The ligand binding domains of the human mineralocorticoid receptor (hMR) and glucocorticoid receptor (hGR) display a high sequence homology. Aldosterone and cortisol, the major mineralocorticoid and glucocorticoid hormones, are very closely related, leading to the cross-binding of these hormones to both receptors. The present study reports on the mechanism by which hMR and hGR are activated preferentially by their cognate hormones. We found that the ability of corticosteroids to stimulate the receptor's transactivation function is depending on the stability of the steroid-receptor complexes. In the light of a hMR structural model we propose that contacts through the corticosteroid C21 hydroxyl group are sufficient to stabilize hMR but not hGR and that additional contacts through the C11-and C17-hydroxyl groups are required for hGR.z 1999 Federation of European Biochemical Societies.

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.

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.

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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 11␤-allenyl group and a C17-ketone function, remain unchanged. In contrast, C11-substituted steroids with a 17␥-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.The mineralocorticoid receptor (MR) belongs to a large family of ligand-activated transcription factors that includes the other steroid receptors as well as thyroid, retinoid, and vitamin D receptors and also orphan receptors whose ligands have not yet been identified. All the members of this large family are characterized by a conserved DNA binding domain and a C-terminal ligand-binding domain (LBD) essential for chaperone protein interaction, receptor dimerization, and hormone-dependent transactivation (Arriza et al., 1987;Evans, 1988). Recently, the crystal structure of ligand-free and liganded LBDs has been solved for several nuclear receptors (NRs) (Bourguet et al., 1995;Renaud et al., 1995;Wagner et al., 1995;Wurtz et al., 1996;Brozowski et al., 1997). These crystal structures reveal a triple-layered antiparallel ␣-helical sandwich fold surrounding the ligand-binding cavity. The major difference between the ligand-free and the agonist-bound LBD is the folding back of the helix H12 toward the LBD core, allowing the binding of transcriptional coactivators (Nichols et al., 1998). Moreover, the helix H12 was demonstrated to be differently positioned after antagonist binding, preventing the coactivator-receptor interaction (Nichols et al., 1998). A three-dimensional model of the human MR (hMR)-LBD, based on the human retinoic acid receptor (hRAR␥-LBD) crystal structure, has recently been proposed that allows the docking of various ligands within the ligand-binding cavity (Fagart et al., 1998). The identification of several amino acid residues involved in the interaction with agonists and antagonists has been made by mutagenesis. Gln-776 and Arg-817, two polar residues highly conserved within the steroid receptor family, anchor the C3-ketone function, common in mineralocorticoid agonist and antagonist ligands. At the opposite side of the ligand-bindin...

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.

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