced release (most likely mediated through Gs{alpha} and cAMP), provides further support for the idea that the GnRH receptor in this cell line was probably coupling through a Gq/11{alpha} protein to PLC to produce secretion.

Another group who have used the same approach to create a model GnRH receptor-containing cell line from GH3 cells did not record the rapid secretory response demonstrated here (12, 31). Instead, they detected an interesting long-term effect of the GnRH receptor on PRL output mediated through cAMP production and protein synthesis. We suggest that the main reason for the discrepancy between our study and theirs is the difference in receptor number per cell expressed in the clonal cell lines created. Our wt28 cells have 3.6-fold more GnRH receptors per cell than their cell line expressing the rat receptor and 16.7-fold more than their cell line expressing the murine receptor. Indeed, a link between the level of [3H]InsP production and GnRH receptor number has been demonstrated in GGH3 cells (32). This may suggest that a minimum number of receptors have to be expressed before the intracellular signaling events leading to secretion occur.

Activation of ERK1/2 in {alpha}T3–1 cells has been shown to involve activation of PKC (11, 16), tyrosine kinase (16), Ca2+ influx through L-type channels (33), and an unidentified pertussis toxin-sensitive component (11), but not cAMP (16). This was borne out by the present study, which emphasized the importance of the route of activation by PKC and the inhibitory role of cAMP. In contrast, activation of ERK1/2 in GH3 cells has been shown to involve PKC, tyrosine kinase, Ca2+, and cAMP (34, 35); interestingly, the study by Kanasaki et al. (35) showed that TRH-induced secretion was independent of ERK1/2 activation. The rat GnRH receptor stably transfected in GH3 cells has been shown to activate ERK1/2 through PKC and PKA (36). Our study supports some of these findings for the murine GnRH receptor in GH3 cells, where the most efficient phosphorylator of ERK1/2 was FSK. We described differences in the time course of ERK1/2 phosphorylation in the two cell types. The inability of the GnRH receptor to stimulate tyrosine phosphorylation in the wt28 cells compared with {alpha}T3–1 cells was also revealed. Increasing tyrosine phosphorylation by inhibiting tyrosine phosphatases with PV resulted in greater ERK1/2 activity over basal and synergistically increased GnRH-induced ERK1/2 phosphorylation in the wt28, but not the {alpha}T3–1, cells. This indicates that there is probably a pathway absent in wt28 cells, relative to {alpha}T3–1 cells, linking GnRH receptor activation to increased tyrosine kinase activity (or inhibiting tyrosine phosphatase activity). As PDBu can activate certain tyrosine kinases in {alpha}T3–1, but not in wt28, cells, and GnRH appears to do the same, it is possible that a PKC isoform or particular tyrosine kinase that is present in {alpha}T3–1 cells is absent in the GH3 parent cells. The route through a tyrosine kinase appears to be necessary for a sustained ERK1/2 response, as we could reproduce it in wt28 cells with addition of PV to GnRH. Sustained ERK1/2 responses in other signaling systems have been shown to rely on tyrosine phosphorylation of intermediary proteins (37) and relative efficacy of receptor agonists (38). The end effect of ERK1/2 activation depends on whether it is acutely or chronically activated (39). Thus, the GnRH receptor could exert different cellular effects through ERK1/2 depending upon the tissue in which it is expressed. A cell type-specific response of the ERK1/2 pathway has been shown for the GH receptor (40). In this study it was demonstrated that there was an all (3T3-F442A cells) or none (IM-9 lymphocytes) activation of ERK1/2, and one of the isoforms of the adapter protein, Shc, was absent in the IM-9 cells.

Phosphorylation of ERK1/2 in wt28 cells was particularly robust using FSK to raise cAMP levels, whereas in {alpha}T3–1 cells FSK was inhibitory at 5–10 min on either GnRH- or PDBu-induced ERK1/2 phosphorylation. These data suggest that in addition to the difference in tyrosine phosphorylation, there is also a difference in the signaling proteins (involved in ERK1/2 activation) expressed by the different cell types. A plausible explanation is that the complement of Raf isoforms and/or ERK pathway-related small G proteins may be different in {alpha}T3–1 and wt28 cells. It has been shown (41) that cAMP has opposing effects on ERK1/2 activation in neurons (cAMP activates ERK1/2) and astrocytes (cAMP inhibits ERK1/2), and it has been suggested that this difference is dependent on the expression of B-Raf in neurons, but not in astrocytes. In cases where raised cAMP results in ERK1/2 activation, the pathway can involve regulation of B-Raf by Rap-1 (a small G protein) (41, 42). An alternative pathway can be through Ras-dependent Raf-1 (43, 44). It has also been shown that where signaling to ERK1/2 is mediated via Ras/Raf-1, raised cAMP levels can be inhibitory (42), consistent with our observations in {alpha}T3–1 cells. However preliminary findings in our laboratory using antibodies to Raf-1 and B-Raf in Western blotting indicate that both of our cell types express Raf-1 abundantly and both the 95- and 68-kDa isoforms of B-Raf at lower levels (compared with rat frontal cortex and SH-SY5Y cells). Thus, if the identity of the Raf isoform activated is important in determining the phosphorylation of ERK1/2 in {alpha}T3–1 cells vs. wt28 cells, the cell type difference does not appear to be at the level of Raf isoform expression.

In sum, we have created a stable cell line in GH3 cells containing the GnRH receptor. GnRH can bind to its receptors in these cells to activate G{alpha}q/11 leading to raised inositol phosphate production and secretion of PRL and GH. However ERK1/2 and tyrosine phosphorylation patterns in response to GnRH (together with their responses to activation of several other signaling pathways) were not the same in the two cell types. This probably results from differences in the expression of particular signaling proteins in the host cell. Therefore, activation of the GnRH receptor has the potential to cause diverse cellular events depending on the tissue in which it is expressed.


   Acknowledgments
 
We thank John Bennie and Sheena Carroll for assistance with the RIAs, Drs. Stuart Sealfon and Wei Zhou for providing the cDNA for the GnRH receptor, Drs. G. D. Niswender, L. E. Reichert, Jr., and the Pituitary Hormone Distribution Agency of the NIDDK (Baltimore, MD), Dr. S. Raiti of the National Hormone and Pituitary, University of Maryland School of Medicine (Baltimore, MD), as well as the Scottish Antibody Production Unit (Carluke, Scotland) for the gift of RIA materials, and Marianne Eastwood for secretarial assistance.


   Footnotes
 
1 This work supported by a research fellowship from the European Community (to W.B.W.). Back

2 Present address: Department of Bioscience and Biotechnology, University of Strathclyde, 204 George Street, Glasgow, United Kingdom G1 1XW. Back

3 Present address: Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, Glasgow, United Kingdom G4 0NR. Back

Received February 17, 2000.


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 Discussion
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