The Ligand-Dependent Interaction of Mineralocorticoid Receptor with Coactivator and Corepressor Peptides Suggests Multiple Activation Mechanisms

Monica L. Hultman, Nataliia V. Krasnoperova, Suzhen Li, Sarah Du, Chunsheng Xia, Jessica D. Dietz, Deepak S. Lala, Dean J. Welsch and Xiao Hu

St. Louis Laboratories, Biological Sciences, Pfizer Global Research & Development, St. Louis, Missouri 63017

Address all correspondence and requests for reprints to: Xiao Hu, Ph.D, Mail Zone AA3G, Pfizer Global Research and Development, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017. E-mail: Xiao.Hu{at}pfizer.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We investigated the coregulator (coactivator and corepressor) interactions with the mineralocorticoid receptor (MR) that lead to activation and inhibition of the receptor in the presence of agonist and/or antagonist. Our results indicate that MR ligand binding domain (LBD) interacts strongly with only a few specific coactivator peptides in the presence of the agonist aldosterone and that these interactions are blocked by the antagonist eplerenone. We also discovered that cortisol, the preferred physiological ligand for the glucocorticoid receptor in humans, is a partial MR agonist/antagonist, providing a possible molecular explanation of the tissue-selective effects of glucocorticoids on MR. However, when we examined the coactivator and corepressor peptide interactions in the presence of cortisol, we found that MR bound with cortisol or aldosterone interacted with the same set of peptides. Thus, the partial agonism shown by cortisol is unlikely to be the result of differential interaction with known coactivators and corepressors. On the other hand, we have identified coactivator binding groove mutations that are critical for cortisol activation but not for aldosterone activation, suggesting that the two steroids induce different MR LBD conformations. In addition, we also show that cortisol becomes full agonist when S810L mutation is introduced in the LBD of MR. Interestingly, MR antagonists, such as eplerenone and progesterone, become partial agonist/antagonist of S810L but are still able to recruit LXXLL peptides to the mutant receptor. Together, these findings suggest a model to explain the MR activation by various ligands.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE RENIN-ANGIOTENSIN-aldosterone system is the major hormonal system that regulates blood pressure by controlling salt and water homeostasis. Aldosterone, through binding to mineralocorticoid receptors (NR3C2), promotes renal sodium reabsorption and potassium secretion in the distal nephron, distal colon, and salivary and sweat glands. Reabsorption of sodium and water then elevates blood pressure indirectly by expanding intravascular volume. Aldosterone can also regulate blood pressure by actions in the brain and directly on the vascular wall. Intracerebroventricular infusion of a selective MR antagonist, at doses that are ineffective when administered systemically, inhibits the development of the hypertension produced by the sc infusion of aldosterone or deoxycorticosterone in normotensive rats (1). Recent evidence also indicates that inappropriate levels of aldosterone, in the presence of moderate to high salt, mediate significant damage in nonepithelial tissues. Examples include endothelial dysfunction, vascular inflammation, and myocardial fibrosis (2).

MR antagonists, such as spironolactone, have been used to treat hypertension. However, spironolactone is not very selective and blocks androgen receptors and activates progesterone receptors, causing unwanted antiandrogenic and progestational side effects that limit its use. These sex hormone-related side effects have been eliminated with the more selective MR antagonist, eplerenone (3). Clinical trials have demonstrated the beneficial effects of eplerenone in the treatment of hypertension (4, 5, 6, 7). In these trials, eplerenone, either alone or in addition to other standard therapy, has been demonstrated to effectively lower blood pressure in hypertensive patients, suggesting that eplerenone is also useful as an add-on therapy. The recently completed Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) trial indicated that the addition of eplerenone to optimal medical therapy results in improved survival and reduced hospitalization among patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure (8).

MR has highest homology to glucocorticoid receptor (GR) in terms of primary amino acid sequence. Both aldosterone and cortisol can bind to MR, with cortisol having approximately 30-fold higher affinity for MR than for GR. In vitro, both aldosterone and cortisol activate MR. In epithelial cells in vivo, MR are protected from cortisol activation by 11ß-hydroxysteroid dehydrogenase (HSD2), the enzyme that converts cortisol into inactive cortisone. Inactivation of 11ß-HSD2, as occurs in the syndrome of apparent mineralocorticoid excess, allows cortisol to function as an MR agonist to increase sodium reabsorption. In some of the nonepithelial tissues, including the heart and the specific regions of the brain, 11ß-HSD2 is not coexpressed with MR so that MR is unprotected. Unprotected MRs are presumably occupied by the much higher level of circulating glucocorticoids and in these circumstances, glucocorticoids antagonize MR (9, 10, 11). Thus, glucocorticoids, such as cortisol, are tissue-selective modulators of MR function.

The ligand binding domain (LBD) of nuclear receptor contains the ligand-dependent activation domain. Agonist-occupied nuclear receptors recruit coactivator proteins to increase transcription and a number of coactivator proteins have been identified (Ref. 12 and references therein). These coactivator proteins interact with nuclear receptors via small peptide motifs called nuclear receptor (NR)-boxes that contain an LXXLL sequence in an {alpha}-helical structure and bind the coactivator binding groove formed by helices 3–5 and 12 of the LBD (13, 14). Among these coactivators, three so-called p160 family coactivators, steroid receptor coactivator (SRC) 1 (NCoA1), GR-interacting protein (GRIP) 1 [NCoA2, TIF2, SRC2], and amplified in breast cancer (AIB) 1 NCoA3, ACTR, P/CIP, RAC3, SRC3) have been studied extensively. Two of these coactivators, SRC1 and GRIP1, have been shown to interact with MR in an agonist-dependent manner in yeast two-hybrid assay (15). The p160 family coactivators are generally ubiquitously expressed and can interact with most nuclear receptors. The roles of other, more tissue-specific coactivators as well as corepressors in MR activation and repression remain to be defined.

In this study, we performed detailed analysis of coactivator and corepressor interactions in response to various ligands using the MR LBD. We discovered that cortisol functions as a partial agonist/antagonist of MR, thus providing mechanistic clues to the tissue-specific effects of cortisol on MR. We also found that cortisol fully activates the MR S810L mutant and MR antagonists, such as eplerenone, partially activates the S810L mutant. Our results suggest a model to explain the MR activation in response to various ligands.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coactivators and corepressors interact with nuclear receptors through NR-box motifs and CoRNR box motifs (13, 16). These motifs are necessary and sufficient to interact with nuclear receptors. To gain a thorough understanding of the interaction of MR with coactivators and corepressors, we used a mammalian two-hybrid assay in human embryonic kidney 293 cells to detect the interaction of MR LBD with peptides containing the NR-box or CoRNR-box motifs. We constructed a panel of 50 coregulator peptide fusions with Gal4 DNA binding domain. These coregulator peptides are derived from 23 known coactivators and corepressors.

The complete interaction profile is summarized in Table 1Go, with Fig. 1AGo showing the results of peptides from p160 family and peroxisome proliferator-activated receptor {gamma} coactivator (PGC1) family coactivators. Overall, only a few peptides showed strong interactions with the MR LBD in the presence of aldosterone. These include peptides from coactivator SRC1, activating signal cointegrator 2 (ASC2), PGC1{alpha}, and PGC1ß (17, 18, 19, 20). One common feature of these strongly interacting peptides is the presence of an extra L before the LXXLL motif in NR-box (LLXXLL, Fig. 1BGo). However, not all LLXXLL containing peptides are capable of strong interactions with MR LBD because SRC1–3, GRIP1–3, and AIB1–3 all have LLXXLL motifs but fail to interact with MR LBD.


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Table 1. Summary of MR LBD and Coactivator and Corepressor Peptide Interactions in Mammalian Two-Hybrid Assay

 


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Fig. 1. The Interaction of MR LBD with Coactivator/Corepressor Peptides in the Presence and Absence of Aldosterone

A, Mammalian two-hybrid interaction of MR LBD with p160 family and PGC1 family coactivator peptides. "Relative Luc Activity" is relative to the basal activity of the vector (Gal4). B, The sequence of coactivator peptides that have strong interactions with MR LBD. C, GST-MR LBD interaction with selected peptides in TR-FRET assay. Left, Interaction assay was done with 100 nM peptide. Vehicle is dimethyl sulfoxide (DMSO). Aldosterone is at 10 nM. Right, TR-FRET assay was done with various concentrations of peptides in the presence of aldosterone. The estimated EC50s and 95% confidence intervals (CI) based on the maximal interaction of each individual peptide are 132 nM (CI: 79–219 nM), >1000 nM, 116 nM (CI: 59–226 nM), 4 nM (CI: 3–5 nM) and 7 nM (CI: 5–8 nM), respectively, for SRC1–1, SRC1–2, SRC1–3, SRC1–4a, and PGC1a.

 
One of these strongly interacting peptides SRC1–4a is present only in one SRC1 isoform (SRC1a) but not in another (SRC1e) (17). Interestingly, even in the SRC1a isoform, there is a polymorphism that changes LLQQLL into LRQQLL (21), and a peptide derived from this polymorphism (SRC1–4b) does not interact with MR (Table 1Go). As a control, peptides that do not interact with MR LBD are able to interact with other nuclear receptors (data not shown). In addition, coactivator interaction domain fragments containing multiple LXXLL motifs, such as those from SRC1e, GRIP1 and AIB1, did not shown enhanced interaction with MR LBD (data not shown), suggesting that single peptide mimics the entire interaction domain of coactivators.

To confirm the interactions we observed in the mammalian two-hybrid system, we used a glutathione-S-transferase (GST) fusion of MR LBD (GST-MRLBD) to detected interactions with individual peptides in a time resolved-fluorescent resonance energy transfer (TR-FRET) based biochemical assay. As shown in Fig. 1CGo, left panel, at 100 nM peptide concentration, SRC1–4a and PGC1a (a peptide fragment from coactivator PGC1{alpha}) interact strongly with GST-MRLBD. Under the same conditions, SRC1–1 and SRC1–3 show very weak interactions, and SRC1–2 does not interact with GST-MRLBD. The relative affinity of each peptide was also measured by varying the peptide concentrations. As clearly shown in Fig. 1CGo, right panel, SRC1–4a and PGC1{alpha} not only have higher affinity for GST-MRLBD but also show higher maximal binding than other peptides tested. Detectable interactions can be observed with as low as 1 nM SRC1–4a or PGC1a peptide, with an estimated EC50 of 4 nM and 7 nM, respectively. In contrast, much higher concentrations (>100-fold) of SRC1–1 or SRC1–3 peptide are required to show comparable interaction with GST-MRLBD. We also found a very weak SRC1–2 peptide interaction at the highest concentration tested. Moreover, peptides from GRIP1 and AIB1 show very weak interactions with the MR LBD in the TR-FRET assay (data not shown). By comparison, no detectable interactions were observed between these peptides and MR LBD under the assay conditions in the mammalian two-hybrid system (Fig. 1AGo). Overall, these TR-FRET results are generally consistent with the mammalian two-hybrid data, except that the TR-FRET assay is much more sensitive and can detect weak interactions.

Having established which cofactor peptides interact with MR LBD, we next examined the ligand dependence of these interactions. As shown in Fig. 2Go, aldosterone recruits SRC1–4a and PGC1{alpha} peptides in a dose-dependent fashion in cells (Fig. 2AGo) and in the test tube (Fig. 2BGo). The EC50 of aldosterone is around 0.5 nM, which is consistent with the EC50 of MR activation and the dissociation constant of aldosterone (22). These results indicate that the aldosterone-dependent conformational change in the LBD of MR allows recruitment of coactivator peptides.



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Fig. 2. Dose-Response Curves of Aldosterone Induced MR LBD Interaction with SRC1–4a and PGC1a Peptides

A, In mammalian two-hybrid assay. B, In TR-FRET assay.

 
The MR-selective antagonist eplerenone completely blocks aldosterone-induced Gal4-MR LBD transcription activity (Fig. 3AGo). As expected, eplerenone by itself does not recruit any coactivator peptides (Fig. 3BGo and data not shown). However, it does block aldosterone-recruited coactivator peptides and does so in a dose-dependent fashion (Fig. 3BGo, right panel). It should be pointed out that every peptide recruited by aldosterone can be blocked by eplerenone. Moreover, eplerenone does not actively recruit any corepressor peptides from nuclear receptor corepressor (N-CoR) or silent mediator of retinoic acid and thyroid receptor (SMRT) (Fig. 3CGo and data not shown), indicating that eplerenone changes the conformation of MR LBD to a neutral state that recruits neither coactivators nor corepressors.



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Fig. 3. Eplerenone Blocks Coactivator Peptide Interactions and Does Not Recruit Corepressors

A, Eplerenone dose-dependently blocks aldosterone-induced activation on Gal4-MR LBD. The transcriptional activity in the absence of eplerenone, but presence of aldosterone is set at 100%. B, Eplerenone blocks coactivator peptide interaction in mammalian two-hybrid assay. Left, Eplerenone bocks interaction with each individual peptide. Right, Eplerenone blocks interaction with SRC14a peptide in a dose-dependent fashion. C, Eplerenone does not recruit corepressors N-CoR. N-CoRID, The C-terminal receptor interaction domain of N-CoR. Aldosterone concentration is at 1 nM and eplerenone concentration is at 10 µM. Fold interaction is the activity in the presence of ligand divided by that in the absence.

 
Glucocorticoids and mineralocorticoids can bind both GR and MR, and glucocorticoids can either be an agonist or antagonist of MR in different tissues. To further investigate the effect of glucocorticoids via MR, we examined the effects of cortisol on the transcriptional activity of Gal4-MR LBD. Figure 4AGo indicates that both aldosterone and cortisol activate MR LBD with EC50 of each ligand very close to the reported dissociation constant (22). However, the maximal inductions of aldosterone and cortisol are different. Cortisol induces a lower level of activation at saturating concentrations. This difference is not due to limited access of the receptor by cortisol because cortisol can partially antagonize the effect of aldosterone. Cortisol can be converted to inactive cortisone by enzyme 11ß-HSD2. Cortisone, on the other hand, may act as an MR antagonist as has been shown for 11-dehydrocorticosterone, the major metabolite of the rodent glucocorticoid, corticosterone (23). One possible explanation of the partial activity is that cortisone contributes to the reduced activity when cells were incubated with cortisol. Although 11ß-HSD2 is not present in 293 cells at significant levels, traces of 11ß-HSD2 could complicate our interpretation. To completely rule out the possibility that the partial activity of cortisol on MR is due to conversion of some cortisol by 11ß-HSD2 into cortisone in 293 cells, we compared the activity of cortisol in the absence and presence of 11ß-HSD2 inhibitor carbenoxolone. As shown in Fig. 4BGo, carbenoxolone has no effect on the activity of cortisol on MR. Moreover, structurally related synthetic glucocorticoids such as prednisolone and dexamethasone are also partial MR agonists/antagonists (data not shown). Together, these results suggest that cortisol is a bona fide partial MR agonist/antagonist.



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Fig. 4. Cortisol Is a Partial MR Agonist/Antagonist

A, Dose response curves of the effects of cortisol on Gal4-MR LBD in the absence (left) and presence of 1 nM aldosterone (right). Aldosterone curve is included for comparison. B, The partial activity of cortisol is not due to inactivation by 11ß-HSD2. The partial effects of cortisol (1 µM) are assayed in the absence or presence of 11ß-HSD2 inhibitor carbenoxolone (10 µM).

 
We hypothesize that the partial agonistic/antagonistic activity of cortisol may be due to differential interactions with coactivators or corepressors. We then tested coactivator and corepressor peptide interactions in the presence of cortisol. To our surprise, we did not see any qualitative differences in coactivator and corepressor peptide interactions. The peptides that interact with MR in the presence of aldosterone can also be recruited by cortisol in both the mammalian two-hybrid assay (Fig. 5AGo) and the TR-FRET recruitment assay (Fig. 5BGo). In addition, the peptides that showed no interaction with MR LBD in the presence of aldosterone are also not recruited by cortisol (data not shown).



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Fig. 5. Cortisol Interacts with the Same Set of Coactivator Peptides as Aldosterone but Requires Different Residues for Activation

A and B, Comparison of aldosterone and cortisol-induced coactivator peptide interactions in mammalian two-hybrid assay (A) and in TR-FRET assay (B). Peptide concentration is at 100 nM. Fold interaction is the activity in the presence of ligand divided by that in the absence. C, Positions of mutations. *, STOP codon at residue 957 of human MR. D, Effects of aldosterone and cortisol on the transcriptional activity of Gal4-MR LBD mutations. Note the scale difference of y-axis in the left and right portion of the graph. Aldosterone is at 10 nM, and cortisol is at 1 µM. E, Cortisol is an antagonist of Gal4-MR LBD E962A or Gal4-MR LBD K785A mutant. Aldosterone is at 1 nM, and cortisol is at 1 µM. The activities of Luc reporter under various conditions were normalized to that of control vector (Gal4) to obtain the relative Luc activity value.

 
The above results indicate that the partial agonist cortisol does not differentially recruit the tested coactivator/corepressor peptides when compared with the full agonist aldosterone. However, there are very small quantitative differences with some of the coactivator peptides. We reason that the difference between the aldosterone and cortisol-induced LBD conformations is very subtle and could involve different LBD residues. Thus, we made a series of mutations of the MR LBD and focused our efforts on the coactivator/corepressor binding groove that is normally formed by helices 3, 4, and 5 (H3–5) and 12 (H12) of the LBD (Fig. 5CGo). As shown in Fig. 5DGo, activation of Gal4-MR LBD by aldosterone or cortisol requires the presence of intact H12 as removal of H12 (Gal4-MRLBD-dAF2) completely disrupted the activation by either hormone. Mutations in several of the surface residues had various effects on MR activation. The I799R mutant (Gal4-MR LBD I799R) has minimal effect on activation. V780R reduces the overall activation induced by either aldosterone or cortisol. The most interesting among these mutations are K785A and E962A, which had quite different effects on the activity induced by aldosterone or cortisol. Activation by aldosterone is reduced but still significantly above vehicle control, whereas the activation by cortisol is completely abolished. This is not due to loss of cortisol binding because published results indicate that E962A mutation does not disrupt cortisol binding (24). Moreover, cortisol can also block aldosterone-induced activation of the E962A or K785A mutant (Fig. 5EGo). Together, these results indicate that the differential activation by aldosterone and cortisol is not due to the inability of these mutants to bind cortisol. Thus, activation of MR LBD by aldosterone and cortisol involves different residues on MR LBD. The two residues K785 and E962 are highly conserved in nuclear receptors and presumably form a salt bridge upon agonist binding as is the case for other nuclear receptors (25). Our results clearly suggest that salt bridge formation is absolutely critical for activation by cortisol but not aldosterone.

We next examined the effects of cortisol and eplerenone on the activity of Gal4-MR S810L mutant. This mutation was identified in patients with early-onset hypertension exacerbated by pregnancy. Progesterone and spironolactone, which normally antagonize MR, activate the S810L mutant (26). As shown in Fig. 6AGo, both aldosterone and cortisol activate the S810L mutant LBD and the maximal inductions by both ligands are the same, indicating that cortisol is a full agonist of the S810L mutant. Interestingly, when we examined the effects of MR antagonists on the S810L mutant, we observed submaximal inductions (Fig. 6BGo, top) at concentrations that allow saturated binding to wild-type MR LBD. These partial effects are not due to the loss of binding affinities to the S810L mutant because the antagonists are able to block the maximal induction by aldosterone (Fig. 6BGo, bottom), which is further validated by analyzing the dose-response curves of these antagonists in the absence and presence of aldosterone (Fig. 6CGo and data not shown). RU486, which has recently been identified as an antagonist of MR S810L (27), is used for comparison. Together, these results suggest that MR antagonists become partial agonists/antagonists of the S810L mutant.



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Fig. 6. The Effects of Cortisol and MR Antagonists on Gal4-MR LBD S810L Mutant

A, Cortisol is a full agonist of MR S810L. The ligand-dependent induction of Gal4-MR LBD S810L was evaluated in the presence of various ligands. B, Top, Effects of MR antagonists on S810L in the absence of aldosterone. Bottom, Same assay was performed in presence of aldosterone except in the vehicle treatment. The activity of either wild type (WT) or S810L in the presence of 10 nM alodsterone is set as maximal activation. Aldosterone is at 10 nM and eplerenone at 10 µM. Cortisol, progesterone, spironolactone, and RU486 are at 1 µM. C, Dose-dependent activation (in the absence of aldosterone) or inhibition (in the presence of 0.1 nM aldosterone) of S810L activity by eplerenone (top) and progesterone (bottom).

 
Because the effects of MR antagonists on S810L are similar to that of cortisol on wild-type MR, we wonder whether they have a similar mechanism to cortisol in terms of partially activating the receptor. We examined the coactivator and corepressor peptide interactions with the S810L mutant in the presence of aldosterone, cortisol, or eplerenone. First of all, as shown in Fig. 7Go, aldosterone, cortisol, and eplerenone are able to recruit the same peptides despite aldosterone and cortisol being agonists and eplerenone being partial agonist of the S810L mutant. These results suggest that the partial agonist effect of MR antagonists on the S810L mutant is also not due to differential LXXLL coactivator interactions.



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Fig. 7. Aldosterone, Cortisol, and Eplerenone Recruit the Same Coactivator Peptides to the MR S810L Mutant

Mammalian two-hybrid assay was used to detect the interaction between coactivator peptides and MR S810L. Aldosterone is at 10 nM, cortisol at 1 µM, and eplerenone at 10 µM. Fold interaction is the activity in the presence of ligand divided by that in the absence.

 
Second, it is noteworthy that the S810L mutant recruits a distinct set of cofactor peptides. A few peptides that do not bind MR LBD interact strongly with S810L, suggesting the mutant LBD adopts a different conformation in the coactivator binding groove. This is not totally surprising because a reported molecular modeling study indicated that the S810L mutation induces a bending of the helix 3 (27), which potentially also changes the coactivator binding surface formed by helices 3, 4, 5, and 12.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We conducted detailed analysis of the interactions between MR and coactivator/corepressor peptides. Our results indicate that MR LBD interacts strongly with relatively few coactivator peptides in the presence of aldosterone. This is in sharp contrast to a few other nuclear receptors, which interact with many of these coactivator/corepressor peptides (data not shown). The peptides that MR LBD interacts with are derived from p160 family coactivators, coactivator PGC1{alpha} and ß, ASC2, TIF1, NSD1, CIA and RIP140 with SRC1, PGC1{alpha}, PGC1ß, and ASC2 being the stronger interacting coactivators.

The p160 family coactivators (SRC1, GRIP1, and AIB1), shown to interact with a large number of nuclear receptor LBDs, do not appear to interact strongly with MR LBD. The only exception is the SRC1–4a peptide derived from an isoform of SRC1 (SRC1a). The two isoforms SRC1a and SRC1e contain three NR-box peptides (SRC1–1, SRC1–2, and SRC1–3) and differ only in the fourth peptide (17). Interestingly, SRC1a and SRC1e are expressed in distinct regions in the brain, including MR- containing areas of the hypothalamus where SRC1a not SRC1e is highly expressed (28). In general, SRC1, GRIP1, and AIB1 are ubiquitously expressed in many tissues and appear to have certain overlapping functions. SRC1 knockout animals are viable and exhibit only partial sex hormone resistance, accompanied by increased expression of GRIP1 (29). GRIP1 knockout, SRC1/GRIP1 double knockout, and AIB1 knockout have defects in reproduction (30, 31, 32). However, the fact that these knockout animals do not exhibit any defects reminiscent of MR knockout phenotype suggests either that the loss of one or two p160 family coactivators can be compensated by other coactivators or that these coactivators are not critical for MR function in the kidney (33). It remains to be determined whether MR function is affected in other tissues of these coactivator knockout mice.

Coactivators PGC1{alpha} and PGC1ß are highly expressed in MR target tissues including kidney and heart (19, 20). PGC1{alpha} is involved in mitochondrial biogenesis, thermogenesis, and gluconeogenesis (34). The effects of PGC1{alpha} on mitochondrial biogenesis and fatty acid oxidation are observed in cardiac cells and tissues. Overexpression of PGC1{alpha} in heart under a cardiac-specific promoter resulted in massive proliferation of enlarged mitochondria with dilated cardiomyopathy (35). Coincidentally, transgenic mice overexpressing human MR in the heart and kidney also developed dilated cardiomyopathy (36). These results imply that PGC1{alpha} and MR have overlapping functions in the heart. PGC1ß has not been studied extensively. Based on its expression pattern and its ability to interact with MR, it is likely that PGC1ß can also coactivate MR in the kidney and heart.

Coactivator ASC2 (NCoA6, RAP250, AIB3, PRIP, TRBP, NRC) is also highly expressed in MR expressing tissues including heart, kidney, and brain (37). Knockout of ASC2 result in embryonic lethality with defects in placenta, blood vessels, and heart (38, 39). Transgenic mice expressing a dominant-negative fragment (containing the ASC2–1 peptide) of ASC2 are viable but have multiple abnormalities including hypertrophy in the heart and hypoplasia in the kidney (40). Given the strong interaction of ASC2–1 and MR in the presence of aldosterone, it would be interesting to see whether these defects are the results of abnormal MR activation.

Class II nuclear receptors, such as thyroid receptors and retinoic acid receptors repress transcription in the absence of any ligand by recruiting corepressors, N-CoR and SMRT. This is normally not the case in class I nuclear receptors (steroid receptors), which do not appear to repress transcription or interact with corepressors in the absence of ligand. However, steroid receptors other than MR have been shown to interact with corepressors in the presence of antagonists. Examples include tamoxifen-bound estrogen receptor (41), cyproterone acetate-bound androgen receptor (42), and RU486 bound GR and progesterone receptor (43, 44).

Although eplerenone is capable of antagonizing the deleterious effects of aldosterone in animal models and in clinical trials, the molecular mechanism of eplerenone in blocking aldosterone-induced MR activation has not been documented. We show here that eplerenone antagonizes aldosterone-induced MR activation and blocks aldosterone-induced interaction with coactivator peptides. All of the peptide interactions induced by aldosterone, strong or weak, can be blocked by eplere-none, suggesting that eplerenone and aldosterone induce distinct MR LBD conformations. Moreover, corepressor CoRNR peptides from N-CoR or SMRT do not interact with MR LBD in the presence of antagonist eplerenone (Fig. 3CGo and data not shown). Thus, unlike GR antagonist RU486, eplerenone only bocks coactivator interactions but does not actively recruits corepressors. Because eplerenone does not have a bulky side chain that could "push" helix 12 into an inactive conformation, it is plausible that eplerenone binding simply destabilizes the active agonist conformation and shifts the equilibrium to the inactive helix 12 conformation, as postulated based on the tetrahydrochrysene-bound ERß structure (45). However, in contrast to some other small antagonists, such as cyproterone acetate, eplerenone does not seem to stabilize a repressive conformation that allows corepressor binding. Instead, it stabilizes a neutral conformation that is transcriptionally inert, perhaps similar to the unliganded MR conformation.

Glucocorticoids such as cortisol have various effects via MR in different tissues (9, 10, 11). In light of the tissue-specific effects of glucocorticoids, it is interesting to note that cortisol functions as a partial agonist/antagonist of MR LBD. The LBD of nuclear receptor mediates the ligand-dependent effect by recruiting coregulators and the N-terminal region can modulate the ligand response by providing additional contact with coregulator complexes. Although current study uses MR LBD and the N-terminal region and the DNA binding domain of the MR could potentially modulate the cofactor interaction therefore affecting the ligand agonism/antagonism, a number of published studies suggest that cortisol can be a partial agonist/antagonist of full-length MR. The most direct evidence came from work done by Quinkler et al. (46). Using full-length MR transfection and mouse mammary tumor virus promoter-driven reporter in CV-1 cells, they have shown that cortisol can partially antagonize aldosterone-induced transactivation. Other evidence indicates that aldosterone and cortisol show differences in the promotion of N-terminal region and LBD communication (24) and in the recruitment of cAMP response element binding protein-binding protein (CBP)/RNA helicase complex to the N-terminal region of MR on a target gene promoter (47). Together, these results support that cortisol can also function as a partial agonist/antagonist in the context of full-length MR.

Lessons from selective estrogen receptor modulators and other tissue selective modulators of nuclear receptors indicate that these ligands turned out to be partial agonists/partial antagonists, and exert their different effects depending on the particular cellular levels of coactivators and corepressors (48, 49). Our finding that cortisol is a partial agonist/partial antagonist of MR may at least in part explain the tissue-specific effects of cortisol. It is to our surprise that we did not observe qualitatively different coactivator or corepressor peptide interactions with cortisol and aldosterone because numerous examples of other nuclear receptor partial agonists/antagonists pointed in this direction. However, the partial activity of cortisol we observed could be a result of differential interactions with other coactivators or corepressors that we have not yet tested, such as those that do not have LXXLL motifs. Alternatively, it could be a result of accumulation of small quantitative differences in peptide interaction with MR LBD. Nonetheless, aldosterone and cortisol must induce different conformations of MR LBD. This is supported by the finding that, when two residues that presumably form a salt bridge in the coactivator binding groove are mutated, cortisol fails to activate MR, whereas aldosterone still does albeit at a much reduced level (Fig. 5DGo). Both ligands bind well to the mutant receptor (24). Cortisol is able to completely block the aldosterone activation (Fig. 5EGo).

The salt bridge mutations are not the only mutations that can distinguish aldosterone and cortisol activation. It has been reported that cortisol failed to activate Q776A (helix 3) and R817A (helix 5) at concentrations that allow maximal binding (22). Interestingly, these residues are also in the vicinity of the coactivator binding groove, suggesting that cortisol and aldosterone induce distinct conformations in the coactivator binding groove of MR LBD.

Contrary to the salt bridge mutations, the S810L mutation converts cortisol from a partial agonist to a full agonist. The serine to leucine substitution at codon 810 was initially identified in patients with early-onset hypertension that is exacerbated by pregnancy (26). The underlying mechanism of the pregnancy effect is that progesterone, which antagonizes MR function, activates the mutant receptor. It has been reported that other antimineralocorticoid steroids, such as spironolactone, also activate the MR S810L mutant (26). We show here that these steroidal MR antagonists are partial agonists/antagonists of the MR S810L mutant, similar to the effect of cortisol on wild-type MR LBD. Moreover, these ligands are as good as aldosterone (and cortisol) in recruiting LXXLL coactivator peptides to the S810L mutant receptor (Fig. 7Go), also similar to the effect of cortisol on cofactor interactions with wild-type MR LBD.

The interesting switch of cortisol from antagonist (salt bridge mutations) to partial agonist (wild type) and then to full agonist (S810L mutation), and the interaction of MR LBD with cofactor peptides in the presence of various ligands led us to propose that there are multiple mechanisms to activate MR (Fig. 8Go). These mechanisms include interaction with 1) the LXXLL-containing coactivators; 2) other unidentified cofactors that differentiate the effects of aldosterone and cortisol; and 3) both.



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Fig. 8. Schematic Representation of the Proposed Mechanisms of MR Activation

LXXLL, Represents the LXXLL-containing coactivators that interact with MR. Other, Represents other unidentified cofactors that are differentially recruited by aldosterone and cortisol. Differential LXXLL, Represents the differential recruitment of LXXLL containing coactivators. The text around each conformation indicates the conditions under which the receptors adopt each corresponding conformation. MR ANT, MR antagonist.

 
In the unliganded state or in the antagonist- (such as eplerenone) bound state, MR adopts a transcriptionally neutral conformation. We should point out that there are certainly conformational differences between the unliganded receptor and the antagonist bound receptor because unliganded MR resides in the cytoplasm and antagonist-bound MR is translocated into nucleus (Ref. 50 and data not shown). However, from the transcriptional activity point of view, their LBDs are in a basal/neutral conformation without activation or repression functions.

We propose that, to fully activate MR LBD, both LXXLL-containing coactivators and the unidentified cofactors need to be recruited. Failing to recruit either class results in partial activation. When aldosterone binds wild-type MR LBD or cortisol binds S810L mutant, the resulting conformation allows the receptor to interact with both LXXLL coactivator and the unidentified cofactors to fully activate the receptor.

In the cortisol-bound MR or MR antagonist-bound S810L mutant MR, the LBDs recruit LXXLL containing coactivators but not the unidentified cofactors, therefore are only partially activated. On the other hand, aldosterone binding to the salt bridge mutants resulted in partial activation (Fig. 5DGo, compare mutant with wild type). Because we could not detect significant interactions of the salt bridge mutant receptors with the LXXLL peptides (data not shown) and because our peptide collection represents most of the known LXXLL containing coregulators, it is plausible to hypothesize that these mutants when bound with aldosterone but not cortisol recruit the unidentified cofactors to partially activate transcription. In addition, although we did not observe differential interaction with LXXLL peptides using the tested steroidal ligands, we cannot rule out this possibility. Based on studies done with other nuclear receptors, we propose that other MR ligands, for example, nonsteroidal MR ligands, could induce differential interactions with the LXXLL containing coactivators to partially activate MR.

In summary, we have shown that cortisol and aldosterone induce distinct MR conformations and require different residues for activation. We propose that several mechanisms exist to partially or fully activate MR. In our model, cortisol is unable to recruit the unidentified cofactors to MR. Further studies to identify the unknown cofactors that are differentially recruited by aldosterone and by cortisol, and to determine the relative cofactor levels in different epithelial and nonepithelial tissues may allow us to decipher the tissue-specific mechanisms of cortisol. Understanding such mechanisms will undoubtedly help the identification of tissue selective MR antagonists.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Constructs
The Gal4 peptide fusion constructs were made by inserting double-stranded oligonucleotides into vector pBind (Promega, Madison, WI). VP16-MR LBD was constructed by inserting the LBD coding sequence (residues 686–984) into pACT vector (Promega). MR mutations and deletions were constructed using QuikChange mutagenesis kit (Stratagene, La Jolla, CA) or PCR. The sequences of the coactivator and corepressor peptides are:

SRC1–1: KYSQTSHK LVQLL TTTAEQQL
SRC1–2: SLTARHKI LHRLL QEGSPSDI
SRC1–3: KESKDHQL LRYLL DKDEKDLR
SRC1–4a: PQAQQKSL LQQLL TE
SRC1–4b: PQAQQKSL RQQLL TE
GRIP1–1: HDSKGQTK LLQLL TTKSDQME
GRIP1–2: SLKEKHKI LHRLL QDSSSPVD
GRIP1–3: PKKKENAL LRYLL DKDDTKDI
AIB1–1: LESKGHKK LLQLL TCSSDDRG
AIB1–2: LLQEKHRI LHKLL QNGNSPAE
AIB1–3: KKKENNAL LRYLL DRDDPSDA
PGC1a: QEAEEPSL LKKLL LAPANTQL
PGC1b: PEVDELSL LQKLL LATSYPTS
PRC: VSPREGSS LHKLL TLSRTPPE
TRAP220–1: SKVSQNPI LTSLL QITGNGGS
TRAP220–2: GNTKNHPM LMNLL KDNPAQDF
ASC2–1: DVTLTSPL LVNLL QSDISAGH
ASC2–2: AMREAPTS LSQLL DNSGAPNV
CBP-1: DAASKHKQ LSELL RGGSGSSI
CBP-2: KRKLIQQQ LVLLL HAHKCQRR
P300: DAASKHKQ LSELL RSGSSPNL
CIA: GHPPAIQS LINLL ADNRYLTA
ARA70–1: TLQQQAQQ LYSLL GQFNCLTH
ARA70–2: GSRETSEK FKLLF QSYNVNDW
TIF1: NANYPRSI LTSLL LNSSQSST
NSD1: IPIEPDYK FSTLL MMLKDMHD
SMAP: ATPPPSPL LSELL KKGSLLPT
Tip60: VDGHERAM LKRLL RIDSKCLH
ERAP140: HEDLDKVK LIEYY LTKNKEGP
Nix1: ESPEFCLG LQTLL SLKCCIDL
LCoR: AATTQNPV LSKLL MADQDSPL
CoRNR1 (N-CoR): MGQVPRTHRLITLADH ICQII TQDFARNQV
CoRNR2 (N-CoR): NLG LEDII RKALMG
CoRNR1 (SMRT): APGVKGHQRVVTLAQH ISEVI TQDTY-RHHPQQLSAPLPAP
CoRNR2 (SMRT): NMG LEAII RKALMG
RIP140-C: RLTKTNPI LYYML QKGGNSVA
RIP140-1: QDSIVLTY LEGLL MHQAAGGS
RIP140-2: KGKQDSTL LASLL QSFSSRLQ
RIP140-3: CYGVASSH LKTLL KKSKVKDQ
RIP140-4: KPSVACSQ LALLL SSEAHLQQ
RIP140-5: KQAANNSL LLHLL KSQTIPKP
RIP140-6: NSHQKVTL LQLLL GHKNEENV
RIP140-7: NLLERRTV LQLLL GNPTKGRV
RIP140-8: FSFSKNGL LSRLL RQNQDSYL
RIP140-9: RESKSFNV LKQLL LSENCVRD
PRIC285-1: ELNADDAI LRELL DESQKVMV
PRIC285-2: YENLPPAA LRKLL RAEPERYR
PRIC285-3: MAFAGDEV LVQLL SGDKAPEG
PRIC285-4: SCCYLCIR LEGLL APTASPRP
PRIC285-5: PSNKSVDV LAGLL LRRMELKP

The NCBI accession numbers of these cofactor proteins are: SRC1 (NP_003734), GRIP1 (NP_006531), AIB1 (NP_006525), PGC1{alpha} (NP_037393), PGC1ß (NP_573570), PRC (NP_055877), TRAP220 (NP_004765), ASC2 (NP_054790), CBP (NP_004371), P300 (NP_001420), CIA (NP_066018), ARA70 (NP_005428), TIF1 (NP_003843), NSD1 (NP_071900), SMAP (NP_006687), Tip60 (NP_006379), ERAP140 (NP_861447), Nix1 (NP_113662), LCoR (NP_115816), N-CoR (NP_006302), SMRT (NP_006303), RIP140 (NP_003480) and PRIC285 (NP_208384).

Cell Culture and Transfection
293 Cells were maintained in DMEM plus 10% fetal bovine serum. Transfection was performed in 96-well plate with 30 ng Gal4 fusion, 60 ng VP16 fusion, 20 ng UASX5-luciferase (Luc) and 5 ng CMV-gal per well unless otherwise specified. After transfection, compounds were added in DMEM plus 10% heat-inactivated and charcoal dextran-stripped fetal bovine serum (Hyclone, Logan, UT). Cells were harvested 20 h later for Luc and ß-Gal activity, and the ratio is taken as the Luc activity. Each data point is the average of the ratio from six wells (or three wells for dose response curves).

TR-FRET Peptide Interaction Assay
TR-FRET peptide interaction assay is a homogeneous biochemical nuclear receptor cofactor recruitment assay using the time-resolved fluorescence technique. The GST-MRLBD proteins are expressed by baculovirus and crude lysate is used in this assay. The assay is performed in 384-well, 50 µl format at conditions of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM MgCl2, 5 mM dithiothreitol, 2 nM of Eu-GST antibody, 60 nM allophycocyanin-streptavidin, and 100 nM of biotin-coactivator peptide or otherwise as described in the figure legends. The plate is read by LJL Analyst (Molecular Devices, Sunnyvale, CA), 330/665 Ex/Em (400 msec integration, 65 msec delay) after a 4-h incubation at room temperature.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. John Funder for his critical comments on the manuscript and his supportive input. We thank Drs. Marcia Heron, Ellen McMahon, Silvia Pomposiello, and Amy Rudolph for their helpful discussion and support.


    FOOTNOTES
 
Present address for D.S.L.: Pfizer PGRD, Building 26 Room 251N, 2800 Plymouth Road, Ann Arbor, Michigan 48105.

First Published Online March 10, 2005

Abbreviations: AIB, Amplified in breast cancer; ASC2, activating signal cointegrator 2; CBP, cAMP response element binding protein-binding protein; GR, glucocorticoid receptor; GRIP, GR-interacting protein; GST, glutathione-S-transferase; HSD2, hydroxysteroid dehydrogenase; LBD, ligand binding domain; Luc, luciferase; MR, mineralocorticoid receptor; N-CoR, NR corepressor; NR, nuclear receptor: PGC1, peroxisome proliferator-activated receptor {gamma} coactivator 1; SMRT, silent mediator of retionic acid and thyroid receptor; SRC, steroid receptor coactivator; TR-FRET, time resolved-fluorescent resonance energy transfer.

Received for publication December 27, 2004. Accepted for publication March 2, 2005.


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