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Section of Nephrology, Yale University School of Medicine, PO Box 208029, New Haven, Connecticut 06520–8029, USA
1 Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030–3498, USA
(Requests for offprints should be addressed to D S Geller; Email: david.geller{at}yale.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The availability of high-resolution, three-dimensional structural information for the LBD of all the five steroid hormone receptors has provided a detailed mechanistic understanding of the steroid receptor agonism and antagonism (Brzozowski et al. 1997, Williams & Sigler 1998, Matias et al. 2000, Bledsoe et al. 2002, 2005, Kauppi et al. 2003, Fagart et al. 2005, Li et al. 2005). The LBD consists essentially of 12 
-helices that surround a central hydrophobic ligand-binding pocket. Upon ligand binding, helix 12 rotates towards the rest of the molecule and, together with helices 3 (H3) and 5 (H5), forms a pocket where transcriptional co-activators can bind (Beato et al. 1995). We became interested in the interactions among these helices in trying to understand the mechanism by which a mutation in the mineralocorticoid receptor (MR) causes severe hypertension exacerbated by pregnancy in humans (Geller et al. 2000). The mutation, a substitution of leucine for S810 human mineralocorticoid receptor (hMRS810L), alters the specificity of the receptor, allowing progesterone and other mineralocorticoid antagonists to function as agonists (Geller et al. 2000). To determine the structural basis for the altered specificity, we created an atomic model of the MR ligand-binding pocket based on its homology with the previously determined crystal structure of the progesterone receptor (PR) LBD (Williams & Sigler 1998). We found that L810 is in close proximity to A773 on H3, and hypothesized that a novel van der Waals (vdW) interaction between L810 and A773 allowed progesterone-mediated activation of hMRS810L (Geller et al. 2000). Crystal structures of MR have yielded support for this model (Bledsoe et al. 2005), although L810 may interact with other H3 residues in addition to A773 (Bledsoe et al. 2005, Fagart et al. 2005).
The crystal structures of the LBD of PR (Williams & Sigler 1998) and of estrogen receptor (ER) (Li et al. 2005), in the presence of ligand show that the homologous residues (hPR:G722 and M759; hER:A350 and L387) are in contact with vdW constant. Furthermore, these residues are frequently co-conserved in the nuclear receptor (NR) family. Receptors containing a leucine or isoleucine at the H5 position generally have an alanine or serine at the H3 position, while receptors with a methionine on H5 have a glycine at the H3 position (Zhang et al. 2005). The wide conservation of this interaction suggested a possible functional role. Hence, we investigated the activity of a number of human glucocorticoid receptor (hGR) and hPR mutants with substitutions at these residues. Intriguingly, alteration of these residues produced a gain-of-function mutation in all four steroid hormone receptors thus far tested, suggesting a critical role of these residues for the specificity and sensitivity of steroid hormone receptors (Geller et al. 2000, Chen et al. 2001, Zhang et al. 2005). Taken together, these data suggest a critical role of the H3–H5 interaction as a ‘molecular switch’ that regulates the activity and specificity of steroid hormone receptors and perhaps other nuclear receptors.
The importance of this H3–H5 interaction with steroid hormone receptor activity is further supported by the finding that the H3 residues we have identified are critical to the action of receptor antagonists in steroid hormone receptors. In the androgen receptor (AR), alteration of G708 to alanine abolishes the partial agonist activity of steroidal anti-androgens (Terouanne et al. 2003). Similarly, synthetic C-11 substituted spirolactones that normally inhibit MR are potent agonists of an hMRA773G mutant (Auzou et al. 2000). hPRG722A or hPRG722C mutants have normal receptor activity, but are completely insensitive to the inhibitory action of RU486 (Benhamou et al. 1992, Lim-Tio et al. 1996), while the corresponding hGRG567A mutant has been reported to fail to bind RU486 (Warriar et al. 1994). As glucocorticoid receptor (GR), PR, and AR, which have a glycine at the H3 position, are sensitive to RU486, while MR possesses an alanine at this position and is insensitive to RU486, it has been proposed that only glycine at this position is permissive for RU486 binding, suggesting that the side chains of other amino acids might sterically interfere with the 11-dimethylaminophenyl group/moiety of RU486 (Warriar et al. 1994, Kauppi et al. 2003).
These data imply a major importance of the H3 residue involved in an H3–H5 interaction to steroid hormone receptor antagonist binding. Because of the proximity of H5–H3 at this site, we propose that a H5 interaction with either the ligand (RU486) or the H3 residue might play a role in the activity of receptor antagonists as well. To better understand the amino acid requirements of RU486-mediated inhibition of these receptors, we screened a variety of hPR, hGR, and hMR mutants with mutations at the respective H3 and H5 residues for their response to RU486.
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Materials and methods |
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Cos-7 cells were provided by ATCC (Manassas, VA, USA). Cell culture media and sera were purchased from GIBCO/Life Technologies. All chemicals were from Sigma-Aldrich unless otherwise stated. All steroids (progesterone, dexamathasone, and RU486) were obtained from Sigma. 3H dexamethasone for receptor binding was from Dupont-New England Nuclear (Boston, MA, USA). DNA purification kits for the preparation of supercoiled plasmids used in transfection were from Qiagen. The luciferase assay system was obtained from Promega and Quikchange Site-Directed Mutagenesis Kit was obtained from Stratagene Co. (La Jolla, CA, USA).
Construction of hPR, hGR, and hMR mutants
The human plasmid pRShGRNX (Rupprecht et al. 1993) was the kind gift of Dr Ronald M Evans. It contains the full-length coding region on human GR and constitutively active Rous Sarcoma Virus promoter. The PR expression plasmid for the full-length human PR isoform B has been described previously (Vegeto et al. 1992). Mutants of GR and PR were constructed with a Quikchange Site-Directed Mutagenesis Kit (Stratagene Co.) using the wild-type pRShGRNX plasmid or the wild-type hPR-B plasmid as a template. All primers used for mutations are listed in Table 1
. PCR conditions were as described previously (Geller 2001). Plasmid DNA was purified, and each resulting mutant receptor gene was sequenced in its entirety to ensure that no undesired mutations were introduced. The plasmid pMTV-Luc (Rupprecht et al. 1993) encoded luciferase, which was under the control of the GR-sensitive mouse mammary tumor virus (MMTV) promoter and was used for assays of GR activity. The progesterone response element (PRE)-luciferase reporter used in this work has been described previously; it contains two copies of the consensus PRE linked to the TATA element from E1b (Nawaz et al. 1999). pSV2 (Promega) encodes ß-galactosidase (ß-gal) from the SV40 early promoter.
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Cos-7 cells were grown in Dulbecco’s minimal essential medium (DMEM: Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin under an atmosphere of 5% CO2 at 37 °C. On the night prior to transfection, cells were seeded on six-well tissue culture plates (2x 105 cells/well). For GR and MR transfections, cells in each well were transfected with 1 µg pMTV-Luc, 1 µg receptor plasmid, and 5 ng pSV2. For PR transfections, cells were transfected with 500 ng PR receptor plasmid, 1 µg PRE-Luc, and 10 ng pSV2. Transfection was performed using cationic liposomes (Lipofectamine-2000; Life Technologies). Following transfection, cells were incubated in DMEM, supplemented with 10% fetal calf serum overnight. Cells were washed with PBS, and then serum-free DMEM containing the test steroid was added. All steroids were dissolved in 100% ethanol, and added to media at 1:1000 dilution so that the total ethanol concentration was never higher than 0.1%. The cells were incubated for an additional 16–24 h prior to assay.
Luciferase reporter assays
Luciferase and ß-gal activities were measured as described previously (Geller et al. 2000). The cells were washed with PBS, and then lysed by incubation for 20 min at room temperature in 300 µl reporter lysis buffer. Lysed cells were scraped into 1.5 ml microcentrifuge tubes and shaken for 2 min. After brief centrifugation at 13 000 g to remove cell debris, 10 µl supernatant was transferred into 12x75 mm plastic tubes (BD Bioscience Franklin Lakes, NJ, USA). The reaction was initiated by rapid addition of 100 µl luciferase assay reagent and light output was integrated over 10 s at room temperature using an Opticomp I photon-counting luminometer (MGM Instruments, Inc., CT, USA). Luciferase activity was normalized to ß-gal activity to correct for transfection efficiency and is expressed as a percentage of the wild-type receptor activity at 10 nM dexamethasone (GR) or 10 nM progesterone (PR). All results are the mean of at least nine independent transfections.
Receptor-binding studies
Cos-7 cells, 1x106 were transfected in 100 mm plates with 5 µg receptor plasmid using Lipofectamine 2000 (Life Technologies). On the day after transfection, serum-free media were substituted and the cells were grown for an additional 24 h. Cells were harvested in 40 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA and lysed by freeze–thaw treatment in hypotonic buffer containing 10 mM Tris–HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA, 10 mM Na2MoO4, 5 mM dithio-threitol (DTT), antipain (5 µg/ml), leupeptin (5 µg/ml), chymostatin (5 µg/ml), pepstatin A (5 µg/ml), and 500 µM phenylmethylsulphonyl fluoride. After centrifugation at 15 000 g for 15 min, extracts were adjusted to 100 mM NaCl and 5% glycerol (binding buffer). Extracts were incubated overnight with [3H]dexamethasone (New England Nuclear) and competitor steroid (Sigma) at 0 °C in a total volume of 200 µl, and then incubated with 100 µl of a 50% slurry of hydroxyapatite in binding buffer. Samples were spun, washed twice in binding buffer, then resuspended in ethanol and prepared for scintillation counting. The value of 100% binding was determined by subtracting the number of counts per minute bound in the presence of 500-fold excess of unlabeled dexamethasone from the counts bound in the absence of competitor. Non-specific binding was determined with a 500-fold excess of unlabeled dexamethasone. No specific binding was seen in mock-transfected cells.
Data analysis
All data are represented as means ± S.E.M. and were subjected to statistical analysis by two-way ANOVA with GraphPad Prism 3.0 software (GraphPad Software, Inc., San Diego, CA, USA). All experiments represent the mean of at least eight independent experiments. The level of statistical significance was set at P<0.05.
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Results |
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We first tested the response of PR receptor mutants to RU486. To do so, we generated a PR mutant in which we changed alanine to glycine on H3 (hPRG722A). As previously reported, (Lim-Tio et al. 1996, Zhang et al. 2005), hPRG722A has no activity in the absence of ligand and has activity identical to hPRWT (WT, wild type) in the presence of progesterone but is completely insensitive to RU486, even at 1 µM concentrations (Fig. 1A and data not shown). This is thought to be due to an inability of the A722 to accommodate the bulky 11-dimethylamino-phenyl functional group of RU486, perhaps as the result of steric hindrance (Benhamou et al. 1992), and illustrates the importance of this residue for RU486 binding to PR. As M759 is in vdW contact with G722 (Williams & Sigler 1998), we asked whether alteration of residues at position 759 would affect RU486 binding. We first tested derivatives of hPRG722A, altering the H5 residue from methionine to leucine (hPRM759L/G722A) or alanine (hPRM759A/G722A). We showed previously that these receptors have some constitutive activity and are further activated by progesterone (Zhang et al. 2005). We found that all receptors bearing a G722A mutation were insensitive to RU486-mediated inhibition in our transcriptional assay, regardless of the H5 residue (Fig. 1A). Transcriptional activity of receptors bearing isoleucine (hPRM759I) or valine (hPRM759V) at position 759 in conjunction with the G722A mutation were also not inhibited by RU486 (data not shown). Hence, all hPR mutants containing the G722A mutation were uniformly insensitive to RU486-mediated inhibition, regardless of the H5 residue.
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). Intriguingly, we found that hPRM759A is insensitive to RU486, requiring a 100-fold higher RU486 concentration for receptor inhibition than hPRWT (Fig. 1B). As the alanine side chain is much smaller than that of leucine or methionine, this result cannot be explained by steric interference with the 11-dimethylaminophenyl group of RU486. Instead, it rather suggests that binding of RU486 to PR requires a vdW interaction with the side chain of the critical H5 residue. However, as both hPRM759A and hPRM759L have constitutive activity, which is possibly due to activation by an unknown cellular sterol (Zhang et al. 2005), we could not rule out the resistance of these receptors to RU486 due to the competition with this sterol.
To clarify this result, we created two additional PR mutants containing either serine (M759S) or glycine (M759G) on H5. The progesterone-mediated activity of hPRM759S was indistinguishable to that of hPRWT, and the addition of the G722A substitution caused only a minor decrease in receptor activity in the presence of 1 and 10 nM progesterone (Fig. 2A). In contrast, hPRM759G had high levels of constitutive activity and was further activated by progesterone (Fig. 2B); addition of the G722A mutation to this receptor caused significant reductions in receptor activity compared to hPRM759G, but it was still significantly more active than hPRWT (Fig. 2B).
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) or 1000-fold (hPRM759G, Fig. 2D) higher concentrations of RU486 than hPRWT for equivalent inhibition. We found that similar levels of RU486 were required to inhibit the constitutive activity of hPRM759G (Fig. 2E). Importantly, RU486 did not stimulate transcription in any of these receptors in the absence of progesterone (data not shown), indicating that the effect we are observing is not due to RU486-mediated agonism of these receptors. These data support the proposal that RU486-mediated inhibition of PR is dependent on a constructive interaction between the side chain of residue 759 on H5 and RU486. Moreover, as hPRM759S has no constitutive activity and yet remains resistant to RU486, these data rule out the possibility that competition with an unidentified cellular sterol plays a role in the resistance of H5-altered receptors to RU486. H3 substitution does not affect RU486 inhibition of GR
Our results with PR suggest that both H3 and H5 residues play an important role in RU486-mediated inhibition. To better understand the effect of H3 and H5 substitutions on antagonist activity, we examined whether mutations at these positions would also affect GR sensitivity to RU486. We first tested hGRG567A, a GR mutant that has been reported previously to be inactive in the presence of dexamethasone (Warriar et al. 1994). To our surprise, we found that hGRG567A possessed significant transcriptional activity in the presence of dexamethasone and cortisol, albeit somewhat reduced from that of hGRWT (Fig. 3A and data not shown). In light of the previous reports on the inactivity of this receptor mutant (Warriar et al. 1994), we repeated our activity assay for hGRG567A using a freshly isolated and sequenced receptor plasmid construct, and we obtained identical results. Our attempt to measure the binding affinity of hGRG567A for dexamethasone, however, was unsuccessful, as it was below the level of detection for our assay (see Materials and methods, data not shown).
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, data not shown). This stands in sharp contrast to hPR, in which the G722A mutation renders hPR entirely resistant to RU486. H5 residues impact GR sensitivity to RU486
Since the H3 substitution did not have any effect on the activity of RU486 on hGR antagonism, we asked whether H5 mutations would affect the sensitivity of hGR to RU486. Therefore we tested a variety of GR constructs with substitutions at residue 604 for the effect on RU486 sensitivity. hGRM604L has increased affinity and activity in the presence of dexamethasone when compared with hGRWT, which is thought to be due to a novel H3–H5 interaction between the leucine side chain and the carbonyl group of G567 (Zhang et al. 2005). We found that RU486 is a potent inhibitor of hGRM604L, suggesting that the leucine side chain does not interfere with RU486 binding (Fig. 4A). Consistent with our results from Fig. 3, addition of the G567A mutation to this construct did not inhibit RU486 inhibition of this receptor.
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). This result is consistent with our findings of the analogous mutant in PR. Once again, we found that addition of the G567A mutant had no effect on RU486-mediated inhibition of this receptor (Fig. 4B).
As with PR, we suspected that the decreased activity of RU486 on hGRM604A might be due to the small side chain of alanine. To verify this, we created mutant hGRs bearing substitutions of either glycine or serine at position 604 on H5. hGRM604G and hGRM604S were activated by similar concentrations of dexamethasone as hGRWT (Fig. 4C), but these receptors, like hGRM604A, were resistant to RU486, requiring 10- to 100-fold higher RU486 concentrations than hGRWT for maximal inhibition (Fig. 4D). RU486 did not stimulate significant transcriptional activity from any of these receptors in the absence of dexamethasone (data not shown), indicating that these results are not due to RU486-mediated agonism of the mutant receptors. As with hPR, these data suggest that RU486-mediated inhibition of hGR is dependent on the side chain length of residue 604. This raises the possibility whether a vdW interaction between RU486 and the side chain of the H5 residue is necessary for efficient RU486 binding.
MR insensitivity to RU486-mediated inhibition is not mediated by the H3 alanine
Unlike GR, PR, and AR, MR has an alanine in place of glycine at the critical H3 residue that we found to regulate RU486 sensitivity in PR. Based on this lack of sequence conservation, Benhamou et al.(1992), proposed that the insensitivity of MR to RU486 is caused by the presence of alanine at this critical position (Benhamou et al. 1992). Our data obtained with GR suggest that the interaction of RU486 with the steroid hormone receptor LBD is more complex than this, and to this end, we tested Benhamou’s proposal directly via analysis of an A773G MR mutant (hMRA773G) that, according to Benhamou’s proposal, should be able to bind RU486. Consistent with previous reports (Pinon et al. 2004), we found that hMRA773G is insensitive to the action of RU486 (Fig. 5). We found that hMRA773G showed only minor sensitivity to the antagonistic action of RU486 at the highest RU486 concentrations (0.1–1 µM) we tested (Fig. 5). Again, it should be noted that we observed no agonistic activities of RU486 on either of these receptors at any concentration tested (data not shown), suggesting that our inability to detect receptor antagonism was not caused by an agonistic effect of the ligand. These data suggest that the insensitivity of hMRWT to RU486 cannot be solely due to the presence of A773 on H3, but must be due to other structural differences between the LBD of MR and GR.
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Discussion |
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This study was performed to better delineate the molecular mechanism responsible for steroid hormone receptor antagonism. Previous studies on PR mutants led to the suggestion that RU486-mediated inhibition of steroid hormone receptors is made possible by the presence of glycine at a critical H3 residue. This suggestion was based on the finding that substitution of alanine (or cysteine) for G722 in PR resulted in a receptor that was entirely resistant to RU486, perhaps due to an inability to accommodate the bulky 11-dimethylaminophenyl group of RU486 within the PR’s ligand binding pocket (Benhamou et al. 1992, Lim-Tio et al. 1996). In contrast to previous studies, however, we found that GR, containing a G567A single or double mutation, is transcriptionally active, albeit with reduced activity when compared with hGRWT. Furthermore, this hGRG567A mutant is as sensitive as hGRWT to the action of RU486. Unlike PR, however, we found that the inhibition of hGR by RU486 is not affected by a G567A mutation. This suggests that the underlying stereochemistry by which RU486 binds to PR and GR is different, and that the ligand-binding pocket of GR is able to adopt a different structure to accommodate the additional moiety on RU486, which is not possible in PR. Taken together, our findings suggest a potential mechanism to alter RU486 in order to create a more specific glucocorticoid antagonist.
At first glance, it is surprising that the hGRG567A mutation is permissive for RU486 binding and activity as the crystal structure of the GR-LBD in association with RU486 does not show space for an alanine in this position. However, it must be remembered that an F602S mutation was introduced into GR toincrease receptorstabilityinthe published crystal structures (Bledsoe et al. 2002, Kauppi et al. 2003). Given the close proximity of the F602S mutation to M604 on H5 (and therefore to G567 on H3), our data raise the possibility that the F602S substitution may have altered the position of H3 withrespect to RU486. Consistent with thisnotion, Kauppi et al.(2003) noted that the F602S mutation causes a series of side chain movements in the vicinity of residue 602 with respect tothe wild-type receptor (Kauppi et al. 2003). The coordinates of wild-type GR-LBD have not been deposited and, consequently, are not available to us for comparison purposes.
The studies outlined here demonstrate another facet of RU486 antagonism, which may prove useful in the design of a more specific steroid antagonist. As noted above, previous studies have focused on the interaction of RU486 with H3 residues and have noted that alterations in the specific H3 residue alter receptor binding of RU486 (Benhamou et al. 1992, Warriar et al. 1994). Our studies also point to a critical interaction between RU486 and H5 residues. We found that GRs and PRs bearing short side chain amino acids such as alanine and glycine at the H5 position are profoundly resistant to RU486 inhibition, requiring 100-fold greater RU486 concentrations than wild-type receptors. With hPRG722A, it has been suggested that steric hindrance of the alanine side chain with the 11-dimethylaminophenyl group of RU486 is responsible for the lack of efficacy of RU486 (Benhamou et al. 1992). However, steric hindrance cannot explain the insensitivity of hPRs and hGRs containing alanine, serine, or glycine to RU486, as the side chains of these residues are substantially smaller than that of the wild-type methionine. We propose that the binding of RU486 to steroid hormone receptors is dependent on a constructive interaction between the receptor’s H5 residue and RU486, and that this required interaction is lost with substitution of alanine (as well as glycine or serine) for methionine (Fig. 6).
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Altogether, our data suggest that H5 residues play a crucial role in determining GR and PR sensitivity to RU486-mediated antagonism, and also suggest an important distinction in the way RU486 binds to these two receptors. Our work may help to provide a basis for the design of a more-specific steroidal and perhaps non-steroidal receptor agonists and antagonists.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Auzou G, Fagart J, Souque A, Hellal-Levy C, Wurtz JM, Moras D & Rafestin-Oblin ME 2000 A single amino acid mutation of ala-773 in the mineralocorticoid receptor confers agonist properties to 11beta-substituted spirolactones. Molecular Pharmacology 58 684–691.
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Received in final form 5 May 2006
Accepted 31 May 2006
Made available online as an Accepted Preprint 9 June 2006
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P<0.01 vs hPRG722A, hPRM759L/G722A).
