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a Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201
b Oregon Regional Primate Research Center, Beaverton, Oregon 97006
c University of Wyoming, Department of Molecular Biology, Laramie, Wyoming 82071
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Gs, and Gq/11 in a single cell type. To examine this possibility we measured palmitoylation of G proteins in response to GnRH receptor occupancy, since this event is a measure of G-protein activation by cognate receptors. GnRH stimulated time (0–120 min)- and dose (10-12–10-6 g/ml)-dependent palmitoylation of both Gi and Gs. Palmitoylation is G-protein activation dependent; accordingly, pertussis toxin (100 ng/ml; PTX), phorbol myristic acid (100 ng/ml), and Antide (50 nM; a GnRH antagonist) did not stimulate palmitoylation of Gi or Gs above basal levels. However, cholera toxin (5 µg/ml), an activator of Gs, stimulated palmitoylation of Gs but not Gi. We used a lactotrope-derived cell line expressing the GnRH receptor (GGH3) to examine whether the ability of the receptor to couple multiple G proteins is gonadotroph specific. GGH3 cells were transfected with specific cDNA coding for different G proteins, and agonist-stimulated second messenger production was assessed. Buserelin (a GnRH agonist) stimulated increased cAMP release in Gs cDNA-transfected GGH3 cells, whereas in Gi cDNA-transfected cells, both inositol phosphate (IP) production and cAMP release were decreased in response to buserelin. Transfection of Gq, G11, G14, and G15 cDNA into GGH3 cells resulted in an increased IP production in response to buserelin, indicating that GnRH receptor couples to this PTX-insensitive G-protein family. The observations presented in this study provide evidence for GnRH receptor coupling to multiple G proteins in a single cell type.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toxin studies in dispersed pituitary cell cultures have shown that G proteins are present in the gonadotroph and are coupled to effector systems. Pertussis toxin (PTX) pretreatment of rat pituitary cell cultures, which catalyzes the adenosine diphosphate ribosylation of the Gi family to prevent activation by receptors, inhibits GnRH-stimulated inositol phosphate (IP) production [2–4]. In addition, in human reproductive tract tumors, GnRH receptors couple to Gi proteins, indicating an intrinsic ability of the receptor to couple to this family of proteins [5]. However, in gonadotroph-derived cell lines (T3–1 cells) and in lactotrope-derived cell lines stably expressing the GnRH receptor (GGH3 cells), Gq/11 couples to the GnRH receptor [6, 7]. More recent evidence suggests that Gq/11 is palmitoylated in a dose- and time-dependent manner in rat pituitary cell cultures [8] and that it is redistributed in response to treatment with GnRH, providing more direct evidence for the activation of Gq/11 by the GnRH receptor [9]. In addition to Gi and Gq/11 coupling to the GnRH receptor, evidence from cholera toxin (CTX) pretreatment studies indicates that Gs may also be coupled to the GnRH receptor. Pretreatment of rat pituitary cell cultures with CTX (an activator of Gs) results in the movement of LH to a releasable pool, potentiating GnRH-stimulated LH release [10].
The coupling of 7-TMS receptors to different signal transduction systems could depend on several factors, such as receptor/G-protein affinity [11] and receptor density [12]. Depending on the type of cell, the concentration of G proteins, receptor density, compartmentalization of signal transduction machinery, and accessibility of G proteins to receptors may change, leading to unexpected interactions. Indeed, transfection of alpha 1D adrenergic receptor cDNA or neuropeptide Y receptor cDNA in different cell lines resulted in differential coupling to second messenger systems [13, 14]. Therefore GnRH receptor/G-protein coupling in primary pituitary cell cultures may be more indicative of physiological interactions than is such coupling measured in cell lines.
Agonist-stimulated second messenger production can be used to indicate which G proteins are involved in signal transduction. However, different G proteins can give rise to the same second messenger. For example, both Gi and Gq/11 proteins can stimulate IP production. Another complication in using second messengers to identify G proteins is that the second messenger production may be too low to be detectable. For example, in the rat pituitary, CTX (Gs activator) potentiates GnRH-stimulated LH release, although it is difficult even with the most sensitive RIA to detect GnRH-stimulated cAMP [10], resulting in an ambiguity about the involvement of Gs in GnRH receptor action. Therefore, it is important that a more direct method be used to investigate Gi and Gs coupling to GnRH receptor.
Previous studies performed to identify the G proteins coupled to receptors have utilized photoreactive guanosine triphosphate (GTP) analogues, [-32P]GTP azidoanilide [15], or ADP-ribosylation [16] of membrane G proteins. By necessity these studies were done in membrane preparations and not in intact cells. The protocols used to isolate membranes can disrupt membrane organization and produce an environment for receptor-G protein interactions that would not normally occur in an intact cell. Furthermore, the enzymatic steps used in membrane preparations may lead to partial receptor or G-protein degradation, which may result in a loss of specificity between receptor-G protein interactions. For example, in HEK 293 cells, a severely truncated parathyroid hormone receptor had differential signaling characteristics when compared with the full-length receptor [17], suggesting that cleavage of receptors can alter G-protein coupling specificity. In the present study, G proteins coupled to the GnRH receptor were identified by GnRH receptor-evoked palmitoylation of these proteins; this technique had minimal impact on the intracellular environment, as it did not rely on membrane preparations or transfected cell lines.
Palmitoylation of G-protein subunits, near the N-terminal Cys, through a thioester linkage is a dynamic process. G-protein activation results in palmitic acid turnover [18,19]. Except for G proteins of the transducin family, all known G proteins have an N-terminus consensus sequence for palmitoylation [18]. Receptor-evoked palmitoylation of G proteins is a well-characterized phenomenon that occurs in a time- and ligand dose-dependent manner [19–21]. G-protein palmitoylation may have physiological significance. For example, palmitoylation of G proteins appears to be required for membrane association [19] and furthermore has been shown to correlate with Gq/11 relocalization in response to GnRH in rat gonadotrophs [9]. These studies suggest that palmitoylation is integral to G-protein activation and that it can serve as an excellent marker for receptor-mediated G-protein activation.
This study was undertaken to identify the G proteins that are activated by GnRH receptor in the rat gonadotroph. As mentioned earlier, different cell types expressing the GnRH receptor have been shown to couple different kinds of G proteins. However, this is the first study of this kind to show multiple G-protein coupling to the GnRH receptor in the same cell type. In this study we used a method, selected because it minimally disrupted the intracellular environment of the gonadotroph, to label G proteins that are activated by the GnRH receptor. Taken together with previous findings [8], the present data show that the GnRH receptor couples to Gq/11, Gi, and Gs proteins in the rat gonadotroph.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Pituitary Cell Cultures
Pituitary cell cultures were prepared and maintained for 48 h prior to experiment as previously described [22].
Metabolic Labeling of G proteins with [9,10-3H]Palmitic Acid and Immunoprecipitation
Pituitary cell cultures were washed twice with Medium 199/BSA 2 h before labeling with [9,10-3H]palmitic acid (specific activity 30–60 Ci/mmol, 0.5 mCi/ml of Medium 199/BSA; DuPont NEN, Boston, MA) containing the indicated compounds for the indicated times as described before [8]. In parallel experiments, gels of resolved G proteins were treated with 1 M hydroxylamine (pH 7.0), after a 15-min fixative period, before fluorography and exposure to autoradiography film [23]. Treatment with hydroxylamine cleaves the thioester bonds of palmitic acids to G proteins, but not the amide bonds of myristic acids, indicating palmitate labeling of G proteins as opposed to myristate labeling [23]. Densitometric analyses were performed with NIH (Bethesda, MD) Image 1.47 software to obtain band density.
Cell Culture and Transfection
GGH31' cells were derived from GH3 cells stably transfected with the rat GnRH receptor cDNA as previously reported [24]. The GGH31' cells were maintained in an atmosphere of 5% CO2 at 37°C in Dulbecco's Modified Eagle's medium (DMEM; Gibco, Grand Island, NY) containing 10% fetal calf serum and 20 µg/ml gentamicin. Cells were grown to confluence in 162-cm2 or 75-cm2 T-flasks (Costar, Cambridge, MA), then scraped and plated at a density of 50 000–100 000 cells per well in a 24-well culture plate for 24 h at 37°C in 5% CO2. Cells were washed once in OPTI-MEM (Gibco) before transfection with the cDNA for specific G proteins or control plasmid. Approximately 24 h after seeding, cells were washed with OPTI-MEM (pH 7.4), and 0.4 µg of DNA mixed with 2 µl of lipofectamine (Gibco) in 0.25 ml of OPTI-MEM was added to wells in triplicate. After 5 h at 37°C, 0.25 ml of DMEM containing 20% fetal calf serum was added to each well. After 24 h from the start of transfection, transfection medium was replaced for another 24 h with DMEM/10% fetal calf serum/20 µg/ml gentamicin. Approximately 48 h after start of transfection, plates were washed twice with DMEM/0.1% BSA/20 µg/ml gentamicin before examination of buserelin-stimulated (GnRH agonist) IP, cAMP, or prolactin production.
Measurement of IP Accumulation
After plates were washed twice with DMEM/0.1% BSA/20 µg/ml gentamicin to remove serum and unattached cells, cellular inositol lipids were labeled in DMEM (inositol free) supplemented with [3H]myo-inositol (specific activity 30–60 Ci/mmol, 4 µCi/ml; Dupont NEN) for 18 h. Buserelin-stimulated inositol IP was measured as described previously [7, 25].
Measurement of Prolactin and cAMP Accumulation
After plates were washed twice with DMEM/0.1% BSA/20 µg/ml gentamicin to remove serum and unattached cells, prolactin and cAMP accumulation was measured by RIA [26–30] following a 24-h incubation in a 1-ml volume of DMEM/0.1% BSA/20 µg/ml gentamicin containing the indicated concentrations of Buserelin. To prevent degradation of cAMP by phosphodiesterases, 0.2 mM methyl isobutylxanthine was included in the medium. After stimulation of GGH3 cells, the samples were collected in tubes containing a final theophylline concentration of 1 mM. The samples were heated (95°C) for 5 min to destroy phosphodiesterase activity. There was no detectable effect on the prolactin RIA when the samples were heated in this fashion.
Production of Polyclonal Gi and Gs Antisera
Antisera were raised in rabbits, using the C-terminal amino acid sequence for the subunit of Gi2 and Gi3 (KENLKDCGLF) and the C-terminal amino acid sequence for the subunit of Gs (RMHLRQYELL), and coupled to keyhole limpet hemocyanin. The antisera were characterized by probing the immunoblots of cell extracts with previously characterized antisera (kindly provided to us by Dr. Allen Spiegel; NIH, Bethesda, MD), along with our own, and looking for comigrating stained bands. The Gi antiserum recognized one major band at an apparent molecular mass of 40 kDa. In immunoblots probed with the Gs antiserum, two major bands at 45 and 52 kDa, respectively, were detected. These bands corresponded to the short and the long form of Gs [31].
Expression Vectors
Complementary DNAs corresponding to G-protein subunits Gq, G11, G14, G15, and Gi were carried by the cytomegalovirus vector pCIS [32]. The Gs was in pCMV. A ß-galactosidase construct inserted into pCIS was used as a transfection control (lac z).
Statistical Analyses
The results are presented as the mean ± SEM of the indicated number of samples. Data were analyzed by one-way ANOVA, followed by Student's t-test with Bonferoni correction for multiple comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Gs in pituitary cell cultures is shown in Figures 1 and 2, respectively. Immunoprecipitation of Gi and Gs with antisera directed against the individual G proteins showed an increase in [3H]palmitate incorporation with GnRH treatment (Figs. 1A and 2A). The earliest detectable incorporation of the [3H]palmitic acid was measurable 20 min after the addition of GnRH. Shorter time intervals (5 and 10 min) did not show any difference from medium-treated values (data not shown). GnRH-stimulated incorporation of the label was detectable up to 120 min, the longest time examined. Gi and Gs proteins in Figures 1A and 2A, respectively, were detected in immunoblots with our antisera, and these bands comigrated with a standard antiserum obtained from Dr. A. Spiegel. Our own Gi antiserum and standard antiserum for Gi each recognized a comigrating band at an apparent molecular mass of 40 kDa. Two major bands at 45 and 52 kDa, respectively, were detected in immunoblots probed with our own Gs antiserum and standard antiserum for Gs. Figures 1B and 2B show the arbitrary optical density of the bands in the autoradiographs.
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In the presence of GnRH, palmitic acid incorporation into Gi and Gs was increased (Figs. 3 and 4, respectively). Pituitary cell cultures were treated with medium or 10-12, 10-10, 10-8, or 10-6 g/ml of GnRH in the presence of [3H]palmitate for 60 min, and [3H]palmitic acid incorporation was assayed by autoradiography as described in Materials and Methods (Figs. 3A and 4A). A concentration of approximately 10-9 g/ml GnRH produced a half-maximal incorporation of palmitate label to either Gi or Gs. Figures 3B and 4B show the arbitrary optical density of the bands in the autoradiographs.
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Rat pituitary cell cultures showed increased incorporation of the palmitate label above control levels in Gi and Gs when stimulated with GnRH (10-6 g/ml; Figs. 5 and 6, respectively). Figures 5A and 6A show the autoradiographs of Gi and Gs proteins, respectively. Only Gs showed increased incorporation of palmitate label when treated with CTX (5 µg/ml; Fig. 6). Rat pituitary cell cultures were incubated with several reagents for 60 min, and Gi and Gs were then immunoprecipitated as described above. Incubation with PTX (100 ng/ml), PMA (a protein kinase C activator; 100 ng/ml), or Antide (GnRH antagonist; 50 nM) did not result in an increased palmitoylation of Gi or Gs compared to medium-treated levels (Figs. 5 and 6, respectively). Figures 5B and 6B show the arbitrary optical density of the bands in the autoradiographs.
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Treatment with 1 M hydroxylamine solubilized all measurable radioactivity from gels, indicating an alkali-sensitive characteristic of a thioester-linked palmitate moiety (not shown).
We used lactotrope-derived GGH3 cells to examine whether the ability of the receptor to couple multiple G proteins is gonadotroph specific. GGH3 cells were transfected with specific cDNA coding for different G proteins, and agonist-stimulated second messenger production was assessed. Figure 7 shows Buserelin dose-dependent stimulation of IP production in GGH3 cells transfected with the cDNA of the Gq/11 family (Gq, G11, G14, and G15), Gi2, and Gs. GGH3 cells were transiently transfected as described in Materials and Methods and stimulated for 2 h with the indicated concentrations of Buserelin. Stimulation of cells with Buserelin resulted in an enhanced IP production compared to that in lac z-transfected cells (control plasmid [32]). However, in cells transfected with the Gi2 cDNA, Buserelin-stimulated IP production was decreased from control levels. As expected, cells transfected with cDNA for Gs resulted in no significant change in Buserelin-stimulated IP production over that in lac z-transfected cells (Fig. 7). Figures 8 and 9 show the effect of transfection of indicated G-protein cDNA on Buserelin-stimulated prolactin and cAMP release, respectively. Transfections were performed as described in Materials and Methods, and the cells were stimulated with the indicated concentrations of Buserelin for 24 h before assay for the released prolactin and cAMP by RIA. Transient transfection of the cDNA for Gq/11 family, Gi2, and Gs did not show a significant change in Buserelin-stimulated prolactin release in these cells (Fig. 8) compared to that in lac z-transfected cells. Only Gs cDNA-transfected cells showed an increased level of cAMP production compared to the level in lac z-transfected cells when stimulated with the indicated doses of Buserelin (Fig. 9). Gi2 cDNA-transfected cells showed a decrease in Buserelin-stimulated cAMP production compared to the control lac z-transfected cells; however, transfection of cDNA from the Gq/11 family resulted in no significant change in the Buserelin-stimulated cAMP production when compared to that in control cells (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Gs in rat pituitary cell cultures. The data demonstrate that GnRH receptor occupancy by the releasing hormone results in a time- and dose-dependent palmitoylation of Gi and Gs. Antide, a GnRH receptor antagonist that binds but does not activate the receptor, did not measurably stimulate palmitoylation above background levels. Increasing the GnRH concentration resulted in a dose-dependent increase in palmitoylation of Gi and Gs. CTX, an activator of Gs, stimulated palmitoylation of Gs in the absence of GnRH. These observations taken together with previous work [8] suggest that the GnRH receptor couples to Gi, Gs, and Gq/11 in the rat gonadotroph.
We have used a measure of G-protein activation, i.e., palmitoylation, to identify regulation of Gi and Gs by the GnRH receptor in the rat pituitary. Palmitic acid turnover on G proteins occurs when the protein is activated by a receptor or by a pharmacological agent. Therefore, regulation of palmitic acid turnover on Gi and Gs when primary pituitary cell cultures are stimulated by GnRH suggests that the GnRH receptor couples to these proteins. The observation that CTX-evoked palmitoylation was seen in Gs and not in Gi suggests that palmitoylation of G proteins is activation dependent and not a nonspecific event.
The ability of the GnRH receptor to couple multiple G proteins is not specific to the gonadotroph. In this study we show that overexpression of G proteins in GGH3 cells results in changes in the cognate signal transduction cascades. This suggests the ability of the GnRH receptor to couple to the respective G proteins. Previous studies have shown that in GGH3 cells, GnRH receptor activation results in dose-dependent production of prolactin, cAMP, and IP [7,26]. Transfection with Gs cDNA resulted in an increased Buserelin (GnRH agonist)-evoked cAMP production. An increase in the free Gs proteins would enable more of these proteins to be activated by the GnRH receptor, thereby increasing the production of cAMP. Conversely, transfection of Gi inhibited Buserelin-stimulated cAMP. This indicates that GnRH receptor occupancy results in the activation of Gi to inhibit cAMP production by the adenylate cyclase enzyme. Transfection of Gi cDNA resulted in a decrease in GnRH-evoked IP turnover. G proteins of the Gi family work through the associated ß
subunits to activate phospholipase Cß (PLCß) and to produce IPs and diacylglycerols [33]. Transfecting with Gi cDNA presumably increased levels of this protein, which in turn would compete with PLCß for the ß subunits, resulting in a decrease in IP production. These observations suggest that GnRH receptor is able to couple Gs to produce an increase in GnRH-evoked cAMP production in GGH3 cells, but also to couple Gi to inhibit adenylate cyclase to produce a decrease in cAMP levels in GGH3 cells.
In this study we were able to lend support to the view that the GnRH receptor couples to Gq/11 by showing that the GnRH receptor was able to activate PLCß through all the known members of the Gq/11 family. The subunit of these G proteins activates PLCß directly when the trimeric G protein is activated by a receptor [34]. Buserelin stimulation of cells transfected with the cDNA of Gq, G11, G14, and G15 resulted in an increased IP production over that in control cells.
In GGH3 cells, GnRH-evoked prolactin release is mediated through a cAMP-dependent signal transduction pathway [26]. However, we did not observe an increase in Buserelin-evoked prolactin release in cells transfected with Gs cDNA, even though Buserelin-evoked cAMP levels were higher than in control cells. Moreover, in Gi-transfected cells, although the Buserelin-stimulated cAMP levels were below control levels, there was no significant difference in the prolactin level. The lack of change seen with Buserelin-stimulated prolactin release may be due to the fact that the amount of cAMP needed to stimulate prolactin release is low. Therefore an increase or decrease in cAMP seen in transiently transfected cells may still be above the levels of the second messenger needed to stimulate prolactin release.
Overexpression of G proteins in the transfected cells was not clearly detectable in immunoblots. This is due to the fact that when the maximum attainable transfection rate is 30% of the total cell population, overexpression of G proteins in transfected cells is technically hard to detect. However, the expression of these plasmids was verified in a previous publication [32].
Receptor-stimulated palmitoylation of G proteins is a convenient in vivo method to identify G-protein activation. However, due to low specific activity of 3H, the exposure time for autoradiographs is prolonged. The long exposure times result in high and varied background exposure levels. For these reasons, and also because of experimental artifacts arising from cell culture preparations, differences in labeling medium, and differences in other experimental procedures, combining data from different experiments was not appropriate.
The studies in the GGH3 cells were undertaken to verify observations in the gonadotroph and to examine whether the ability of the GnRH receptor to couple multiple G proteins is restricted to the gonadotroph. GGH3 cells were an ideal model because the progenitor cell line, GH3, does not express GnRH receptor endogenously, and these cells have been characterized extensively [7, 24, 26]. This enabled us to examine whether the ability to couple multiple G proteins is receptor specific or gonadotroph specific. The answer to this question would explain the different observations investigators have seen with different cell lines with respect to GnRH receptor/G-protein coupling. Our paradigm used one cell type to show GnRH receptor coupling to Gi, Gs, and Gq/11. Moreover, we provide evidence to show that this ability is not gonadotroph specific but is more a receptor-specific ability. The gonadotroph may control the availability of G proteins to the receptor, but as far as the GnRH receptor is concerned, it has the ability to couple to multiple G proteins. The ability to couple multiple G proteins enables the GnRH receptor to activate multiple signal transduction pathways. The activation of multiple signal transduction pathways with different endpoints may be useful in order for GnRH to stimulate multiple responses from the gonadotroph, such as LH and FSH release.
The ability of 7-TMS receptors to couple multiple G proteins is documented for other members of this family. The human thyrotropin receptor is known to couple to all four classes of G proteins, i.e., Gi, Gs, Gq/11, and G12 [35], and the 2A-adrenergic receptor couples to Gi and to Gs [16]. This is the first study of this nature to identify the G proteins that are activated by the GnRH receptor in its native environment and to show multiple G-protein coupling to the GnRH receptor in a single cell type.
The mechanism by which multiple G proteins interact with the GnRH receptor is unknown. However, the second and the third intracellular loops appear to be involved in signal transduction [8, 36], suggesting that multiple sites on the receptor may interact with G proteins. Another possibility is that receptors in different cells may interact with different G proteins; but cell-by-cell heterogeneity in the same cell population, with respect to GnRH receptor G-protein coupling, has not been identified. These studies do not indicate whether the GnRH receptor is able to couple to multiple G proteins simultaneously or couple each protein one at a time. The propensity of the GnRH receptor to couple multiple G proteins may explain how the activation of the GnRH receptor by the releasing hormone can regulate multiple cellular events in a coordinate fashion.
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FOOTNOTES |
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2 Correspondence. FAX: (503) 690–5569; connm{at}ohsu.edu
Accepted: April 21, 1998.
Received: March 19, 1998.
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R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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