30 YEARS of the MINERALOCORTICOID RECEPTOR Coregulators As Mediators of Mineralocorticoid Receptor Signalling Diversity

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p j fuller and others MR coactivators 234:1 T23–T34 Thematic Review

30 YEARS OF THE MINERALOCORTICOID RECEPTOR Coregulators as mediators of mineralocorticoid receptor signalling diversity

Peter J Fuller, Jun Yang and Morag J Young Correspondence should be addressed Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research and the Monash to P J Fuller University Department of Molecular Translational Science, Clayton, Victoria, Australia Email [email protected]

Abstract

The cloning of the mineralocorticoid receptor (MR) 30 years ago was the start of a new era of research into the regulatory processes of MR signalling at target genes in the distal nephron, and subsequently in many other tissues. Nuclear receptor (NR) Key Words signalling is modified by interactions with coregulatory proteins that serve to enhance ff cortisol or inhibit the gene transcriptional responses. Over 400 coregulatory proteins have ff aldosterone been described for the NR super family, many with functional roles in signalling, f Endocrinology f mineralocorticoid receptor of cellular function, physiology and pathophysiology. Relatively few coregulators have however been described for the MR although recent studies have demonstrated both ff coregulators ligand and/or tissue selectivity for MR-coregulator interactions. A full understanding Journal of the cell, ligand and promoter-specific requirements for MR-coregulator signalling is an essential first step towards the design of small molecular inhibitors of these protein-protein interactions. Tissue-selective steroidal or non-steroidal modulators of the MR are also a desired therapeutic goal. Selectivity, as for other steroid hormone receptors, will probably depend on differential expression and recruitment of coregulatory proteins. Journal of Endocrinology (2017) 234, T23–T34

Introduction

The cloning of the mineralocorticoid receptor (MR) functional characterization of the receptor including its by Jeff Arriza working in the laboratory of Ron Evans ability to bind both aldosterone and cortisol (Evans & (Arriza et al. 1987) marked a critical inflexion point for Arriza 1989). These transcription factors enhanced gene research on aldosterone action. It followed the cloning of expression in ‘trans’, which is to say they interacted with the other steroid hormone receptors (SHRs) and indeed response elements in the chromatin, which were not other members of the nuclear receptor (NR) superfamily contiguous with the promotor being at some distance of ligand-dependent transcription factors. The from the transcriptional start site. As a consequence, determination of the amino acid sequences of the SHR it became clear that a mechanism was required for the enabled the characterization of shared structural domains, receptor to interact with the transcriptional machinery and the cloned MR was used in ‘cis-trans’ assays to provide assembled on the promoter and thus, by analogy with

http://joe.endocrinology-journals.org © 2017 Society for Endocrinology This paper is part of a thematic review section on 30 Years of the DOI: 10.1530/JOE-17-0060 Published by Bioscientifica Ltd. Mineralocorticoid Receptor. The guest editors for this section were Printed in Great Britain John Funder and Maria Christina Zennaro.Downloaded from Bioscientifica.com at 09/25/2021 03:49:39PM via free access

10.1530/JOE-17-0060 Thematic Review p j fuller and others MR coactivators 234:1 T24

other transcription factors, there must exist coregulators flexibility for protein-protein interactions (McEwan et al. that bridge the activation function in the NR to the 2007); its structure is therefore effectively determined by promoter complex. its interacting partner. Various studies have identified The search for SHR coactivators was on, and in at least three regions in the NTD, termed activation 1995, the O’Malley group (Onate et al. 1995) cloned the functions (AFs), which are thought to recruit coactivators: first coactivator for SHR, steroid receptor coactivator-1 AF1a (amino acids 1–169), middle domain (MD, amino (SRC-1). Other groups identified co-repressors which acids 247–385) and AF1b (amino acids 451–602) modulated NR (Chen & Evans 1995, Horlein et al. 1995) (Govindan & Warriar 1998, Fuse et al. 2000, Pascual-Le and other coactivators including SRC-2 and SRC-3, both Tallec et al. 2003, Fischer et al. 2010). However, AF-1b has of which have multiple aliases reflecting each laboratory’s been reported to adopt a stable secondary structure and desire to place their imprimatur on the molecule (or more can interact with protein targets in the absence of induced charitably, reflecting the disparate contexts in which each folding (Fischer et al. 2010). A central inhibitory region laboratory identified these molecules). SRC-1, SRC-2 and (amino acids 163–437) has also been described (Pascual-Le SRC-3 comprise the extensively characterized p160 family Tallec et al. 2003) although it may reflect the ability of of coactivators. Coactivators both transduce and integrate MD to recruit corepressors (Fischer et al. 2010). The fact signalling well beyond merely being coactivators of steroid that the regions identified may vary between studies may receptors (York & O’Malley 2010). The coregulators also reflect the intrinsic structural plasticity and therefore serve as docking platforms for further coregulator binding context dependence of the NTD. The NTD also interacts to form larger transcriptional complexes (McInerney et al. with the LBD in an N-C interaction to stabilize receptor 1998). These complexes are recruited to the target gene conformation (Rogerson & Fuller 2003). by the activated, DNA-bound, receptors to enable many The DBD (66 amino acids) is highly conserved. It of the actions needed for gene expression. These include is characterized by two ‘zinc fingers’ each containing a chromatin remodelling, histone modification, initiation of zinc ion tetrahedrally coordinating four cysteine residues. transcription, elongation of RNA chains, RNA splicing and It determines the specificity of DNA recognition and

Endocrinology termination of transcriptional responses (Auboeuf et al. binding, as well as having a role in receptor homo- and of 2007, O’Malley 2007). Corepressors, conversely, reverse hetero-dimerization (Pippal & Fuller 2008). It is linked many of these processes; for instance, while coactivators to the LBD by a hinge region (61 amino acids) that also promote acetylation of histones, coregulators inhibit plays a role in receptor homo-dimerization. The structure Journal gene expression by inducing histone deacetylase activity. of the DBD has been solved using both nuclear resonance There are now over 400 published molecules designated spectroscopy and crystallography for many of the NRs as coregulators (O’Malley 2016) which have been including recently, the MR (Hudson et al. 2014). recognized to play a central role in modulating gene The LBD (251 amino acids), the crystal structure of expression mediated by NR, and are thought to impart which was solved independently by 3 groups in 2005 tissue and ligand specificity to receptor activity Lonard( & (Bledsoe et al. 2005, Fagart et al. 2005, Li et al. 2005), O’Malley 2006). is a complex structure organized in eleven α-helices (labelled by convention 1 to 12; helix 2 is unstructured in the SHR) and four β-strands forming three anti- Structural determinants in the MR parallel layers (Bledsoe et al. 2005, Fagart et al. 2005, Li et al. 2005). It contains a ligand-dependent activation The cloning of the MR confirmed that, as with other function 2 (AF-2) made up of helices 3, 4, 5 and 12. The members of the NR superfamily, the MR has three major LBD interacts with chaperone proteins in the absence functional domains: an N-terminal domain (NTD), a of ligand and undergoes conformational change central DNA-binding domain (DBD) and a C-terminal upon ligand binding such that helix 12 forms a stable ligand-binding domain (LBD) (Fig. 1) (Arriza et al. 1987). interaction that creates a hydrophobic cleft on the The MR-NTD is the longest among all the receptors surface of the LBD (Huyet et al. 2007) which serves as a (602 amino acids) and is the least conserved domain docking platform (AF-2) for transcriptional coregulators with respect to both sequence and structure across the (Fig. 2). Many coactivators bind to the AF-2 region via steroid receptors (Lavery & McEwan 2005). The NTD is a conserved ‘NR-box’ that contains one or more LxxLL considered to be intrinsically disordered in the absence (where L is leucine and x is any amino acid) motifs of other binding proteins, thereby providing structural (Heery et al. 1997, Darimont et al. 1998).

http://joe.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-17-0060 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 03:49:39PM via free access Thematic Review p j fuller and others MR coactivators 234:1 T25

16602 70 984

NH2 NTD DBDHinge LBD COOH

AF1a MD AF1b AF2

Figure 1 Schematic representation of the human mineralocorticoid receptor (MR) structure. The MR, as with other nuclear receptors, can be divided into three principle domains: the N-terminal domain (NTD), the DNA-binding domain (DBD) and the ligand-binding domain (LBD). The DBD 11and LBD are joined by a relatively poorly conserved hinge region. Activation Functions (AFs): 1a, 1b and 2 are indicated as is the putative repressive function termed the middle domain (MD). The amino acid numbers are above the schematic with NH2 and COOH representing the N- and C-termini of the molecule, respectively.

Coregulators for the MR with the AF-2 region of the MR via their LxxLL motifs. p300/CREB-binding protein (CBP) is able to potentiate Although over 400 coregulatory molecules have been MR-induced transcription via both AF-1 and AF-2. identified (O’Malley 2016), only a relatively small number Although a direct interaction with the AF-1 region was have been characterized for the MR (reviewed in Yang & not initially observed (Fuse et al. 2000) for SRC-2, SRC-3 Fuller 2012). Of the relatively ‘generic’ coactivators, SRC-1, and CBP, they were found to bind directly to the MR-NTD SRC-2 and peroxisome proliferator-activated receptor after stabilization of the NTD structure with the natural gamma coactivator 1-alpha (PGC-1α) interact strongly osmolyte trimethyl N-oxide (TMAO) (Fischer et al. 2010). Endocrinology of Journal

Figure 2 Schematic representation of mineralocorticoid receptor (MR)-mediated transactivation. Ligand binding to the MR leads to a conformational change that permits nuclear transfer. Within the nucleus, MR monomers dimerize at the hormone response element (HRE). Coactivators, such as the steroid receptor coactivators (SRC) and p300/CREB-binding protein (CBP), are then recruited, and they interact with the basal transcriptional apparatus to promote expression of the target gene.

Zennaro and coworkers (Zennaro 2001) addressed this mutant MR lacking an AF-2 region. This finding suggests issue using a mutant MR that is unable to form AF-2. They that these coactivator-MR interactions likely involve examined three previously described AF-2 interacting more than just the interaction of the LxxLL motifs in steroid receptor coactivators, SRC-1, RIP140 (140-kDa the coactivator with AF-2 in the MR-LBD; evidence from receptor-interacting protein) and TIF1α (TRIM24: tripartite other systems points to the involvement of the NTD motif containing 24), which were known to interact in an (Zennaro et al. 2001). agonist-dependent fashion with the MR (Hellal-Levy et al. Unlike NR, which are structurally conserved, 2000). Zennaro and coworkers (Zennaro et al. 2001) coregulators are structurally and functionally diverse, identified an interaction of the three coactivators with the and are often recruited in a ligand- and cell type-specific

Table 1 Summary of coregulators that interact with the MR (modified from Yang & Fuller 2012).

Sites of interaction with Coregulator the MR Known functions References Coactivators SRC-1 AF-2; AF-1 by SRC-1e Recruits histone acetylation complex to (Hultman et al. 2005, Meijer et al. 2006, isoform initiate transcription; weak intrinsic histone Wang et al. 2004, Zennaro et al. 2001) acetyltransferase activity. SRC-2, TIF2, AF-1, AF-2 Enhances transactivation. (Fuse et al. 2000, Hong et al. 1997, GRIP1 Wang et al. 2004) p300/CBP AF-1, AF-2 Exerts histone acetyltransferase activity; (Fuse et al. 2000) recruits RNA polymerase II to target gene promoter PGC-1α AF-2 Recruits histone acetyltransferase complex; (Hultman et al. 2005, Knutti et al. 2000) facilitates binding of NR to transcription initiation complex ELL AF-1b RNA polymerase II elongation factor; prevents (Pascual- Le Tallec et al. 2005) premature arrest and transient pausing of Endocrinology polymerase II of FLASH AF-1 Regulates cell apoptosis (Obradovic et al. 2004) FAF-1 AF-1 Regulates cell apoptosis (Obradovic et al. 2004) Ubc9 NTD SUMO E2-conjugating enzyme; forms (Yokota et al. 2007) Journal coactivation complex with SRC-1 TIF1α NTD Transcriptional coactivator / corepressor (Zennaro et al. 2001) RIP140 NTD NR coactivator (Zennaro et al. 2001) (Rogerson et al. 2014) Tesmin LBD Ligand-specific coactivator (Yang et al. 2014) EEF1A1 ? Growth and proliferation (Yang et al. 2014) XRCC6 ? DNA repair Corepressors SMRT LBD Recruits histone deacetylase (Wang et al. 2004) NCoR LBD Recruits histone deacetylase (Wang et al. 2004) DAXX NTD Regulates cell apoptosis; represses MR (Obradovic et al. 2004) transactivation in some cell lines PIAS1 NTD SUMO E3 ligase; exact mechanism of (Pascual-Le Tallec et al. 2003) ? LBD repressive action unclear NF-YC AF-1 Inhibits aldosterone-induced MR N-C (Murai-Takeda et al. 2010) interaction Gemin 4 ? LBD Nuclear organelle associated transcription (Yang et al. 2015) elongation factor SSRP1 ? (Yang et al. 2014)

AF (activation function), CBP (cAMP-response element-binding protein-binding protein), DAXX (death-associated protein), DBD (DNA-binding domain), ELL (eleven-nineteen lysine-rich leukaemia), FLASH (Fas-associated death domain-like IL-1β-converting enzyme -associated huge), FAF-1 (Fas-associated factor 1), GRIP1 (glucocorticoid receptor-interacting protein 1), LBD (ligand-binding domain), MR (Mineralocorticoid receptor), NCoR (nuclear receptor corepressor), NTD (N-terminal domain), PGC1 (peroxisome proliferators-activated receptor gamma coactivator 1), PIAS (protein inhibitor of activated signal transducer and activator of transcription), RHA (RNA helicase A), RIP140 (receptor-interacting protein 140), SMRT (silencing mediator of retinoid and thyroid hormone receptor ), SRC-1(steroid receptor coactivator 1), SUMO-1 (small ubiquitin-related modifier-1), TIF1α (transcriptional intermediary factor 1α), TIF2 (transcriptional intermediary factor 2), Ubc9 (Ubiquitin-like protein SUMO-1 conjugating enzyme), Gem (nuclear organelle)-associated protein 4, eukaryotic elongation factor 1A1 (EEF1A1), structure-specific recognition protein 1 (SSRP1), X-ray repair cross-complementing protein 6 (XRCC6).

manner as demonstrated for the estrogen receptor Corepressors and the MR (ER), androgen receptor (AR), peroxisome proliferators- activated receptor γ (PPAR γ) and a range of other receptors The corepressors nuclear receptor corepressor (NCoR) (Kodera et al. 2000, Kraichely et al. 2000, Bramlett et al. and silencing mediator of retinoid and thyroid hormone 2001, Klokk et al. 2007, McKenna 2011). receptor (SMRT) interact with the MR-LBD via their The crystal structure of the ligand-bound LBD C-terminal corepressor-NR interaction (CoRNR) boxes associated with a LxxLL motif containing coactivator- containing a core I/L-xx-I/V-I motif to attenuate derived peptide was solved for the ER (Brzozowski et al. aldosterone-dependent MR-mediated transactivation by 1997, Shiau et al. 1998) and subsequently for many inducing histone deacetylase activity (Wang et al. 2004). other NRs. Obtaining crystal structures for full- These corepressors were originally identified through length receptors has been more elusive, but structural their role in transcriptional repression of NRs in the information has recently been published for a series of absence of ligand or when bound to synthetic antagonists ligand-activated NR heterodimers, bound to DNA and (Chen & Evans 1995, Horlein et al. 1995). However, coactivator peptides (Khorasanizadeh & Rastiejad 2016). NCoR is recruited by the MR upon aldosterone binding In these structures, the NTD is not however visualized, and not upon binding of spironolactone, the steroidal consistent with its unstructured state (Chandra et al. MR antagonist, or finerenone, a newly synthesized non- 2008). Yi and coworkers (Yi et al. 2015) have used steroidal MR antagonist (Fagart et al. 2010). SMRT and cryoelectron microscopy to visualize the structure of an NCoR serve as key corepressors for many different NRs ER-coactivator complex bound to DNA. This shows that and transcription factors. The amino acids in the core I/L- each ER molecule in the homodimer recruits one SRC-3 xx-I/V-I motif as well as the flanking sequences are critical via AF-2 and that these two SRC-3 bind to different determinants of receptor preference (Hu & Lazar et al. regions of one p300 protein. Using this technology, they 1999). Both corepressors are subjected to extensive mRNA were able to identify AF-1 and provide evidence that it splicing events which produce ‘custom tailored’ variants has a role in SRC-3 recruitment as previously predicted, with unique CoRNR boxes, resulting in different affinities for the various NRs (Goodson et al. 2005). The splice Endocrinology but until now not formally demonstrated. of PGC-1α is a tissue-specific coactivator involved variant that interacts with the MR is not known. in metabolic regulation and energy homeostasis (Knutti et al. 2000). The physiological significance of Journal PGC-1α as a putative coactivator in MR signalling MR-interacting proteins is unclear, especially in the kidney where it is not co-expressed with the MR (Lombes et al. 1990, A series of putative coregulatory molecules that interact Portilla et al. 2002). However, the high level of with the MR (Table 1) are described in detail below. These expression of PGC-1α in brown fat may be relevant have been identified using a range of approaches, which given increasing evidence of a key role of the MR in fundamentally identify a protein-protein interaction. adipocyte differentiation (Armani et al. 2014). Most have used yeast 2-hybrid (Y-2-H) screens in which Hultman and coworkers (Hultman et al. 2005) either the full-length MR or more often a fragment of used a mammalian two-hybrid (M-2-H) system to the MR is expressed in yeast as ‘the bait’ with a cDNA identify further MR-LBD interacting coregulators using library as ‘the prey’. The bait is fused to the GAL4 DBD, 50 coregulator peptides derived from 23 known NR and the prey is fused to the GAL4 activation domain. A coactivators and corepressors. Each peptide contained the productive interaction between bait and prey brings these LxxLL motif (coactivators) or the CoRNR box motif found two elements of the GAL4 transcription factor together in corepressors. In a similar approach, Li and coworkers and allows transcriptional activation. The yeast contains a (Li et al. 2005) screened 38 peptides in an alpha screen GAL4 responsive construct containing the GAL4 response for competitive binding to ligand-bound MR-LBD against elements and a reporter gene LacZ whose product, an established LxxLL motif. Neither study identified β-galactosidase, changes a chromogenic substrate (X-gal) additional MR binding coregulator proteins beyond to a blue colour enabling selection of the yeast clone of those already described. Both studies also sought ligand interest. The system has limitations in that yeast although specificity for the interactions but found no differences similar are not identical to mammalian cells so that ligand between aldosterone and cortisol, the two physiological specificity may not be robust. It is also not 100% efficient MR ligands. so that the entire ‘interactome’ is not captured, and it can

have a high false positive rate as only 1 in 6 of the clones attached to its protein target (Seeler & Dejean 2003). While in the cDNA library will be in frame and in the correct sumoylation of NRs has generally been shown to have a orientation. The M-2-H assay follows the same principles repressive effect on gene expression (Bulynko & O’Malley but assesses the interaction in mammalian cells, and it is 2011), Ubc9 interacts with the MR-NTD to potentiate not used for screening but ideal for more detailed analysis aldosterone-dependent MR transactivation, independent of the interaction. We have also used phage display, a of its sumoylation activity (Yokota et al. 2007). No technique in which the bait is fixed to a solid surface interaction was observed between Ubc9 and the MR-LBD. and the library is incorporated into a cell surface protein Interestingly, SRC-1 is recruited simultaneously and can of the phage. The phage are then washed across a plate synergistically potentiate MR-mediated transcription containing the bait; the adherent phage are eluted and with Ubc9. This observation suggests that Ubc9 mediates the relevant fragment from the library is retrieved. This is its effect in part by binding SRC-1 and recruiting other a powerful technique particularly for identifying surface coactivators, which is supported by the co-localization of interactions; an obvious limitation is that the interaction MR, Ubc9 and SRC-1 in mouse kidney collecting duct cell is in vitro rather than intracellular. Initially, M13 phage nuclei (Yokota et al. 2007). were used, which contained short oligonucleotide Another sumoylation enzyme, protein inhibitor of fragments harbouring a known core sequence, e.g. LxxLL, activated signal transducer and activator of transcription or random sequences. However, all peptides that were 1 (PIAS1), interacts with the MR-NTD but as a corepressor isolated contained the LxxLL motif. Subsequent studies (Pascual-Le Tallec et al. 2003). PIAS1 inhibits aldosterone- have used T7 phage, which express cDNA fragments dependent MR-mediated transactivation. The repressive derived from tissues or cells (Yang et al. 2014). effect of PIAS1 is partly mediated by promoter-dependent sumoylation of the MR; however, PIAS1 contains two overlapping CoRNR boxes, which do not mediate the MR-specific coregulators repression. PIAS1 also contains three LxxLL motifs and has been shown to enhance transactivation mediated by

Endocrinology In view of the high structural conservation in the LBD the AR and GR (Tan et al. 2000). PIAS3, like PIAS1, also of and the finding that AF-2-interacting coactivators such as interacts with the MR-NTD and represses MR-induced SRC-1 appear to interact with most if not all of the steroid transactivation in a neural cell line, but does not affect receptors, various investigators focused on identifying the transcriptional activity of GR (Tirard et al. 2004). Journal putative MR-specific coregulators by looking for NTD The overlap between protein-modifying enzymatic interactions. The MR-NTD, which shares less than 15% activity and coactivation has also been reported for identity with other NRs, has been used as bait in pull- the histone deacetylases (HDACs). Lee and coworkers down and two-hybrid studies (Viengchareun et al. 2007). (Lee et al. 2015) have reported that the class II HDAC, This approach identified the elongation factor ELL HDAC4 mediates an interaction between HDAC3 (eleven-nineteen lysine-rich leukaemia) as an MR-specific and the MR, which coactivates aldosterone-induced, interacting partner, which enhances RNA polymerase MR-mediated transactivation. Obradovic and coworkers II activity (Shilatifard et al. 2003, Pascual-Le Tallec et al. (Obradovic et al. 2004) sought to address the role of 2005). ELL interacts exclusively with the AF-1b region of coregulators in conferring MR vs GR specificity in neurons the MR to potentiate MR-mediated transactivation while where both receptors are responding to the same ligand, conversely repressing GR-mediated transactivation. ELL cortisol. They also used a Y-2-H screen with the NTD as is co-expressed with the MR in the cortical collecting bait with a human brain cDNA library. Although only a duct cells of the human kidney and is upregulated by small number of interacting peptides was identified which aldosterone (Pascual-Le Tallec et al. 2005). The ability of did not overlap with previous studies, they did identify ELL to differentially regulate the MR and GR may thus three proteins, DAXX (death-associated protein), FLASH be particularly relevant in those tissues that coexpress the (FLICE-associated huge) and FAF-1 (Fas-associated factor two receptors. 1), previously characterized for their role in Fas receptor- Yokota and coworkers (Yokota et al. 2007) found that mediated apoptosis. In a transactivation assay using a ubiquitin-like protein SUMO-1 conjugating enzyme (Ubc9) hippocampal cell line, DAXX corepressed both MR- and potentiates aldosterone-dependent MR transactivation. GR-mediated, ligand (aldosterone and dexamethasone, Ubc9 is involved in the sumoylation process whereby respectively)-dependent transactivation, whereas FLASH SUMO-1 (small ubiquitin-related modifier-1) is covalently coactivated both MR and GR. FAF-1 (Fas-associated

factor 1) weakly coactivated MR but not GR-mediated cortisol equivalently (Arizza et al. 1987). Subsequently, transactivation. The results were however different when in 1988, the distinction ‘mineralocorticoid’ vs ‘type these assays were repeated in a neuroblastoma cell line, 1 corticosteroid’ was found to be conferred at a tissue highlighting the importance of cellular context. level by the enzyme 11β hydroxysteroid dehydrogenase Nuclear transcription factor Y subunit gamma (NF-YC) type 2 (HSD2) (Edwards et al. 1988, Funder et al. 1988). was identified in a Y-2-H screen using full-length MR as bait In epithelial tissues, the vasculature and discrete (Murai-Takeda et al. 2010) as a specific corepressor of the subpopulations of hypothalamic neurones (Geerling & MR. NF-YC, a subunit of the heterotrimeric transcription Loewy 2009) where HSD2 is co-expressed with the MR, factor NF-Y, recognizes a CCAAT box motif found in the HSD2 confers aldosterone specificity by virtue of its ability RNA polymerase II initiation site in many eukaryotic to convert cortisol to its inactive metabolite cortisone (or promoters and activates transcription (Nakshatri et al. corticosterone to 11-dehydrocorticosterone) (Odermatt 1996). It interacts with the MR-NTD and represses MR & Kratschmar 2012). The MR is however expressed in a transactivation in a dose- and agonist concentration- diverse range of non-epithelial tissues where it plays a dependent manner. NF-YC is selective for the MR with fundamental role in cellular function and pathology; in no effect on GR, AR or PR-mediated transactivation. many of these tissues, there is little or no HSD2, so the Endogenous NF-YC is recruited to the MR response MR is undoubtedly acting as a receptor for cortisol (Fuller element of the human ENaC gene promoter a short time & Young 2005). In epithelial cells and in vascular smooth after MR and SRC-1 recruitment, suggesting that it may muscle cells, when HSD2 is blocked, both aldosterone and limit the transactivation induced by coactivators. cortisol act as MR agonists (Young et al. 2003, Wilson et al. These studies therefore provide compelling evidence 2009). In non-epithelial cells such as neurons and that for a number of these coregulatory molecules, there cardiomyocytes, glucocorticoids can antagonize the effects are important differences in their interactions between of aldosterone (Gomez-Sanchez et al. 1990, Sato & Funder the MR and the GR. In tissues such as the hippocampus 1996, Mihailidou 2006). These observations, together or in macrophages where both receptors are abundant with evidence of differences between the interaction of

Endocrinology and co-expressed, respond to the same ligand (cortisol aldosterone and cortisol with the MR in ligand binding of or corticosterone) and potentially interact with the same (Rogerson et al. 1999, Rogerson et al. 2007) and the response elements in the same genes, coregulators may be N-C interaction (Rogerson & Fuller 2003, Pippal et al. important determinants of the distinct, receptor-specific 2009) supports the concept of tissue- and ligand-specific Journal integrated cellular responses to ligand. Changes in interactions. It follows that ligand-discriminant activation coregulator profiles and/or coregulator activation status of the MR may be mediated by differential coregulator may integrate other signalling pathways that interact interactions, as is well described for the selective estrogen with corticosteroid signalling (Fuller & Young 2016). receptor modulators (SERM), tamoxifen and raloxifene (Shang & Brown 2002). Rogerson and coworkers (Rogerson et al. 2014) Ligand- or cell-specific MR coactivators described the first ligand-specific coactivator for the MR. This study was also based on a Y-2-H screen, but The MR was originally identified as the type 1, in this, they used the MR-LBD as bait (Rogerson et al. corticosteroid receptor (Feldman et al. 1973) reflecting the 2014). Several of the interactions represented known sometimes overlooked fact that both the MR and the GR coactivators, including SRC-1 and PGC-1α. The majority bind cortisol (corticosterone in rodents) although only of interactions identified were seen with both aldosterone the MR binds aldosterone (Rogerson et al. 1999). Prior to and cortisol, but Y-2-H clones that appeared to be ligand the cloning of the MR, there remained uncertainty as to discriminant were identified and one has been fully whether there was a renal, ‘aldosterone-preferring’ MR characterized. This Y-2-H clone encoded a fragment of and a ‘hippocampal cortisol-preferring’ MR. An earlier tesmin or metallothionein-like 5 (MLT5), which is a 508 study by the late Zig Krozowski working with John Funder amino acid protein with a cysteine-rich region that binds (Krozowski & Funder 1983) had provided compelling zinc. This clone showed aldosterone specificity in the Y-2-H evidence that the ‘two’ MR without cellular factors were the assay; an interaction was also observed in the presence same, a conclusion that was unambiguously confirmed by of cortisol in a M-2-H assay, but the M-2-H interaction the cloning of the MR, which enabled the demonstration was 12-fold more active in the presence of aldosterone in vitro that the cloned MR bound both aldosterone and than cortisol. When full-length tesmin was analysed

in a transactivation assay, it was found to coactivate uniquely transcriptionally active in H9c2 cells. SSRP1 aldosterone-induced MR-mediated transactivation but displayed distinct cell-specific effects as it repressed not cortisol-induced MR-mediated transactivation; the MR-mediated transactivation in the HEK293 cells but interaction was antagonized by spironolactone. Tesmin selectively upregulated aldosterone-induced MR activity and MR were co-immunoprecipitated in the presence in the H9c2 cells. Gene expression studies of three MR of aldosterone but not cortisol arguing for a direct target genes, GILZ, SGK1 and CNKSR3, also revealed cell- interaction; inactivation of the AF-2 region in the LBD and gene-specific differences. Overexpression of EEF1A1 inhibited the interaction, again consistent with a direct enhanced transcription of all three MR target genes in LxxLL-mediated interaction. Tesmin contains two LxxLL H9c2 cells, but its effect in HEK293 cells was limited to motifs; mutation of the two LxxLL motifs eliminated the CNKSR3 expression. XRCC6 increased GILZ and CNKSR interactions in the co-immunoprecipitation, M-2-H and but not SGK1 expression in HEK293 cells, and had no transactivation assays. The actual structural determinants effect in H9c2 cells. Possible mechanisms for these specific of the coactivation per se remain to be determined effects are postulated below. The biologic function of each although clearly the LxxLL motifs are essential for the coactivator in the setting of MR activation remains to be primary interaction. The structural basis of the ligand elucidated although each has known functions in addition discrimination, aldosterone vs cortisol, also remains to be to receptor coactivation: EEF1A1 plays an important role determined. in growth, cellular proliferation, signal transduction In parallel studies using recombinant full-length and nuclear export (Becker et al. 2013); SSRP1 acts as a MR (Clyne et al. 2009) with phage display, Yang and transcription elongation factor; and XRCC6 plays a role coworkers (Yang et al. 2011) identified novel, LxxLL in specific pathways for DNA repair (Boulton & Jackson motif-constrained, 19-mer peptides that interact with the 1998, Winkler & Luger 2011). MR in a ligand-dependent manner. A unique consensus binding motif, MPxLxxLL, was demonstrated in peptides that displayed robust interactions with the MR in M-2-H Gene specificity

Endocrinology assays (Yang et al. 2011). Sequence alignment of the of consensus motif within a protein database identified Gem The nature of the coactivator complex assembled by an (nuclear organelle)-associated protein 4 (GEMIN4) as a activated DNA-bound NR complex in a given cell type potential molecular partner of the MR (Yang et al. 2015). Journal will vary with the response element and its context. Gene- When cotransfected with the MR, Gemin4 repressed specific effects of coregulators for NRs are well described agonist-induced, MR-mediated transactivation in a cell- (O’Malley 2003, Won Jeong et al. 2012). This may reflect specific manner. This repressive effect was observed in a changes in receptor conformation induced by distinct human embryonic kidney (HEK293) cell line, but not in DNA response elements, which may in turn alter the a rat cardiomyocyte (H9c2) cell line, again highlighting binding of coactivators (Wood et al. 2001, Klinge et al. the importance of cellular context for coregulator 2004). Pascual-Le Tallec and coworkers (Pascual-Le function (Yang et al. 2015). Gemin4 also displayed Tallec et al. 2003) reported promoter dependence for gene specificity in its corepressive activity whereby its MR corepression by PIAS1, and Yang and coworkers co-expression significantly decreased the expression of (Yang et al. 2014, 2015) reported gene-specific effects for four well-characterized, endogenous MR target genes GEMIN4, XRCC6, EEF1A1 and SSRP1. These differences (Yang et al. 2015). may be mediated by other transcription factors including In a study using T7-phage cDNA libraries derived so-called pioneer/licensing factors such as FOXAI which from either human heart or kidney RNA and full-length have been extensively characterized for steroid receptors human MR as bait, Yang and coworkers (Yang et al. 2014) particularly the AR (Pihlajamaa et al. 2015). Large-scale identified several additional putative coregulators that gene expression profiling and coactivator recruitment differ between cell type, ligand and promoter in their assays for novel progesterone receptor modulators coregulation of the MR response. These include eukaryotic have also found that regulation is highly gene specific elongation factor 1A1 (EEF1A1), structure-specific despite a common coactivator recruitment profile for recognition protein 1 (SSRP1) and X-ray repair cross- certain compounds (Berrodin et al. 2009). This level complementing protein 6 (XRCC6). XRCC6 selectively of detail has not yet been achieved for MR biology, enhanced cortisol-induced MR transactivation of three and indeed cistromic information remains limited for promoter constructs in HEK293 cells, while EEF1A1 was the MR. A recent study from Le Billan and coworkers

(Le Billan 2015) did not identify an association of determine whether an ER ligand is agonist (estradiol), mineralocorticoid response elements (MRE) with binding a SERM (tamoxifen) or an antagonist (fulvestrant). The motifs for other transcription factors, although these were MR is an important therapeutic target in cardiovascular found in putative binding sites that lacked MRE, a finding disease, but the current MR antagonists are limited by of uncertain significance. their lack of tissue specificity resulting in hyperkalaemia (Juurlink et al. 2004) due to concurrent renal MR blockade. Antagonists that block the MR in cardiovascular tissues, Tissue specificity but not in the distal nephron, would seem highly desirable, but have yet to emerge. As the role of the MR As noted above, the cellular repertoire of coregulatory in an expanding range of tissues becomes more fully molecules will determine the cellular response to elucidated, it is tempting to speculate that new MR activation of the MR. Similarly, the impact of a given ligands may emerge, selective MR modulators, which coregulatory molecule may be modified by this cellular exploit the ligand- and tissue-specific MR coactivators milieu as, for instance, in the above studies of Obradovic to provide tissue specificity, not just in cardiovascular and coworkers (Obradovic et al. 2004) and Yang et al. disease but across a range of conditions. (2014, 2015) which highlight a clear context dependence for the transactivation assay. Conversely, tesmin shows equivalent ligand-specific coactivation of the MR in two disparate cell lines. Some of the studies discussed have Declaration of interest The authors declare that they have no conflict of interest. been confined to one cell line so that no conclusions can be drawn. Ultimately it will be critical to understand the role of coregulatory molecules in vivo, particularly for the MR where appropriate cellular model systems with Funding endogenous MR expression are generally lacking (Fuller This work is supported by grants-in-aid from the National Health & Medical Research Council of Australia through a Project Grant (#1058336) & Young 2014). and a Senior Principal Research Fellowship to PJF (#1002559); The Endocrinology Hudson Institute is supported by the Victorian Government’s Operational of Infrastructure Scheme. Conclusions Journal

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Received in final form 10 April 2017 Accepted 3 May 2017