Fraser M. Rogerson

Interdomain interactions in the mineralocorticoid receptor

The potential for interaction between the N-terminal domain and the C-terminal region (hinge and ligand-binding domain) of the mineralocorticoid receptor (MR) was examined using the mammalian-2-hybrid assay. The MR C-terminal region was fused to the GAL4 DNA-binding domain (GAL4-MRC). To examine if the AF-2 is involved in the interaction, as has been reported for other steroid hormone receptors, it was inactivated by point mutation (E962A). The N-terminal domain was fused to the VP16 transactivation domain (VP16-MRNT). In the mammalian-2-hybrid assay both GAL4-MRC and GAL4-MRC(E962A) interact with VP16-MRNT in an aldosterone-dependent manner. The GAL4-MRC(E962A) construct was used in subsequent experiments to examine the AF-2-independent N/C-interaction. The MR antagonist spironolactone inhibits the aldosterone-mediated association of the two domains. GAL4-MRC(E962A) interacts weakly with the GR or AR N-terminal domains in the presence of aldosterone. No dimerization between GAL4-MRC(E962A) and VP16-MRC is observed. Interestingly, cortisol produces a much weaker N/C-interaction than aldosterone, and it is possible that the N/C-interaction may contribute to observed functional differences in the MR bound to the two ligands.

Structural Determinants of Aldosterone Binding Selectivity in the Mineralocorticoid Receptor

The structural determinants of aldosterone binding specificity in the mineralocorticoid receptor (MR) have not been determined. The MR has greatest sequence identity with the better characterized glucocorticoid receptor (GR), which is reflected in their overlapping ligand binding specificities. There must be subtle sequence differences that can account for the MR-specific binding of aldosterone and the shared binding of cortisol. To characterize ligand binding specificity, chimeras were made between the human MR and GR ligand-binding domains (LBDs). Three points were chosen as break points to generate a total of 16 different constructs. These chimeric LBDs were placed in a human GR expression vector containing the GR DNA-binding and N-terminal domains and assayed by co-transfection into CV-1 cells with the mouse mammary tumor virus-luciferase reporter plasmid. Binding of [3H]aldosterone and [3H]dexamethasone was also measured. All of the constructs that are potently activated by aldosterone contain amino acids 804–874 of the MR. The results of the ligand binding experiments using [3H]aldosterone were consistent with the transactivation assay. Cortisol activation of the chimeras was surprisingly complex. Constructs that are activated by cortisol contain either amino acids 804–874 and 932–984 of the MR or amino acids 598–668 and 726–777 of the GR. However, all of the chimeras retained the ability to bind the synthetic glucocorticoid [3H]dexamethasone, and cortisol was able to displace [3H]dexamethasone binding, suggesting that the differential effects of cortisol on transcriptional activation are caused by an effect that occurs downstream of ligand binding. These results identify a subregion of the MR LBD that confers specificity of aldosterone binding, which contrasts with cortisol binding where differential effects between chimeras appear to be mediated by interactions distal to ligand binding.

Mineralocorticoid receptor binding, structure and function.

The isolation of aldosterone 50 years ago was a critical first step in elucidating the mechanism by which corticosteroids regulate electrolyte homeostasis. The broad principles of this mechanism involving an intracellular receptor acting on specific genes to induce the expression/repression of aldosterone-induced proteins (AIP) were established 30 years ago. The cloning of the mineralocorticoid receptor (MR) has enabled studies of the subcellular mechanisms of aldosterone action, including the molecular dissection of structure-function relationships in the receptor. We have exploited the close structural and functional similarity of the MR with the glucocorticoid receptor to identify the regions in the MR that confer ligand-binding specificity. The critical region is located, not as might be expected in the ligand-binding pocket but rather on the surface of the molecule. These studies have been extended to an analysis of the interactions between the N-terminal and ligand-binding domains of the MR. In the last decade, AIP have been identified; the regulation of the genes encoding these AIP are discussed.

Structural and functional characterization of the interdomain interaction in the mineralocorticoid receptor.

The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterize the MR N/C-interaction. By using a range of natural and synthetic MR ligands in a mammalian two-hybrid assay we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone and cortisol weakly promote the interaction but predominantly inhibit the aldosterone-mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonize the interaction. In contrast, the synthetic agonist 9alpha-fludrocortisol robustly induces the interaction. The ability of the N/C interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C interaction is not cell specific, consistent with the evidence from a glutathione-S-transferase pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands, which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR that has been reported in vivo.

Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor

1.u2002The importance of mineralocorticoid receptor (MR) antagonists in the treatment of cardiovascular disease has been emphasised by two recent clinical trials, one using spironolactone and the other using a new selective MR antagonist, namely eplerenone.

Cortisol resistance in the New World revisited

Insights into the molecular basis of glucocorticoid action have been obtained from the analysis of cortisol resistance. The glucocorticoid receptor (GR) in both New World primates and guinea pigs has a decreased affinity, in vivo, for cortisol; this is achieved by two distinct mechanisms. In the New World primates recent studies have identified a key role for co-chaperones. The amino acids responsible for cortisol resistance in the guinea pig GR lie not in the ligand-binding pocket but on the surface of the receptor. We hypothesize that this region might be the site of interaction between the co-chaperones and the GR, and hence that the resistance occurs through the same mechanism, albeit from opposite sides.

Dissecting mineralocorticoid receptor structure and function.

The molecular mechanisms by which aldosterone regulates epithelial sodium transport in the distal colon and the distal nephron remain to be fully elucidated. Aldosterone acts via the mineralocorticoid receptor (MR) to induce the expression of genes whose products are involved in sodium transport. The structural basis of MR interactions with aldosterone has been examined by creating chimeras of the MR and the closely related glucocorticoid receptor; we have exploited differences in ligand-binding specificity to determine the region(s) of the MR that confer aldosterone-binding specificity. These findings have been related to a three-dimensional model of the MR based on the crystal structure of the progesterone receptor. These studies have been extended to include the characterisation of interactions between the N- and C-termini of the MR. We have characterised six genes that are regulated in vivo in the distal colon and/or kidney of the rat that are directly and acutely regulated by aldosterone administration: the three subunits of the epithelial sodium channel, serum and glucocorticoid-induced kinase, channel-inducing factor and K-ras2A. These studies provide insights into the molecular pathways that mediate aldosterone-induced amiloride-sensitive epithelial sodium transport.

Identification and characterization of a ligand-selective mineralocorticoid receptor coactivator

The mineralocorticoid receptor (MR) is unique in responding to 2 physiological ligands: aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11 β‐hydroxysteroid dehydrogenase type 2. In other tissues, cortisol is the primary ligand. To understand the structural determinants of ligand‐specific MR activation, we sought to identify coregulatory molecules that interact with the ligand‐binding domain (LBD) of the MR. A yeast‐2‐hybrid (Y2H) kidney cDNA library was screened with the human MR‐LBD in the presence of aldosterone and cortisol. One clone, identified as aldosteronespecific in the Y2H assay, exhibited a 7‐fold greater response, aldosterone vs. cortisol, in a mammalian‐2‐hybrid (M2H) assay. This clone encodes the region of the tesmin gene that has 2 leucine‐x‐x‐leucine‐leucine (LxxLL) motifs. Full‐length tesmin coactivates (>2‐fold) MR‐mediated transactivation in the presence of aldosterone, but not of cortisol; this specificity is observed with a range of promoters. GST pulldown and coimmunoprecipitation of the MR by tesmin supports a direct interaction, mediated by the 2 LxxLL motifs. Tesmin thus represents a novel MR coregulator that exhibits a differential interaction, providing further evidence of the adoption of ligand‐dependent conformations by the MR‐LBD.—Rogerson, F. M., Yao, Y.‐Z., Young, M. J., Fuller, P. J., Identification and characterization of a ligand‐selective mineralocorticoid receptor coactivator. FASEB J. 28, 4200‐4210 (2014). www.fasebj.org