Molecular determinants of the recognition of ulipristal acetate by oxo-steroid receptors.

The human progesterone receptor (PR) plays a key role in reproductive function in women. PR antagonists have numerous applications in female health care including regular and emergency contraception, and treatment of hormone-related pathological conditions such as breast cancer, endometriosis, and leiomyoma. The main factor limiting their long-term administration is the fact that they cross-bind to other oxo-steroid receptors. Ulipristal acetate (UPA), a highly potent PR antagonist, has recently come onto the market and is much more selective for PR than the other oxo-steroid receptors (androgen, AR, glucocorticoid, GR, and mineralocorticoid, MR receptors) and, remarkably, it displays lower GR-inactivating potency than RU486. We adopted a structural approach to characterizing the binding of UPA to the oxo-steroid receptors at the molecular level. We solved the X-ray crystal structure of the ligand-binding domain (LBD) of the human PR complexed with UPA and a peptide from the transcriptional corepressor SMRT. We used the X-ray crystal structure of the GR in its antagonist conformation to dock UPA within its ligand-binding cavity. Finally, we generated three-dimensional models of the LBD of androgen and mineralocorticoid receptors (AR and MR) in an antagonist conformation and docked UPA within them. Comparing the structures revealed that the network of stabilizing contacts between the UPA C11 aryl group and the LBD is responsible for its high PR antagonist potency. It also showed that it is the inability of UPA to contact Gln642 in GR that explains why it has lower potency in inactivating GR than RU486. Finally, we found that the binding pockets of AR and MR are too small to accommodate UPA, and allowed us to propose that the extremely low sensitivity of MR to UPA is due to inappropriate interactions with the C11 substituent. All these findings open new avenues for designing new PR antagonist compounds displaying greater selectivity.

11-deoxycorticosterone is a potent agonist of the rainbow trout (Oncorhynchus mykiss) mineralocorticoid receptor.

The teleost fish are thought to lack the mineralocorticoid hormone aldosterone but possess mineralocorticoid receptor (MR) homologs. Here we describe the characterization of two rainbow trout (Oncorhynchus mykiss) MRs, called rtMRa and rtMRb. The open reading frame of rtMRa cDNA encoded a protein of 1041 amino acids. The rtMRb predicted protein sequence is similar, differing in only 10 amino acids in the nonconserved A/B domain and lacking a three-amino acid insertion between the two zinc fingers of the C domain. Expression of rtMR mRNA (sum of both forms), measured in juvenile trout by real-time RT-PCR, shows that the transcripts are ubiquitous. Expression was significantly higher in brain than the other tissues studied (eye, trunk kidney, head kidney, gut, gills, liver, spleen, ovary, heart, white muscle, skin). Hormonal stimulation of receptor transactivation activity was studied in COS-7 cells transiently cotransfected with receptor cDNA and a mouse mammary tumor virus-luciferase reporter. The mineralocorticoids 11-deoxycorticosterone and aldosterone were more potent enhancers of rtMRa transcriptional activity (EC50 = 1.6 +/- 0.5 x 10(-10) and 1.1 +/- 0.4 x 10(-10) M, respectively) than the glucocorticoids cortisol and 11-deoxycortisol (EC50 = 1.1 +/- 0.3 x 10(-9) and 3.7 +/- 1.9 x 10(-9) M, respectively). A similar response was observed in transactivation assays with rtMRb. These results are discussed in the view of reported circulating levels of corticosteroids in trout.

[Agonists and antagonists of mineralocorticoids. The relation between structure and activity]. / Agonistes et antagonistes minéralocorticoïdes. Relation structure-activité.

The mechanism of action of aldosterone and its links with the mineralocorticoids receptor (MR) are described. The physiologic importance of the MR structure is emphasized, in relation with the preferential activation of the receptor by aldosterone.

Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Multiple aspects of mineralocorticoid selectivity.

Aldosterone regulates renal sodium reabsorption through binding to the mineralocorticoid receptor (MR). Because the glucocorticoid receptor (GR) is expressed together with the MR in aldosterone target cells, glucocorticoid hormones bound to GR may also intervene to modulate physiological functions in these cells. In addition, each steroid can bind both receptors, and the MR has equal affinity for aldosterone and glucocorticoid hormones. Several cellular and molecular mechanisms intervene to allow specific aldosterone regulatory effects, despite the large prevalence of glucocorticoid hormones in the plasma. They include the local metabolism of the glucocorticoid hormones into inactive derivatives by the enzyme 11beta-hydroxysteroid dehydrogenase; the intrinsic properties of the MR that discriminate between ligands through differential contacts; the possibility of forming homo- or heterodimers between MR and GR, leading to differential transactivation properties; and the interactions of MR and GR with other regulatory transcription factors. The relative contribution of each of these successive mechanisms may vary among aldosterone target cells (epithelial vs. nonepithelial) and according to the hormonal context. All these phenomena allow fine tuning of cellular functions depending on the degree of cooperation between corticosteroid hormones and other factors (hormonal or tissue specific). Such interactions may be altered in pathophysiological situations.

A single amino acid mutation of ala-773 in the mineralocorticoid receptor confers agonist properties to 11beta-substituted spirolactones.

Sequence analysis revealed a strong homology between the ligand-binding domain (LBD) of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR). Nevertheless, steroids with bulky C11-substituents bind to hGR, unlike hMR. In this report, a mutant hMR, in which the residue Ala-773 facing the C11 steroid position was replaced by a glycine (A773G), was assayed for its capacity to bind steroids, to interact with receptor coactivators, and to stimulate transcription. The capacity of A773G to bind aldosterone and C11-substituted spirolactones was the same as that of the wild-type receptor. The agonist properties of aldosterone, as well as the antagonist feature of compounds bearing a 11beta-allenyl group and a C17-ketone function, remain unchanged. In contrast, C11-substituted steroids with a 17gamma-lactonic ring displayed antagonist properties with hMR and acted as potent agonists with A773G. An agonist-dependent hMR interaction with SRC-1 was observed for both the wild-type and the mutant receptors. The hMR activation process is discussed in the light of the hMR-LBD homology model based on the structural data of the human progesterone receptor LBD.

Crucial role of the H11-H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor.

The crystal structures of ligand-free and agonist-associated ligand-binding domain (LBD) of nuclear receptors (NRs) reveal that the amphipathic helix H12 is folded back toward the LBD core in the agonist-associated conformation, allowing the binding of coactivators. We used alanine scanning mutagenesis to explore the role of the residues of the loop connecting H11 and H12 in the activation of the human mineralocorticoid receptor (hMR), a member of the NRs family. H950A retained the ligand binding and transcriptional activities of the wild-type receptor and interacted with coactivators. In contrast F956A had no receptor functions. Aldosterone bound to the mutant hMRs (L952A, K953A, V954A, E955A, P957A) with nearly the same affinity as to the wild-type receptor and caused a receptor conformational change in these mutant hMRs as it does for the wild-type receptor. But the aldosterone-induced transcriptional activity of the mutant hMRs was lower (L952A, E955A, P957A) than that of the wild-type receptor or completely abolished (K953A, V954A) and their interaction with coactivators was impaired (E955A) or suppressed (L952A, K953A, V954A, P957A). In the light of a hMR-LBD model based on the structure of the progesterone-associated receptor-LBD, we propose that the integrity of the H11-H12 loop is crucial for folding the receptor into a ligand-binding competent state and for establishing the network of contacts that stabilize the active receptor conformation.

Mechanistic aspects of mineralocorticoid receptor activation.

Aldosterone exerts its biological effects through binding to mineralocorticoid receptor (MR). Ligand binding induces a receptor transconformation within the ligand-binding domain and dissociation of associated proteins from the receptor. The ligand-activated receptor binds as a dimer to the response elements present in the promoter region of target genes and initiates the transcription through specific interactions with the transcription machinery. The glucocorticoid hormone cortisol binds to the human MR (hMR) with the same affinity as aldosterone, but is less efficient than aldosterone in stimulating the hMR transactivation. The antimineralocorticoid spirolactones also bind to the hMR but induce a receptor conformation that is transcriptionally silent. In this report, we describe the key residues involved in the recognition of agonist and antagonist ligands and propose a two-step model with a dynamic dimension for the MR activation. In its unliganded state, MR is in an opened conformation in which folding into the ligand-binding competent state requires both the heat shock protein 90 and the C-terminal part of the receptor. An intermediate complex is generated by ligand binding, leading to a more compact receptor conformation. This transient complex is then converted to a transcriptionally active conformation in which stability depends on the steroid-receptor contacts.

Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.

The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (R(T) approximately 4700 ohm x cm2) and potential difference (P(D) approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 microA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10(-10) to 10(-6) M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5x10(-7) M)-induced Isc (fourfold) was associated with a three- to fivefold increase in alpha-ENaC mRNA (but not in those for beta- or gamma-ENaC) and three- to 10-fold increases in alpha-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine.

Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.

[Corticosteroid hormones: mechanisms involved in the recognition of aldosterone by mineralocorticoid receptors]. / Les hormones corticostéroïdes: mécanismes impliqués dans la reconnaissance de l'aldostérone par le récepteur aux minéralocorticoïdes.

Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones in humans, are structurally very closed. Both hormones bind to the mineralocorticoid receptor (MR) with the same affinity. Nevertheless MR is preferentially activated by aldosterone, suggesting that the binding of these two hormones to MR involved some distinct contacts. We constructed a tridimensional model of the ligand-binding domain of the human MR, by taking as a template the structural data of the retinoid receptor associated with its ligand. The MR model allowed the identification of several residues involved in the interaction with aldosterone and cortisol. The residues Gln 776 and Arg 817 make hydrogen bonds with the 3-keto function and the residue Asn 770 with the C21-hydroxyl group. Analyses of the wild type and mutant MRs activities in response to corticosteroids bearing hydroxyl groups at various steroid skeleton position led to the following conclusions: 1) the interaction between the residue Asn 770 and the C21-hydroxyl group of corticosteroids is determinant for stabilizing the active MR conformation and 2) the stability of this conformation is enhanced by the 11-18 hemiketal group of aldosterone whereas it is decreased by the 11 beta- and 17 alpha-hydroxyl groups of cortisol. These results are discussed in the light of a model for the MR activation process.

Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.

The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.

[Aldosterone receptors: new insights in hormonal signal transduction]. / Récepteurs de l'aldostérone: données récentes sur la transduction du signal hormonal.

Aldosterone exerts its effects by binding to an intracellular receptor, the mineralocorticoid receptor (MR), that belongs to the superfamily of ligand-regulated transcription factors. This family includes receptors for steroid and thyroid hormones, vitamin D and retinoids and also orphan receptors with unidentified ligands. All the members of this family display a modular structure composed of a transactivation domain, a DNA binding domain and a ligand binding domain. Upon hormone binding, the receptor undergoes a series of structural modifications leading to its interaction with DNA sequences and transcriptional modulation of specific genes. Since the level of circulating glucocorticoids is 100-1000 fold higher than that of aldosterone and the affinity of the MR is identical for glucocorticoids and aldosterone, the question of the mechanisms responsible for the in vivo selectivity of aldosterone for its receptor arises. The mechanisms of cellular and tissular selectivity involved in the physiological response of aldosterone are discussed in this review.

Folding requirements of the ligand-binding domain of the human mineralocorticoid receptor.

The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a series of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus-truncated hMRs showed that the ligand-binding domain (LBD: amino acids 734-984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (Kd) 2.9 vs. 0.47 nM] and does not interact with hsp90. Addition of the five-amino acid sequence (729-733) upstream from the LBD is necessary for interaction with hsp90, but a larger region is needed for high aldosterone affinity. Deletions at the C-terminal end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids reduced the affinity for aldosterone to 1/20 that of the entire protein, and deletion of the last four amino acids completely abolished binding, although the interaction with hsp90 was not affected. These effects can be explained by misfolding of the receptor, since limited proteolysis assays showed that deletions at the C-terminal end of hMR affect the accessibility of the cleavage sites within the DNA-binding domain and the N-terminal part of the hinge region to trypsin. Thus, our results support the idea that a short sequence upstream of the LBD is essential for the interaction of hMR with hsp90 and that the C terminus of hMR and hsp90 are both essential for folding of the receptor in a high-affinity hormone-binding state.

Antagonism in the human mineralocorticoid receptor.

Key residues of the human mineralocorticoid receptor (hMR) involved in the recognition of agonist and antagonist ligands were identified by alanine-scanning mutagenesis based on a homology model of the hMR ligand-binding domain. They were tested for their transactivation capacity and ability to bind agonists (aldosterone, cortisol) and antagonists (progesterone, RU26752). The three-dimensional model reveals two polar sites located at the extremities of the elongated hydrophobic ligand-binding pocket. Mutations of Gln776 and Arg817 in site I reduce the affinity of hMR for both agonists and antagonists and affect the capacity of hMR to activate transcription, suggesting that the C3-ketone group, common to all ligands, is anchored by these two residues conserved within the nuclear steroid receptor family. In contrast, mutations of Asn770 and Thr945 in the opposite site only affect the binding of agonists bearing the C21-hydroxyl group. The binding of hMR antagonists that exhibit a smaller size and faster off-rate kinetics compared with agonists is not affected. In the light of the hMR homology model, a new mechanism of antagonism is proposed in which the AF2-AD core region is destabilized by the loss of contacts between the antagonist and the helix H12 region.

Pleiotropic effects of dihydrotestosterone in immortalized mouse proximal tubule cells. Technical note.

Dihydrotestosterone (DHT) binding studies and the effects of DHT on the expression of beta-glucuronidase (Gus) and kidney androgen-regulated protein (KAP) genes and cell growth were investigated in immortalized early PKSV-PCT and late PKSV-PR proximal tubule cells, derived from transgenic mice carrying the L-pyruvate kinase/SV40 hybrid gene. [3H]DHT binding studies indicated that both cell lines have conserved substantial amounts of androgen receptors. The levels of KAP and Gus transcripts in PKSV-PCT cells, and those of KAP transcripts in PKSV-PR cells, decreased when cells were shifted from a serum-supplemented to a steroid-free medium. The addition of 30 nM DHT to the steroid-free medium resulted in a slight increase in Gus and in a more marked increase in KAP transcripts in both cell lines. Dihydrotestosterone also affected the growth of PKSV-PCT and PKSV-PR cells, since this hormone added to the steroid-free medium stimulated the incorporation of [3H]thymidine in a dose-dependent manner and induced the formation of domes, which represent indicators of ionic transport processes. Thus, because these early and late mouse proximal tubule cells have conserved androgen receptors, they represent attractive cell systems to analyze the action of androgens on specific functions of the mouse proximal tubule.

Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody.

To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Léger and Auzou (1996) J. Steroid Biochem. Mol. Biol. 57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones.

Cell-specific, promoter-dependent mineralocorticoid agonist activity of spironolactone.

The agonist activity of the antimineralocorticoid spironolactone was evaluated in various cell lines through the use of transfection experiments. The target promoters were derived from the deltaMTV promoter in which one or several glucocorticoid-responsive elements (GRE) were inserted in tandem. Spironolactone at 100 nM activated by 6-fold the GRE/deltaMTV promoter in the human hepatoma HepG2 cell line and only partially prevented the 10-fold activation of this promoter by 0.1 nM aldosterone. Both effects were completely dependent on the cotransfection of an expression vector for the mineralocorticoid receptor. The half-maximal agonist effect of spironolactone was similar to its half-maximal antagonist effect (approximately 10 nM). For the GRE-2/deltaMTV, GRE-4/deltaMTV, and wild-type MMTV promoters, the activation by aldosterone was much more potent (70-, 100-, and 110-fold, respectively), whereas spironolactone elicited a 10-, 24-, and 25-fold activation, respectively. Thus, the effect of both compounds and the relative efficiency of spironolactone, compared with that of aldosterone, were dependent on the number of GREs present in the regulatory region of the promoter. The agonist effect of spironolactone was cell specific. Indeed, although spironolactone agonist activity was observed in H5 kidney tubule cells, none could be detected at concentrations of < or = 1 microM in the CV1 monkey fibroblast cells. In contrast, the antagonist effect was observed in all cells. Furthermore, other antimineralocorticoids, such as RU 26752 and progesterone, also displayed mineralocorticoid receptor-dependent agonist activity in the HepG2 cells. The antiprogesterone RU 486 and the antiandrogen cyproterone acetate were ineffective at < or = 1 microM. In conclusion, we show that under certain experimental conditions, several antimineralocorticoids display significant agonist activity in a cell-specific and promoter-dependent manner.

New steroidal diazo ketones as potential photoaffinity labeling reagents for the mineralocorticoid receptor: synthesis and biological activities.

Three diazo ketones in the progesterone series were synthesized as potential photoaffinity reagents. The diazo ketone group was introduced at the C17 (21-diazopregn-4-ene-3,20-dione, 1) or C13 (18-(diazomethyl)-20-hydroxypregn-4-ene-3,18-dione, 2, 18-(diazomethyl)pregn-4-ene-3, 18,20-trione, 3) position of the pregnene skeleton. Whereas compound 1 could be easily obtained from the corresponding acid chloride, preparation of 2 and 3 required a less straightforward route involving reaction of tosyl azide on the formyl derivative of methyl ketone 5. The affinity of the diazo ketones for the human mineralocorticoid receptor (hMR), expressed in Sf9 insect cells using the Baculovirus system, was estimated by competition experiments using [3H]aldosterone as specific ligand. The affinity of 1 for hMR was almost identical with that of aldosterone. The affinities of 2 and 3 were 1, order of magnitude lower than that of aldosterone. The mineralocorticoid activity of the diazo ketones was measured in cis-trans cotransfection assays in CV-1 cells with the mouse mammary tumor virus as DNA target sequence. Compound 1 exhibits an agonist activity (ED50 = 6 x 10(-9) M) with no antagonist activity. In contrast 2 and 3 behave as antagonists, displaying an IC50 of approximately 10(-6) M whether the substituent at the C20 position is a hydroxy (2) or an oxo (3) group.

Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure.

To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) or hMR (hMR 711-984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone-hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1-944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711-984 region is sufficient for hsp90-MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand.