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Endocrinology Vol. 144, No. 10 4354-4365
Copyright © 2003 by The Endocrine Society

Gonadotropin-Releasing Hormone-Desensitized LßT2 Gonadotrope Cells Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling

Fujun Liu, Darrell A. Austin and Nicholas J. G. Webster

Department of Medicine (F.L., N.J.G.W.) and the University of California San Diego Cancer Center (N.J.G.W.), University of California, San Diego, California 92093; and the Medical Research Service (N.J.G.W., D.A.A.), San Diego Veterans Healthcare System, San Diego, California 92161

Address all correspondence and requests for reprints to: Nicholas Webster, Department of Medicine 0673, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: nwebster{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of gonadotropin release, but the mechanisms involved are poorly understood. It is known that desensitization is associated with decreased GnRH receptor and Gq/11 levels in {alpha}T3-1 cells, but it is not known whether downstream signaling is impaired. We have shown previously that chronic stimulation of signaling via expression of an active form of G{alpha}q causes GnRH resistance in LßT2 cells. In this study we investigated whether chronic GnRH treatment could down-regulate protein kinase C (PKC), cAMP, or Ca2+-dependent signaling in LßT2 cells. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. Desensitization was observed for activation of ERK and p38 MAPK and induction of c-fos and LHß protein expression. Activation of individual signaling pathways was able to partially mimic the desensitizing effect of GnRH on ERK, p38 MAPK, c-fos, and LHß but not on Gq/11. Chronic stimulation with phorbol esters reduced GnRH receptor expression to the same extent as chronic GnRH. Sustained GnRH also desensitized PKC signaling by down-regulating the {delta}, {epsilon}, and {theta} isoforms of PKC. We further show that chronic GnRH treatment causes heterologous desensitization of other Gq-coupled receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC DECAPEPTIDE GnRH is the central regulator of the mammalian reproductive system. It acts mainly in the anterior pituitary via a specific GnRH receptor (GnRH-R) on the plasma membrane of gonadotrope cells, in which it triggers the secretion of LH and FSH, which in turn stimulate production of gonadal steroids (1, 2). The GnRH-R is a member of the G protein-coupled receptor (GPCR) family. Binding of GnRH activates phospholipase C (PLC) via the Gq/11 proteins, causing hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol, and also activates adenylate cyclase via the Gs protein, leading to production of cAMP. These second messengers are able to mobilize Ca2+ from intracellular stores and activate protein kinase C (PKC) and protein kinase A (PKA), respectively, thereby propagating a signaling cascade that accounts for many of the biological effects of GnRH (3, 4, 5).

Gonadotrope responsiveness is modulated both by the GnRH concentration and the frequency or pattern of its administration. Physiologically, GnRH is secreted in the pulsatile fashion by hypothalamic neurons, but sustained exposure of gonadotrope cells to an equivalent dose of GnRH markedly impairs their responsiveness to an acute GnRH stimulus (6, 7). This phenomenon termed homologous desensitization is a common feature of many seven-transmembrane domain GPCRs (8). Homologous desensitization causes a profound reduction in circulating gonadotropin and gonadal steroid levels and provides the rationale for the major clinical application of GnRH agonists in the treatment of endometriosis and steroid-dependent mammary and prostate carcinoma (9, 10). Extensive studies, particularly of the ß-adrenergic receptors, have revealed a specific mechanism for rapid homologous desensitization in which the active conformation of the receptor becomes rapidly phosphorylated by GPCR-specific kinases. ß-Arrestin binds the phosphorylated receptor, preventing further interaction with G proteins and causing internalization of the receptor. This mechanistic scheme has been extended to encompass many other GPCRs, which couple to Gs, Gi, and Gq/11 (11, 12, 13). The residues within these GPCRs that are phosphorylated by GPCR-specific kinases usually lie within the C-terminal tail of the receptor or the third intracellular loop (14, 15, 16). Cloning of the first mammalian GnRH-R revealed that it lacks C-terminal tail and has a comparatively short third intracellular loop, which is unique among known GPCRs. These unexpected structural features, particularly the lack of the C-terminal tail, has raised the question of whether mammalian GnRH receptors are able to desensitize rapidly (17).

In {alpha}T3-1 cells, the GnRH-R is coupled exclusively via the Gq/11 protein to PLC, and its activation promotes a rapid increase of inositol phosphates (IPs) and diacylglycerol (DAG) production (18). Activation of the GnRH-R induced a linear accumulation of IPs for up to 30 min (19, 20). This result indicates that the GnRH-R does not undergo homologous desensitization within this time frame. However, sustained GnRH treatment of these cells resulted in substantial loss of GnRH-evoked IP responses, a significant decrease in the number of GnRH-Rs, and reductions in both the initial Ca2+ spike and the plateau phase (21, 22). Another, more recent study showed that sustained GnRH pretreatment resulted in inhibition of IP formation, attenuation of phospholipase D activation, and suppression of GnRH-induced arachidonic acid release (23).

The mechanism underlying this chronic GnRH desensitization is not yet clear. GnRH-R does not interact with ß-arrestin (24), so the mechanism of desensitization must differ from other GPCRs. It is known that desensitization is associated with decreased GnRH-R and Gq/11 levels in {alpha}T3-1 cells, so gonadotrope desensitization might occur at the proximal steps of the GnRH-signaling pathways (25, 26). Our previous study showed that chronic activation of G{alpha}q signaling via expression of a constitutively active form of G{alpha}q (Q209L) results in a state of GnRH resistance (5), so in this study we investigated whether chronic GnRH treatment could down-regulate more distal downstream signaling pathways that are activated by the GnRH-R in LßT2 cells (27, 28).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
GnRH and cholera toxin (CTX) were purchased from Sigma Chemical Co. (St. Louis, MO). Phorbol-12-myristate-13-acetate (PMA) and forskolin were from Calbiochem (La Jolla, CA). Rabbit polyclonal anti-phospho-p44/42 ERK (Thr202/Tyr204) antibodies and rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibodies were from Cell Signaling Technology (Beverly, MA). PKC{alpha}, PKCß, PKC{gamma}, PKC{delta}, PKC{eta}, PKC{lambda}, PKC{theta}, and PKC{iota} monoclonal antibodies were purchased from BD Transduction Labs (San Diego, CA). Rabbit polyclonal anti-c-fos antibodies (sc-52), rabbit polyclonal anti-G{alpha}q/11 (C-19), anti-G{alpha}i-3 (C-10), anti-Gß (T-20), anti-G{alpha}s/olf (C-18), goat polyclonal anti-GnRH-R antibodies, and horseradish peroxidase-linked antimouse, antirabbit, and antigoat antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-LHß antibodies kindly provided by Dr. A. F. Parlow at the National Hormone Pituitary Program (NHPP), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated antirabbit antibodies were purchased from Jackson ImmunoResearch Laboratory, Inc. (West Grove, PA). All other reagents were purchased from either Sigma or Fisher Scientific (Pittsburgh, PA).

Cell culture
LßT2 cells were maintained in monolayer cultures in DMEM supplemented with 10% fetal bovine serum and antibiotics in humidified 10% CO2 at 37 C. Cells were pretreated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl in serum-free DMEM for 48 h, and then cells were stimulated acutely with 100 nM GnRH, 100 nM PMA, 10 µM forskolin, or 50 mM KCl 5 min for ERK and p38 MAPK activation, 1 h and 4 h for c-fos induction, or 8 h for LHß induction.

Western Blotting
LßT2 cells were grown to confluence in six-well plates, washed once with PBS, and pretreated then acutely stimulated as above. Thereafter, cells were washed with ice-cold PBS, and lysed on ice in sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris, 5% glycerol, 2% SDS, 0.005% bromophenol blue, 84 mM dithiothreitol, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate, pH 6.8), boiled for 5 min to denature proteins and sonicated for 5 min to shear the chromosomal DNA. Equal volumes (30–40 µl) of these lysates were separated by SDS-PAGE on 10% gels, and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dried milk in TBS-Tween [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20]. Blots were incubated with primary antibodies in blocking buffer for 60 min at room temperature and then incubated with horseradish peroxidase-linked secondary antibodies followed by chemiluminescent detection. Anti-phospho-p44/42 ERK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-c-fos antibodies, anti-PKC antibodies, anti-Gq/11, anti-Gs, anti-Gi, or anti-Gß antibodies were used at a dilution of 1:1000. Anti-GnRH-R antibodies were used at a dilution of 1:500. To verify protein loading, the PVDF membranes were immediately stripped by placing the membrane in stripping buffer (0.5 M NaCl and 0.5 M acetic acid) for 10 min at room temperature. The membrane was then washed once for 10 min in TBS-Tween, reblocked, and blotted with antibodies to the unphosphorylated form of the enzyme. The intensities of the bands corresponding to phospho-ERK, phospho-p38MAPK, and c-fos were quantified using an EDAS290 gel documentation system (Kodak, Rochester, NY) with an Agfa Arcus II scanner. Band intensity for the phospho-protein was corrected for intensity of the native protein and then expressed as the percentage inhibition, compared with the agonist treatment alone. Immunoblotting individual PKC isoforms was performed in a slot-blotter apparatus (Hoefer Scientific, San Francisco, CA). Equal amounts of lysate were separated in parallel lanes and transferred to a PVDF membrane. Individual lanes on the filter were aligned in the slot-blotter apparatus allowing each to be incubated with a separate antibody. Antibody incubations were performed at a dilution of 1:1000 as above.

Immunostaining
LßT2 cells were plated on 10-mm acid-washed glass coverslips and pretreated with agonists and stimulated acutely as above. Cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. Following two washes in PBS, the cells were permeabilized and blocked in PBS containing 5% BSA and 0.5% Nonidet P-40 for 10 min. Coverslips were incubated with the rabbit anti-LHß antibody (1:1200 dilution) for 60 min at room temperature, washed once in PBS, and then incubated with TRITC-conjugated antirabbit IgG antibody (1:100 dilution) in PBS with 5% BSA and 0.5% Nonidet P-40 for 30 min at room temperature. Following a wash with PBS, coverslips were incubated with a DNA intercalating dye (Hoechst 33258, Sigma) diluted 1:250 for 60 min to stain nuclei. Finally, the coverslips were extensively washed with PBS, rinsed with water, and mounted in PBS containing 15% gelvatol (polyvinyl alcohol), 33% glycerol, and 0.1% sodium azide. Staining was visualized with an Axiophot fluorescence microscope (Zeiss, Gottingen, Germany) and photographed using the ISEE imaging system (Inovision, Raleigh, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chronic GnRH down-regulates ERK activation by PKC and cAMP but not by Ca2+ signaling
Chronic GnRH treatment causes GnRH resistance in primary pituitary cells and {alpha}T3-1 cells (29, 30). Part of this resistance can be explained by decreases in GnRH receptor expression, but the degree of resistance is greater than the decrease in receptor levels, suggesting that downstream signaling might also be impaired. Decreases in a number of signaling proteins after chronic GnRH treatment are consistent with this notion. We have shown previously that induction of c-fos and LHß protein expression by GnRH requires the MAPK kinase (MEK)-ERK cascade in LßT2 cells (31). Therefore, these experiments were designed to test whether acute activation of ERK is impaired in LßT2 cells treated chronically with GnRH. Initially, cells were treated with 100 nM GnRH for increasing times and assayed for ERK phosphorylation to determine the optimum conditions for down-regulation. Activation of ERK was still detectable at 4, 8, and 24 h of treatment, but by 48 h no ERK activation was detectable (data not shown). Based on this result, 48 h was chosen as the time for chronic treatment with GnRH.

LßT2 cells were treated with 100 nM GnRH for 48 h and then stimulated acutely with 100 nM GnRH, 100 nM PMA to activate PKC, or 10 µM forskolin to elevate cAMP for 5 min. Whole-cell lysates were immunoblotted for the dually phosphorylated forms of ERK. Pretreatment with GnRH completely blocked the subsequent acute stimulation of ERK by GnRH in agreement with the desensitization observed in {alpha}T3-1 cells (Fig. 1AGo). More importantly, chronic GnRH also blocked the acute stimulation of ERK by forskolin or PMA. This finding shows that both PKC- and cAMP-dependent signaling is suppressed in the GnRH-down-regulated cells. In light of this, we tested whether chronic activation of individual signaling pathways could mimic the effect of GnRH. LßT2 cells were pretreated with 100 nM PMA or 100 ng/ml CTX, to activate Gs and elevate cAMP, for 48 h, then stimulated acutely with GnRH, PMA, or forskolin. We were unable to use forskolin for the chronic studies on LßT2 cells because of toxicity (data not shown). As expected, chronic PMA and CTX caused desensitization of acute PKC and cAMP signaling, respectively (Fig. 1AGo). In addition, pretreatment with PMA completely (>90%) blocked GnRH stimulation of ERK, and pretreatment with CTX reduced activation by 36%. These results are consistent with GnRH signaling via both PKC and cAMP to stimulate ERK, as we have proposed previously (5).



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  		FIG. 1. Effect of GnRH, PMA, CTX, or KCl pretreatment on agonist-induced ERK activation. A, Effect of GnRH, PMA, and CTX pretreatment on agonist-induced ERK activation. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, and 100 ng/ml CTX for 48 h before acute treatment with 100 nM GnRH, 100 nM PMA, and 10 µM forskolin (Fors) for 5 min. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an antibody phospho-ERK1/2. B, Effect of depolarization with KCl on ERK activation. LßT2 cells were pretreated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX or 50 mM KCl for 48 h and then stimulated by 50 mM KCl for 5 min, or cells were pretreated with 50 mM KCl for 48 h, followed by 100 nM GnRH, 100 nM PMA, 10 µM forskolin (Fors), or 50 mM KCl for 5 min. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with the antibody to phospho-ERK1/2. Blots were stripped and re-blotted for ERK1/2 to verify protein loading. The blots are representative of three independent experiments. C, Summary of desensitization: +, total inhibition (>90%); -, no inhibition (<10%); and ±, partial inhibition. Numbers in parentheses represent percentage of inhibition ± SD vs. agonist stimulation alone (P < 0.05).

 
We showed previously that calcium signaling was not important for ERK activation by GnRH, although elevation of calcium by depolarization of cells with KCl was able to activate ERK (31). Consequently, we tested whether chronic treatment with the various agonists would desensitize calcium signaling also. Chronic treatment with GnRH, PMA, or CTX had no effect on acute calcium signaling (Fig. 1BGo). Chronic depolarization of cells with 50 mM KCl inhibited the acute activation of ERK by KCl but had no effect on PMA or forskolin activation of ERK (Fig. 1BGo). Surprisingly, chronic depolarization partially (47%) blocked the GnRH activation of ERK, although calcium signaling is not required for the GnRH effect. These desensitization results are summarized in Fig. 1CGo. The PKC, cAMP, and calcium signaling pathways activate ERK independently and do not show any cross-desensitization on chronic treatment. Chronic GnRH treatment desensitizes cells to a subsequent GnRH challenge and also desensitizes cells to both PKC and cAMP signaling but has no effect on calcium signaling. These results agree with our previous conclusion that GnRH activation of ERK proceeds via PKC and cAMP but not calcium signaling pathways. Chronic activation of each signaling pathway was able to desensitize cells to GnRH to differing extents. The impairment with chronic cAMP or calcium signaling was only partial (36% and 47%, respectively), but chronic PMA blocked completely. We have shown previously that PKC signaling accounts for approximately 50% of the effect of GnRH on ERK activation in whole cells but is absolutely required for nuclear translocation of ERK. The effect of chronic PMA is greater than the effect of PKC inhibition, so down-regulation of PKC signaling alone cannot account for observed desensitization.

Chronic GnRH down-regulates p38 MAPK activation by PKC, cAMP, and Ca2+ signaling
Our previous study showed that GnRH stimulates the p38 subfamily of MAPKs with a similar time course to the ERKs (31). In this section, we investigated whether activation of this subfamily is similarly desensitized by long-term exposure with GnRH. LßT2 cells were pretreated with 100 nM GnRH for 48 h as before and then stimulated with 100 nM GnRH, 10 µM forskolin, or 100 nM PMA for 5 min. Whole-cell lysates were immunoblotted with an antibody to the dually phosphorylated, activated form of p38 MAPK. Pretreatment with GnRH completely blocked the subsequent acute stimulation of p38 MAPK by GnRH as expected. Acute forskolin treatment activated p38 MAPK as strongly as GnRH, but PMA was a much weaker agonist. Chronic GnRH blocked the acute forskolin- and PMA-stimulated p38 MAPK as we had observed for ERK (Fig. 2AGo), confirming that PKC- and cAMP-dependent signaling is suppressed. Here again, we tested whether chronic activation of individual signaling pathways could mimic the effect of GnRH. Chronic stimulation of cells with 100 nM PMA or 100 ng/ml CTX for 48 h desensitized cells to PKC and cAMP signaling, respectively. Chronic PMA treatment was also able to partially (31%) reduce the acute GnRH activation of p38 MAPK, despite the very weak activation of p38 MAPK by PMA alone.



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  		FIG. 2. Effect of GnRH, PMA, CTX, or KCl pretreatment on agonist-induced p38 MAPK activation. A, Effect of GnRH, PMA, and CTX pretreatment on agonist-induced p38 MAPK activation. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX for 48 h and then stimulated acutely with 100 nM GnRH, 100 nM PMA, or 10 µM forskolin (Fors) for 5 min. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an antibody to phospho-p38 MAPK. B, Effect of depolarization with KCl on p38 MAPK activation. LßT2 cells were pretreated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h and then stimulated by 50 mM KCl for 5 min, or cells were pretreated with 50 mM KCl for 48 and stimulated by 100 nM GnRH, 100 nM PMA, 10 µM forskolin (Fors), or 50 mM KCl for 5 min. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an antibody to phospho-p38 MAPK. Blots were stripped and reblotted with an antibody to p38 MAPK to verify protein loading. The blots are representative of two independent experiments. C, Summary of desensitization: +, total inhibition (>90%); -, no inhibition (<10%); and ±, partial inhibition. Numbers in parentheses represent percentage of inhibition ± SD vs. agonist stimulation alone (P < 0.05).

 
We also tested whether depolarization of cells with KCl would activate p38 MAPK. Acute treatment of LßT2 cells with 50 mM KCl activates p38 MAPK robustly (Fig. 2BGo). Desensitization of cells by chronic GnRH blocked the acute calcium effect, as did chronic depolarization of cells with KCl. Surprisingly, chronic CTX partially (54%) reduced the acute KCl stimulated p38 MAPK, but chronic PMA had no effect. Conversely, calcium levels were chronically elevated by treatment with 50 mM KCl, and cells were stimulated acutely (Fig. 2BGo). Chronic depolarization of cells with KCl inhibited the subsequent acute GnRH- and forskolin-stimulated but had no effect on PMA activation of p38 MAPK. The desensitization results for p38 MAPK activation are summarized in Fig. 2CGo. Unlike activation of ERK, GnRH appears to activate p38 MAPK primarily via calcium signaling because chronic PMA and CTX do not substantially impair activation. Pretreatment of cells with BAPTA-AM to chelate intracellular calcium also blocks the GnRH stimulation of p38 MAPK (data not shown). Desensitization of cells with chronic GnRH suppresses PKC, cAMP, and Ca2+ signaling to p38 MAPK. This is discordant with activation of ERK. Chronic GnRH has no effect on calcium activation of ERK but completely blocks calcium activation of p38 MAPK. It is possible that different calcium-dependent signals are involved in the two processes. The cross-desensitization experiments also uncovered unexpected cross-talk between the cAMP- and calcium-signaling pathways that was not seen for ERK activation.

Chronic GnRH down-regulates c-fos induction by PKC, cAMP, and Ca2+ signaling in LßT2 cells
We have shown previously that GnRH induced c-fos protein expression is MEK- and calcium-dependent but PKC-independent (31). Therefore, we investigated the effect of chronic GnRH on acute induction of c-fos. LßT2 cells were pretreated with 100 nM GnRH for 48 h and stimulated acutely with 100 nM GnRH, 100 nM PMA, or 10 µM forskolin for 60 min. Whole-cell lysates were then immunoblotted for c-fos (Fig. 3AGo). Chronic treatment with GnRH blocked the acute GnRH- and PMA-induced c-fos protein expression. Forskolin treatment for 1 h was unable to induce c-fos expression, however. As before, cells were treated chronically with PMA or CTX and then stimulated acutely with GnRH, PMA, or forskolin. Chronic CTX treatment partially reduced GnRH- and PMA-induced c-fos protein expression (40% and 44%, respectively). Similarly, chronic PMA treatment partially blocked GnRH-induced c-fos expression (60%). Our previous data showed that stimulation of c-fos expression by forskolin is detectable after 4 h, so the experiment was repeated using acute stimulation with forskolin for 4 h. Under these conditions, forskolin is able to induce c-fos expression (Fig. 3BGo). As expected, pretreatment with GnRH or CTX blocked forskolin-induced c-fos expression, but PMA had no effect (Fig. 3BGo).



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  		FIG. 3. Effect of GnRH, PMA, CTX, or KCl pretreatment on agonist-induced c-fos expression. A, Effect of GnRH, PMA, and CTX pretreatment on agonist-induced c-fos expression. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX for 48 h and then stimulated acutely with 100 nM GnRH, 100 nM PMA, or 10 µM forskolin (Fors) for 60 min. Whole-cell lysates were separated by SDS-PAGE and blotted with an antibody to c-fos. The blots were then stripped and reblotted with an antibody to ERK1/2 to determine total protein loading. B, Effect of GnRH, PMA, and CTX pretreatment on forskolin-induced c-fos expression. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX for 48 h, followed by 10 µM forskolin (Fors) for 4 h. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an antibody to anti-c-fos. The blots were then stripped and reblotted with an antibody to ERK1/2 to determine total protein loading. C, Effect of depolarization with KCl on c-fos expression. Cells were pretreated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h and stimulated with 50 mM KCl for 60 min or cells incubated in serum-free DMEM with 50 mM KCl for 48 h, followed by 100 nM GnRH, 100 nM PMA, 10 µM forskolin (Fors), or 50 mM KCl for 60 min. Whole-cell lysates were separated by SDS-PAGE and blotted with an antibody to c-fos. The blots were then stripped and reblotted with an antibody to ERK1/2 to determine total protein loading. The blots are representative of three experiments. D, Summary of desensitization: +, total inhibition (>90%); -, no inhibition (<10%); and ±, partial inhibition. Numbers in parentheses represent percentage of inhibition ± SD vs. agonist stimulation alone (P < 0.05). ND, Not determined.

 
Because we had shown previously that GnRH-induced c-fos expression is calcium dependent, we investigated whether chronic GnRH would inhibit acute calcium-induced c-fos expression and vice versa (Fig. 3CGo). Chronic treatment with GnRH, PMA, or KCl but not CTX, blocked the acute KCl-induced c-fos expression. Conversely, chronic depolarization of cells with KCl completely blocked c-fos induction by GnRH, PMA, and KCl. The desensitization results for c-fos expression are summarized in Fig. 3DGo. As we had observed for activation of p38 MAPK, chronic GnRH desensitizes PKC, cAMP, and Ca2+ signaling to c-fos. Down-regulation of any of the three signaling pathways impairs the acute GnRH effect. The desensitization by chronic cAMP or calcium is consistent with our previous results showing that GnRH induction of c-fos is partially blocked by inhibition of Gs and calcium signaling (5, 31). The desensitization by chronic PMA, however, cannot be explained simply by down-regulated PKC signaling, as induction of c-fos by GnRH is independent of PKC signaling (31). These results also uncovered an unexpected cross-talk between PKC and calcium signaling. This could be at the level of the calcium-dependent conventional PKC isoforms, although we have shown previously that calcium induction of c-fos is not inhibited by the general PKC inhibitor bisindolylmaleimide I (31).

Chronic GnRH down-regulates LHß protein expression by PKC, cAMP, and Ca2+ signaling
The LßT2 cells express both LHß and FSHß mRNAs and secrete mature LH in response to GnRH. The mRNAs for LHß and FSHß, as well as the GnRH-R, but not the common {alpha}-subunit are induced by GnRH (28). A previous study had shown that the suppression of gonadotropin secretion by chronic GnRH treatment in primary pituitary cells was due in part to decreased gonadotropin ß-subunit gene expression, but the {alpha}-subunit gene, in contrast, was not altered. Consequently, we investigated whether LHß protein expression is desensitized in chronically treated LßT2 cells. LßT2 cells were plated on coverslips; pretreated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h in serum-free medium; and then stimulated acutely with 100 nM GnRH for 8 h. Cells were fixed and stained for LHß, counterstained for DNA, visualized by fluorescence microscopy, and counted (Fig. 4AGo). The chronic treatment of LßT2 cells with GnRH completely blocked the acute GnRH-induced LHß protein expression without affecting basal LHß expression. Chronic desensitization with PMA, CTX, or KCl also significantly inhibited the acute GnRH induction. The effect of CTX was weaker than PMA or KCl, however. The desensitization by chronic PMA again cannot be explained by down-regulation of PKC signaling because induction of LHß by GnRH is independent of PKC signaling, as is induction of c-fos. Desensitization of LßT2 cells by chronic GnRH also blocks the subsequent induction of LHß by PMA, forskolin, or KCl, confirming that GnRH signals via all three pathways (Fig. 4BGo).



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  		FIG. 4. Effect of GnRH, PMA, CTX, or KCl pretreatment on agonist-induced LHß protein expression. A, Effect of GnRH, PMA, CTX, or KCl pretreatment on GnRH-induced LHß expression. LßT2 cells on coverslips were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h and then stimulated with 100 nM GnRH for 8 h. LHß protein expression was visualized using a rabbit-anti-LHß antibody, followed by a TRITC-conjugated secondary antibody. Nuclei were counterstained with Hoechst 33258 DNA dye. Cells with perinuclear LHß protein expression were counted from a minimum of three independent fields. Results are presented as percent of cells positive for LHß fluorescence. Error bars, SEM. Asterisks, statistical significance (P < 0.05) vs. acutely stimulated cells. B, Effect of GnRH pretreatment on agonist-induced LHß expression. LßT2 cells on coverslips were incubated in serum-free DMEM with 100 nM GnRH for 48 h and then stimulated acutely with 100 nM PMA, 10 µM forskolin, or 50 mM KCl for 8 h. Cells were fixed and stained as above. The results are the mean ± SE of three experiments.

 
Chronic GnRH or PMA treatment down-regulates the GnRH-R
Homologous desensitization is common feature of GPCRs. It is mediated acutely by uncoupling of the receptor from heterotrimeric G-proteins and by internalization and degradation of the receptor and at later time points by down-regulation of receptor gene expression. The long-term decreased responsiveness of a cell often is due to this reduction in receptor gene expression (8). Although the GnRH-R does not undergo rapid desensitization at the protein level, sustained exposure of primary rat gonadotropes and {alpha}T3-1 cells to GnRH does cause down-regulation of the GnRH-R gene. Because of the observed discordance between PMA desensitization and PKC-independent GnRH signaling to p38 MAPK, c-fos, and LHß signaling, we explored whether chronic PMA treatment down-regulates GnRH-R expression. LßT2 cells were treated with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX for 48 h and whole-cell lysates were immunoblotted for the GnRH-R (Fig. 5Go). Chronic GnRH treatment markedly reduced GnRH-R protein (63%) as expected, as did treatment by PMA (57%). Chronic CTX had no effect on GnRH-R (data not shown). These data offer a potential explanation for the loss of GnRH responsiveness when cells are chronically treated with GnRH or PMA. They also suggest that signaling by PKC is important for down-regulation of the GnRH-R in LßT2 cells.



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  		FIG. 5. Down-regulation of GnRH-R by chronic GnRH and PMA. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX (not shown) for 48 h. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against GnRH-R. The blots were stripped and reblotted for ERK1/2 protein, demonstrating equivalent loading. These blots are representative of two experiments.

 
Chronic GnRH treatment specifically down-regulates Gq/11
Not only are GPCRs subject to homologous desensitization, but heterotrimeric G proteins are also subject to down-regulation. A previous study (32) showed that prolonged GnRH treatment reduced the level of the Gq/11 protein. We had observed down-regulation of the GnRH-R with chronic GnRH or PMA treatment, so we then investigated whether the G protein subunits might be decreased also. LßT2 cells were treated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h, and then whole-cell lysates were immunoblotted with antibodies to Gq/11, Gs, Gi, or Gß. Chronic GnRH treatment specifically reduced the level of the Gq/11 protein and had no effect on Gs, Gi, or Gß proteins (Fig. 6AGo). Chronic stimulation with PMA, CTX, or KCl did not alter the levels of any of these proteins. This result shows that GnRH specifically down-regulates the Gq/11 protein but not the Gs protein, although the GnRH receptor couples to both. The down-regulation of Gq/11 is not mimicked by PMA, cAMP, or calcium signaling. The reduction in the level of Gq/11 also suggests that activation of PLC by other Gq/11-coupled receptors would be impaired in GnRH-desensitized cells. To test this proposition, LßT2 cells were treated with 100 nM GnRH for 48 h and then stimulated acutely with a mixture of agonists known to signal via G{alpha}q (Gqmix: 50 nM bombesin, 50 nM bradykinin, and 10 nM endothelin-1) (5). Immunoblotting with antibodies to phospho-ERK confirmed that chronic treatment of GnRH reduced Gqmix-induced ERK activation by 48% (Fig. 6BGo).



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  		FIG. 6. Down-regulation of G protein subunits by chronic agonist treatment. A, LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h. Basal and chronically treated whole-cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against Gq/11, Gs, Gi, or Gß. These blots are representative of two experiments. B, Chronic GnRH treatment desensitizes Gqmix-induced ERK activation. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH for 48 h and then stimulated with a mixture of Gq agonists (Gqmix: 50 nM bombesin, 50 nM bradykinin, and 10 nM endothelin-1) for 5 min. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against phospho-ERK1/2. Then blots were stripped and reblotted for ERK1/2 protein to determine equal total protein loading. Blots are representative of two experiments with similar results.

 
Chronic GnRH treatment reduces novel PKC isoforms
Acute activation of PKCs is often followed by desensitization of the associated cellular responses. This desensitization is due to the loss of specific PKC isoforms, depending on the duration of the stimulus by either phorbol esters or neuropeptides (23, 33, 34). Chronic GnRH treatment of LßT2 cells impairs subsequent PMA stimulation of ERK, p38 MAPK, c-fos, and LHß. Consequently, we investigated whether expression of PKC isoforms were altered under conditions of chronic desensitization. LßT2 cells were treated with 100 nM GnRH, 100 nM PMA, 100 ng/ml CTX, or 50 mM KCl for 48 h. Whole-cell lysates were immunoblotted with antibodies to PKC isoforms. Blots were stripped and reblotted for ERK1/2 to control for protein loading. The {alpha}, ß, {delta}, {epsilon}, {iota}, {lambda}, and {theta} isoforms of PKC were observed in LßT2 cells, but the PKC{gamma} and -{eta} were not detected (Fig. 7Go). Chronic GnRH treatment selectively eliminated expression of PKC{delta} and reduced the {epsilon} and {theta} isoforms but had no effect on other isoforms. Chronic PMA treatment reduced the expression of all DAG-dependent isoforms including {alpha}, ß, {delta}, {epsilon}, and {theta} but had no effect on the atypical PKC{iota} and -{lambda}. Chronic treatment by CTX and KCl did not alter PKC isoform expression. These data suggest that GnRH-induced down-regulation of PKC{delta}, -{epsilon}, and -{theta} are sufficient to block PMA activation of ERK and p38 MAP, and induction of c-fos of LHß.



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  		FIG. 7. Down-regulation of PKCs by chronic GnRH, PMA, CTX, or KCl treatment. LßT2 cell were incubated in serum-free DMEM with 100 nM GnRH, 100 nM PMA, or 100 ng/ml CTX, or 50 mM KCl for 48 h in separate experiments. Whole-cell lysates were separated by SDS-PAGE and blotted for the {alpha}, ß, {gamma}, {delta}, {eta}, {epsilon}, {iota}, {lambda}, and {theta} isoforms of PKC. The blots were then stripped and reblotted with an antibody to ERK1/2 to verify protein loading.

 
GnRH responses are suppressed for up to 4 h after removal of GnRH
We also investigated the time required for recovery for GnRH-induced desensitization in LßT2 cells (Fig. 8Go). LßT2 cells were pretreated with 100 nM GnRH for 48 h, and then the GnRH was removed and cells allowed to recover for increasing times before an acute GnRH stimulation for 5 or 60 min. Whole-cell lysates were immunoblotted for phospho-ERK, c-fos, Gq/11, and Gs protein levels. GnRH activation of ERK started to reappear in cells after 90 min but was still only 47% of the maximal acute effect after 4 h of recovery. Addition of fresh medium without the subsequent acute stimulation by GnRH had no effect on ERK activation (data not shown). In contrast, c-fos expression was still refractory to GnRH stimulation after 4 h. In agreement with the c-fos induction, Gq/11 protein levels were still reduced after the 4-h recovery, but Gs protein levels did not change throughout the experiment. These data demonstrate that desensitization induced by chronic GnRH treatment persists for more than 4 h after removal of the GnRH, with activation of ERK being the first observable event. This recovery experiment also demonstrates that the lack of response to the acute agonist is not due simply to depletion of a component of the cell culture medium during the pretreatment.



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  		FIG. 8. Time course of recovery of GnRH-induced desensitization. LßT2 cells were incubated in serum-free DMEM with 100 nM GnRH for 48 h, and then the medium containing GnRH was removed and cells washed and refed with new medium without GnRH for increasing times. Cells were then stimulated acutely with 100 nM GnRH for 5 min for ERK activation or 60 min for c-fos induction. Whole-cell lysates were separated by SDS-PAGE and blotted for phospho-ERK, c-fos, Gq/11, and Gs, respectively. The blots were then stripped and reblotted with an antibody to ERK1/2 to verify protein loading. The blots are representative of two independent experiments. Addition of fresh medium alone had no effect on ERK activation (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has long been known that GnRH-stimulated gonadotropin secretion is desensitized during sustained stimulation of gonadotropes with GnRH. The multiple mechanisms contributing toward this effect include depletion of intracellular gonadotropin pools, internalization of GnRH receptors, and GnRH receptor down-regulation. Homologous desensitization of the gonadotrope cells has been studied using primary pituitary cell culture (6, 7, 30, 35) as well as the {alpha}T3-1 pregonadotrope cell line (19, 20, 36). After long-term exposure of the gonadotrope cell to high concentrations of the neuropeptide, the number of GnRH receptors is diminished, Gq/11 protein levels are reduced, production of inositol phosphates is impaired, and IP3 receptors are down-regulated (35, 36, 37). The finding that signaling proteins such as Gq/11 and IP3 receptor are reduced has led to the suggestion that desensitization may involve impairments in downstream signaling as well as receptor expression.

In the present study, we investigated the mechanisms of GnRH-induced desensitization of the LßT2 gonadotrope cell line. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. The suppression was observed whether assaying for ERK and p38 activation or induction of c-fos and LHß protein expression. Thus, chronic GnRH treatment not only results in homologous desensitization but also could potentially cause heterologous desensitization via receptors that use similar signaling pathways. There was one notable exception, however. GnRH desensitization did not alter depolarization-induced activation of ERK, but activation of p38 MAPK and induction of c-fos and LHß proteins were severely impaired. GnRH treatment of LßT2 cells does cause increases in intracellular calcium and activation of calcium signaling, although our previous study showed that GnRH-induced ERK activation is calcium independent. The discrepancy between activation of ERK and the other end points during KCl-induced depolarization of GnRH-desensitized LßT2 cells suggests that different calcium signaling pathways are involved in the responses, and only the pathways leading to p38 MAPK, c-fos, and LHß are subject to down-regulation.

Most signaling pathways are subject to negative feedback. We determined whether the GnRH desensitization could be mimicked by stimulation of a specific pathway. GnRH stimulates PKC, cAMP, and calcium-dependent signaling in LßT2 cells, so these three pathways were stimulated chronically before acute agonist treatment. Chronic stimulation of any of the three pathways caused partial resistance to a subsequent GnRH challenge. The degree of resistance correlated very well with our previous findings on the involvement of specific signaling pathways in the individual GnRH response. Therefore, PMA caused the greatest resistance for ERK activation as GnRH signals primarily through PKC to activate ERK, and KCl caused the greatest resistance for p38 MAPK, c-fos, and LHß because GnRH signals primarily via calcium for these responses. We did not observe any cross-desensitization of signaling to ERK, indicating that these pathways activate ERK independently. Because activation of ERK by these signaling pathways is MEK dependent, the desensitization for each pathway must occur upstream of the point of convergence in the ras-Raf-MEK-ERK cascade. We did observe cross-desensitization for activation of p38 MAPK and induction of c-fos. In particular, cAMP and calcium signaling may use a common pathway to stimulate p38 MAPK. The c-fos promoter contains many regulatory elements including serum, cAMP, and stat-response elements and an activator protein-1 (AP-1) site that are the target of different pathways. Activation of PKC or calcium signaling may therefore target a common response element to induce c-fos.

The finding that PMA could cause GnRH resistance for induction of c-fos and LHß was unexpected as blockade of PKC signaling with bisindolylmaleimide I had no effect on induction of these genes (31). This was explained by the observation that GnRH-R expression was down-regulated by chronic PMA, thus all GnRH responses were partially impaired irrespective of the signaling pathways involved. The effect of PMA to down-regulate the GnRH receptor is likely mediated at the level of gene expression because it has been shown that phorbol esters suppress the human GnRH receptor promoter via an AP-1 site located at -1000 to -994 in the promoter (38). An AP-1 site is also found in the mouse GnRH-R promoter at -330 to -360, and this site partially mediates the induction of promoter activity by GnRH (39). Chronic down-regulation with either GnRH or PMA eliminates the subsequent acute induction of the mouse promoter, but it was not shown whether the AP-1 site mediates this effect. It is interesting to speculate that the AP-1 site in the mouse GnRH-R promoter may coordinate both up-regulation because of acute PMA and down-regulation with chronic PMA, as has been shown for the human promoter. How much of the desensitization is caused by reductions in receptor expression vs. impairments in signaling? The GnRH-induced activation of p38 MAPK is reduced only 31% by PMA down-regulation. PMA is a weak activator of p38 MAPK in these cells, and PKC is not involved in the GnRH induction of p38MAPK, so the reduction in receptor expression may account for only a fraction of the observed resistance.

There are two families of GnRH-Rs; one family contains GnRH-Rs from nonmammalian vertebrates (catfish, goldfish, chicken, Xenopus) that possess a C-terminal tail, and the other family contains the mammalian GnRH-Rs that lack this cytoplasmic extension (40). Studies have revealed that nonmammalian GnRH-Rs show rapid desensitization, whereas mammalian GnRH-Rs do not (41, 42). Similarly, nonmammalian GnRH-Rs undergo rapid agonist-induced internalization, but mammalian GnRH-Rs internalize at much slower rates (43). The resistance of mammalian GnRH-Rs to desensitization and internalization has been attributed to the lack of C-terminal tail phosphorylation sites that bind ß-arrestin (44). However, we find that sustained stimulation of LßT2 cells with GnRH does cause down-regulation of the GnRH-R protein and desensitization. This observation is consistent with studies in other cells (19, 21). GnRH regulates the expression of its receptor in cultured pituitary cells, and the sensitivity of gonadotropes to GnRH correlates with the number of GnRH-Rs expressed on the cell surface (45, 46). The loss of receptors by homologous desensitization is attributable to decreased receptor gene expression (36). Thus, one mechanism of homologous desensitization by GnRH may be receptor loss. We have estimated that for PMA-induced desensitization, GnRH-R loss may account for only a fraction of the observed GnRH desensitization. If the same is true for GnRH-induced desensitization, how can we explain the remaining resistance?

The GnRH-R primarily uses the Gq protein for downstream signaling but can also interact with the Gs protein in selected cells, including the LßT2 cell line. We observed that sustained GnRH treatment of LßT2 cells results in the selective decrease of the Gq/11 protein level and has no effect on the levels other G protein subunits. This result confirms an earlier study in {alpha}T3-1 cells in which sustained exposure of LHRH-ethylamide resulted in the posttranslational down-regulation of Gq/11 protein levels (32). The selective down-regulation was not mimicked by activation of PKC, cAMP, or calcium signaling, suggesting that it is the direct result of activation by the receptor and likely reflects the enhanced proteosomal degradation of activated Gq/11 (47). The down-regulation of Gq/11 might explain the loss of PLC-dependent IP production and suppression of GnRH signaling that has been reported (48). Alternatively, activation of PKC may exert a direct negative regulatory effect on PLC stimulation by GnRH (18, 23). Acute activation of PKCs is followed by desensitization of associated cellular responses, however (49, 50). Depending on the duration of the stimulus by either phorbol esters or agonists, selective isoforms are down-regulated.

We observed the expression of several PKCs in LßT2 cells, including the {alpha}, ß, {delta}, {epsilon}, {iota}/{lambda}, and {theta} isoforms. Chronic GnRH treatment selectively down-regulated the novel PKC{delta}, {epsilon}, and {theta}, whereas PMA down-regulated all the DAG-dependent isoforms. The selective loss of PKC{delta} and {epsilon} with GnRH confirms a previous study in both {alpha}T3-1 and LßT2 cells (51). It is interesting that GnRH only down-regulates the novel PKC isoforms but does not alter the conventional PKC{alpha} and PKCß isoforms, yet GnRH completely suppresses the ability of PMA to activate ERK and p38 MAPK, or induce c-fos and LHß. This implies that conventional PKC isoforms are not involved in these responses. It has been shown that PKC{delta} mediates the activation of ERK downstream of the nerve growth factor receptor via direct phosphorylation of Raf (52). It remains to be determined whether GnRH uses a similar pathway. The GnRH-induced desensitization of cAMP signaling may result from decreases in the levels of PKA subunits. Both GnRH and pituitary adenylate cyclase activating polypeptide cause a dose-dependent decrease in the catalytic subunit and regulatory subunits RI and RII of PKA in {alpha}T3-1 cells (53). This effect is mimicked by forskolin, which implicates cAMP signaling. Similarly, 8-Br-cAMP causes a dose- and time-dependent decrease in PKA catalytic subunit in rat pituitary cultures (54). Of course, the effect on cAMP signaling may not be related to PKA but could result from changes in adenylate cyclase and phosphodiesterase isoform expression or other cAMP-dependent pathways.

The finding that the Gq/11 protein is decreased and downstream signaling pathways are impaired in GnRH-desensitized LßT2 cells raises the possibility of heterologous desensitization. We verified that heterologous desensitization does indeed occur by demonstrating that activation of ERK by other Gq-coupled receptors was impaired under these conditions. This may have physiological importance as the pituitary integrates many hormonal signals in vivo. Although the primary stimulator of the gonadotrope is GnRH, there are a number of other inputs that modulate the final hormonal output. Androgens and estrogens regulate gonadotropin secretion both at the level of the hypothalamus, in which they modulate GnRH pulse rate and at the level of the pituitary in which they suppress LHß and FSHß transcription because of gonadal feedback (55, 56, 57). Activin, inhibin, and follistatin also modulate gonadotropin synthesis, although the effect is more pronounced on FSH rather than LH (58, 59). {gamma}-Aminobutyric acid receptors have been found in {alpha}T3-1 cells and {gamma}-aminobutyric acid agonists increased the transient calcium elevation in the cells in response to GnRH (60). Insulin and IGF-I enhance the ability of GnRH to stimulate LH release from rat and bovine pituitary cultures (61, 62). Leptin stimulates LH secretion from gonadotropes by stimulating nitric oxide synthase (63). Recently, pituitary adenylate cyclase activating polypeptide was found to potentiate the GnRH activation of nitric oxide-dependent cGMP production in rat anterior pituitary cultures (64). The C-type natriuretic peptide receptor can also generate cGMP in pituitary cells, and C-type natriuretic peptide can inhibit GnRH-stimulated increases in intracellular calcium (65). Although some of these effects are evidently at the level of the gonadotropin subunit genes, others result from cross-talk between signaling pathways, which underlines the importance of understanding the mechanisms of heterologous desensitization.

In summary, we demonstrated that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. This impairment in signaling was also shown with other Gq-coupled receptors, demonstrating that heterologous desensitization occurs in pituitary cells. The loss of the GnRH-R appears to be mediated by PKC signaling, but the loss of Gq/11 is not dependent on signaling. This desensitization was observed for activation of both ERK and p38 and induction of c-fos and LHß protein expression. We also showed that the novel PKC isoforms {delta}, {epsilon}, and {theta} are specifically down-regulated in GnRH-desensitized cells.


   Acknowledgments
 
We thank Dr. A. F. Parlow (NIDDK) for the gift of LHß antibody and Dr. Pamela Mellon (University of California, San Diego, UCSD) for the LßT2 cells.


   Footnotes
 
This work was supported by a U54 Center Grant from the National Institutes of Health (HD-12303).

N.J.G.W. is a faculty member of the UCSD Biomedical Sciences Graduate Program.

Abbreviations: AP-1, Activator protein-1; CTX, cholera toxin; DAG, diacylglycerol; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptor; IP, inositol phosphate; MEK, MAPK kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; TRITC, tetramethylrhodamine isocyanate.

Received February 12, 2003.

Accepted for publication June 17, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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