The establishment of the long terminal repeat of the mouse mammary tumor virus into CV-1 cells allows a functional analysis of steroid receptors.

To analyze in situ the effects of mineralocorticoid receptor (MR) on the nucleo-protein organization of the target MMTV promoter, we have established a new cell line by integrating in CV-1 cells a construct containing the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). The MMTV-LTR contains glucocorticoid response elements (GREs), known to interact with MR. CV-1 cells were selected because they lack glucocorticoid receptor (GR). The absence of GR in the host cell line allows the selective analysis of transcription activation by aldosterone in cells expressing MR transiently. The CV-1 cells were transfected with the construct pMAMneoCAT, a plasmid containing the MMTV promoter driving the chloramphenicol acetyl transferase (CAT) gene and a gene for neomycin selection. A neomycin-resistant clone (M8), which contains two copies of the unrearranged construct was characterized. The integrated MMTV promoter is functional, as demonstrated by the induction of the CAT activity upon addition of aldosterone, dexamethasone, and R5020 to M8 cells transiently transfected with MR, GR, and progesterone receptor (PR) expression vectors, respectively. Induction of the CAT activity by dexamethasone or progesterone was 2 to 3-fold higher than by aldosterone. These differences in CAT activities were not related to differences in the levels of receptor expression. In the transiently transfected M8 cells, MR and PR contents were similar (50-70 fmol/mg protein) while GR content was higher (250 fmol/mg protein). Thus, this new cell line M8, provides a useful tool for selectively studying the effect of MR on a target promoter organized into chromatin.

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.

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.

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.

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.

Spironolactone, an aldosterone antagonist, acts as an antiglucocorticosteroid on the mouse mammary tumor virus promoter.

The ability of the glucocorticosteroid receptor to bind mineralocorticosteroids suggests that spironolactone, a potent aldosterone antagonist, may also interact with the glucocorticosteroid receptor, resulting in an agonist or antagonist glucocorticosteroid activity. We have investigated the effect of this drug on the activity of the glucocorticosteroid-regulated mouse mammary tumor virus (MMTV) promoter. For these studies we used the mouse fibroblast cell line 1471.1. It contains about 200 copies of a permanently established chimeric DNA construct comprising a transcription unit [MMTV long terminal repeat (LTR)] driving the reporter gene chloramphenicol acetyltransferase linked to the 69% transforming fragment of the bovine papilloma virus genome. This cell line has a high level of glucocorticosteroid receptor (1200 fmol/mg protein) and no detectable mineralocorticosteroid receptor. Competition experiments showed a binding of spironolactone to glucocorticosteroid receptor, with an affinity 50-fold lower than that of dexamethasone. In these cells, spironolactone behaves as an antiglucocorticosteroid, inhibiting in a dose-dependent fashion dexamethasone-induced chloramphenicol acetyltransferase activity, with an ED50 of 8 microM. The absence of agonist activity, even at a high concentration of this compound (10 microM), demonstrates that spironolactone is a pure antiglucocorticosteroid in this cell line. MMTV LTR DNase-I hypersensitivity studies demonstrated that spironolactone, when administered in combination with dexamethasone, inhibits formation of the hormone-induced hypersensitive site located about 160 basepairs up-stream of the MMTV cap site. Furthermore, spironolactone alone failed to induce this DNase-I-hypersensitive site, suggesting that the antagonist-receptor complex does not interact productively with MMTV LTR chromatin.

The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11 beta-hydroxysteroid dehydrogenase.

To investigate the mechanisms involved in the in vivo aldosterone selectivity of the mineralocorticoid receptor (MR), we studied the respective contribution of the receptor and the enzyme 11 beta-hydroxysteroid dehydrogenase (11HSD), which converts glucocorticoids into inactive metabolites. Using a cotransfection assay in CV-1 cells, aldosterone activated mouse mammary tumor virus promoter through human MR (hMR) with an ED50 of 0.01 nM. An at least 100-fold higher concentration of cortisol (F), corticosterone (B), or dexamethasone was required to obtain half-maximum transactivation, indicating a functional preference of hMR for aldosterone over glucocorticoids. The catalytic activity of 11HSD was analyzed using HPLC by measuring the tritiated metabolites produced in CV-1 and COS cells. Both cell types displayed a significant dehydrogenase activity (20 fmol/10 min.10(3) cells) inhibitable by carbenoxolone, but no detectable reductase activity. In this model, B was more rapidly metabolized than F. Carbenoxolone treatment of hMR-transfected CV-1 cells did not result in a shift of the dose-response transactivation curves of B and F toward lower concentrations, ruling out the implication of 11HSD in the aldosterone MR selectivity of these conditions. Despite similar affinity constants of aldosterone and glucocorticoids for the hMR, kinetic experiments showed that the off-rate of aldosterone from hMR was 5 times lower than that of glucocorticoids, pointing to an intrinsic discriminating property of the receptor. Therefore, we propose that in addition to 11HSD, MR plays an active role in the mechanism of aldosterone selectivity.

The mineralocorticoid activity of progesterone derivatives depends on the nature of the C18 substituent.

To investigate the role of the C18 substituents in the agonist/antagonist properties of mineralocorticoids, the activities of certain C18-substituted progesterone (P) derivatives were examined. These compounds were characterized by an unsaturated side-chain in the case of 18-vinylprogesterone (18VP) and 18-ethynylprogesterone (18EP) and by an enone group in the case of 18-oxo-18-vinylprogesterone (18OVP). P and its 18-substituted derivatives bind to the recombinant human MR (hMR) overexpressed in Sf9 cells with the following hierarchy of affinity: P > aldosterone > 18VP > 18EP >> 18OVP. Functional cotransfection assays in CV-1 cells, using mouse mammary tumor virus promoter as a steroid receptor-inducible DNA target sequence, indicated that the mineralocorticoid activity depends on the nature of the C18 substituent. 18VP and 18EP retained the antimineralocorticoid feature of P, with the following order of activity: P = 18VP > 18EP. The antagonist potency of 18VP was higher (IC50, approximately 10(-8) M) than that of spironolactone (IC50, approximately 7 x 10(-8) M), the most widely used aldosterone antagonist. Interestingly, introducing an oxo function at C18 conferred agonist mineralocorticoid properties; 18OVP behaves as a full agonist (ED50, approximately 10(-7) M) with no antagonist activity. In contrast to what was observed when the three 18-substituted P derivatives acted through hMR, they retained the agonist feature of P through the human P receptor, with the following order of potency: P > 18VP = 18OVP > 18EP. The activity of the 18-substituted P derivatives through the human glucocorticoid receptor was only detected at concentrations higher than 10(-6) M; P and 18VP displayed a partial antagonist activity, whereas 18OVP had a full agonist activity (ED50, approximately 2 x 10(-6) M). Thus, the presence of an oxo group at C18(18OVP) does not change the agonist feature of P through human P receptor, but confers to the ligand an agonist activity through hMR, suggesting that the C18 carbonyl group of aldosterone plays a crucial role in its agonist activity.

Mechanism of action of a new antialdosterone compound, prorenone.

Two new aldosterone antagonists, K-prorenoate [potassium 3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl)propionate] and prorenone [3(17 beta-hydroxy-6 beta, 7 beta-methylen-3-oxo-4-androsten-17 alpha-yl) propionic acid gamma-lactone], its lactonic form, were studied in rat kidney using in vitro systems. Study of [3H]prorenone binding by a recently developed computer method indicated a high affinity, low capacity class of sites which are, seemingly, mineralocorticoid receptors. In competition experiments performed on [3H]aldosterone- and [3H]dexamethasone-binding sites, prorenone appeared to be a good competitor for mineralocorticoid-binding sites and a poor competitor for glucocorticoid-binding sites. The specificity of this molecule was further confirmed by its poor ability to displace [3H]dihydrotestosterone from rat prostate androgenic receptors compared to spironolactone [3-(3-oxo-7 alpha-acetylthio-17 beta-hydroxy-4-androsten-17 alpha-yl) propionic acid gamma-lactone]. In the same experiments, K-prorenoate demonstrated a very low affinity for the two types of receptors. The behavior of [3H]prorenone cytosolic complex was also studied in kidney mince experiments, which showed that the [3H]prorenone complex was not able to translocate into the nucleus. Prorenone inhibited the binding of [3H]aldosterone to the receptor and, consequently, the nuclear binding of aldosterone was not observed.

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.

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.

Differences between aldosterone and its antagonists in binding kinetics and ligand-induced hsp90 release from mineralocorticosteroid receptor.

We have previously reported that mineralocorticosteroid receptor (MR) is a 8-9 S heterooligomeric complex that includes the 90 kDa heat shock protein (hsp90). To elucidate how antagonist-receptor complexes are biologically inactive in terms of transcriptional regulation, we analyzed the binding of mineralocorticosteroid agonists and antagonists with MR and the ligand-induced transformation of its heterooligomeric structure. This study was performed in the cytosol of adrenalectomized rat kidney and of COS cells transiently transfected with human MR cDNA. Although aldosterone antagonists (SC9420 and RU26752) bind MR with the same affinity as aldosterone, they dissociate much more rapidly from the 8-9 S form of both rat and human MR than does aldosterone. Using sedimentation gradient analysis, we showed that the interaction between hsp90 and the steroid binding subunit of MR is highly dependent upon the nature of the steroid ligand since the binding of aldosterone antagonists results in an easy release of hsp90. We propose that both rapid dissociation of ligand and weakened hsp90-receptor interaction play a key role in the mechanism of mineralocorticosteroid antagonism. In the COS cell model, cortisol, described as a weak mineralocorticosteroid agonist, dissociates also more rapidly from human MR than does aldosterone. Our results suggest that ligand binding kinetics and ligand dependent modification in receptor structure are important modulators of MR function as a transcriptional regulatory factor.

Sulfhydryl groups are involved in the binding of agonists and antagonists to the human mineralocorticoid receptor.

To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4 degrees C inhibited the binding to hMR of both [3H]aldosterone and [3H]RU26752 (a synthetic aldosterone antagonist). At 4 degrees C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4 degrees C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20 degrees C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hmR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action.

Mineralocorticoid receptors in SV40-transformed tubule cell lines derived from rabbit kidney.

The presence of mineralocorticoid (MR) and glucocorticoid (GR) receptors was investigated in two renal tubular cell lines, derived from primary cultures of isolated rabbit kidney cortical cells infected with the wild-type SV40 virus, which exhibit thick ascending limb (RC.SV2) and collecting tubule (RC.SV3) phenotypes (Vandewalle et al. J. Cell. Physiol. 141, 1989, 203-221). MR and GR were quantified, in cell monolayers and cell cytosolic fractions, with [3H]aldosterone, [3H]dexamethasone and [3H]RU486, an antiglucocorticoid with no affinity for MR. Cytosolic receptors from RC.SV2 and RC.SV3 cells labeled with [3H]aldosterone, [3H]dexamethasone or [3H]RU486 sedimented at approximately 8 S in a 15-40% glycerol gradient. All steroids displaced bound [3H]dexamethasone to the same extent, suggesting that dexamethasone bound to both MR and GR: under the conditions of assay, [3H]aldosterone binds exclusively to MR, and [3H]RU486 to GR. In both RC.SV2 and RC.SV3 cells, [3H]aldosterone bound to one class of high affinity sites (Kd 0.14-0.8 nM; Nmax 8 to 22 fmol/mg protein). In both cell lines, the number of high affinity binding sites for [3H]dexamethasone ranged from 9 to 18 fmol/mg protein with an affinity of 0.5-1.3 nM. Compared to renal cortex, the most striking observation was a marked decrease in [3H]dexamethasone binding in primary cultures and SV40-transformed cells. These results indicate that MR and GR are expressed in two established mammalian kidney tubular cell lines providing new models of cultured renal cells for studies on the physiological effects of corticosteroid hormones.

Tritiated 9alpha-fluorocortisol metabolism and binding in rat kidney.

Metabolism of 9alpha-fluorocortisol (9alpha-F) has been studied in rat kidney slices, homogenate, and in the isolated perfused kidney. These studies show that the rate of 9alpha-F metabolism varies depending upon the experimental conditions. The major metabolite formed, identified by mass spectrometry, is 20(xi)-dihydro-9alpha-fluorocortisol. The kidney slice experiments show that only 3H-9alpha-F and none of the metabolites bind to cytosolic receptors. In competition experiments performed with tritiated and unlabeled 9alpha-fluorocortisol, aldosterone (A), dexamethasone (DM) and triamcinolone acetonide (TA), 9alpha-F was found to bind to mineralocorticoid receptors with a lower affinity than A but also to glucocorticoid receptors with a higher affinity than A. The Scatchard plot analysis indicated that 3H-9alpha-F is characterized by KD : 8.6 X 10(-9)M and N : 1.9 x 10(-13)moles/mg of protein. In conclusion it is felt that 9alpha-F would not be a better "marker" than aldosterone for the renal mineralocorticoid receptors.

[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.

[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.

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.

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.