Structural determinants of ligand binding to the mineralocorticoid receptor

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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 MRS810L, which is responsible for a familial form of hypertension, has made it possible to elucidate the peculiar mechanism of activation of MRS810L and established a clear structure/activity relationship for steroidal and non-steroidal MR antagonists.

Highlights

► Importance of hsp90 for ligand-binding competent state of mineralocorticoid receptor. ► Activation of mineralocorticoid receptor based on structural approaches. ► Steroidal and non-steroidal antagonists of mineralocorticoid receptor.

Introduction

Aldosterone is the main mineralocorticoid hormone in mammals. It plays a key role in sodium homeostasis and in regulating blood pressure (Horisberger and Rossier, 1992, Bonvalet, 1998, Rossi et al., 2005). Aldosterone acts mainly by binding to the mineralocorticoid receptor (MR), a transcription factor belonging to the superfamily of nuclear receptors. The mechanism by which MR is activated in target cells is well documented. In its inactive form, MR is involved in a multiprotein complex that also includes the heat shock protein, hsp90. This complex is predominantly found in the cytoplasm (Robertson et al., 1993, Lombes et al., 1994). When activated by the binding of an agonist ligand, MR dissociates from the multiprotein complex, and is transferred into the nucleus. It then binds to the hormone response element (HRE), which is located in the transcription region of target genes, where it recruits transcriptional coactivators, thus enhancing the transcription of genes involved in sodium reabsorption (Fuller and Young, 2005).

As a result of its activation by agonist binding, MR is implicated in several diseases, including hypertension and heart failure, and MR antagonists have been clinically evaluated for treating such diseases (Pitt et al., 1999, Pitt et al., 2003, Garthwaite and McMahon, 2004, Zannad et al., 2011). The MR antagonists currently available are all steroidal molecules. Most of them display high affinity and potency for MR, but they are not very selective and cross-react with other steroid receptors (De Gasparo et al., 1986, Losert et al., 1986, Nedelec et al., 1986). Spironolactone, for example, displays very high affinity and potency for MR, but also binds to the androgen receptor (AR), leading to significant side effects (Corvol et al., 1976). The main competitor of spironolactone on the market is eplerenone. It is more MR selective, but also displays a lower affinity for MR and is far less potent than spironolactone (Garthwaite and McMahon, 2004). To find a way round the selectivity problem, the focus of the researchers has recently shifted to non-steroidal molecules, with the aim of designing molecules that would bind to MR in a different way from steroids.

MR is a member of the nuclear receptor superfamily and is composed of four domains (Fig. 1). The N-terminal domain, which is characterized by two activation functions, known as AF1a and AF1b, is involved in binding coregulators. No structural information is yet available about this domain. The central domain is the DNA-binding domain (DBD), the X-ray crystal structure of which has not been determined for MR, but has for the glucocorticoid receptor (GR) (Luisi et al., 1991), the steroid receptor with which MR shares the greatest sequence identity. The DBD is linked to the C-terminal domain by a small hinge region, the role of which remains unknown. The C-terminal domain constitutes the ligand-binding domain (LBD). It harbors an activation function, known as AF2, which is directly regulated by the binding of a ligand. This domain is also involved in binding coregulators (Hultman et al., 2005) and in interactions with the N-terminal domain (Rogerson and Fuller, 2003). There is evidence that this interaction is aldosterone-dependent, and that it is further enhanced by the activation of AF2, suggesting cooperation between AF1 and AF2 (Rogerson and Fuller, 2003).

Over the years, a plethora of studies of MR has provided clues about the structural requirements of ligand-binding. They concern both the shape of the receptor itself, and the structure of the ligand. In this paper, we review the structural determinants of ligand binding to MR.

Section snippets

Binding of hsp90 to MR

In the absence of ligand, steroid receptors have been shown to interact with many chaperone proteins, such as hsp90, hsp40, Hop/hsp60, hsp70, p23, p70, and cochaperone proteins, such as the immunophilins FKBP51, FKBP52, and protein phosphatase 5 (pp5). These complexes appear to be transcriptionally silent (see Smith and Toft, 2008, Galigniana et al., 2010, for reviews). In the 1990s, numerous studies provided evidence that the binding of hsp90 to MR is required to maintain the receptor in a

Binding of steroidal ligands to MR

The binding properties of agonist and antagonist ligands to MR have been intensively explored in vitro. The first very important conclusion emerging from these studies was that the binding of a ligand stabilizes MR. It has actually been shown that MR expressed in vitro rapidly loses its binding capacity and is degraded. The binding of a ligand to MR stabilizes it, and protects it from degradation (Rafestin-Oblin et al., 1977). This observation has also been reinforced by limited proteolysis

Binding of non-steroidal ligands to MR

Recently, by means of high-throughput screening, dihydropyridine and pyrazoline derivatives have been shown to be able to bind to MR and inhibit its aldosterone-induced activity (Dietz et al., 2008, Arhancet et al., 2010, Fagart et al., 2010, Kosaka et al., 2010, Meyers et al., 2010). On the basis of docking experiments within the MR-LBD X-ray crystal structure, it has been proposed that pyrazoline compounds are unable to contact the Asn770 residue, thus explaining their antagonist feature

Critical regions for maintaining MR in its ligand-binding competent state

3D homology models of MR-LBD together with sequence alignment have shown that MR is characterized by a 10-residue C-terminal extension after the H12 helix (residues 975–984 in hMR, after the H12 helix), which is highly conserved only among steroid receptors, but not in the estrogen receptor. This C-terminal extension has been proved to be essential for maintaining the highest affinity of the receptor for its ligand. A mutagenesis study involving deletion mutants has shown that the last four

Ligand-specific structural adaptations of MR

In response to agonist binding, MR undergoes conformational changes that trigger the recruitment of transcriptional coregulators characterized by an LXXLL motif, where L stands for leucine and X for an unspecified residue (Heery et al., 1997). In the structure of MR-LBD complexed with corticosterone and a coactivator peptide, the LXXLL motif adopts an α-helical conformation, and is accommodated in a hydrophobic groove formed by the H3, H4, H5 and H12 helices, which forms the AF2 sub-domain (Li

Conclusion

MR is rather unstable and rapidly degraded in the absence of ligand. The association of chaperone and cochaperone proteins to MR in the cytoplasm helps to maintain the receptor in a ligand-binding competent state. When a ligand binds to MR, it induces a conformational change that triggers its dissociation from the multiprotein complex and its translocation into the nucleus. More importantly, the binding of a ligand to MR stabilizes the receptor. However, although all ligands stabilize MR, they

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    1

    Current address: Department of Biological Sciences, Birkbeck College, University of London, United Kingdom.

    2

    Current address: Inserm U693, Faculté de Médecine Paris-Sud 11, 63 rue Gabriel Péri, 94276 Le Kremlin-Bicêtre, France.

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