Regulation of Gq/11{alpha} by the Gonadotropin-Releasing Hormone Receptor

Dinesh Stanislaus, Jo Ann Janovick, Shaun Brothers and P. Michael Conn

Department of Physiology and Pharmacology (D.S., P.M.C.),Oregon Health Sciences University, Portland, Oregon 97201,
Oregon Regional Primate Research Center,(D.S., J.A.J., S.B., P.M.C.), Beaverton, Oregon 97006


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of Gq/11{alpha} subunits(Gq/11{alpha}). First, we used GnRH-stimulated palmitoylation of Gq/11{alpha} to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-3H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of Gq/11{alpha}. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0–120 min) labeling of Gq/11{alpha}. GnRH (10-12, 10-10, 10-8, or 10-6 g/ml) for 40 min resulted in dose-dependent labeling of Gq/11{alpha} compared with controls. Cholera toxin (5 µg/ml; activator of Gs{alpha}), pertussis toxin (100 ng/ml; inhibitor of Gi{alpha} actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of Gq/11{alpha} above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of Gq/11{alpha} above basal levels, but not to the same extent as 10-6 g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of {alpha}1B-adrenergic receptor ({alpha}1B3i) couples to Gq/11{alpha}, it can inhibit Gq/11{alpha}-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to Gq/11{alpha}. Transfection (efficiency 5–7%) with {alpha}1B3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples toGq/11{alpha}), resulted in 10–12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of {alpha}2A-adrenergic receptor, M2-acetylcholine receptor (both couple to Gi{alpha}), and D1A-receptor (couples to Gs{alpha}) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of Gq/11{alpha} in a PRL-secreting adenoma cell line (GGH31') expressing the GnRH receptor. Stimulation of GGH31' cells with 0.1 µg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15–20% decrease in total Gq/11{alpha} at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10-6 g/ml). Neither Antide (10-6 g/ml, 24 h) alone nor phorbol myristic acid (0.33–100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of Gq/11{alpha} immunoreactivity. The loss of Gq/11{alpha} immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of Gq/11{alpha} by the GnRH receptor in primary pituitary cultures and in GGH3 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH receptor, like other receptors in the 7-transmembrane segment receptor superfamily, couples to multiple G proteins (1, 2). In dispersed pituitary cell cultures, pertussis toxin (PTX) pretreatment results in decreased inositol phosphate (IP) turnover compared with medium-pretreated levels in response to GnRH (3), suggesting that a PTX-sensitive G protein (such as Gi or Go) couples the receptor to IP turnover. However, in GGH312' cell cultures (GH3 cells stably transfected with the rat GnRH-receptor cDNA), GnRH agonist-evoked IP turnover is insensitive to PTX (4), indicating that a PTX-insensitive G protein may be involved in signal transduction and the receptor may be coupled differently in different cells.

Gq/11{alpha} immune-depletion studies show that in membranes derived from {alpha}T3–1 cells, GnRH receptor is coupled to Gq/11{alpha} (5). Furthermore, PRL synthesis in GGH3 cell cultures in response to GnRH is mediated by cAMP, implicating Gs{alpha} in this signal transduction pathway (6), although cAMP does not mediate GnRH-stimulated hormone release from the gonadotrope (7). It is evident from these studies that GnRH receptor is able to couple to different G proteins in different cell lines. Therefore, to investigate the G proteins that couple to the GnRH receptor, it is important to undertake these studies in primary pituitary cultures.

In the gonadotrope, the little that is known about G proteins involved in GnRH receptor-mediated signal transduction has been obtained from toxin studies and from second messenger studies (1, 2, 3). PTX-sensitive G proteins have been implicated in the GnRH receptor/G protein coupling. In addition, the observation that cholera toxin (CTX) pretreatment enhances GnRH stimulated LH release (8) has implicated Gs{alpha} in modulation of GnRH action. Such studies provide only indirect evidence as to the identity of the G proteins involved in GnRH receptor-mediated signal transduction. Complicating this further is the observation that both protein kinase C (PKC) and protein kinase A, regulated by different G proteins, are capable of regulating IP turnover by phosphorylating phospholipase C (9). Therefore, the sole use of IP turnover as a marker for a specific G protein activation would lead to unclear results. Furthermore, cross-talk between CTX-sensitive G protein and PKC (10) can further complicate the identification of the G proteins that couple to the GnRH receptor.

Palmitoylation (i.e. the addition of a 16-carbon fatty acid to a cysteine residue through a thioester link) of G protein {alpha}-subunits is a dynamic process that is regulated after receptor activation. Receptor-evoked palmitoylation of Gq/11{alpha} and Gs{alpha} is a well characterized phenomenon (11, 12, 13) and occurs in a time- and dose-dependent manner (11, 12). Furthermore, G proteins that do not associate with a specific receptor do not incorporate [3H]palmitic acid into their {alpha}-subunits when that receptor is stimulated (11). In addition, mutationally activated Gs{alpha} turns over [3H]palmitic acid labeling more rapidly than wild type Gs{alpha} (14). These studies suggest that activation of G protein is required for the {alpha}-subunit to undergo palmitoylation. In the present study, in order to identify the moieties that are affected when cells are stimulated with GnRH, we used the ability of G protein-coupled receptors to stimulate the palmitoylation of G protein {alpha}-subunits they activate.

Several reports (15, 16) have shown that the cellular expression of the third intracellular loop of G protein-coupled receptors can inhibit receptor- evoked second messenger production. This effect is greatest when the peptide is derived from the same receptor, although expression of heterologous third intracellular loops inhibits receptor-stimulated second messenger production to a lesser extent as long as the intracellular loop and the receptor couple to a common G protein. These studies demonstrate that peptides derived from the third intracellular loop of G protein-coupled receptors produce G protein-specific inhibition of receptor-mediated signal transduction (15). We used the ability of the third intracellular loop to act as an intracellular inhibitor to corroborate our findings from the palmitoylation studies.

To examine further the regulation of G proteins by the GnRH receptor, we examined receptor-evoked down-regulation of G protein {alpha}-subunits. Agonist-induced reduction in total cellular G protein {alpha}-subunits has been observed for members of the G protein family (Gs{alpha}, Gi{alpha}, Gq/11{alpha}; 17). This reduction is observed for G proteins that interact with the activated receptor (17) and can be used as a marker for receptor regulation of G proteins. Therefore, we used an RIA developed in our laboratory to assess GnRH agonist-evoked reduction of total cellular Gq/11{alpha} in a rat pituitary adenoma cell line stably expressing the GnRH receptor. We opted to use a homogeneous cell line instead of a dispersed rat pituitary cell culture, because gonadotropes are only about 20% of cells obtained from a rat pituitary cell dispersion (female weanlings), and changes in total G protein content may be masked against a relatively high background from nongonadotrope cells.

In this study, we assess evidence to indicate GnRH receptor regulation of Gq/11{alpha}. Evidence for regulation of Gq/11{alpha} by the receptor is present in rat pituitary cell dispersions and also in a cell line stably expressing the GnRH receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The time course of GnRH-stimulated palmitoylation of Gq/11{alpha} in pituitary cell cultures is shown in Table 1Go and Fig. 1Go. Rat pituitary cell cultures were treated with 10-6 g/ml GnRH or cell culture medium in the presence of [3H]palmitate for 0, 20, 40, 60, 90, and 120 min. Immunoprecipitation of Gq/11{alpha} showed an increase in [3H]palmitate incorporation with GnRH treatment. The earliest detectable incorporation of the label is measurable at 20 min after the addition of GnRH. GnRH-stimulated incorporation of the label is detectable up to 120 min. The basal incorporation of [3H]palmitate label increases with time, although in the presence of GnRH there is an increased incorporation over basal levels.


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Table 1. The Time Course for GnRH-Stimulated Incorporation of [3H]palmitic Acid into Gq/11{alpha}

 


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Figure 1. The Time Course for GnRH-Stimulated Incorporation of [3H]Palmitic Acid into Gq/11{alpha}

Dispersed rat pituitary cell cultures were incubated in the presence or absence of 10-6 g/ml GnRH for the indicated times. Incorporation of the label into Gq/11{alpha} was assayed by immunoprecipitation then resolved by 10% SDS-PAGE, fluorography, and densitometry. Data show band density in arbitrary optical density units. The data are from one representative experiment. Three separate experiments showed similar results.

 
The palmitate incorporation to Gq/11{alpha} was dose dependent with GnRH (Fig. 2Go). Pituitary cell cultures were treated with medium and GnRH (10-12, 10-10, 10-8, and 10-6 g/ml) in the presence of [3H]palmitate for 60 min. GnRH concentration of approximately 10-9 g/ml produced a half-maximal incorporation of palmitate label on Gq/11{alpha}.



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Figure 2. The Dose-Response for GnRH-Stimulated Incorporation of [3H]Palmitic Acid into Gq/11{alpha}

Dispersed rat pituitary cell cultures were incubated for 60 min with the indicated doses of GnRH. Incorporation of the label into Gq/11{alpha} was assayed by immunoprecipitation then resolved by 10% SDS-PAGE, fluorography, and densitometry. Data show band density in arbitrary absorbance units. The data are from one representative experiment. Three separate experiments showed similar results.

 
To further assess the specificity of GnRH receptor-stimulated palmitoylation of Gq/11{alpha}, we examined the ability of CTX (5 µg/ml), PTX (100 ng/ml), phorbol myristic acid (PMA, a PKC activator; 100 ng/ml), and Antide (GnRH antagonist; 50 nM) to evoke palmitoylation of Gq/11{alpha} (Fig. 3Go). Rat pituitary cell cultures were incubated in the presence of the above agents for 40 min, and Gq/11{alpha} was immunoprecipitated. Only PMA and GnRH increased the incorporation of [3H]palmitate label on Gq/11{alpha} above basal incorporation levels. The level of Gq/11{alpha} incorporation of palmitate when treated with CTX, PTX, and Antide was not significantly different from basal levels.



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Figure 3. The Effect of the Indicated Agents on Gq/11{alpha} Incorporation of [3H]Palmitic Acid

Dispersed rat pituitary cell cultures were incubated in the presence of the indicated agents at the indicated concentrations for 40 min. Incorporation of the label into Gq/11{alpha} was assayed by immunoprecipitation then resolved by 10% SDS-PAGE, fluorography, and densitometry. Data show band density in arbitrary absorbance units. The data are from one representative experiment. Three separate experiments showed similar results.

 
Gels treated with 1 M hydroxylamine before autoradiography did not show any residual radioactivity (data not shown), indicating that the radiolabel was alkali sensitive, as would be expected from a thioester-linked palmitate label.

Transfection of primary cell cultures with the cDNA for the third intracellular loops of the {alpha}1B-adrenergic receptor ({alpha}1B3i), {alpha}2A-adrenergic receptor ({alpha}2A3i), M1-muscarinic receptor (M13i), M2-Muscarinic receptor (M23i), and D1A-dopamine receptor (D1A3i) did not inhibit GnRH-stimulated LH release from vector-transfected levels (Fig. 4Go). Primary cell cultures were transiently transfected using lipofectamine, and GnRH-stimulated LH release was measured by RIA. Transfection efficiency was measured to be 5–7%.



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Figure 4. GnRH Stimulated LH Release in Transiently Transfected Dispersed Rat Pituitary Cell Cultures

Cell cultures were transiently transfected, as described in Materials and Methods, with the indicated third intracellular loops of G protein-coupled receptors in pRK5 expression vector. Twenty four hours after transfection, cells were stimulated for 2 h with the indicated concentrations of GnRH, and LH released into the medium was assayed by RIA. The data are the mean of triplicate transfections, and error bars show the SEM. Three separate experiments showed similar results.

 
Although {alpha}1B3i did not inhibit GnRH-stimulated LH release, transfection of this loop resulted in approximately 10% inhibition of GnRH-stimulated IP turnover as compared with vector-transfected values in rat pituitary cell cultures. IP turnover was measured as described in Materials and Methods. Transfection of cDNA for {alpha}2A3i, M13i, M23i, and D1A3i resulted in no significant inhibition of GnRH-stimulated IP turnover (Fig. 5Go).



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Figure 5. GnRH Stimulated IP Turnover in Transiently Transfected Dispersed Rat Pituitary Cell Cultures

Cell cultures were transiently transfected, as described in Materials and Methods, with the indicated third intracellular loops of G protein-coupled receptors in pRK5 expression vector. Twenty four hours after transfection, the cellular inositol was labeled with [3H]-myo-inositol for 18 h, after which cells were stimulated for 2 h with the indicated concentrations of GnRH, and total 3H-labeled IPs were determined by Dowex anion exchange chromatography and liquid scintillation spectroscopy. The data are the mean of triplicate transfections, and error bars show the SEM. Three separate experiments showed similar results.

 
Cellular expression of {alpha}2A3i was determined by immunoblotting cell lysates with the peptide-specific antisera (Fig. 6Go). Cell lysates were prepared from rat pituitary cell cultures transiently transfected with the cDNA for {alpha}2A3i. A single band was seen at the apparent molecular mass of approximately 21 kDa. This band was absent in cell lysates obtained from pRK5-transfected rat pituitary cell cultures.



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Figure 6. Protein Immunoblot Depicting Expression of the Third Intracellular Loop of the {alpha}2A-Adrenergic Receptor

Rat pituitary cell cultures were transiently transfected with the third intracellular loop of the {alpha}2A-adrenergic receptor or the empty expression vector pRK5. Twenty-four hours after transfection, cell cultures were lysed and expression of the peptide was detected with immunoblotting using peptide-specific antisera as described in Materials and Methods.

 
The time course of Buserelin-stimulated net loss of Gq/11{alpha} immunoreactivity is shown in Fig. 7Go. GGH31' cell cultures (GH3-derived cell line stably expressing the GnRH receptor) were treated for the indicated times with 0.1 µg/ml of Buserelin or medium alone, and the total cellular Gq/11{alpha} was measured by RIA as described in Materials and Methods. Consistent with a cell-doubling time of approximately 1 day, the total amount of Gq/11{alpha} increased with time both in control cells and in cells treated with 0.1 µg/ml of Buserelin. However, in cells treated with Buserelin, total levels of Gq/11{alpha} were consistently 15–20% less than in control cells at 24 h. The reduction in total cellular Gq/11{alpha} after Buserelin treatment was first observed at 6 h.



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Figure 7. Time Course of Gq/11{alpha} Proteins from GGH31' Cells Treated with Medium Alone (•) or 0.1 µg/ml Buserelin ({blacksquare})

The Gq/11{alpha} proteins were measured by RIA. The data shown are the means ± SEM of quadruplicate determinations. A molar correction factor of 0.03 should be used to adjust the values obtained to account for the ratio of the molecular weight of the standard (CTS) to that of Gq/11{alpha}. Three separate experiments showed similar results.

 
The loss of Gq/11{alpha} immunoreactivity was dose-dependent with Buserelin (Fig. 8Go). GGH31' cells were treated with the indicated doses of Buserelin for 24 h, and the total cellular Gq/11{alpha} immunoreactivity was assayed by RIA. Buserelin concentration of 10-9 g/ml produced a half-maximal loss of Gq/11{alpha} immunoreactivity. The effect of 10-9 g/ml Buserelin on assayable Gq/11{alpha} was blocked by 10-6 g/ml Antide (a GnRH antagonist; 340 ± 8 pg/60 µl), as compared with medium-treated levels (339 ± 13 pg/60 µl). Furthermore, 10-6 g/ml Antide alone (322 ± 8 pg/60 µl) did not produce a loss of Gq/11{alpha} immunoreactivity compared with medium-treated levels (339 ± 13 pg/60 µl).



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Figure 8. Dose-Response Curve of Gq/11{alpha} Proteins from GGH31' Cells Treated 24 h with Buserelin (•)

Antide (10-6 g/ml) in the presence of 10-9 g/ml Buserelin (340 ± 8 pg/60 µl) or in the absence (322 ± 8 pg/µl) did not produce a loss of immunoreactivity as compared with medium-treated levels (339 ± 13 pg/60 µl). The Gq/11{alpha} proteins were measured by RIA. The data shown are the means ± SEM of triplicate determinations. A molar correction factor of 0.03 should be used to adjust the values obtained to account for the ratio of the molecular weight of the standard (CTS) to that of Gq/11{alpha}. Three separate experiments showed similar results.

 
PMA did not mimic this action of Buserelin treatment (Fig. 9Go). GGH31' cells were treated with 0.33–100 ng/ml of PMA for 24 h, and total cellular Gq/11{alpha} was assayed by RIA. Treatment of GGH31' cells with PMA for 24 h did not result in any significant loss of immunoreactive Gq/11{alpha}, as compared with medium-treated levels.



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Figure 9. Dose-Response Curve of Gq/11{alpha} Proteins from GGH31' Cells Treated for 24 h with PMA

The Gq/11{alpha} proteins were measured by RIA. The data shown are the means ± SEM of triplicate determinations. A molar correction factor of 0.03 should be used to adjust the values obtained to account for the ratio of the molecular weight of the standard (CTS) to that of Gq/11{alpha}. Three separate experiments showed similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data presented here demonstrate that Gq/11{alpha} is palmitoylated in a time- and dose-dependent manner when the GnRH receptor is occupied by an agonist. Antagonist occupancy of the receptor does not lead to this effect. Stimulation of hormone release from dispersed pituitary cell cultures with increasing concentrations of GnRH resulted in the concurrent increase in incorporation of [3H]palmitic acid label into Gq/11{alpha}. This effect was time- and dose-dependent. Similarly, CTX and PTX did not increase the incorporation of the label into Gq/11{alpha}. Gq/11{alpha} is CTX- and PTX-insensitive, and, as expected, these agents do not stimulate the incorporation of [3H]palmitic acid. PMA, a PKC activator, stimulated palmitate labeling of Gq/11{alpha}, although not to the same extent as GnRH-stimulated levels. We also show that the transfection of the {alpha}1B3i loop cDNA resulted in the partial inhibition of GnRH receptor-mediated IP turnover in dispersed rat pituitary cell cultures. The expression of this peptide, however, did not significantly inhibit GnRH-stimulated LH release in these cultures. Transfection of the cDNA sequences for {alpha}2A3i loop, M13i loop, M23i loop, and D1A3i loop did not significantly inhibit GnRH-stimulated IP turnover or LH release. The expression of one of the plasmids containing the cDNA sequence for the {alpha}2A3i loop was confirmed by Western analysis. We were unable to perform Western analysis to confirm the expression of other third intracellular loops due to the unavailability of antisera against these peptides. Because the same expression vector (pRK5; 18 was used for all studies, it is reasonable to believe that these would be expressed at similar levels as the {alpha}2A3i loop. The regulation of Gq/11{alpha} by the GnRH receptor extends toward the GGH31' cells. Buserelin treatment of these cells resulted in the loss of total cellular Gq/11{alpha} as assessed by a RIA. This effect of Buserelin was time- and dose-dependent and was antagonized by GnRH antagonist, Antide. Neither Antide alone nor PMA mimicked the actions of Buserelin.

This study was designed to investigate the G proteins that are regulated by the GnRH receptor. G protein involvement in GnRH action in the gonadotrope has been suggested by studies that show stimulation of LH release by stable GTP analogs and by IP accumulation in ATP-permeabilized cells (1, 21). Although ample evidence is available to suggest G protein involvement in the gonadotrope, specific G protein(s) are yet to be identified. In this study, we used G protein-coupled receptor-evoked palmitoylation of G{alpha} to identify specific moieties that are regulated by the GnRH receptor. This study demonstrates that GnRH receptor regulates the palmitoylation of Gq/11{alpha}. Because palmitoylation of Gq/11{alpha} is dependent on this moiety being activated by a receptor, it suggests that GnRH receptor is coupled to Gq/11{alpha}. PMA, a PKC activator, stimulated Gq/11{alpha} incorporation of [3H]palmitic acid, albeit less than GnRH-stimulated levels. The effect of PMA on Gq/11{alpha} incorporation of [3H]palmitic acid is puzzling, as PMA did not have any significant effect on the Buserelin-evoked loss ofGq/11{alpha} immunoreactivity in GGH3 cells. The effect of PMA on Gq/11{alpha} palmitoylation may be the result of activation of G protein palmitoyltransferase, the enzyme responsible for addition and removal of palmitate from G proteins. Alternatively, PMA may activate Gq/11{alpha} directly or through PKC, thereby presenting it as an activated G protein for palmitoyltransferase to palmitoylate, although there is no prior evidence for this action of PMA. However, GnRH-evoked palmitoylation is greater than PMA alone. This may indicate that GnRH receptor-evoked palmitoylation reflects a direct activation of G proteins, while the action of PMA (mediated through PKC) may be pharmacological. The receptor-mediated component demonstrates that the GnRH receptor is coupled to Gq/11{alpha} in the gonadotrope.

Inhibition of GnRH receptor-evoked IP turnover by transfecting the cDNA for the {alpha}1B3i loop, whose cognate receptor mediates IP turnover through a member of the Gq/11{alpha} family, demonstrated that the GnRH receptor is coupled to Gq/11{alpha} and further confirmed the palmitoylation studies. Luttrell et al. (15) showed that the {alpha}1B3i loop inhibited heterologous receptor-mediated IP turnover, which is consistent with the data presented in this study. Although M1Ach receptor mediates IP turnover through a PTX-insensitive G protein, transfection of M13i loop cDNA did not inhibit GnRH receptor-mediated IP turnover. This may indicate that this loop is less effective at inhibiting GnRH receptor-mediated IP turnover, because the inhibition of heterologous receptor-mediated activity by third intracellular loop peptides can vary (16). The lack of inhibition may also be due to the fact that this loop is expressed to a lesser extent than {alpha}1B3i loop, although this is unlikely as both the {alpha}1B3i loop and the M13i loop are translated by the same regulatory elements, namely, the CMV promoter of the pRK5 vector. Transfection of {alpha}2A3i loop cDNA and M23i loop cDNA did not inhibit GnRH receptor-mediated signal transduction, although the cognate receptors of these loops couple to Gi{alpha} (16), which has been implicated in GnRH receptor-mediated PTX-sensitive IP turnover (3). The lack of inhibition may be due to the same reasons that were mentioned earlier, including the fact that GnRH receptor may not couple to these G proteins. As would be expected, D1A3i loop, which inhibits Gs{alpha}-mediated actions, did not inhibit GnRH-evoked IP turnover or LH release because these events are not thought to involve Gs{alpha}. The decrease in maximal effects of GnRH-evoked IP turnover seen with {alpha}1B3i loop transfection has also been observed by other investigators (16). They have shown that the peptide- mediated inhibition of receptor-evoked IP turnover is due to competition between the hormone-receptor complex and the peptide for the common binding site on G{alpha}-subunit. This competition can be overcome by increasing the hormone-receptor coupled by increasing its transfected receptor cDNA.

Previous work done in cell lines has shown that activation of Gs{alpha} induces a conformational change that allows a loss of membrane avidity and increases its degradation rate (19). Furthermore, in {alpha}T3–1 cell lines GnRH agonist treatment results in an increased degradation of Gq/11{alpha} (20). These observations support our findings in the GGH3 cells; Buserelin treatment resulted in the time- and dose-dependent decrease in cellular Gq/11{alpha}. Using the increased degradation of activated G{alpha} as a marker, our results show that Gq/11{alpha} is activated by the GnRH receptor. For technical reasons, these studies were done in an immortalized cell line, because gonadotropes are only about 20% of the cells in culture prepared from female weanling rat pituitaries, and changes in G protein content can be masked by a relatively high background from nongonadotrope cells. Although this study was done in a cell culture, it corroborates well with our previous findings to show that the GnRH receptor regulates Gq/11{alpha}. Furthermore, the fact that PMA treatment did not mimic the actions of Buserelin on loss of Gq/11{alpha} may indicate that PKC is not involved, and this action of GnRH agonist is mediated through a direct GnRH-receptor/Gq/11{alpha} interaction.

This study provides evidence for GnRH receptor regulation of Gq/11{alpha}. We have shown that Gq/11{alpha} incorporates [3H]palmitic acid in a dose- and time-dependent manner when treated with GnRH, and the third intracellular loop of the {alpha}1B-adrenergic receptor, which couples to Gq/11{alpha}, acts as an intracellular inhibitor of GnRH receptor-mediated IP turnover. We have also shown that GnRH agonist treatment results in a dose- and time-dependent loss of Gq/11{alpha} immunoreactivity in GGH31' cells. The observation made in this study suggest that GnRH receptor regulates the activity of Gq/11{alpha} in rat gonadotropes and also in the GGH31' cell line. The ability of the GnRH receptor to regulate Gq/11{alpha} activity indicates that it is able to couple to this G protein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Suppliers of materials used in this study were as follows: horse serum and FCS, Hyclone Laboratories (Logan, UT); BSA, Irvine Scientific (Santa Ana, CA); HEPES, United States Biochemical (Cleveland, OH); collagenase, Worthington Biochemical (Freehold, NJ); formic acid, Mallinkrodt (McGraw Park, IL); ammonium formate, sodium deoxycholate, and EDTA, Fisher Scientific (Fairlawn, NJ); Nonidet P-40, Particle Data Laboratory (Elm Hurst, IL); gentamicin sulfate, Gemini (Bio-products, Calabasas, CA), hyaluronidase, DNAse I, and PMA, Sigma (St. Louis, MO); and Antide (Ares-Serono, Geneva). Other reagents were obtained at the highest grade available from commercial vendors as indicated. The G protein-coupled receptor third intracellular loops expression plasmids — pRK{alpha}1B3i, pRK{alpha}2A3i, pRKM13i, pRKM23i, and pRKD1A3i — were a gift from Dr. R. J. Lefkowitz (Duke University, Durham, NC) (3).

Preparation of Pituitary Cell Cultures
Pituitary cell cultures were prepared as previously described (7). Briefly, pituitary glands were removed from 28-day-old female Sprague-Dawley rats (B&K Universal, Inc, Kent, WA) and placed in medium 199 (Irvine Scientific) containing 0.3% (wt/vol) BSA and 10 mM HEPES, pH 7.4 (M199/BSA). The pituitaries were minced and incubated in sterile M199/BSA containing 0.125% (wt/vol) collagenase and 0.1% (wt/vol) hyaluronidase in a 37 C shaking water bath for 15 min. The dissociated cells were filtered through organza cloth, and the remaining tissue was incubated a second time with a similar enzyme solution for another 15 min. The combined cells were collected by centrifugation (10 min at 200 x g) and resuspended in M199/BSA containing 10% (vol/vol) horse serum, 2.5% (vol/vol) FCS, and 20 µg/ml gentamicin sulfate and filtered through an organza cloth. For palmitoylation studies, cell suspensions were plated at a cell density of 2.5 x 106 cells per well in six-well culture plates (Costar, Cambridge, MA). Single cell suspensions were obtained for transfection studies by resuspending the cell pellet with M199/BSA/4 mM EDTA containing 0.2% (wt/vol) collagenase and 0.2% (wt/vol) hyaluronidase and incubating for 30 min at 37 C, and adding DNAse I (100 µg/ml) for the last 5 min of the incubation. The dissociated cells were filtered through a 10-µm mesh cloth and incubated for another 15 min at 37 C with M199/BSA/4 mM EDTA containing 0.2% collagenase and 0.2% hyaluronidase. The cells were collected by centrifugation (15 min at 200 x g; 4 C) and resuspended in cold M199/BSA containing 10% horse serum, 2.5% FCS, and 20 µg/ml gentamicin sulfate. The cell suspensions were plated at cell density of 15 x 104 cells per well in 24-well culture plates (Costar, Cambridge, MA). Cells were maintained for approximately 48 h at 37 C in a water-saturated atmosphere before experiments were begun.

Metabolic Labeling of G Proteins with \[9,10-3H\]Palmitic Acid and Immunoprecipitation
Pituitary cell cultures were washed twice with M199/BSA (pH 7.4), 2 h before labeling with \[9,10-3H\]palmitic acid (specific activity 30–60 Ci/mmol, 0.5 mCi/ml of M199/BSA; DuPont NEN, Boston, MA) containing the indicated compounds for the indicated times. Labeling was stopped at the appropriate times by aspirating the labeling medium and washing once with cold Dulbecco’s-PBS, and the cells were lysed for 1 h on ice with 750 µl cold RIPA buffer \[50 mM HEPES, pH 7.4, 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 1 mM EDTA and 2.5 mM MgCl2\]. The insoluble material and nuclei were removed by centrifugation at 12,000 x g (Eppendorf microcentrifuge) for 3 min. Nonspecific binding was removed by rocking the cell extract in 1.5-ml Eppendorf tubes containing 75 µl Protein A-Sepharose 6MB (Pharmacia Biotech, Piscataway, NJ), previously coupled to IgG from normal rabbit serum, for 30 min at 4 C. After this step, the cell extract was transferred to new 1.5-ml Eppendorf tubes containing 75 µl Protein A-Sepharose coupled to our Q7 rabbit polyclonal antibody specific for Gq/11{alpha} and immunoprecipitated overnight at 4 C. The cell extract was centrifuged gently, the supernate was discarded, and the beads were washed three times with 750 µl of cold RIPA buffer. Finally the beads were resuspended in SDS-PAGE sample buffer (reducing agents were omitted to prevent the hydrolysis of thioester-linked fatty acids) and heated at 100 C for 2 min. The immunoprecipitates were resolved by 10% SDS-PAGE, fixed, and prepared for fluorography with Fluoro-Hance (RPI, Mt. Prospect, IL). The gels were exposed to Kodak X-OMAT autoradiography film (Eastman Kodak, Rochester, NY) for approximately 30 days at -70 C. In parallel experiments, gels were treated with 1 M hydroxylamine (pH 7.0) after a 15-min fixing period, before fluorography and exposure to autoradiography film (22). Treatment with hydroxylamine cleaves the thioester bonds of palmitic acids to G proteins, and not the amide bonds of myristic acids, indicating palmitate labeling of G proteins as opposed to myristate labeling (22).

Transfection of Primary Pituitary Cell Cultures
Transfection of primary cell cultures was done in 24-well plates (Costar). Approximately 48 h after cell dispersion, cells were washed with M199 (pH 7.4), and 0.4 µg DNA mixed with 2 µl lipofectamine (GIBCO BRL, Gaithersburg, MD) in 0.25 ml of M199/BSA was added to each well in triplicate. After 5 h at 37 C, 0.25 ml M199 containing 20% horse serum and 5% FCS was added to each well. After 24 h from start of transfection, medium was removed and plates prepared for IP assays or LH RIA.

Measurement of IP Accumulation
After the transfection medium was removed, plates were washed twice with a balanced salt solution (BSS) containing 0.3% BSA to remove serum and unattached cells. Cellular inositol lipids were labeled with [3H]myo-inositol (specific activity 30–60 Ci/mmol, 4 µCi/ml; Dupont NEN) for 18 h. After inositol labeling, cells were washed twice with BSS containing 5 mM LiCl (BSS/LiCl) and stimulated for 2 h with the indicated GnRH concentrations prepared in BSS/LiCl. The treatment solutions were removed, and 1 ml of 0.1 M formic acid was added to each well. The cells were freeze-thawed once to disrupt the cell membranes, and the total 3H-labeled IPs were determined by Dowex anion exchange chromatography and liquid scintillation spectroscopy (23).

Quantification of LH Release
After the transfection medium was removed, cells were washed twice with M199/BSA and stimulated with the indicated GnRH concentrations for 2 h. The medium was collected from the culture wells. LH released was determined by RIA.

The RIA used a highly purified rat LH for iodination (24) and a reference preparation (RP3) obtained from the NIDDK (Baltimore, MD). LH antisera (C102) was prepared and characterized as previously described (25). Bound and free hormone were determined with immobilized protein A (26).

Western Blots
SDS-polyacrylamide gels (12% acrylamide) and Western transfers to nitrocellulose paper (Hoefer Scientific Instruments, San Francisco, CA) were performed as previously described (27). Polyclonal antisera (Ref. 28; a gift from Dr. Hitoshi Kurose, University of Tokyo, Tokyo, Japan) made against the third intracellular loop of the {alpha}2A-adrenergic receptor was used at 1:500 titer. Color was developed on Western blots using 4-chloro-1-napthol (horseradish peroxidase) color development reagent (Bio-Rad Laboratories, Richmond, CA). Standards were color-stained proteins (rainbow markers, Amersham) with the following molecular weights (including the dye): myosin (200K), phosphorylase (92.5K), BSA (69K), ovalbumin (46K), carbonic anhydrase (30K), trypsin inhibitor (21.5K), and lysozyme (14.3K).

Cell Culture and Transfection
GGH31' cells were derived from GH3 cells stably transfected with the rat GnRH receptor cDNA as previously reported (29). The GGH31' cells were maintained in an atmosphere of 5% CO2 at 37 C in DMEM (GIBCO, Grand Island, NY) containing 10% FCS and 20 µg/ml gentamicin. Cells were grown to confluency in 162-cm2 T flasks (Costar), then scraped and plated at a density of 350,000 cells per well in a 24-well culture plate for 36–40 h at 37 C in 5% CO2. Cells were washed twice in DMEM, 0.1% BSA, and 20 µg/ml gentamicin and treated with the indicated secretogogues for the indicated times.

Production of Polyclonal Gq/11{alpha} Antisera and Gq/11{alpha} RIA
Antisera were raised in rabbits using the C-terminal sequence ("CTS", QLNLKEYNLV) for the {alpha}-subunit of the Gq/11 family of guanyl nucleotide-linked proteins coupled to keyhole limpet hemocyanin. This same CTS was radioiodinated to serve as the immunoligand in the RIA. Unlabeled CTS was the standard; accordingly, a molar correction factor of 0.03 should be used to adjust the values obtained to account for the ratio of the mol wt of the standard (CTS) to that of Gq/11{alpha}. The sensitivity and lower limit of quantification values for this assay were <5 pg/tube at a final antiserum titer of 1:100,000. The RIA was set up by disproportionation for 12 h. The cellular Gq/11{alpha} proteins were measured after solubilization of the cells in 0.1% Triton X-100. Bound and free proteins were separated using the second antibody technique (26).

DNA Quantification
GGH31' cells were plated at 350,000 cells per well in a 24-well plate and maintained at 37 C, 5% CO2, for 36–40 h. Cells were treated with medium alone (control) or with the GnRH agonist, Buserelin (Hoescht-Roussel Pharmaceuticals, Somerville, NJ), 0.1 µg/ml for 0, 1, 2, 3, 4, 5, 6, and 24 h. The supernate was removed and the cells were frozen. The previously frozen cells were scraped, washed, and assayed in 0.44 N perchloric acid. The DNA content per well for each treatment and time point were assessed using the mini-diphenylamine assay (30).


    ACKNOWLEDGMENTS
 
We thank Linda Wolf for preparing the manuscript and Dr. Brian Hawes and Dr. Alfredo Ulloa-Aguirre for helpful suggestions.


    FOOTNOTES
 
Address requests for reprints to: Michael Conn, Oregon Regional Primate Research Center, 505 Northwest 185th Avenue, Beaverton, Oregon 97006.

This study was supported by NIH Grants HD-19899, HD-00163, and HD-18185

Received for publication January 31, 1997. Accepted for publication March 14, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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