A Single Amino Acid Mutation of Ala-773 in the Mineralocorticoid Receptor Confers Agonist Properties to 11β-Substituted Spirolactones

Abstract

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...

Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor

“…This greater instability of the antagonist -hMR complex could lead to faster dissociation of progesterone from the receptor (8). The antagonistic potency of progesterone has been described with IC 50 values ranging from 2 to 10 nmol/l (6,33,44). Our result, with a half-maximal inhibition of the aldosterone-induced activity of hMR by progesterone of 11 nmol/l (Table 1), is in agreement with these data.…”

“…In order to study the steroid-binding characteristics, the lysate was diluted after translation with 1 vol icecold buffer containing 20 mmol/l Tris -HCl pH 7.4, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, 20 mmol/l sodium tungstate and 10% glycerol (33 Unbound steroids were separated from bound steroids by using dextran-coated charcoal (DCC): 1 ml DCC suspension (0.5%) was added to each aliquot (25 ml), shaken for 7 min on ice and centrifuged at 14 000 g for 10 min at 4 8C.…”

The Met852 Residue Is a Key Organizer of the Ligand-Binding Cavity of the Human Mineralocorticoid Receptor

“…Three-dimensional models of the MR-LBD have been generated using the crystallographic data of nuclear receptors as the template Auzou et al, 2000). In these models, the ligand-binding pocket is delineated by the H3, H5, H7, H11, and H12 helices, the first ␤-turn, and the H6-H7 and H11-H12 loops.…”

“…The three-dimensional MR model generated by using the crystallographic data from nuclear receptors as a template Auzou et al, 2000) predicted that the steroid C7 substituent faces Met852, a residue of the H7 helix. This finding led us to generate a mutant receptor by substituting alanine for methionine (MR A852 ), which we used to test the role of this residue in the accommodation of spirolactones in the ligand-binding pocket of the receptor.…”

“…The crystal structure of the MR-LBD has not yet been solved. However, three-dimensional homology models of the hMR-LBD have been generated from the crystal structures of nuclear receptors, making it possible to predict the threedimensional organization of the MR-LBD and to see how agonist and antagonist ligands are docked within the ligandbinding cavity Auzou et al, 2000).…”