ABSTRACT
The first and critical step in the mechanism of aldosterone action is its binding to the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Over the last 40 years, numerous studies have attempted to determine the structural determinants of ligand-binding to MR. An initial set of data showed that hsp90 is bound to the receptor via specific regions and maintains it in a ligand-binding competent state. Site-directed mutagenesis and functional studies guided by a 3D model of the MR ligand-binding domain (LBD) made it possible to identify the residues responsible for the high affinity and selectivity for aldosterone, and to characterize the mechanisms of MR activation and inactivation. The recent determination of the X-ray crystal structures of the LBD of the wild-type MR and MR(S810L), which is responsible for a familial form of hypertension, has made it possible to elucidate the peculiar mechanism of activation of MR(S810L) and established a clear structure/activity relationship for steroidal and non-steroidal MR antagonists.
Subject(s)
Receptors, Mineralocorticoid/chemistry , Receptors, Mineralocorticoid/metabolism , Aldosterone/chemistry , Aldosterone/metabolism , Animals , Crystallography, X-Ray , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/metabolism , Humans , Ligands , Mineralocorticoid Receptor Antagonists , Models, Biological , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs/physiology , Receptors, Mineralocorticoid/agonists , Structure-Activity RelationshipABSTRACT
Spirolactones harboring various C7 substituents are aldosterone antagonists, and some of them are used in the treatment of essential hypertension. They bind to the human mineralocorticoid receptor and render it transcriptionally inactive. Structural analysis using a three-dimensional homology model of the ligand-binding domain of the receptor has revealed that the Met852 residue of the ligand-binding cavity faces the C7 substituent of spirolactones. We therefore tested the binding capacities of C7-substituted spirolactones in an in vitro system expressing either the mutant receptor, in which Met852 was replaced by alanine, or the wild-type receptor. The M852A mutation had almost no effect on the binding of C7-substituted spirolactones to mineralocorticoid receptor but dramatically reduced the capacity of the receptor to bind steroids with no C7 substituent (aldosterone, cortisol, deoxycorticosterone, and canrenone). cis-trans Cotransfection assays revealed that two spirolactones characterized by having a propyl group [7 alpha-propyl-17 alpha-hydroxy-3-oxo-preg-4-ene-21-carboxylic acid gamma-lactone (RU26752)] or a thioacetyl group (spironolactone) at the C7 position acquired agonist properties when bound to the mutant receptor. In contrast, mexrenone and eplerenone, both of which harbor an acetyl group at the C7 position, retained antagonist properties when bound to the mutant receptor. Overall, these findings indicate that Met852 acts as an organizer residue that plays two major roles: 1) it allows steroids with no substituent at the C7 position to be accommodated within the ligand-binding cavity; and 2) it is involved in the steric hindrance that prevents C7-substituted spirolactones from folding the receptor in its active state.
Subject(s)
Ligands , Methionine/metabolism , Receptors, Mineralocorticoid/metabolism , Animals , COS Cells , Chlorocebus aethiops , Dose-Response Relationship, Drug , Humans , Methionine/chemistry , Mice , Protein Binding/physiology , Rabbits , Receptors, Mineralocorticoid/chemistryABSTRACT
The ability of steroid ligands to inactivate the human mineralocorticoid receptor (MR(WT)) has been shown to be due to their inability to contact Asn770, a residue of the H3 helix involved in stabilizing contacts with the H11-H12 loop region. However, all steroid ligands that display antagonist properties when bound to MR(WT), have been shown to activate a mutant receptor (MR(L810)) associated with a severe form of hypertension. Biochemical studies revealed that S810L mutation induces a change in the receptor conformation and increases the steroid-receptor complexes stability. From a three-dimensional model of the MR ligand-binding domain, it is likely that the S810L mutation causes a steric hindrance between the side chains of Leu810 (H5) and Gln776 (H3) that provokes a bending of the H3 helix. As a consequence, the positioning of MR(WT) antagonists within the ligand-binding cavity is modified in such a way that they can activate the mutant MR(L810). The results from biochemical studies also revealed that 5alpha-pregnan-20-one, 4,9-androstadiene-3,17-dione and RU486, unable to bind MR(WT), acted as potent MR(L810) antagonists.
