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-Mediated Signaling in Xenopus laevis Oocytes by Signaling through Classical Steroid Receptors
Department of Internal Medicine (K.E., M.J., S.R.H.), Division of Endocrinology and Metabolism, Department of Pharmacology (K.E., M.J., S.R.H.), and Department of Obstetrics and Gynecology (B.B.), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-8857
Address all correspondence and requests for reprints to: Stephen R. Hammes, M.D., Ph.D., Department of Internal Medicine, Division of Endocrinology and Metabolism, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: stephen.hammes{at}utsouthwestern.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and other G proteins maintains oocytes in meiotic arrest. Steroids (androgens in vivo, and androgens and progesterone in vitro) overcome this inhibition to promote meiotic resumption. To test this model, we used G protein-regulated inward rectifying potassium channels (GIRKs) as markers of Gß activity. Overexpression of GIRKs 1 and 2 in Xenopus oocytes resulted in constitutive potassium influx, corroborating the presence of basal Gß signaling in resting oocytes. Testosterone and progesterone rapidly reduced potassium influx, validating that steroids attenuate Gß activity. Interestingly, reduction of classical androgen receptor (AR) expression by RNA interference abrogated testosterone’s effects on GIRK activity at low, but not high, steroid concentrations. Accordingly, androgens bound to the Xenopus progesterone receptor (PR) at high concentrations, suggesting that, in addition to the AR, the PR might mediate G protein signaling when androgens levels are elevated. In contrast, progesterone bound with high affinity to both the Xenopus PR and AR, indicating that progesterone might signal and promote maturation through both receptors, regardless of its concentration. In sum, these studies introduce a novel method for detecting nongenomic steroid effects on G proteins in live cells in real time, and demonstrate that cross talk may occur between steroids and their receptors during Xenopus oocyte maturation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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One well-established and biologically relevant model of nongenomic steroid signaling is Xenopus laevis oocyte maturation (5, 6, 7). Xenopus oocytes are held in meiotic arrest at prophase I until just before ovulation, when gonadotropins stimulate steroid production. Steroids, in turn, trigger oocytes to progress through the cell cycle until they arrest again at metaphase II of meiosis.
Progesterone has long been considered the physiological mediator of Xenopus oocyte maturation, perhaps signaling via classical Xenopus progesterone receptors (PRs) (5, 8, 9). However, evidence suggests that this model is incomplete. First, the classical PR antagonist RU486 does not inhibit progesterone-mediated oocyte maturation (8, 10). Second, elevation or reduction of PR expression in oocytes reveals only small changes in progesterone sensitivity (8, 9). Third, oocytes contain high cytochrome P450 (CYP)17 activity that converts progesterone to its equally potent androgen metabolite androstenedione (11, 12, 13); thus, incubating oocytes with progesterone is equivalent to adding two different ligands. Finally, both in vitro (14) and in vivo (12) measurements of gonadotropin-induced ovarian steroidogenesis reveal extremely low progesterone production but high testosterone secretion, suggesting that the more potent testosterone is the true physiological mediator of Xenopus oocyte maturation. In fact, testosterone appears to be promoting maturation, at least in part, through the classical androgen receptor (AR), as reduction of AR expression by RNA interference attenuates androgen-mediated oocyte maturation under conditions of low (
50 nM) testosterone concentrations (15). Interestingly, high testosterone concentrations (500 nM range) still trigger maturation in oocytes with reduced AR expression (15), suggesting that additional receptors are used to promote meiotic progression when steroid concentrations are elevated.
How do androgens and progesterone trigger Xenopus oocyte maturation? The current Release of Inhibition model proposes that constitutive G protein signaling in resting oocytes maintains meiotic arrest. Addition of steroids attenuates this inhibitory signal, allowing meiotic progression (7, 16, 17). Although both G
s and Gß contribute to maintaining meiotic arrest (7, 16, 17, 18, 19), Gß appears to be essential for this process. Overexpression or sequestration of Gß inhibits or enhances steroid-triggered oocyte maturation, respectively (16, 20), and stimulation of the Gß-coupled muscarinic 2 receptor (M2R) inhibits steroid-mediated maturation (15).
Although changes in Gß expression alter steroid-mediated maturation (16, 20), steroids themselves have not been shown to directly modify Gß-mediated signaling. To address this question and to test the release of inhibition model, we studied the effects of steroids on G protein-regulated inward rectifying potassium channels (GIRKs). GIRK1/GIRK2 heterodimers are extremely sensitive to changes in Gß activity, with elevated Gß signaling promoting increased GIRK activity, or potassium flux, at the plasma membrane (21, 22, 23, 24) (Fig. 1
). These channels can therefore be expressed in X. laevis oocytes and used as markers of Gß signaling.
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activity occurred in resting Xenopus oocytes, and that testosterone and progesterone rapidly reduced this baseline activity. Similar to earlier maturation studies, reduction of AR expression abrogated androgen-mediated inhibition of GIRK activity at low, but not high, agonist concentrations. Interestingly, androgen bound to the Xenopus AR at low concentrations, but bound to both the Xenopus AR and PR at high concentrations. In contrast, progesterone bound to both the Xenopus AR and PR with equally high affinities. Together, these results suggest that cross talk may occur between steroids and their receptors during nongenomic steroid-triggered processes. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. Basal inward potassium current was detected in unstimulated oocytes (Fig. 2, A and C), confirming that resting X. leavis oocytes contained constitutive Gß signaling. Notably, the general potassium channel blocker, barium chloride, completely inhibited this inward signal, verifying that the detected current was indeed due to potassium flux (data not shown). Addition of 100 nM testosterone led to a 30% decrease in potassium current at 5 min (Fig. 2D vs. 2C), whereas addition of the carrier 0.01% ethanol reduced the potassium current by only 5% (Fig. 2B vs. 2A).
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. Ethanol mildly suppressed GIRK activity in a linear time-dependent fashion throughout the 10 min, reaching a maximum of approximately 10% inhibition. In contrast, testosterone suppressed GIRK activity in a logarithmic pattern, with the greatest change in inhibition occurring by 5 min (30%). This very rapid effect of testosterone on GIRK activity supports the concept that testosterone is signaling in a nongenomic fashion in Xenopus oocytes.
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dependent; thus, any effects on GIRK-mediated potassium influx should be directly mediated by Gß. The specific sensitivity of GIRKS to Gß is well established (21). However, to confirm that testosterone was not simply suppressing all potassium currents in a nonspecific fashion, the constitutive potassium channel ROMK (renal outer medullary potassium channel) was overexpressed in Xenopus oocytes, and potassium currents were measured in the presence of ethanol or testosterone. Whereas testosterone inhibited potassium influx in oocytes from the same batch that were expressing GIRK1/2, the steroid had no effect on potassium influx in oocytes expressing ROMK (Fig. 3B
). This result confirmed that the androgen effects were specific to GIRK channels, and that Gß signaling was therefore being altered in response to testosterone.
If testosterone is indeed altering Gß signaling, then overstimulation of endogenous Gß should further increase GIRK activity and block the suppressive effects of testosterone. To test this hypothesis, the Gß-coupled muscarinic 2 receptor (M2R) was coexpressed with GIRK1/2 in oocytes, and potassium currents were measured. M2R expression was confirmed by Western blot (Fig. 3D). As expected, testosterone suppressed baseline GIRK activity by approximately 30% at 5 min. Similarly, as anticipated, the M2R agonist carbachol increased GIRK activity by approximately 30% at 2 min. Interestingly, subsequent addition of testosterone to the carbachol-treated oocytes did not suppress GIRK activity at 5 min, suggesting that the potent stimulatory effect of carbachol on Gß signaling through the M2R was preventing the suppressive effects of testosterone (Fig. 3C). The ability of M2R signaling to block androgen effects on GIRK activity mirrors previous studies where stimulation of the M2R blocked steroid-mediated maturation (15), supporting the hypothesis that interplay occurs between steroids, maturation, and Gß signaling in Xenopus oocytes.
The Pharmacology of Androgen-Mediated Suppression of Gß Signaling and Maturation Are Similar
If testosterone’s actions are mediated by a specific receptor, then the steroid’s effects on GIRK activity should be dose dependent and saturable. Accordingly, testosterone inhibited GIRK activity in a dose-dependent fashion, reaching a maximum inhibition of 30% at 100 nM, with an EC50 around 10 nM (Fig. 4A). Notably, testosterone concentrations higher than 100 nM had no greater inhibitory effects on GIRK activity (data not shown). Interestingly, the dose response for testosterone-mediated inhibition of GIRK activity was nearly identical to that of testosterone-triggered maturation of oocytes from the same batch on the same day, suggesting that testosterone may be regulating both processes via the same receptor (Fig. 4A, bottom).
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The Classical AR Is Necessary for Testosterone-Mediated Suppression Gß Signaling When Steroid Concentrations Are Low
Reduction of AR expression by RNA interference abrogates MAPK activation and oocyte maturation triggered by low concentrations of testosterone, suggesting that the classical AR mediates nongenomic signaling under these conditions (12, 15, 25). To determine whether the classical AR similarly regulates testosterone-mediated inhibition of GIRK activity, potassium currents were measured in oocytes where AR expression was reduced by RNA interference (15, 26). In oocytes injected with double-stranded AR cRNA, AR levels were lowered by approximately 60–70%, whereas expression of MNAR (modulator of nongenomic actions of steroid receptors), a nonspecific protein also involved in regulating Gß signaling (26), was unaffected (Fig. 5A). In accordance with maturation studies (15), low concentrations of testosterone (50 nM) were no longer sufficient to inhibit GIRK activity in oocytes with reduced AR expression when compared with mock-injected oocytes, whereas high concentrations of testosterone (500 nM) still suppressed GIRK activity (Fig. 5B). These results confirm that the classical AR is required to regulate nongenomic Gß signaling at low androgen concentrations, whereas additional receptors may also be involved in mediating nongenomic signaling when androgen levels are high.
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). Importantly, the Kd for testosterone binding to the AR was 0.8 nM, but was 170 nM for binding to the PR. These values may explain why the AR was required for 50 nM, but not 500 nM, testosterone to suppress Gß signaling and trigger oocyte maturation. Perhaps at higher concentrations, testosterone can bind to and signal via the Xenopus PR. As proof in principle that androgens can activate a signal via the Xenopus PR, the androgens testosterone and androstenedione were shown to indeed activate PR-mediated transcription, albeit at lower levels relative to progesterone (Fig. 6A).
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). Given that progesterone binds equally well to both the Xenopus PR and AR, and promotes AR-mediated transcription, progesterone may likewise mediate nongenomic AR-mediated signals. Notably, similar to testosterone, progesterone rapidly attenuated Gß-mediated GIRK activity in oocytes (Fig. 5C), perhaps via signaling through both the PR and AR.
RU486 Binds Poorly to the Xenopus PR
As mentioned, one of the conundrums of progesterone-mediated oocyte maturation is that the classical PR antagonist RU486 does not block progesterone-triggered maturation. This ineffectiveness of RU486 appears to be due to its extremely low affinity for the Xenopus PR (Table 1). Interestingly, the Xenopus PR contains a cysteine rather than glycine at residue 376 (Fig. 7A), and this cysteine substitution is known to decrease RU486 binding in the chicken and hamster PRs (3, 27). In contrast, the Xenopus AR contains a glycine at the equivalent position (579), likely explaining why RU486 bound with high affinity to the AR (Table 1). As would be expected, when this glycine in the AR is mutated to cysteine, the receptor’s affinity for RU486 is markedly reduced (data not shown).
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Blockade of Both the AR and PR Significantly Reduces Progesterone-Mediated Oocyte Maturation
As mentioned, a big problem studying progesterone-mediated oocyte maturation in vitro is that traditional PR antagonists have minimal inhibitory effects. Our binding studies may explain this problem because no one ligand sufficiently blocks both the AR and the PR. To address this issue, we attempted to block progesterone-mediated maturation using the high-affinity AR ligand R1881 and the high-affinity PR ligand RU5020 as competitive antagonists. Addition of R1881 and RU5020 together only promoted a small amount of oocyte maturation (Fig. 7B), indicating slight partial agonistic activity. R1881 slightly inhibited progesterone-mediated maturation, most likely due to specific blockade of progesterone and its metabolite, androstenedione, binding to the Xenopus AR. RU5020 inhibited progesterone-mediated maturation slightly more than R1881, likely because of its ability to bind tightly to the PR but still moderately to the AR. Finally, the combination of R1881 and RU5020 inhibited progesterone-mediated maturation by 50–60%, probably the result of significant blockade of both classical receptors (Fig. 7B). Notably, maturation could not be reduced to zero by the inhibitors, most likely due to the partial agonist qualities of R1881 and RU5020, as well as the extended incubation times used for a maturation assay (16 h).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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signaling in real time using intact cells. This system involves measuring currents in oocytes overexpressing the G protein-coupled inward-rectifying potassium channels GIRK1 and GIRK2, which have previously been characterized to be very specific and sensitive to Gß-mediated signaling (23, 24, 28). We have then used this system to test and confirm the validity of the release of inhibition hypothesis regarding steroid-mediated maturation of Xenopus oocytes.
First, we confirmed that resting oocytes contained constitutive GIRK activity. Earlier studies showed that this constitutive GIRK activity could be reduced by overexpression of proteins that sequester Gß and enhanced by overexpression of Gß (29). Similarly, we verified here that stimulation of the overexpressed Gß-linked G protein-coupled receptor M2R enhanced constitutive GIRK activity (Fig. 3). Together, these studies corroborate that GIRK activity is regulated tightly by Gß signaling in Xenopus oocytes, and that measurement of potassium current through GIRK1/2 is a sensitive and specific indicator of Gß signaling in our hands.
Second, we demonstrated that testosterone, the physiological mediator of Xenopus oocyte maturation, attenuated resting GIRK activity in a dose-dependent fashion (Fig. 3). Given that, as mentioned, GIRK activity is mediated almost exclusively by Gß, these androgen-induced changes are very likely to be due to specific alterations in Gß-mediated signaling. In support of this interpretation, increasing Gß signaling by stimulating the M2R with carbachol blocked the inhibitory effects of testosterone on GIRK activity (Fig. 3). Furthermore, testosterone had no effect on ROMK-mediated potassium currents, ruling out the possibility that the steroid was nonspecifically suppressing potassium influx (Fig. 3B).
Notably, each batch of Xenopus oocytes can have significantly different dose-response curves for testosterone-induced maturation. We found that the dose responses for testosterone-mediated GIRK inhibition and maturation matched nearly perfectly for every preparation tested (Fig. 4A and data not shown), suggesting that both testosterone-regulated events are part of the same process and are being mediated by the same receptor.
What, then, are the receptors that regulate testosterone- and progesterone-mediated inhibition of Gß signaling and eventual oocyte maturation? Based on previous work and on our functional and binding data, we propose the following model depicted in Fig. 7C. In vivo, androgens (mainly testosterone) are the primary regulators of oocyte maturation (12). In fact, inhibition of androgen production markedly reduces and delays gonadotropin-mediated oocyte maturation and ovulation, respectively (25). Testosterone mediates maturation primarily via its high-affinity interactions with the AR (thick line); however, when androgen levels are high enough, androgens may promote maturation via binding to the PR (thin dotted line). In contrast, addition of progesterone to oocytes in vitro is far more complicated. First, progesterone is partly metabolized to androstenedione, which binds to the Xenopus AR at low to moderate concentrations, but can cross-react with the PR at high levels. In addition, progesterone itself binds with equally high affinity to both the Xenopus PR and AR and even promotes significant transcription mediated by either receptor. Progesterone may therefore promote maturation via either receptor, regardless of whether it is metabolized to androstenedione.
This proposed model is compelling, because it reconciles our current data with the previous work described in the introduction. 1) Our data explain why RU486 does not block progesterone-mediated maturation by demonstrating that RU486 binds to the Xenopus PR with extremely low affinity, likely due to a cysteine to glycine substitution in position 376 of the ligand-binding domain. In contrast, RU486 binds with high affinity to the Xenopus AR, which contains the required glycine at the equivalent position 579. The ability of RU486 to bind with high affinity to the AR may explain why it is actually a weak agonist of oocyte maturation (10) when added in vitro. 2 and 3) Our data explain why reduction or overexpression of the Xenopus PR in oocytes has real but only small effects on progesterone-mediated maturation. In addition to CYP17-mediated conversion of progesterone to the androgen androstenedione, progesterone itself binds with high affinity to the Xenopus AR. Thus, reduction of PR expression lowers PR-, but spares AR-mediated maturation, resulting in only a partial reduction of progesterone-mediated signaling and maturation. 4) Our data confirm and explain why reduction of AR expression by RNA interference blocks androgen-mediated effects on Gß, MAPK signaling, and maturation only at steroid concentrations in the 50 nM range. At higher concentrations, androgens can bind to, and likely activate, the PR to regulate nongenomic signaling and maturation in oocytes.
Notably, RU5020 does not promote nongenomic steroid-triggered oocyte maturation, nor does it activate any other transcription-independent pathways in oocytes. However, RU5020 is known to activate nongenomic human PR-induced Src and MAPK signaling in somatic cells (30). One difference could be that Xenopus PR lacks the important PXXP motif that is found in the human PR and, in fact, is necessary for Src activation. In addition, Src is not activated by steroid in oocytes and does not appear to be important for steroid-triggered maturation (26). These differences highlight that classical steroid receptors can trigger multiple nongenomic signals, depending upon both the cell type and the receptor sequence/structure. Whereas Src activation may predominate in somatic cells, regulation of G protein signaling is most important in oocytes. In addition, classical steroid receptors can use multiple means of activating G proteins, including direct interactions (31), indirect interactions via the scaffold molecule striatin (32), or, as may be the case in Xenopus oocytes, indirectly via the MNAR protein (26).
As one final note, progesterone-mediated inhibition of Gß signaling could still be due to activation via a nonclassical steroid receptor, such as the recently described mPR (33). This possibility seems unlikely, however, as the progestin RU5020 does not bind to mPR (34), yet it partially blocks progesterone-mediated maturation in Xenopus oocytes. Furthermore, as mentioned, manipulation of Xenopus PR levels does, in fact, result in small but reproducible changes in progesterone-mediated maturation (8, 9), suggesting that this classical receptor plays at least a partial role in regulating nongenomic progesterone signaling. Finally, the evidence from RNA interference and pharmacology studies are relatively strong that the classical AR regulates androgen-mediated maturation at 10–100 nM concentrations of ligand; therefore, the likelihood that progesterone uses a completely different family of receptors to regulate oocyte maturation seems low. Notably, to date, no mPR isoforms have been shown to significantly bind to any androgen (33, 34).
In sum, these studies introduce a novel method for detecting rapid nongenomic steroid effects on G protein signaling at the membrane. Furthermore, these data demonstrate that considerable cross talk may occur between steroids and their receptors during Xenopus oocyte maturation, underscoring the importance of redefining the novel pharmacology of nongenomic vs. genomic steroid signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oocyte Preparation and RNA Injection
Stage V–VI oocytes were isolated from unprimed female X. laevis (Nasco, Fort Atkinson, WI) and treated as previously described using collagenase A (35, 36). For every preparation, dose-response curves with testosterone were performed to determine their steroid sensitivities (16).
Equal concentrations of the cRNAs encoding GIRK1 and GIRK2 were mixed, and approximately 25 ng of total cRNA was injected into each oocyte. For the studies using M2R, approximately 10 ng of cRNA encoding the HA-tagged M2R was injected into each oocyte.
For the AR RNA interference studies, cRNAs were generated that encoded the forward and reverse AR sequences. Equal amounts of the two RNAs were mixed together and heated to 90 C, followed by gradual cooling to room temperature. Then, 15 ng of the double-stranded RNA was injected into oocytes, followed 1 h later by injection of cRNAs encoding GIRKs 1 and 2. Assays were then performed 35–48 h later.
Two-Electrode Voltage-Clamp Recording
Oocytes were injected with cRNA for GIRK1/GIRK2 and/or M2R as indicated above. Two-electron voltage-clamp readings were taken 2 d after cRNA injections. Oocytes were bathed at 23 C in a solution of 96 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.5 by KOH). Current-voltage relationships (–100 to +100 mV, in 25-mV steps) were measured in oocytes as described (37) by two-electrode voltage clamp using an OC-725C oocyte clamp amplifier (Warner Instruments, Hamden, CT), pCLAMP7 software (Molecular Devices, Inc., Sunnyvale, CA), and a Digidata 1200A digitizer (Molecular Devices). The resistance of current and voltage microelectrodes (filled with a 3 M KCl solution) was 1–2 M
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After the initial readings, oocytes were treated with steroid or ethanol at the indicated concentrations for the indicated times, at which point the measurements were repeated. Ethanol concentrations were kept the same at 0.01%. For the experiments using oocytes expressing M2R, 30 µM carbachol (Sigma, St. Louis, MO) was added to oocytes followed shortly by the addition of testosterone.
M2R Expression
HA-tagged M2R expression was confirmed by Western blot using an anti-HA antibody (16). Oocytes were broken apart by pipetting with 20 µl/oocyte of lysis buffer (150 mM NaCl; 2 mM EDTA; 0.5 mM sodium vanadate; 2 mM NaF; 1% Triton X-100; 20 mM Tris, pH 7.6) containing 100 µg/ml phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 1 µg/ml pepstatin (Sigma) at 4 C. Lysates were microcentrifuged at full speed for 10 min, and the supernatant was removed and diluted 1:2 in 2x Laemmli sample buffer with 10% ß-mercaptoethanol (Sigma-Aldrich). Samples were separated on 10% polyacrylamide gels and transferred to Immobilon membranes (Millipore Corp., Billerica, MA). Membranes were blocked with 5% milk in TBST (100 mM NaCl; 0.1% Tween 20; 50 mM Tris, pH 7.4) for 1 h, incubated overnight at 4 C with (1:4000) rabbit anti-HA (Covance, Princeton, NJ), washed three times with TBST, incubated 1 h at room temperature with (1:4000) horseradish peroxidase-conjugated goat antirabbit antibody, and washed another three times with TBST. Blots were then treated with ECL-Plus (GE Healthcare, Piscataway, NJ) to visualize the proteins.
Steroid Binding Studies
Steroid binding studies were performed in COS cells as described previously (15). Progesterone and testosterone affinities were determined by direct binding assays using [1,2,6,7-3H(N)]testosterone or [1,2,6,7-3H(N)]progesterone (PerkinElmer, Boston, MA). RU5020, RU486, and R1881 binding affinities were determined by competition assay using 1 nM radiolabeled testosterone or progesterone with increasing concentrations of unlabeled test steroid. Kd values were then determined using Prism software (Graphpad Software, Inc., San Diego, CA).
Oocyte Maturation Assays
Steroid-mediated maturation assays were performed as described (12). For the inhibition assays, 1 µM RU5020 and/or 1 µM R1881 was added 30 min before the addition of the indicated concentrations of progesterone, and germinal vesicle breakdown was recorded 16 h later.
Transcription Assays
Transcription assays were performed as previously described (12, 15). Briefly, COS and CV1 cells were grown in complete medium consisting of DMEM (Fisher Scientific, Pittsburgh, PA), 10% bovine serum (Invitrogen, San Diego, CA), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen).
For the PR transcription assays, cells were transfected in 12-well plates using lipofectamine (Invitrogen). Cells were transfected with 0.8 µg Xenopus PR (9) in mammalian expression vector pcDNA3.1, 1.2 µg mouse mammary tumor virus-luciferase plasmid, and 5 ng of cytomegalovirus-ß-galactosidase plasmid. Cells were incubated with the mixture for 4 h and placed in complete medium with 5% charcoal-filtered serum and the indicated steroids. After 24 h, cells were incubated with the indicated steroid concentration and lysed after 16 h. Extracts were analyzed by using the Promega luciferase assay system (Promega Corp., Madison, WI) and PerkinElmer Galactostar kit.
For the AR transcription assays, CV1 cells were transfected by calcium phosphate precipitation with the murine mammary tumor virus (MMTV)-luciferase plasmid and pcDNA3.1 containing the Xenopus AR cDNA coding sequence (12). Cells were then incubated in complete medium containing 5% charcoal-stripped fetal bovine serum and the indicated steroids for 48 h, and luciferase and ß-galactosidase expression was measured as above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Author Disclosure Summary: Neither K.E., M.J., B.B., nor S.H. have anything to declare.
First Published Online October 4, 2006
Abbreviations: AR, Androgen receptor; CYP, cytochrome P450; GIRK, G protein-regulated inward rectifying potassium channel; HA, hemagglutinin; M2R, muscarinic 2 receptor; MMTV, murine mammary tumor virus; MNAR, modulator of nongenomic actions of steroid receptors; PR, progesterone receptor; ROMK, renal outer medullary potassium channel.
Received for publication July 24, 2006. Accepted for publication September 28, 2006.
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REFERENCES |
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(i). J Biol Chem 276:27071–27076 subunits inhibit nongenomic progesterone-induced signaling and maturation in Xenopus laevis oocytes. Evidence for a release of inhibition mechanism for cell cycle progression. J Biol Chem 275:41512–41520 and Gß in maintaining G2 arrest in Xenopus oocytes. J Cell Physiol 202:32–40[CrossRef][Medline] S subunit, a component of the plasma membrane and yolk platelet membranes. J Cell Biol 130:275–284 stimulated adenylyl cyclase is involved in Xenopus laevis oocyte maturation. J Cell Physiol 202:223–229[CrossRef][Medline] subunits. Curr Biol 11:405–416[CrossRef][Medline] subunits. Nature 370:143–146[CrossRef][Medline] subunits and function as heteromultimers. Proc Natl Acad Sci USA 92:6542–6546 subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel. J Gen Physiol 99:961–983 binds directly to the G protein-gated K+ channel, IKACh. J Biol Chem 270:29059–29062. Proc Natl Acad Sci USA 101:17126–17131NURSA Molecule Pages Link:
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