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Molecular Endocrinology, doi:10.1210/me.2004-0145
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Molecular Endocrinology 19 (8): 2074-2085
Copyright © 2005 by The Endocrine Society

Constitutively Active Gq Impairs Gonadotropin-Releasing Hormone-Induced Intracellular Signaling and Luteinizing Hormone Secretion in LßT2 Cells

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

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

Address all correspondence and requests for reprints to: Dr. Nicholas J. G. 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
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chronic GnRH treatment causes homologous desensitization by reducing GnRH receptor and Gq/11 expression and by down-regulating protein kinase C (PKC), cAMP, and calcium-dependent signaling. It also causes heterologous desensitization of other Gq-coupled receptors, but the mechanisms involved remain elusive. In this study, we investigated the effect of constitutive activation of Gq signaling on GnRH-induced signaling and LH secretion. We show that adenoviral expression of a constitutively active mutant Gq(Q209L) results in a state of GnRH resistance but does not alter GnRH receptor expression. We observed that Gq(Q209L) reduced expression of phospholipase C (PLC)ß1, a target of Gq in these cells, but not PLCß3 or PLC{gamma}1. Downstream of PLCß1, expression of novel PKC isoforms ({delta} and {epsilon}) was reduced. Adenoviral expression of a kinase-inactive, dominant-negative version of PKC{delta} impaired GnRH activation of ERK, but not induction of c-Fos and LHß proteins, indicating that the novel PKCs signal to the ERK cascade. Despite reductions in PLCß1, calcium responses to GnRH were elevated in Gq(Q209L)-infected cells due to increased calcium influx through L-type calcium channels. Paradoxically, downstream calcium-dependent signaling and LH secretion were impaired. Taken together, these data demonstrate that prolonged activation of the Gq pathway desensitizes GnRH-induced signaling by selectively down-regulating the PLC-PKC-Ca2+ pathway, leading to reduced LHß synthesis and LH secretion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH IS A HYPOTHALAMIC peptide critical for normal mammalian reproductive function. It acts in the anterior pituitary via a specific GnRH receptor (GnRH-R) on the plasma membrane of gonadotrope cells and triggers the secretion of the gonadotropins LH and FSH, which in turn stimulate production of gonadal steroids (1, 2). These two hormones are members of the glycoprotein hormone family that is characterized by a shared {alpha}-subunit and a unique, hormone-defining, ß-subunit. Gonadotrope responsiveness is modulated by GnRH concentration and the frequency or pattern of its administration. GnRH is secreted in a 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 (3, 4). This phenomenon, termed homologous desensitization, is a common feature of many G protein-coupled receptors (GPCRs) (5). Physiologically, 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 (1, 6).

GnRH signaling has been studied in primary pituitary cultures as well as model systems (2, 7, 8). It is generally accepted that the GnRH-R signals primarily through the Gq/11 proteins in pituitary gonadotropes, although some signaling through Gs is apparent in selected cell types (9, 10, 11, 12, 13). Activation of the Gq/11 class of G proteins leads to stimulation of phosphoinositide signaling through phospholipase C (PLC). Increases in PLC activity can occur through direct activation of PLCß1 and PLCß3 by the Gq {alpha}-subunit, by Gß{gamma} activation of PLCß2, or by direct binding of PLC{gamma} isoforms to tyrosine-phosphorylated receptors (14). The hydrolysis of phosphatidylinositol-4,5-bisphosphate by the PLC enzymes results in the generation of two second messengers, diacylglycerol (DAG) and inositol-trisphosphate (IP3). One of the major targets for DAG is the protein kinase C (PKC) family of proteins. The classical calcium-dependent isoforms (PKC{alpha}, PKCß, and PKC{gamma}) and novel calcium-independent isoforms (PKC{delta}, PKC{epsilon}, and PKC{theta}) bind to DAG in the plasma membrane via their C1 domains (15). Activation of these PKCs requires DAG and phosphorylation by the upstream kinase, phosphoinositide-dependent protein kinase 1. Previous studies have shown that chronic GnRH treatment selectively reduces expression of PKC {delta}- and {epsilon}-isoforms (16, 17). The other second messenger, IP3, binds to a specific receptor located in the endoplasmic reticulum and allows calcium efflux from the endoplasmic reticulum. This causes a rapid but transient increase in cytosolic calcium. This transient increase is often followed by a plateau of sustained calcium elevation due to influx of calcium via voltage-gated calcium channels in the plasma membrane. Elevations in cytosolic calcium can activate downstream signaling and are important for triggering secretion from neuroendocrine cells. For example, calcium/calmodulin-dependent kinase II (CaMKII) is an important intracellular mediator of calcium signaling in several cells and tissues, including the pituitary (18, 19). It was recently shown that GnRH stimulates CaMKII phosphorylation within 2 min, reaching a peak at 5–15 min, and then declining in LßT2 cells (20, 21). Interestingly, activation of CaMKII requires both intracellular and extracellular calcium sources.

We have previously shown that GnRH signals via calcium to induce the c-Fos and LHß proteins in LßT2 cells, and that these pathways become refractory to depolarization-induced rises in calcium in cells treated with chronic GnRH. Desensitization occurs not only through reductions in GnRH-R and Gq/11 expression, but also by down-regulation of PKC, cAMP, and calcium-dependent signaling and, more importantly, causes heterologous desensitization of other Gq-coupled receptors (16). Constitutively active G protein subunit mutants have been very useful in the study of desensitization and cellular adaptation, because the activity of such mutants is independent of interaction with a particular GPCR. In this study, we used adenoviral expression of a constitutively active Gq mutant (Q209L) to selectively study the role of the Gq signaling pathway in GnRH responses and desensitization. We show that reduced expression of PLCß1 and PKC{delta}/{epsilon} contribute to impaired GnRH-induced intracellular signaling and LH secretion in LßT2 cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Desensitization of Intracellular Signaling by Overexpression of a Constitutively Active Form of Gq in LßT2 Cells
We have shown previously that expression of Gq(Q209L) induces a state of GnRH resistance in LßT2 cells (16). Under these conditions, GnRH stimulation of ERK phosphorylation and induction of c-Fos were reduced 40–50% by immunofluorescence. These observations were confirmed here by immunoblotting whole-cell extracts. LßT2 cells were infected with wild-type (WT) Gq, the constitutively active mutant Gq(Q209L), or a control virus expressing ß-galactosidase (LacZ) for 16 h, incubated for an additional 60 h at 37 C to allow protein expression, and then serum starved and stimulated acutely with 100 nM GnRH for 5 min for ERK activation, or 1 h for c-Fos expression. Whole-cell lysates were immunoblotted for the dually phosphorylated form of ERK or c-Fos (Fig. 1AGo). Adenoviral expression of WT Gq or ß-galactosidase had no effect on either basal or GnRH-stimulated ERK or c-Fos expression. In contrast, chronic expression of Gq(Q209L) reduced GnRH stimulation of ERK and c-Fos, but had no effect on basal ERK activation and c-Fos expression. GnRH-stimulated ERK phosphorylation was 60 ± 15% (mean ± SD, n =4) compared with cells infected with the control virus. Similarly, c-Fos induction was 70% (n =2) of the maximal response. This result is consistent with our previous immunostaining and confirmed that chronic activation of Gq signaling induces a state of GnRH resistance. To verify the expression of WT Gq and Gq(Q209L), whole-cell lysates from infected cells were immunoblotted with an anti-Gq/11 antibody (Fig. 1BGo). Both Gq proteins were expressed at similar levels, approximately 5-fold over the endogenous protein. Cell extracts were also immunoblotted for Gs, Gi, and Gß (Fig. 1BGo). Adenoviral expression of Gq did not alter the expression of Gs, Gi, and Gß proteins, indicating that desensitization is not caused by alteration in endogenous G protein expression.



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  		Fig. 1. Chronic Gq Activation Impairs GnRH-Induced ERK Activation and c-Fos Protein Expression in LßT2 Cells

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10 and then incubated for an additional 60 h to allow expression of the virally encoded proteins. A, Cells were stimulated with vehicle or 100 nM GnRH for 5 min for ERK activation (n = 4) or 60 min for c-Fos induction (n = 2). Whole-cell lysates were immunoblotted with antibodies to phospho-ERK and c-Fos. Blots were stripped and reblotted for ERK protein to verify equal loading. B, Whole-cell lysates from infected LßT2 cells were immunoblotted with antibodies against Gq/11, Gs, Gi, or Gß. The experiment was repeated three times with similar results.

 
Overexpression of Gq(Q209L) Does Not Alter Expression of GnRH-Rs
A simple explanation for the observed GnRH resistance might be that Gq(Q209L) reduces GnRH-R expression. Therefore, we measured receptor expression by radiolabeled binding assays. LßT2 cells were infected with the constitutively active mutant Gq(Q209L), the WT Gq, or the control (LacZ) adenovirus as before. Infected cells were incubated with [125I]GnRH in the absence or presence of 10–6 M unlabeled GnRH for 1 h and then washed extensively, and the cell-associated radioactivity was counted (Fig. 2Go). Adenoviral expression of Gq(Q209L) did not alter GnRH-R expression and hence cannot explain the observed desensitization.



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  		Fig. 2. Chronic Gq Activation Does Not Alter GnRH-R Expression

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10, and allowed to express the virally encoded proteins for an additional 60 h. Whole-cell binding experiments were performed with [125 I]GnRH in triplicate. Nonspecific binding was measured in the presence of 10–6 M unlabeled GnRH. Data are shown as mean ± SEM from three experiments. Binding is normalized to control (LacZ)-infected cells.

 
Overexpression of Gq(Q209L) Specifically Down-Regulates PLCß1 Expression
We then investigated whether overexpression of Gq(Q209L) altered PLC isozyme expression. LßT2 cells were infected with the constitutively active mutant Gq(Q209L), the WT Gq, or the control (LacZ) adenovirus as before. Whole-cell lysates were immunoblotted with antibodies to the PLCß1, PLCß3, and PLC{gamma}1 isoforms (Fig. 3Go). Blots were stripped and reblotted for ERK1/2 to control for equal protein loading. Overexpression of Gq(Q209L) caused a reduction in PLCß1, but not PLCß3 or PLC{gamma}1. The mean level of PLCß1 in the Q209L-infected cells was 59 ± 6% (mean ± SD; n = 5) compared with control infected cells. This suggested that PLCß1 is the direct downstream target of Gq signaling in these cells.



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  		Fig. 3. Chronic Gq Activation Causes Down-Regulation of PLCß1, But Not PLCß3 or PLC{gamma}1

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10, and allowed to express the virally encoded proteins for an additional 60 h. Whole-cell lysates were immunoblotted with antibodies to PLCß1, PLCß3, or PLC{gamma}1. Blots were stripped and reblotted for ERK to verify equal protein loading. The experiment was repeated five times with similar results.

 
Overexpression of Gq(Q209L) Down-Regulates Novel PKC Isoforms
Chronic GnRH stimulation reduces selected PKC isoforms. Therefore, we investigated whether chronic activation of Gq signaling is sufficient for this effect. LßT2 cells were infected with a constitutively active Gq mutant (Q209L), the WT Gq, and the control virus (LacZ). Whole-cell lysates were immunoblotted with antibodies to PKC isoforms using a slot-blot apparatus. Blots were stripped and reblotted for ERK1/2 to control for protein loading. The {alpha}-, ß-, {delta}-, {epsilon}-, {iota}-, {lambda}-, and {theta} isoforms, but not the {gamma}- or {eta}-isoforms, of PKC are expressed in LßT2 cells, as we have shown previously (Fig. 4Go). The control adenovirus or the WT Gq virus had no effect on PKC isoform expression, but overexpression of Gq(Q209L) selectively reduced expression of the {delta}- and {epsilon}-isoforms of PKC but had no effect on other isoforms. The mean levels of PKC{delta} and PKC{epsilon} in the Q209L-infected cells were 50 ± 3% and 72 ± 6%, respectively (mean ± SD; n = 3). This is similar to our earlier observations after chronic GnRH treatment.



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  		Fig. 4. Chronic Gq Activation Causes Down-Regulation of Novel PKCs {delta} and {epsilon}

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10, and allowed to express the virally encoded proteins for an additional 60 h. Whole-cell lysates were immunoblotted for the {alpha}-, ß-, {gamma}-, {delta}-, {eta}-, {epsilon}-, {iota}-, {lambda}-, and {theta}-isoforms of PKC using a slot-blot apparatus. The blots were stripped and reblotted with an antibody to ERK1/2 to verify equal protein loading. The experiment was repeated three times with similar results.

 
Desensitization of ERK Signaling by Adenoviral Expression of a Dominant-Negative PKC{delta} (DN-PKC{delta})
The previous experiments suggested that loss of the novel isoforms of PKC might be involved in GnRH resistance induced by expression of Gq(Q209L). To test this hypothesis, we expressed WT PKC{delta} and a kinase-inactive DN-PKC{delta} containing a mutation in the ATP-binding site (K376R) by adenovirus (22). LßT2 cells were infected with the WT PKC{delta} virus, the DN-PKC{delta} virus, or the control LacZ virus at a M.O.I of 10 for 16 h, and then incubated for an additional 60 h to allow protein expression as for the Gq viruses. Whole-cell lysates were immunoblotted with a monoclonal antibody to PKC{delta} to verify protein expression (Fig. 5AGo). Both WT PKC{delta} and DN-PKC{delta} were expressed approximately 5- to 7-fold over the endogenous protein. To confirm the kinase activity of the expressed proteins, whole-cell lysates were immunoblotted with an antibody to phospho-Thr505 in PKC{delta}. This threonine lies on the activation loop of the kinase (23). Threonine 505 was phosphorylated on the WT PKC{delta} protein, consistent with its activity, but this residue was not phosphorylated on the dominant negative DN-PKC{delta} protein. We then investigated whether the DN-PKC{delta} would inhibit intracellular signaling downstream of the GnRH-R. Cells were infected with PKC{delta}, DN-PKC{delta}, or control viruses, serum-starved, and then acutely stimulated with 100 nM GnRH for 5 min. Whole-cell lysates were immunoblotted for phospho-ERK (Fig. 5BGo). Expression of DN-PKC{delta}, but not WT PKC{delta}, reduced GnRH activation of ERK. The mean activation of ERK was 77 ± 4% (mean ± SD; n = 5) compared with control infected cells. We verified this result by immunofluorescence microscopy. Virally infected cells were stimulated with 100 nM GnRH for 5 min for ERK phosphorylation, 1 h for c-Fos, and 8 h for LHß expression. Expression of DN-PKC{delta} reduced nuclear phospho-ERK immunofluorescence by more than 60% (Fig. 6AGo). The control virus or WT PKC{delta} had no effect. The effect of DN-PKC{delta} on ERK phosphorylation was notably greater by immunofluorescence than by immunoblot. This is because nuclear translocation of active ERK requires a PKC-dependent phosphorylation event, and in the absence of PKC, active ERK remains sequestered in the cytoplasm and is not detected by the antibody (24). In contrast to its effect on ERK, expression of DN-PKC{delta} had no effect on the induction of c-Fos or LHß (Fig. 6Go, B and C).



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  		Fig. 5. Expression of a Kinase-Inactive DN-PKC{delta} Impairs GnRH-Induced ERK Activation

LßT2 cells were infected with recombinant adenoviruses expressing WT PKC{delta} (PKC{delta}), DN-PKC{delta}, or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10 and allowed to express the virally encoded proteins for an additional 60 h. A, Whole-cell lysates were immunoblotted with antibodies against PKC{delta} or an antibody to phospho-PKC{delta} (Thr505). B, Infected cells were stimulated with 100 nM GnRH for 5 min. Whole-cell lysates were immunoblotted with an antibody to phospho-ERK. In all cases, blots were stripped and reblotted for ERK protein to verify equal protein loading. The experiment was repeated five times with similar results.

 


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  		Fig. 6. Expression of a Kinase-Inactive DN-PKC{delta} Impairs Nuclear Translocation of ERK, But Does Not Impair Induction of c-Fos or LHß

LßT2 cells on coverslips were infected with recombinant adenoviruses expressing WT PKC{delta} (PKC{delta}), DN-PKC{delta}, or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10, and allowed to express the virally encoded proteins for an additional 60 h. A, Cells were stimulated with 100 nM GnRH for 5 min and then fixed and processed for immunofluorescent staining. Active-ERK was visualized with an antibody against dually phosphorylated ERK (Thr183/Tyr185). B, Cells were stimulated with 100 nM GnRH for 60 min and then fixed and stained. Nuclear c-Fos expression was visualized using a rabbit anti-c-Fos antibody. C, Cells were stimulated with 100 nM GnRH for 8 h and then fixed and stained. LHß protein expression was visualized using a rabbit anti-LHß antibody. In all cases, the primary antibody was visualized using a TRITC-conjugated secondary antibody. Nuclei were counterstained with Hoechst 33258 DNA dye to facilitate identification of individual cells. The percentage of cells positive for immunofluorescence was counted from multiple fields. White and gray bars indicate basal and GnRH-treated cells, respectively. Results are the mean ± SEM of three experiments. The asterisk indicates statistical significance (P < 0.05) vs. GnRH-stimulated control (LacZ)-infected cells.

 
Expression of Gq(Q209L) Alters the Acute Calcium Response upon GnRH Stimulation
The observation of decreased levels of PLCß1 led us to examine whether calcium signaling might be impaired in cells expressing Gq(Q209L). Virally infected cells were loaded with Fluo 3-AM for 45 min, and calcium fluorescence was measured over a period of 5 min after GnRH stimulation (Fig. 7AGo). Resting calcium concentrations were similar in all cells. In LacZ and WT infected cells, GnRH caused an acute rise in cytosolic calcium followed by a slow decline to basal levels over 5 min. In Gq(Q209L)-infected cells, the acute rise in calcium was enhanced, but the calcium fluorescence decayed with similar kinetics. This was quantified by measuring the area under the curve and subtracting the basal calcium level. The area under the curve was increased 40% in Gq(Q209L)-infected cells (Fig. 7BGo). The enhanced calcium response was mediated by calcium influx via L-type calcium channels (LTCCs) as it was blocked by either EGTA or nimodipine (Fig. 7Go, C and D). IP3-sensitive calcium pools were unaltered as 2 µM thapsigargin caused similar increases in cytosolic calcium in all infected cells (Fig. 7EGo). This is supported by the observation that intracellular release of calcium in the absence of influx through calcium channels was not altered by viral infection. We also verified that IP3 receptor expression was unaltered (data not shown). Passive elevation of calcium by application of 1 µM ionomycin caused a similar increase in all infected cells (Fig. 7FGo).



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  		Fig. 7. Chronic Gq Activation Increases Calcium Influx through LTCCs

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10 and allowed to express the virally encoded proteins for an additional 60 h. Cells were loaded with 4 µM Fluo3-AM for 45 min. After washing, the cells were aliquoted at 107 cells per well into a black-walled 96-well plate. Fluorescence was measured in a microplate fluorescence spectrophotometer at 538 nm with excitation at 485 nm. Measurements were taken every 6 sec over a period of 5 min. A, Intracellular calcium concentration in virally infected cells. GnRH (100 nM) was added at the time indicated by the arrow. Q209L-infected cells are shown in open circles, WT-infected cells are shown in open squares, and LacZ-infected cells are shown in open triangles. Vertical lines indicate SEM. Results represent the mean of seven experiments. B, Area under the calcium curve after GnRH stimulation. Results are the mean and SEM (*, P < 0.05 vs. control). C, Cells stimulated with GnRH in the presence of 10 mM EGTA to chelate extracellular calcium (n = 7). D, Cells stimulated with GnRH in the presence of 10 µM nimodipine to block LTCCs (n = 4). E, Cells stimulated with 2 µM thapsigargin (n = 4). F, Virally infected cells stimulated with 1 µM ionomycin (n = 4).

 
Desensitization of LH Secretion by Overexpression of Gq(Q209L)
LH secretion from primary gonadotropes and LßT2 cells is dependent on calcium signaling. As the calcium response appears to be altered in LßT2 cells infected with the Gq(Q209L) virus, we determined whether LH secretion was affected. Cells were infected with the control (LacZ), WT Gq, or Gq(Q209L) viruses, starved in serum-free medium overnight, washed, and stimulated with 100 nM GnRH for 30 min. The culture medium was collected for measurement of LH secretion by RIA. The remaining cells were lysed with RIPA buffer to measure intracellular content of LH. GnRH caused an increase in LH secretion into the medium in LacZ and WT-infected cells (Fig. 8AGo). Expression of Gq(Q209L) significantly decreased basal secretion of LH and eliminated the GnRH stimulation (Fig. 8AGo). Intracellular content of LH decreased with GnRH stimulation in control-infected cells (Fig. 8BGo). Expression of Gq(Q209L) reduced intracellular content of LH to levels seen after GnRH stimulation of control cells, and there was no further reduction upon GnRH stimulation consistent with the lack of stimulated secretion (Fig. 8BGo).



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  		Fig. 8. Chronic Gq Activation Suppresses Basal and GnRH-Induced LH Secretion

LßT2 cells were infected with recombinant adenoviruses expressing WT Gq, constitutively active Gq (Q209L), or ß-galactosidase (LacZ) for 16 h at a multiplicity of infection of 10 and allowed to express the virally encoded proteins for an additional 60 h. Infected cells were washed and stimulated with 100 nM GnRH for 30 min. Medium was collected and the remaining cells were lysed. LH in the medium and cell lysates was measured by RIA. A, LH secretion into medium. Results are expressed as nanograms/well and represent the mean ± SEM of three experiments. B, Intracellular LH content. LH was measured on cell lysates. Results are expressed as nanograms/ml lysates and represent mean ± SEM. C, Infected cells were treated with 1 µM ionomycin for 30 min. LH secretion into the medium is expressed as nanograms/well (mean ± SEM). Asterisks indicate statistical significance (P < 0.05) vs. vehicle-treated cells; # indicates statistical significance (P < 0.05) vs. control infected (LacZ).

 
The impaired LH secretion in Gq(Q209L)-infected cells might result from the desensitization of signaling or from impairments in the secretory apparatus. Therefore, we tested whether cells had become refractory to calcium-induced LH secretion. Cells infected with the three viruses were treated with ionomycin for 30 min to raise calcium levels passively, and the medium was collected for measurement of LH by RIA. Calcium elevation by ionomycin was identical in all infected cells (Fig. 7FGo). Ionomycin caused a doubling of LH release in control cells (Fig. 8CGo). Basal secretion of LH was reduced in Gq(Q209L)-infected cells as before, but ionomycin-induced LH secretion was unimpaired, indicating that the cells can respond to calcium increases normally. Similar results were obtained by depolarization with KCl (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We previously showed that the GnRH-R signaled via the heterotrimeric G proteins, Gq and Gs, to activate ERK and regulate gene expression in LßT2 cells (25). Selective activation of cAMP signaling downstream of Gs, via treatment with forskolin, partially mimicked the effect of GnRH in these cells. Similarly, acute infection with an adenovirus expressing the constitutively active Gq(Q209L) protein also partially mimicked GnRH. However, we observed that chronic infection with the Gq(Q209L) virus impaired the response of the cells to acute stimulation by GnRH. In particular, chronic Gq(Q209L) expression completely eliminated the induction of LHß protein by GnRH. In this study we used this Gq(Q209L)-induced desensitization to investigate the mechanisms involved in induction of LHß synthesis and LH secretion. This approach has distinct advantages over pharmacological methods to dissect signaling as GnRH-R and G protein expression is not altered; hence the proximal components of the signaling cascades are intact.

It is well known that proteins of the Gq/11 family activate PLCß, with concomitant production of DAG and IP3 leading to activation of PKC and calcium signaling (26). Of the four PLCß isoforms, only ß1 and ß3 are found in the pituitary (27). PLCß1 is activated by Gq/11, but PLCß3 is activated by both Gq/11 and Gß{gamma} subunits (14). We observed that Gq(Q209L) selectively down-regulates the PLCß1 isoform. This implies that PLCß1 is the major target for Gq/11 in gonadotropes and is consistent with the observation that PLCß3 knockout mice are fertile and show normal calcium responses to GnRH in primary pituitary cells (27). Furthermore, PLCß1 is a GTPase-activating protein for Gq/11, and activation of PLC in vivo is via the Gq/11 activation of PLCß1 (28, 29). The link between Gq/11 and PLCß1 activation is corroborated in vitro by the finding that Gq/11 can activate phosphatidylinositol 4,5-bisphosphate hydrolysis in reconstituted vesicles containing the M1 muscarinic receptor and PLCß1 (30).

Downstream of PLC, we observed that the novel PKC isoforms, {delta} and {epsilon}, are reduced by chronic Gq signaling. This is similar to the effect of chronic GnRH treatment, but is distinct from phorbol ester treatment that reduces all DAG-dependent PKCs, including PKC{alpha} and PKCß (16, 17). Activation of DAG-dependent PKCs leads to proteosomal degradation of those PKC isoforms, which serves to terminate signaling in the face of chronic activation and underlies the well-known down-regulation of PKC by phorbol esters (17). Only the novel PKCs, {delta} and {epsilon}, are reduced by chronic GnRH or constitutively active Gq(Q209L), which indicates that DAG production by PLCß1 downstream of Gq is selective for these isoforms and does not activate PKC{alpha} and PKCß.

The reduction in PLCß1 protein level suggested that calcium responses might be impaired in LßT2 cells, but paradoxically we observed that adenoviral expression of Gq(Q209L) enhances the calcium increase in response to GnRH, primarily through an effect on extracellular calcium influx. Resting calcium levels were unaltered, and we found no evidence for alterations in IP3-R or SERCA2 expression, or for alterations in intracellular calcium release. This implies that the IP3-mediated calcium increases are intact in Gq(Q209L)-expressing cells. Our findings appear to be different from study results from the McArdle laboratory. Those studies demonstrated that chronic GnRH treatment results in loss of IP3 receptors and suppression of LH secretion in humans and {alpha}T3–1 cells (31, 32, 33). It is possible that the lack of IP3-receptor down-regulation in our system reflects an inherent difference in desensitization via chronic hormone treatment vs. chronic activation of Gq. Indeed, GnRH-R levels are unaltered in our paradigm but are reduced with chronic GnRH. Alternatively, it is known that the GnRH-R couples only to Gq/11 in {alpha}T3–1 cells, whereas it couples to both Gq/11 and Gs in LßT2 cells. This could allow additional signaling events that might prevent or rescue IP3-R down-regulation. It is somewhat surprising that intracellular calcium release is normal despite a 50% reduction in PLCß1 levels. This may indicate that the remaining PLCß1 is sufficient to generate a normal response or that redundant signaling via the Gß{gamma} activation of PLCß3 can compensate for the loss of PLCß1.

How might chronic activation of Gq translate into enhanced calcium influx through L-type channels? Two possible explanations are increased expression of channel subunits, or increased activity of existing channels. Pituitary LßT2 gonadotropes express the LTCC pore subunits, Cav1.1 and Cav1.2, as well as the auxiliary subunits, {alpha}2-{delta}, ß2, ß3, {gamma}1, {gamma}2, and {gamma}6, by gene expression profiling (data not shown). Although the mechanisms regulating expression of the individual subunits are not known, there is evidence for hormonal regulation of L-type channels. In particular, the expression of Cav1.2 is increased by cAMP signaling in AtT-20 cells (34). Similarly, estradiol increases LTCC calcium influx in response to GnRH in primary pituitary cultures (35). Alternatively, there is also evidence that the activity of L-type channels can be regulated by PKC and cAMP signaling. Pituitary adenylate cyclase activating peptide acutely potentiates LTCC activity via both PKC and MAPK signaling in neurons (36), and orexin-B increases L-type current in primary somatotropes via the PKC pathway (37). In chromaffin cells, LTCCs are under tonic inhibition, possibly through direct interaction with G protein subunits, and this inhibition is reversed by activation of cAMP signaling, either directly or downstream of a GPCR (38). Therefore, both the level of expression and activity of LTCCs may be altered by chronic Gq signaling. Further research will be required to uncover the molecular mechanism underlying this enhanced calcium flux.

Although calcium flux is enhanced in Gq(Q209L)-expressing cells, downstream calcium signaling appears to be impaired as LH secretion is inhibited. This is reminiscent of the desensitization of cells due to chronic depolarization with KCl (14). How could alterations in intracellular signaling alter LH secretion? First, Haisenleder et al. (20, 21) demonstrated that GnRH increases CaMKII phosphorylation and activity 2- to 3-fold in primary pituitary cultures and LßT2 cells, and that activation requires both intracellular and extracellular calcium. They also showed that CaMKII signals to gonadotropin subunit gene expression but does not appear to mediate gonadotropin secretion. Thus, impaired CaMKII activation could reduce LHß expression at the transcriptional level. The acute induction of LHß protein expression by GnRH also requires calcium signaling (25). This induction is a posttranscriptional effect that is mediated by MAPK-dependent phosphorylation of translation initiation factors (39). Expression of Gq(Q209L) impairs activation of MAPK via a reduction in PKC{delta}; therefore, LHß translation could also be reduced. These two effects likely explain the reduced levels of LHß protein in cells expressing Gq(Q209L).

Second, calcium is the major stimulus for gonadotropin secretion in pituitary cells (40). Depolarization of primary pituitary cultures or LßT2 cells, or treatment with calcium channel agonists or ionophores, completely mimics the ability of GnRH to stimulate LH secretion (41, 42, 43). The reduced basal LH secretion seen with overexpression of Gq(Q209L) is likely the result of decreased LHß synthesis, as discussed above, but why is GnRH unable to stimulate secretion in the face of enhanced calcium influx? Cells can still secrete LH in response to calcium ionophores; therefore, the secretory apparatus must be grossly intact. Although the calcium responses to GnRH and ionomycin are of similar magnitudes, they are of very different duration. The GnRH calcium response is transient and decays rapidly to basal levels within 5 min. In contrast, elevation of calcium with ionomycin is rapid but is sustained for more than 5 min. Studies of neuroendocrine secretion have identified at least three pools of vesicles (reviewed in Ref. 44). These include a rapidly released pool of docked and primed vesicles that fuse with the plasma membrane upon calcium elevation, a pool of docked but unprimed vesicles, and a larger cytoplasmic pool of undocked vesicles. Activation of PKC enhances exocytosis in chromaffin cells by increasing the size of the rapidly released pool fraction, possibly through disruption of the cortical actin network, allowing more vesicles to dock, and depletion of PKC reduces the size of this fraction (45, 46). PKC has also been shown to increase the calcium sensitivity of these rapidly released vesicles (47). The loss of PKC{delta} and -{epsilon} upon expression of Gq(Q209L) may result in a smaller pool of docked/primed LH-containing vesicles that fail to respond to the transient increase in calcium with GnRH. Sustained increases in cytoplasmic calcium below the threshold to trigger exocytosis have been shown to allow priming of vesicles and enhance the rapidly released pool fraction in chromaffin cells (48). The sustained elevation of calcium with ionomycin may therefore compensate for the absence of PKC{delta}/{epsilon}, allowing for priming of docked vesicles and seemingly normal LH secretion.

An alternative, but not mutually exclusive, explanation is that LH secretory granule biogenesis is altered. Nicol et al. (49, 50) have shown that LH and FSH are sorted into distinct vesicle populations in both primary gonadotropes and LßT2 cells. In particular, LH that is released in response to GnRH is secreted from secretogranin II (SgII)-containing granules. Immunogold labeling of mouse gonadotropes revealed both LH+/SgII+ and LH+ /SgII– granules (51). The number of LH+/SgII+ granules decreases in response to GnRH with a concomitant increase in plasma levels of both proteins (52). The LH+/SgII– granules that do not respond to GnRH are more likely involved in basal unregulated secretion. In contrast, FSH tends to be more associated with chromogranin A (CgA)-containing granules, although increased secretion of FSH in response to activin and GnRH is not associated with increased plasma CgA, leading to the suggestion that FSH is released from granin-independent vesicles (49). Hormones can modulate the partitioning of gonadotropins between these populations as activin increases LH secretion from the SgII– granules that are thought to mediate the basal secretion. GnRH treatment increases expression of CgA in LßT2 cells (50) and SgII in {alpha}T3–1 cells (53); therefore, the altered secretion that we observed could result from alterations in these two granins and subsequent changes in secretory granule biogenesis and properties.

In conclusion, we have manipulated Gq signaling in LßT2 gonadotrope cells by expression of a constitutively active Gq(Q209L) mutant lacking GTPase activity. Expression of this mutant renders the cells refractory to stimulation with GnRH by decreasing expression of key signaling proteins involved in the PKC- and calcium-signaling pathways. Further genetic studies to selectively deplete or knock out these proteins in both cell lines and primary cells will allow the dissection of the critical signals leading to these events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
GnRH and Fluo 3-AM were purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-ACTIVE MAPK (phosphoThr183/Tyr185) antibodies were from Promega Corp. (Madison, WI). Rabbit polyclonal anti-PLCß1, anti-PLCß3, anti-Gq/11, anti-Gs, anti-Gi, anti-Gß, and anti-c-Fos antibodies and horseradish peroxidase-linked antirabbit antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-PLC{gamma}-1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-LHß antibody was kindly provided by Dr. A. F. Parlow at the National Hormone Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. Antibodies to PKC isoforms were from BD Transduction Laboratories (San Diego, CA). The antibody to phospho-PKC{delta} (Thr505) was from New England Biolabs, Inc. (Beverly, MA). Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antirabbit antibodies were purchased from Jackson ImmunoResearch Laboratory, Inc. (West Grove, PA). Recombinant adenoviruses expressing LacZ, WT, or GTPase-deficient (activated) Q209L mutant Gq have been described elsewhere (54). DMEM and fetal bovine serum (FBS) were purchased from Life Technologies (Gaithersburg, MD). 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 a humidified 10% CO2 atmosphere at 37 C. Cells were starved overnight in serum-free DMEM and then stimulated acutely with GnRH or agonists.

Immunostaining
Immunostaining was performed essentially as described previously (24). LßT2 cells were plated on 10-mm acid-washed glass coverslips and simulated with agonists. For c-Fos and LHß staining, cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. After 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-c-Fos antibody (1:400 dilution) or 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. After 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.

For phospho-ERK staining, cells were washed with PBS, fixed in 3.7% formaldehyde in PBS as above, and then washed with TBS-Triton (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Triton X-100). The cells were permeabilized in 100% methanol at –20 C for 10 min, washed with TBS-Triton, and then blocked with 5% normal horse serum in TBS-Triton for 60 min at room temperature to reduce nonspecific staining. Coverslips were incubated with the anti-ACTIVE MAPK antibody at a 1:400 dilution in 5% BSA in TBS-Triton overnight at 4 C. The cells were washed with 0.1% BSA in TBS-Triton and then incubated with a TRITC-conjugated antirabbit IgG antibody at a 1:100 dilution in 3% BSA in TBS-Triton for 60 min at room temperature. Coverslips were washed with TBS-Triton and incubated with Hoechst 33258 dye (1:250 dilution) in TBS-Triton for 60 min at room temperature. The coverslips were washed and mounted as described above. Staining was visualized on a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Thornwood, NY), and photographed using the ISEE imaging system (Inovision, Raleigh, NC).

Western Blotting
LßT2 cells were grown to confluence in six-well plates, washed once with PBS, and incubated in serum-free medium overnight. Cells were stimulated with agonists for various periods of time at 37 C. Thereafter, cells were washed with ice-cold PBS, then 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 7.5% or 10% gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., 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. For the phospho-specific antibodies, the polyvinylidene difluoride 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 to control for equal protein loading.

Adenovirus Infection
LßT2 cells were transduced at a multiplicity of infection of 10 plaque-forming units/cell for 16 h with either a control recombinant adenovirus containing the lacZ gene, the recombinant adenoviruses expressing WT Gq, GTPase-deficient mutant Gq (Q209L), a kinase-inactive DN-PKC{delta} (K376R), or WT PKC{delta} in DMEM/2% heated-inactivated FBS. Generation of a DN-PKC{delta} adenovirus is described elsewhere (22). To allow recombinant protein expression, infected cells were incubated for 60 h at 37 C under 10% CO2 in high-glucose DMEM with 2% heat-inactivated FBS. The efficiency of adenovirus-mediated gene transfer was greater than 90% as measured by X-gal staining of lacZ-infected cells (data not shown). The survival of LßT2 cells was unaffected by adenoviral infection, because the total amount of cell protein remained the same in infected and uninfected cells.

LH Secretion
The concentration of LH in cell culture media and intracellular protein extracts was measured by RIA. The assays were performed by The University of Virginia Center for Research in Reproduction, Ligand Assay and Analysis Core Facility. All samples from each experiment were assayed in duplicate.

Measurement of Cytosolic Ca2+
LßT2 cells (1 x 107 cells/ml) were suspended in DMEM without fetal bovine serum and loaded with 4 µM Fluo 3-AM, by incubating at 37 C under 5% CO2 for 45 min as described elsewhere (55). After washing three times with PBS, the cells were resuspended in Hanks’ balanced salt solution/0.5% BSA without fetal bovine serum and aliquoted at 107 cells per well into a black-walled 96-well fluorescence plate. Fluorescence was measured in a microplate fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA) at 538 nm with excitation at 485 nm. Fluorescence was measured every 6 sec over a period of 5 min. Cells were then lysed with 1% NP-40 in Hanks’ balanced salt solution for 5 min, and fluorescence was measured for 5 min (Fmax = total fluorescence of dye at saturating Ca2+). Calcium was chelated with 50 mM EGTA and fluorescence was measured for 5 min (Fmin = total fluorescence of dye in the absence of free Ca2+). Calcium concentrations were calculated according to the following formula:

where Kd = 390 nM for Fluo-3

The area under the calcium excursion curve over the 5 min of GnRH stimulation was calculated after subtracting the basal calcium values at time zero.

Receptor-Binding Assay
[125I]Tyr5-GnRH used for binding assays was purchased from PerkinElmer Life Sciences (Boston, MA). LßT2 cells were plated into six-well plates and infected with a control recombinant adenovirus containing the lacZ gene or the recombinant adenoviruses expressing WT Gq, or mutant Gq (Q209L). To avoid interference from growth factors in the FBS, the medium was replaced with serum-free DMEM medium overnight before binding assays. The cells were washed with PBS/1%BSA, and then incubated at room temperature in the binding buffer (DMEM containing 1% BSA and 20 mM HEPES) containing approximately 10–10 M 125I-labeled GnRH in the absence or presence of 10–6 M unlabeled GnRH for 1 h. Then the cells were washed three times with cold PBS/1% BSA and solubilized in 1 ml of 0.2 M NaOH with 1% SDS. Radioactivity bound to the cells was determined in a {gamma}-counter. Nonspecific binding in the presence of 10–6 M GnRH was subtracted from the total counts. All assay points were in triplicate, and experiments were repeated at least three times.


   ACKNOWLEDGMENTS
 
We thank Dr. A. F. Parlow (National Institute of Diabetes and Digestive and Kidney Diseases) for the gift of LHß antibody and Dr. Pamela Mellon (UCSD) for the LßT2 cells. We also thank Dr. Joan Brown (UCSD) for providing the control recombinant adenovirus containing the lacZ gene and the recombinant adenovirus expressing WT G{alpha}q and Q209L mutant G{alpha}q, and Dr. Trevor Biden (Garvan Institute, Sydney, Australia) for the PKC{delta} adenoviruses.


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

First Published Online May 5, 2005

Abbreviations: CaMKII, Calcium/calmodulin-dependent kinase II; CgA, chromogranin A; DAG, diacylglycerol; DN-PKC, dominant-negative PKC; FBS, fetal bovine serum; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptor; IP3, inositol 1,4,5-trisphosphate; LTCC, L-type calcium channel; PKC, protein kinase C; PLC, phospholipase C; SDS, sodium dodecyl sulfate; SgII, secretogranin II; TRITC, tetramethylrhodamine isocyanate; WT, wild type.

Received for publication April 8, 2004. Accepted for publication April 28, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Conn PM, Crowley WFJ 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–405[CrossRef][Medline]
  2. Kaiser UB, Conn PM, Chin WW 1997 Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev 18:46–70[Abstract/Free Full Text]
  3. Weiss J, Cote CR, Jameson JL, Crowley WFJ 1995 Homologous desensitization of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone secretion in vitro occurs within the duration of an endogenous GnRH pulse. Endocrinology 136:138–143[Abstract]
  4. Weiss J, Duca KA, Crowley Jr WF 1990 Gonadotropin-releasing hormone-induced stimulation and desensitization of free {alpha}-subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perifused rat pituitary cells. Endocrinology 127:2364–2371[Abstract/Free Full Text]
  5. Freedman NJ, Lefkowitz RJ 1996 Desensitization of G protein-coupled receptors. Recent Prog Horm Res 51:319–351
  6. Barbieri RL 1993 Gonadotropin-releasing hormone agonists: treatment of endometriosis. Clin Obstet Gynecol 36:636–641[CrossRef][Medline]
  7. Kraus S, Naor Z, Seger R 2001 Intracellular signaling pathways mediated by the gonadotropin-releasing hormone (GnRH) receptor. Arch Med Res 32:499–509[CrossRef][Medline]
  8. Ando H, Hew CL, Urano A 2001 Signal transduction pathways and transcription factors involved in the gonadotropin-releasing hormone-stimulated gonadotropin subunit gene expression. Comp Biochem Physiol B Biochem Mol Biol 129:525–532[CrossRef][Medline]
  9. Stanislaus D, Janovick JA, Ji T, Wilkie TM, Offermanns S, Conn PM 1998 Gonadotropin and gonadal steroid release in response to a gonadotropin-releasing hormone agonist in Gq{alpha} and G11{alpha} knockout mice. Endocrinology 139:2710–2717[Abstract/Free Full Text]
  10. Grosse R, Schmid A, Schoneberg T, Herrlich A, Muhn P, Schultz G, Gudermann T 2000 Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. J Biol Chem 275:9193–9200[Abstract/Free Full Text]
  11. Hawes BE, Barnes S, Conn PM 1993 Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology 132:2124–2130[Abstract/Free Full Text]
  12. Imai A, Takagi H, Horibe S, Fuseya T, Tamaya T 1996 Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. J Clin Endocrinol Metab 81:3249–3253[Abstract]
  13. Stanislaus D, Janovick JA, Brothers S, Conn PM 1997 Regulation of G(q/11){alpha} by the gonadotropin-releasing hormone receptor. Mol Endocrinol 11:738–746[Abstract/Free Full Text]
  14. Rhee SG 2001 Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312[CrossRef][Medline]
  15. Newton AC 2001 Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 101:2353–2364[CrossRef][Medline]
  16. Liu F, Austin DA, Webster NJG 2003 Gonadotropin-releasing hormone-desensitized LßT2 gonadotrope cells are refractory to acute protein kinase C, cyclic AMP, and calcium-dependent signaling. Endocrinology 144:4354–4365[CrossRef][Medline]
  17. Junoy B, Maccario H, Mas JL, Enjalbert A, Drouva SV 2002 Proteasome implication in phorbol ester- and GnRH-induced selective down-regulation of PKC ({alpha}, {epsilon}, {zeta}) in {alpha}T3–1 and LßT2 gonadotrope cell lines. Endocrinology 143:1386–1403[Abstract/Free Full Text]
  18. Hanson PI, Schulman H 1992 Neuronal Ca2+/calmodulin-dependent protein kinases. Annu Rev Biochem 61:559–601[CrossRef][Medline]
  19. Schulman H, Heist K, Srinivasan M 1995 Decoding Ca2+ signals to the nucleus by multifunctional CaM kinase. Prog Brain Res 105:95–104[Medline]
  20. Haisenleder DJ, Ferris HA, Shupnik MA 2003 The calcium component of gonadotropin-releasing hormone-stimulated luteinizing hormone subunit gene transcription is mediated by calcium/calmodulin-dependent protein kinase type II. Endocrinology 144:2409–2416[Abstract/Free Full Text]
  21. Haisenleder DJ, Burger LL, Aylor KW, Dalkin AC, Marshall JC 2003 Gonadotropin-releasing hormone stimulation of gonadotropin subunit transcription: evidence for the involvement of calcium/calmodulin-dependent kinase II (Ca/CAMK II) activation in rat pituitaries. Endocrinology 144:2768–2774[CrossRef][Medline]
  22. Carpenter L, Cordery D, Biden TJ 2001 Protein kinase C{delta} activation by interleukin-1ß stabilizes inducible nitric-oxide synthase mRNA in pancreatic ß-cells. J Biol Chem 276:5368–5374[Abstract/Free Full Text]
  23. Gschwendt M 1999 Protein kinase C{delta}. Eur J Biochem 259:555–564[Medline]
  24. Liu F, Austin DA, Mellon PL, Olefsky JM, Webster NJ 2002 GnRH activates ERK1/2 leading to the induction of c-fos and LHß protein expression in LßT2 cells. Mol Endocrinol 16:419–434[Abstract/Free Full Text]
  25. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, Webster NJG 2002 Involvement of both Gq/11 and Gs proteins in gonadotropin-releasing hormone receptor-mediated signaling in LßT2 cells. J Biol Chem 277:32099–32108[Abstract/Free Full Text]
  26. Berridge MJ 1993 Inositol trisphosphate and calcium signalling. Nature 361:315–325[CrossRef][Medline]
  27. Romoser VA, Graves TK, Wu D, Jiang H, Hinkle PM 2001 Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice. Mol Endocrinol 15:125–135[Abstract/Free Full Text]
  28. Taylor SJ, Chae HZ, Rhee SG, Exton JH 1991 Activation of the ß1 isozyme of phospholipase C by {alpha}-subunits of the Gq class of G proteins. Nature 350:516–518[CrossRef][Medline]
  29. Berstein G, Blank JL, Jhon DY, Exton JH, Rhee SG, Ross EM 1992 Phospholipase C-ß1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell 70:411–418[CrossRef][Medline]
  30. Berstein G, Blank JL, Smrcka AV, Higashijima T, Sternweis PC, Exton JH, Ross EM 1992 Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-ß1. J Biol Chem 267:8081–8088[Abstract/Free Full Text]
  31. McArdle CA, Willars GB, Fowkes RC, Nahorski SR, Davidson JS, Forrest-Owen W 1996 Desensitization of gonadotropin-releasing hormone action in {alpha}T3–1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization. J Biol Chem 271:23711–23717[Abstract/Free Full Text]
  32. McArdle CA, Franklin J, Green L, Hislop JN 2002 Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. J Endocrinol 173:1–11[Abstract]
  33. Willars GB, Royall JE, Nahorski SR, El-Gehani F, Everest H, McArdle CA 2001 Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in {alpha}T3–1 gonadotropes. J Biol Chem 276:3123–3129[Abstract/Free Full Text]
  34. Xie J, Nagle GT, Childs GV, Ritchie AK 1999 Expression of the L-type Ca(2+) channel in AtT-20 cells is regulated by cyclic AMP. Neuroendocrinology 70:1–9[CrossRef][Medline]
  35. Bouali-Benazzouz R, Audy MC, Bonnin M 1993 Estradiol modulation of GNRH-stimulated LH release in rat anterior pituitary cells: involvement of dihydropyridine-sensitive calcium channels. Neuroendocrinology 57:1161–1170[Medline]
  36. Dziema H, Obrietan K 2002 PACAP potentiates L-type calcium channel conductance in suprachiasmatic nucleus neurons by activating the MAPK pathway. J Neurophysiol 88:1374–1386[Abstract/Free Full Text]
  37. Xu R, Roh SG, Gong C, Hernandez M, Ueta Y, Chen C 2003 Orexin-B augments voltage-gated L-type Ca(2+) current via protein kinase C-mediated signalling pathway in ovine somatotropes. Neuroendocrinology 77:141–152[CrossRef][Medline]
  38. Baldelli P, Hernandez-Guijo JM, Carabelli V, Novara M, Cesetti T, Andres-Mateos E, Montiel C, Carbone E 2004 Direct and remote modulation of L-channels in chromaffin cells: distinct actions on {alpha}1C and {alpha}1D subunits? Mol Neurobiol 29:73–96[CrossRef][Medline]
  39. Nguyen KA, Santos SJ, Kreidel MK, Diaz AL, Rey R, Lawson MA 2004 Acute regulation of translation initiation by gonadotropin-releasing hormone in the gonadotrope cell line LßT2. Mol Endocrinol 18:1301–1312[Abstract/Free Full Text]
  40. Petersen OH, Petersen CC, Kasai H 1994 Calcium and hormone action. Annu Rev Physiol 56:297–319[CrossRef][Medline]
  41. Rao K, Paik WY, Zheng L, Jobin RM, Tomic M, Jiang H, Nakanishi S, Stojilkovic SS 1997 Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion. Endocrinology 138:1440–1449[Abstract/Free Full Text]
  42. Thomas P, Mellon PL, Turgeon J, Waring DW 1996 The LßT2 clonal gonadotrope: a model for single cell studies of endocrine cell secretion. Endocrinology 137:2979–2989[Abstract]
  43. Blotner M, Shangold GA, Lee EY, Murphy SN, Miller RJ 1990 Nitrendipine and {omega}-conotoxin modulate gonadotropin release and gonadotrope [Ca2+]i. Mol Cell Endocrinol 71:205–216[CrossRef][Medline]
  44. Martin TF 2003 Tuning exocytosis for speed: fast and slow modes. Biochim Biophys Acta 1641:157–165[Medline]
  45. Yang Y, Udayasankar S, Dunning J, Chen P, Gillis KD 2002 A highly Ca2+-sensitive pool of vesicles is regulated by protein kinase C in adrenal chromaffin cells. Proc Natl Acad Sci USA 99:17060–17065[Abstract/Free Full Text]
  46. Gillis KD, Mossner R, Neher E 1996 Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules. Neuron 16:1209–1220[CrossRef][Medline]
  47. Zhu H, Hille B, Xu T 2002 Sensitization of regulated exocytosis by protein kinase C. Proc Natl Acad Sci USA 99:17055–17059[Abstract/Free Full Text]
  48. von Ruden L, Neher E 1993 A Ca-dependent early step in the release of catecholamines from adrenal chromaffin cells. Science 262:1061–1065[Abstract/Free Full Text]
  49. Nicol L, McNeilly JR, Stridsberg M, McNeilly AS 2004 Differential secretion of gonadotrophins: investigation of the role of secretogranin II and chromogranin A in the release of LH and FSH in LßT2 cells. J Mol Endocrinol 32:467–480[Abstract]
  50. Nicol L, McNeilly JR, Stridsberg M, Crawford JL, McNeilly AS 2002 Influence of steroids and GnRH on biosynthesis and secretion of secretogranin II and chromogranin A in relation to LH release in LßT2 gonadotroph cells. J Endocrinol 174:473–483[Abstract]
  51. Crawford JL, McNeilly AS 2002 Co-localisation of gonadotrophins and granins in gonadotrophs at different stages of the oestrous cycle in sheep. J Endocrinol 174:179–194[Abstract]
  52. Crawford JL, McNeilly JR, Nicol L, McNeilly AS 2002 Promotion of intragranular co-aggregation with LH by enhancement of secretogranin II storage resulted in increased intracellular granule storage in gonadotrophs of GnRH-deprived male mice. Reproduction 124:267–277[Abstract]
  53. Song SB, Rhee M, Roberson MS, Maurer RA, Kim KE 2003 Gonadotropin-releasing hormone-induced stimulation of the rat secretogranin II promoter involves activation of CREB. Mol Cell Endocrinol 199:29–36[CrossRef][Medline]
  54. Imamura T, Vollenweider P, Egawa K, Clodi M, Ishibashi K, Nakashima N, Ugi S, Adams JW, Brown JH, Olefsky JM 1999 G{alpha}-q/11 protein plays a key role in insulin-induced glucose transport in 3T3–L1 adipocytes. Mol Cell Biol 19:6765–6774[Abstract/Free Full Text]
  55. Merritt JE, McCarthy SA, Davies MP, Moores KE 1990 Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem J 269:513–519[Medline]



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GNRH Induces the Unfolded Protein Response in the L{beta}T2 Pituitary Gonadotrope Cell Line
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S. Lariviere, G. Garrel-Lazayres, V. Simon, N. Shintani, A. Baba, R. Counis, and J. Cohen-Tannoudji
Gonadotropin-Releasing Hormone Inhibits Pituitary Adenylyl Cyclase-Activating Polypeptide Coupling to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway in L{beta}T2 Gonadotrope Cells through Novel Protein Kinase C Isoforms and Phosphorylation of Pituitary Adenylyl Cyclase-Activating Polypeptide Type I Receptor
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T. B. Salisbury, A. K. Binder, J. C. Grammer, and J. H. Nilson
Maximal Activity of the Luteinizing Hormone{beta}-Subunit Gene Requires {beta}-Catenin
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S. Lariviere, G. Garrel, V. Simon, J.-W. Soh, J.-N. Laverriere, R. Counis, and J. Cohen-Tannoudji
Gonadotropin-Releasing Hormone Couples to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway through Novel Protein Kinase C{delta} and -{epsilon} in L{beta}T2 Gonadotrope Cells
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