help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0246
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rogerson, F. M.
Right arrow Articles by Fuller, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rogerson, F. M.
Right arrow Articles by Fuller, P. J.
Molecular Endocrinology 21 (4): 817-828
Copyright © 2007 by The Endocrine Society

A Critical Region in the Mineralocorticoid Receptor for Aldosterone Binding and Activation by Cortisol: Evidence for a Common Mechanism Governing Ligand Binding Specificity in Steroid Hormone Receptors

Fraser M. Rogerson, Yi-Zhou Yao, Romi E. Elsass, Nektaria Dimopoulos, Brian J. Smith and Peter J. Fuller

Prince Henry’s Institute of Medical Research (F.M.R., Y.-Z.Y., R.E.E., N.D., P.J.F.), Clayton 3168, Victoria, Australia; and Walter & Eliza Hall Institute (B.J.S.), Parkville 3050, Victoria, Australia

Address all correspondence and requests for reprints to: Professor Peter J. Fuller, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton 3168, Victoria, Australia. E-mail: peter.fuller{at}princehenrys.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The amino acids that confer aldosterone binding specificity to the mineralocorticoid receptor (MR) remain to be determined. We had previously analyzed a panel of chimeras created between the MR and the glucocorticoid receptor and determined that amino acids 804–874 of the MR ligand binding domain are critical for aldosterone binding. In the present study a further series of chimeras was created within this region. The chimeras were analyzed by a transactivation assay and [3H]aldosterone binding, and the critical region was narrowed down to amino acids 820–844. Site-directed mutagenesis was used to create single and multiple amino acid substitutions in this region. These studies identified 12 of the 16 amino acids that differ in the MR and the glucocorticoid receptor in this region as being critical to conferring aldosterone responsivity. The amino acids that differ in the region 820–844 lie on the surface of the molecule and, therefore, it appears that MR ligand binding selectivity is conferred by residues that do not form part of the ligand binding pocket. Other studies have found that the corresponding regions of the androgen and glucocorticoid receptors are critical for the binding of natural and synthetic ligands, suggesting a common mechanism governing ligand binding specificity. The new chimeras also displayed, as previously reported, a dissociation between cortisol binding and transactivation and, intriguingly, only those that bound aldosterone with high affinity were activated by cortisol, suggesting a common mechanism that underlies specificity of aldosterone binding and the ability of cortisol to activate the MR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE STEROID HORMONE aldosterone is an important factor in the control of blood pressure through its promotion of sodium reabsorption in the kidney and colon (1) and through actions in the central nervous system (2). Aldosterone also has direct actions in the heart (3). There is now mounting evidence that hyperaldosteronism is an important factor in cardiac failure (4). In two recent trials, addition of an aldosterone antagonist to the treatment regimen of patients who had heart failure led to a significant decrease in mortality (4). Four known monogenetic causes of hypertension (5), glucocorticoid remediable hyperaldosteronism, apparent mineralocorticoid excess, Liddle’s syndrome (pseudoaldosteronism), and pregnancy-exacerbated hypertension (6) all involve aldosterone, its receptor, or sodium reabsorption. Despite the importance of aldosterone, there is only limited knowledge of the molecular mechanisms that underlie its action.

Aldosterone binds to, and acts through, the mineralocorticoid receptor (MR). This protein belongs to the nuclear hormone receptor superfamily of ligand-dependent transcription factors (7). As with other members of the family, the MR structure can be divided into four major domains (8). The first of these is the N-terminal domain, which contains an activation function involved in transcriptional activation (9). In the middle of the protein is the DNA-binding domain (DBD), which binds to specific DNA sequences on target genes. C-terminal to the DBD is the ligand-binding domain (LBD), which is involved in ligand binding, transcriptional activation, and heat-shock protein binding (10). Between the DBD and the LBD lies the hinge region, the functions of which are unknown in the MR. In the absence of ligand, the MR exists predominantly in the cytoplasm in a multiprotein complex with a number of heat shock proteins and associated factors (11). This complex maintains the LBD in a structural conformation that promotes high-affinity ligand binding (11). The binding of ligand alters the conformation of the receptor and displaces the heat shock proteins, which exposes regions of the receptor involved in dimerization, nuclear localization, and DNA binding. Thus, ligand binding activates the receptor.

Of the nuclear hormone receptors, the MR is most closely related to the glucocorticoid receptor (GR) (8). This is reflected in the overlapping binding specificities of the two receptors. Both receptors bind cortisol and corticosterone with high affinity (12). The synthetic GR agonist dexamethasone binds to the MR with relatively high affinity (8), although it does not activate the MR with the same potency as it does the GR (13). In contrast, aldosterone binds to the MR with a much higher affinity than it does the GR (12).

It is likely that there are sequences of amino acids shared between the two receptors that allow both GR and MR to bind cortisol with the same high affinity and sequences that differ between the two receptors that confer high affinity binding of aldosterone only to the MR. The amino acids involved in MR binding specificity, however, remain to be determined. Using a panel of 16 chimeras created between the LBDs of the MR and GR, we previously determined that amino acids 804–874 of the MR confer aldosterone binding specificity (14). In the present study, using a set of MR:GR chimeras concentrating on the region between amino acids 804–874, we have narrowed the region critical for aldosterone binding to amino acids 820–844 and explored the contribution of the amino acids that differ between the MR and the GR in this region. The results of recent crystallization of the LBD of the MR (15, 16, 17) suggest that this region does not form part of the ligand-binding pocket. This suggests that aldosterone binding specificity is determined by amino acids in the MR that act indirectly, and not by interaction with the steroid. Intriguingly, other studies (18, 19) suggest that the equivalent regions in the GR, progesterone receptor (PR) and androgen receptor (AR) are important for the binding of natural and synthetic ligands, suggesting a common mechanism that governs ligand-binding specificity in steroid hormone receptors.

The panel of chimeras used in our previous study demonstrated an intriguing dissociation between binding and transactivation (14). All of the chimeras bound cortisol, as would be expected given that cortisol binds with high affinity to both parental receptors. Interestingly, however, only those containing sequence from the same receptor (MR or GR) in the second and fourth regions were activated by cortisol in a transactivation assay (14). In the present study we were able to examine this phenomenon further, as the new chimeras were created with MR sequence at the C terminus. Interestingly, although cortisol again binds to all the chimeras, it only activates those chimeras that contain regions of the MR needed for high-affinity aldosterone binding. The reason for this remains unexplained but may point to a common mechanism that underlies aldosterone binding specificity and cortisol activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Creation of Chimeras
Aldosterone binding affinity is conferred by amino acids 804–874 of the MR (14). To narrow down the critical region, we generated a series of MR:GR chimeras between amino acids 804–874 of the MR LBD and the complementary region of the GR LBD (Fig. 1Go). The positions of the break points were made with reference to a published canonical structure of a nuclear hormone receptor LBD (20) before the crystal structures of the steroid receptors becoming available (7). The (804–874) chimeras were assayed using a transactivation assay and by direct [3H]-ligand binding experiments. The transactivation assay was used as an initial screen of the chimeras. For these experiments, the (804–874) chimeras were placed in the context of GxMM [using the four-letter nomenclature described in Materials and Methods, where x is the position of the (804–874) chimera]. Although the identity of the sequence C-terminal to amino acids 804–874 is not important for the affinity of aldosterone binding, it is important for receptor activation by the steroid, because chimeras containing MR sequence in this region are more potently activated by aldosterone in the transactivation assay (14). We reasoned, therefore, that the optimal conditions for examination of the (804–874) chimeras would be in a receptor context where aldosterone activation is at its most potent. Based on the transactivation results, [3H]aldosterone binding studies were then performed on selected chimeras. For the binding studies we wished to examine the importance of the (804–874) chimera for aldosterone binding affinity independent of any possible minor contribution by the C-terminal MR sequence. The chimeras were therefore placed in the context of the GR LBD.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Schematic Representation of Chimeras Used to Examine Aldosterone- and Cortisol-Induced Transactivation

Chimeras were created between amino acids 804–874 of the MR LBD and the corresponding amino acids 598–668 of the GR LBD. These chimeras were placed into the context of GxMM, where G corresponds to amino acids 488–597 of the GR LBD, MM corresponds to amino acids 875–984 of the MR LBD, and x corresponds to the chimera sequence. All the LBD chimeras were placed into an expression vector (pRShGR) containing the DBD and N-terminal domain of the GR. For binding studies the chimeras were placed into context of the GR, i.e. the ligand-binding domain is represented GxGG.

 
Transactivation Assay Using Aldosterone
The contribution of a particular region of the MR LBD to aldosterone binding specificity was assessed in terms of the ability of the corresponding GxMM chimera to lower the EC50 value for aldosterone relative to GGMM. To confirm expression of the chimeras, [3H]dexamethasone binding experiments were performed in transfected COS-1 cells. All of the chimeras bound [3H]dexamethasone at 20 nM (results not shown), suggesting that they are all expressed and are able to bind ligand. Table 1Go shows the EC50 values for this panel of chimeras. The most striking result is that of the MR (804–844)-GGMM chimera, which has an EC50 value equal to that of the MR LBD, suggesting that this chimera contains all the sequences required for high affinity aldosterone binding. Consistent with this result, the MR (848–874)-GGMM chimera is activated very poorly by aldosterone, with an EC50 value greater than 0.3 µM, similar to the EC50 value for the GR. The MR sequences of the other three constructs overlap that of the MR (804–844)-GGMM chimera. Two of these chimeras, MR (804–815)-GGMM and MR (804–828)-GGMM, are activated very poorly by aldosterone, with EC50 values greater than 0.3 µM. This result would suggest that the critical sequences for aldosterone binding lie between amino acids 834–844 (829–833 being conserved between the MR and GR). The result of the MR (834–874)-GGMM chimera, however, indicates a more complex situation. Aldosterone activates this construct with a somewhat greater potency than GGMM; the EC50 value (0.1 µM) is, however, still two orders of magnitude higher than that for the wild-type MR LBD (1 nM). This suggests that sequences between amino acids 834–844 are involved in aldosterone binding but in themselves they are not sufficient to confer high affinity binding of the ligand.


View this table:
[in this window]
[in a new window]

  		Table 1. Results of Transactivation Assays Showing Approximate EC50 Values for the Chimeras Together with the MR (MMMM) and GGMM LBD Chimera

 
For further analysis, we began with the assumption that amino acids 804–815 do not contribute to binding specificity, given that MR (804–815)-GGMM is very weakly activated by aldosterone. Amino acids 816–819 are conserved between the MR and GR, and so we tested if amino acids 820–844 of the MR are involved in high affinity aldosterone binding. We created and examined the chimera MR (820–844)-GGMM. As shown in Fig. 2Go, aldosterone activates this chimera and the MR LBD with equal potency, with the EC50 values both being approximately 1 nM. This strongly suggests that within the 804–844 region, amino acids 820–844 (Fig. 3AGo) are critical for aldosterone binding specificity.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. Aldosterone Dose-Response Curves of the Wild-Type MR LBD (Open Circle) and the MR (820–844) Chimera (Closed Square)

CV-1 cells were transiently transfected with receptor expression plasmid, MMTV-LUC reporter plasmid, and ß-galactosidase (B-GAL) control plasmid. The cells were incubated with aldosterone for 24 h before assay of LUC and ß-galactosidase activities. Results of LUC expression were calculated relative to a constitutively expressed ß-galactosidase reporter and graphed as a percentage of the maximal response to steroid for each chimera. Each point represents the mean ± SEM of duplicates of two different experiments.

 

Figure 3
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 3. Sequence and Structural Information of Amino Acids 820–844 of the MR

A, Comparison of the sequence of amino acids 820–844 of the MR and the corresponding GR sequence. Amino acids that are identical in the MR and GR are shown in bold. B, The MR LBD crystal structure (Ref. 16 ; Protein Data Bank 2AA2) with aldosterone in the ligand-binding pocket. Helices are indicated. The region containing amino acids 820–844 is indicated. C, Comparison of the sequence of amino acids 820–844 with the corresponding GR, AR, and PR sequences. Residues identical to those in the MR sequenced are indicated by the asterisks.

 
Scatchard Analysis of [3H]Aldosterone Binding
The results of the transactivation assays revealed that MR (804–844)-GGMM and MR (820–844)-GGMM were activated by aldosterone with a potency very similar to that of the full-length MR LBD. The contribution of the (804–844) and (820–844) regions to the binding of [3H]aldosterone was then determined. For reasons described above, the chimeras MR (804–844)-GR and MR (820–844)-GR were created, in which amino acids 804–844 and 820–844, respectively, were inserted into the GR LBD in place of the corresponding GR sequence. The calculated Kd values for [3H]aldosterone binding to the MR LBD, MR (804–844)-GR, and MR (820–844)-GR are shown in Table 2Go. The Kd value of MR (820–844)-GR is arguably lower than that of MR (804–844)-GR, suggesting that amino acids 804–819 of the MR are not involved in determining binding specificity. The Kd value of MR (820–844)-GR is similar to that of the full-length MR LBD, confirming the importance of amino acids 820–844 in aldosterone binding specificity. Statistical comparison of the Kd values for the MR LBD and MR (820–844)-GR reveals that there is a significant difference (P < 0.005) between the two, suggesting that this region is not the only determinant of aldosterone binding specificity. We attempted to measure the Kd value for [3H]aldosterone binding to the GR. This is technically difficult to perform using our whole-cell binding assay, because of the very high concentrations of [3H]aldosterone that would be required to obtain an accurate measurement. A value of 0.1 µM was obtained in one experiment, although it is very likely to be only an approximate measurement. Nevertheless, comparison of this value with that for MR (820–844)-GR suggests that swapping amino acids 820–844 into the GR confers at least a 2-order of magnitude increase in aldosterone binding affinity.


View this table:
[in this window]
[in a new window]

  		Table 2. Results of Scatchard Analysis of [3H]Aldosterone Binding

 
Amino Acids 820–844 in the MR LBD
The x-ray crystal structure of the MR in complex with aldosterone has recently been reported (15, 16, 17). The ligand-binding pocket consists of 23 amino acids from {alpha}-helices 3, 5, 6, 7, 11, and 12, and the ß-turn. Strikingly, amino acids 820–844 lie predominantly on the surface of the receptor, encompassing the C terminus of helix 5, the ß-turn, and helix 6 (Fig. 3BGo). Amino acids 829–833, which are completely conserved between the MR and GR, form the loop between the two ß-sheets of the ß-turn. There is only one residue in the (820–844) region that forms part of the ligand binding pocket, Phe (829), which lies in the ß-turn. This residue is conserved between the MR and GR. It therefore appears that aldosterone binding specificity in the MR is conferred by residues that do not interact directly with the ligand.

Of the 25 amino acids, nine are found in both the MR and the GR (Fig. 3AGo). There are thus 16 amino acids that may confer specificity. To identify which of these 16 amino acids is critical, we examined the equivalent sequence in both the AR and PR (Fig. 3CGo) and the position of the residues in the MR models. Neither offered much guidance. The greatest dyshomology is clearly in the regions 821–826 and 841–844. In addition, the previous chimeras (Fig. 1Go) suggested that amino acids 841–844 in the MR were likely to be more important. These amino acids [His (841), Gln (842), Ser (843), Ala (844)] were substituted into the chimera GGMM individually and as a block of four. The single substitutes were without effect, all four together yielded a small response with an EC50 of more than 300 nM (Table 3Go). In these studies, a maximum concentration of 1 µM aldosterone was used so that in those chimeras whose response was poor, a precise EC50 could not be calculated, so the values are reported as lower limits.


View this table:
[in this window]
[in a new window]

  		Table 3. Results of Transactivation Assays Showing Approximate EC50 Values for Substitution of Amino Acids within the MR820–844 Region with the GMMM Chimeras

 
Given the daunting number of possible combinations, even with only 16 possible point alterations, we further analyzed the contribution of amino acids in MR 820–844 to aldosterone binding specificity in six blocks: [Lys (820) His (821); Thr (822) Asn (823); Ser (824) Gln (825); Phe (826) Tyr (828); Val (834) Phe (835); Glu (838) Lys (839)] and the previous block [His (841) to Ala (844)]. The results of various combinations of substitutions into the region of the GR 614 to 638 in the context of the chimera GXMM are shown in Table 3Go. It should be noted that all of these latter chimeras bound [3H]dexamethasone as well or better than GGMM or MR (820–844) GGMM, confirming that the chimeras are expressed at an appropriate level and in an appropriate conformation (data not shown). The pairs Lys (820) His (821) and Glu (838) Lys (839) did not add any increase in response even in the context of the two larger substitutions, Thr (822) to Gln (825) with His (841) to Ala (844). However, the other paired substitutions, Phe (826) Tyr (828) and Val (834) Phe (835) did increase the response, so these were pursued further. Although the insertion of Thr (822) to Gln (825) adds little in most contexts, it is still required for full activity. Thus, a combination of up to 12 of the 16 amino acids that differ between the MR and the GR in this region confers aldosterone binding and transactivation.

Structural superposition (21) of the MR and GR structures reveals very close structural similarity (Fig. 4Go). The largest deviation in structure between MR and GR exists in the region of MR from 823 to 863, encompassing {alpha}-helices H6 and H7, and the ß-sheet of the ß-turn; the average distance in this region is 1.4 Å (Fig. 4AGo), whereas outside this region the average distance is 0.8 Å. In all published structures of the MR (15, 16, 17), the region from 823 to 863, whether complexed with agonist or antagonist, is essentially identical. This also applies to region 617 to 657 of the GR (22, 23). Thus, the difference in conformation in this region between MR and GR is not ligand related, but an intrinsic property of the polypeptide. The superposition of the 823–863 region of MR with the corresponding region of GR, 617–657, is shown in Fig. 4BGo. The displacement of the helices is apparent, as is the different conformation of the loop region connecting these helices. The side-chain groups of the conserved Leu (827) and Phe (829) in the MR occupy identical positions to Leu (621) and Phe (623) in the GR. The loop between the end of helix H5 and ß-strand ß 3 (823–826) in the MR is displaced away from helix H7 in comparison to the GR to accommodate the Ser (824) in MR [Ala (618) in GR]. The ß-turn in the MR is displaced from its position in the GR to accommodate Tyr (828) [c.f. Cys (622) in GR]. The side-chain carboxylate of Glu (837) forms hydrogen bonds with the side-chain groups of Gln (850) and Tyr (846) in MR, whereas Glu (631) in GR projects outward from the protein to the solvent. Although the side-chain groups of Phe (835) in MR and Leu (629) in GR occupy similar positions, the conformation of the side-chain of Phe (835) places it such that it would clash with the side-chain atoms of Met (845) if they were to adopt the same orientation as observed for Met (639) in the GR. The side-chain groups of the Met (840) in MR and Met (634) in GR occupy similar space despite their {alpha}-carbon atoms being separated by 2.3 Å. In the complex of the MR with aldosterone, Met (845) forms contact with the ligand. In the GR, this conserved residue is separated in the superposition by approximately 6 Å and unable to contact the ligand.


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 4. Structural Superposition of the Structures of the GR and the MR in the Region of Interest

A, Distance between {alpha}-carbons of MR and GR in the superposition (MR numbering). Highlighted are the residues in the region of 823 to 863 of MR where the largest deviation between the structures is observed. B, Ribbon diagram of the region 823–863 of MR (green ribbon) (Ref. 16 ; Protein Data Bank 2AA2), and 617–657 or GR (cyan ribbon) (Ref. 23 ; Protein Data Bank 1P93) in the superposition. The difference in the paths of helix-7 (H7) and the loop immediately preceding H7 in the two structures is apparent. The position of side-chains of Leu (827) and Phe (829) or MR, and Leu (621) and Phe (623) of GR, respectively, are identical. The side-chain of Phe (835) of MR is in close proximity to Met (639) of GR. The side-chains of Ser (824) and Tyr (828) are larger than the equivalent residues in GR, AL (618), and Cys (622), respectively, resulting in differences in polypeptide path locally.

 
Cortisol Activation of the Chimeras
Both the MR and GR bind cortisol with high affinity, and so it would be expected that all the chimeras would bind cortisol. Indeed every one of the initial panel of chimeras do bind cortisol (14). Curiously, only those chimeras with sequence from the same receptor in the second and fourth regions were activated by cortisol in the functional assay (14). For example, the GGMM chimera is not activated by cortisol, whereas the GMMM chimera is activated by cortisol with the same EC50 value as the MR LBD (14). This contrasts to the aldosterone results, where there was a close correlation between aldosterone binding and transactivation (14). Because the (804–874) chimeras were placed in the context of GxMM, we were able to further investigate this phenomenon. Table 4Go shows the EC50 values for the (804–874) chimeras together with the wild-type GR. The chimeras MR (804–815)-GGMM, MR (804–828)-GGMM, MR (834–874)-GGMM and MR (847–874)-GGMM were activated very poorly by cortisol. In contrast to the above chimeras, the MR (804–844)-GGMM and MR (820–844)-GGMM chimeras were activated by cortisol with EC50 values of 10 nM, equal to that of the wild-type MR LBD. The ability of cortisol to compete for [3H]dexamethasone binding to the chimeras was also tested. As shown in Fig. 5Go, cortisol competes for [3H]dexamethasone binding to the MR LBD and the MR (804–815)-GGMM, MR (804–828)-GGMM, MR (847–874)-GGMM, MR (804–844)-GGMM and MR (820–844)-GGMM chimeras with a similar potency. Although cortisol appears to displace dexamethasone from the wild-type MR with slightly greater potency than from the chimeras, the lack of cortisol-induced transactivation by these chimeras is clearly not due to their inability to bind cortisol. The MR (834–874)-GGMM was not tested because of low levels of expression. Given the apparently parallel binding pattern of aldosterone and cortisol, the chimera containing 12 substitutions (Table 3Go), which yields a wild-type response to aldosterone, was also examined for cortisol binding. Equivalent binding of cortisol was observed to this chimera [MR (822–825, 826. 828, 834. 835, 841–844)- GGMM; Table 3Go] to that observed for MR (820–844)-GGMM. In conclusion, adding amino acids 820–844 of the MR to the GGMM chimera confers full cortisol responsiveness as well as the ability to bind aldosterone.


View this table:
[in this window]
[in a new window]

  		Table 4. Results of Cortisol Transactivation Assays Showing Approximate EC50 Values for the Chimeras Together with the MR LBD and the GMMM and GGMM Chimeras

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 5. Cortisol Competition for [3H]Dexamethasone Binding

Experiments were performed using the MR LBD, MR (804–815)-GGMM, MR (804–828)-GGMM, MR (847–874)-GGMM, MR (804–844)-GGMM, and MR (820–844)-GGMM chimeras. COS-1 cells were transiently transfected with the chimera expression plasmid. The cells were incubated for 1 h with 20 nM [3H]dexamethasone ± increasing concentrations of cortisol. The cells were then washed and lysed and the radioactivity was counted. Each receptor construct was assayed in triplicate, and the results are displayed as mean ± SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The MR is unique among steroid hormone receptors in that it is a physiologically important receptor for two classes of steroid hormones: mineralocorticoids, such as aldosterone, and glucocorticoids, such as cortisol and corticosterone (1, 10). In MR-expressing cells of the kidney and colon the receptor acts predominantly as a mineralocorticoid receptor, as glucocorticoids are enzymatically inactivated (24), whereas in the brain the MR acts predominantly as a high affinity glucocorticoid receptor (25).

Aldosterone Binding
Phylogenetic evidence suggests that the MR and the GR diverged from a common ancestor, probably in jawless fish, that functioned as a glucocorticoid receptor (26). In fish, glucocorticoids have been shown to be involved in a number of osmoregulatory processes, and there is evidence that the MR ortholog is involved in glucocorticoid-mediated adaptation to ion-deficient water (27). Interestingly, however, aldosterone itself first appears as an active steroid in amphibians (26). It is attractive to postulate that the MR originated in fish to regulate ion balance under the control of glucocorticoids and that amphibians evolved mineralocorticoids specifically to bind to this receptor and take over this function. Possibly the requirement for glucocorticoids in other crucial aspects of homeostasis, particularly those involved in adaptation to terrestrial life, were no longer compatible with their having a continuing role in ion balance. The classical GR has a very low affinity for aldosterone. Using [3H]-ligand competition assays, it has been reported that aldosterone has a 700-fold greater affinity for the MR than the GR (Ki values of 0.09 and 63 nM, respectively) (12). The amino acid changes that account for this increased affinity for mineralocorticoids are yet to be determined. We have examined this question using chimeras created between the MR and GR LBDs. Examination of an initial panel of 16 chimeras revealed that amino acids 804–874 of the MR are critical for aldosterone binding (14). The same region is critical for the binding of the MR antagonists spironolactone (28) and eplerenone (29) to the receptor. Martinez et al. (30) created a series of MR:GR LBD chimeras using in vivo homologous recombination; the region they identified as critical for aldosterone binding spans this region. In this study we created a second set of MR:GR chimeras within the 804–874 region to define the critical region.

Examination of the chimeras by transactivation and [3H]aldosterone binding assays revealed that amino acids 820–844 of the MR LBD are critical for the binding of aldosterone to the MR. The results of the two assays were consistent with both showing that swapping amino acids 820–844 of the MR in place of the corresponding GR sequence confers a 2- to 3-order of magnitude increase in aldosterone binding affinity, and the resulting chimeras have an affinity for aldosterone very similar to that of the full-length MR LBD. The Kd value of [3H]aldosterone binding to MR (820–844)-GR was slightly higher than that of the MR LBD, suggesting that other residues in the MR LBD have a minor contribution to aldosterone binding specificity. These amino acids are likely to be Met (777) and Gln (779), identified in a study examining the ability of aldosterone to compete for triamcinolone acetonide (TA) binding to GR mutants (31). Swapping Val (571) of the GR with the corresponding MR residue, Met (777), increased the binding affinity for aldosterone 2.5-fold with no effect on the affinity of TA itself. Swapping both Val (571) and Ala (573) of the GR with the corresponding MR residues [Met (777) and Gln (779)] increased the binding affinity for aldosterone competition for TA binding 5.5-fold, although this double mutant also increased the affinity of the receptor for TA by 2.5-fold. In a subsequent study, using molecular dynamics simulations, these investigators (32) concluded that changing Val (571) to methionine in the GR stabilized both the coactivator binding site and the ligand-binding pocket. The mechanism by which this reduction in the "entropic cost for ligand binding" is achieved is less clear. It may be that residues in the 820–844 region, together with these two more N-terminal amino acids, can fully account for the difference in binding affinity of aldosterone between the MR and GR.

Amino acids 820–844 of the MR share only 36% amino acid sequence identity with the GR (Fig. 5AGo), which is much lower than the overall 57% sequence identity between the two receptors over the entire length of the LBD. The major area of identity in this region occurs between amino acids 829–833, which are completely conserved between the MR and GR. On either side of this conserved sequence there is little sequence similarity between the two receptors in this region. Our results suggest that sequences both N- and C-terminal to amino acids 829–833 are required to confer binding specificity. The MR (804–828) chimera, containing MR sequences N-terminal to the conserved region, is not activated by aldosterone. The MR (834–874) chimera, containing MR sequences C-terminal to the conserved region, is only weakly activated by aldosterone. Therefore, neither the sequences between amino acids 820–828 or 834–844 by themselves are able to confer high-affinity aldosterone binding.

Chimeras have been used to investigate the determinants of ligand binding specificity to other steroid hormone receptors, and the results of these experiments suggest that a very similar region is important in conferring ligand binding specificity in other receptors. Chimeras between the PR and AR were used to examine the binding of both progestins and androgens (18). Swapping amino acids 769–797 of the AR into the PR conferred dihydrotestosterone binding, although the binding affinity was lower than for the full-length AR LBD. This corresponds to amino acids 834–862 of the MR and overlaps the region critical for MR binding specificity. Conversely, swapping amino acids 788–791 of the AR with the corresponding PR residues led to a complete loss of dihydrotestosterone binding (18). In another study, chimeras between the PR and GR were used to examine the binding specificity of synthetic progestins and glucocorticoids (19). Replacement of amino acids 628–655 of the GR with the corresponding PR residues abolished binding of specific glucocorticoids, dexamethasone and RU43044, and allowed binding of the specific progestin RU27987. This region corresponds to amino acids 834–861 of the MR and again overlaps the region critical for MR binding specificity. Binding of RU27987 to the chimeras was conferred by PR residues Ser (792) and Phe (794) that correspond to amino acids positions 843 and 845 of the MR. The loss of glucocorticoid binding was conferred by mutating the GR residues Asp (641), Gln (642), and Leu (647) with the corresponding PR residues. The corresponding residues in the MR lie just C-terminal to the region critical for MR binding specificity. The results of PR:AR and PR:GR chimeras, together with those of our MR:GR chimeras, and a recent study which used homologous recombination to create MR:GR chimeras (30) suggest that this general region is critical for governing both binding affinity and specificity of a number of different ligands to steroid hormone receptors.

Our finding, initially from molecular modeling (33), that the amino acids that differ in the region MR 820–844 lie on the surface of the MR LBD was a surprising and somewhat unexpected result given that the region of the LBD identified in our initial study included many sequences that form the ligand-binding pocket (14). How residues that lie on the surface of the molecule, and do not form part of the ligand-binding pocket, confer binding specificity remains to be determined but there are a number of possibilities. Residues such as Phe (829), which is conserved in both the GR and the MR, interacts with aldosterone as can residues close to the 820–844 region. The region may thus position these residues within the ligand-binding site. Other possible explanations relate to the conformation of the unliganded receptor. The ligand binding pocket of the MR and other nuclear hormone receptors lies in the core of the structure, and thus the ligand has to enter into this core. This region could be at the "entrance" where the ligand first interacts with the receptor protein. Comparison of the related apo- and holo-retinoid X receptor crystal structures, however, suggests that ligand enters the structure on the opposite face of the protein and involves conformational changes in helix 12 (7). This possibility, therefore, is unlikely. It should be noted, however, that presently no crystal structure of a nonliganded steroid hormone receptor has been published. Another possibility is that the conformation of this region may affect the overall conformation of the ligand binding pocket by a distant packing effect. There are precedents for this type of effect in other steroid hormone receptors. In the study described above, using chimeras to identify determinants of binding specificity to the GR and PR (19), binding of the progestin RU27987 was conferred by two residues: a phenylalanine pointing in toward the ligand-binding pocket and a serine on the surface of the molecule. Binding of glucocorticoids to the PR:GR chimeras involved two residues, Asp (641) and Leu (647), that do not appear to interact directly with the ligand. The guinea pig GR has a decreased affinity for glucocorticoids, due to amino acid differences with the human sequence that occur in a region that does not form part of the ligand-binding pocket (34). Similarly in a study using estrogen receptor {alpha}:estrogen receptor ß chimeras, Nettles et al. (35) demonstrated the importance of residues outside of the ligand binding pocket. A variation on the "distant packing effect" involves the interactions with the hsp90 complex (34). Thus an altered interaction with a component of this complex may in turn influence the conformation of the unliganded receptor.

Recently three groups have reported crystal structures for the human MR. Fagart et al. (15) reported the crystal structure of the mutant S810L with both deoxycorticosterone and progesterone bound. This mutation, originally identified in a kindred with severe, early-onset, pregnancy-exacerbated hypertension was first described by Geller et al. (6). The receptor is constitutively active and is further activated by agonists, antagonists, and even the inactive steroid cortisone. Bledsoe et al. (16) obtained a crystal structure for the MR LBD bound to aldosterone, deoxycorticosterone, and progesterone as well as the structure of the S810L mutant bound to progesterone, cortisone, and spironolactone. The third study from Li et al. (17) reports the structure of the MR LBD bound to aldosterone and to corticosterone. Li et al. (17) also cocrystallized the MR-corticosterone complex with an LXXLL motif from the coactivator steroid receptor coactivator-1. In each case the general structure for steroid hormone receptors, consisting of 11 {alpha}-helices within four ß-sheets folded into a three-layer helical sandwich, was confirmed.

The MR ligand-binding pocket is lined by 23 amino acids (17); of these only Phe (829) lies within the 820–844 region and it is conserved between the MR and the GR. Methionine 845 also lies in the pocket but it also is conserved MR to GR. It is possible that minor reorientation of either Phe (829) or Met (845) in response to differences in neighboring amino acids may alter their interactions with the ligand. The ligand-binding pocket is formed by helices 3, 4, 5, 7, and 10 and the first two ß-strands as well as helix 12. The 820–844 region includes the last two residues of helix 5, the ß-strands ß3 and ß4, and all of helix 6. Li et al. (17) identified three key differences between the MR and GR ligand-binding pockets. In the loop between helices H6 and H7, the serine at position 843 in the MR is a proline in the GR (position 637). They report that the proline forces this region outward creating a side pocket into which substitutions of the C17 positions can fit. The second difference involves the impact of leucine at MR848 in place of a glutamine in the GR (position 642), which interacts with the C17 hydroxyl of dexamethasone and cortisol. These changes in the hinge between helices 6 and 7 may impact by altering the opening between helices 7 and 11. The final difference relates to the presence of two hydrophilic residues, at positions 810 and 811, whereas the corresponding region of the other steroid receptors is hydrophobic; the significance of this change is seen in the mutation described by Geller et al. (6). Of these only Ser (843) lies in the 820–844 region; our studies indicate that Ser (843) is among the residues involved in conferring specificity. Li et al. (17) analyzed the consequences of reciprocal substitution at MR843/GR637 in a transactivation assay but not a binding assay, and then only with cortisol and corticosterone (see below).

The region 820–844 represents the greatest structural variation between MR and GR. Comparison of this region in the GR and MR structures (Fig. 4Go) identifies several key residues that are likely to play a role in aldosterone selectivity (via conformational conformity with MR): Ser (824), Tyr (828), Phe (835) and Ser (843). All are among the 12 residues identified (Table 3Go) as potentially required; we therefore speculated that they may be the essential component of the grouped substitutes used. The MR (824, 828, 835, 843)-GGMM chimera did indeed respond to aldosterone in the transactivation assay; however, the EC50 was still one order of magnitude greater than the response of MR (820–844)-GMM measured in the same assay (Table 3Go). By comparing this response to the response of the other constructs in Table 3Go, one might conclude that the degree of left-shift achieved is consistent with three of four changes being correct and/or the need for at least one additional mutation. This again emphasizes the complexity of this type of analysis where multiple interrelated substitutions have occurred in an evolutionary context.

The structural differences between MR and GR in this region allow Met (845) to contact ligand and may be critical in the differential specificity between MR and GR toward binding aldosterone. Li et al. (17) identified Pro (637) in the GR to be likely responsible for an altered form of the loop between H6 and H7 compared with MR. In addition, Phe (835) is unlikely to be compatible with the GR loop structure in this region. Differences in the structure in the preceding region (823–834) may have a role in maintaining the side-chain of Phe (835) in its position observed in the crystal structures.

The observations of Li et al. (17) suggest that the pocket of the GR LBD in this region is larger; failure of aldosterone to bind the GR may therefore reflect on instability generated by increased distance over which ligand must form stable interactions with the pocket. In the case of cortisol, given that the region of the ligand involved in the interaction, steroid rings A and B, do not differ between aldosterone and cortisol, its ability to bind must reflect compensating changes elsewhere in the GR that reposition the ligand relative to the MR and bring it back into an appropriate proximity, albeit not one that is as favorable as in the MR. This is consistent with the requirement for cortisol seen in the original chimeras that the same receptor sequence, i.e. GR or MR, is required at the second and fourth positions (14) for transactivation.

Cortisol Transactivation
In our previous study we described dissociation between the binding of cortisol and the ability of the steroid to activate the chimeras in a transactivation assay (14). Cortisol bound to all the chimeras. However, in the transactivation assay only those chimeras containing sequence from the second and fourth regions of the same receptor were activated by cortisol: i.e. those chimeras with the sequence XMXM or XGXG, where X is either sequence. The GGMM chimera, on which this study was based, has GR sequence in the second region and MR sequence in the fourth region, and is not activated by cortisol. The exact mechanism that underlies this dissociation of binding and activation remains to be determined. In the present study we find that the MR (804–844)-GGMM and MR (820–844)-GGMM chimeras are activated by cortisol, each with an EC50 value of 10 nM, which is equal to the value for the MR LBD. The other chimeras are not activated by cortisol. Further analysis with the substitutions from the 820–844 region confirmed that the only chimeras activated by cortisol are also those that bind aldosterone with high affinity. It appears, therefore, that cortisol transactivation tracks with the ability to bind aldosterone, which may point to a common mechanism underlying both properties. It is possible that cortisol binding to the inactive chimeras is relatively unstable and that rapid dissociation of the ligand leads to inactivation of the transcriptional complex. The amino acids that confer aldosterone binding specificity may be important for stabilizing the binding of both ligands. Because these residues are also required for aldosterone binding affinity as well as stability there will be no dissociation of aldosterone binding and activation in these chimeras. Although this may be a mechanism to account for the general dissociation between cortisol binding and transactivation, it does not explain our original observation (14) that cortisol activation is only retained in chimeras containing sequence from the same receptor in the second and fourth regions. It is difficult to understand how a combination of MR amino acids 820–844 and 932–984 (i.e. the fourth region) is required for cortisol activation. In the x-ray crystal structures of the MR LBD amino acids 932–984 and amino acids 820–844 lie on the opposite faces of the receptor, and there is no indication that there is direct interaction between these two regions of the protein. The structural comparison between MR and GR reveals the different association of helix H7 in the two structures. This association appears to be related to the conformation of the preceding region identified above as being responsible for specificity of aldosterone binding. This helix packs in an anti-parallel manner with helix H10, and may connect the function of MR amino acids 820–844 with activation in the chimeras.

Summary
The binding specificity of aldosterone for the MR compared with the GR is conferred by a 25 amino acids region, MR (820–844) in the LBD. Of the 16 amino acids that differ in this region, 12 appear essential for aldosterone selectivity. This same region, although not critical for cortisol binding, is critical for cortisol-induced transactivation. Similar studies involving AR:PR and PR:GR chimeras also point to the critical role of this region in steroid binding per se, specificity of binding and functionality of binding/transactivation. The residues that differ in the MR (820–844) region are predicted to lie not in the ligand-binding pocket as might be expected, but rather on the surface of the receptor. The mechanism by which this region so profoundly influences steroid binding and its functional consequences remains to be determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Chimeras
A series of MR:GR chimeras was created between amino acids 804–874 of the MR LBD and the corresponding amino acids 598–668 of the GR LBD. Different sets of chimeras were created for transactivation and [3H]-ligand binding assays. For the transactivation assays, the remaining LBD sequence of these chimeras consisted of amino acids 488–597 of the GR (N-terminal to the chimeras) and amino acids 875–984 of the MR (C-terminal to the chimeras) (Fig. 1Go). These chimeras were named using the same general nomenclature employed in the previous study in which the MR and GR LBDs were divided into four sections (14); i.e. the chimera is given a four-letter name based on the sequence in each of four sections, where M is MR sequence and G is GR sequence. Amino acids 804–874 of the MR correspond to the second of these four sections. For [3H]-ligand binding studies, the MR sequence was swapped into the GR. All chimeras were created by overlap extension PCR using pRShGR and the GMGG-pRShGR, GGMM-pRShGR, and GMMM-pRShGR chimeras from our previous study (14) as templates. All four plasmids were linearized using KpnI before PCR. In all cases PCR was performed using Pfu polymerase (Stratagene, La Jolla, CA). The final PCR products were digested with XhoI and BglII and ligated into pSP72 (Promega, Madison, WI) and then fully sequenced. The chimera sequence was subsequently removed from the vector by digestion with XhoI and BglII and ligated into pRShGR digested with XhoI and BamHI. The primers used for PCR are shown in the supplemental data published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. Mutations to incorporate restriction enzyme sites are underlined.

MR (804–815)-GGMM
The 5' end was amplified from the GMGG template using the primers GR1 and MR815R. The 3' end was amplified from the GGMM template using the primers MR815F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (804–828)-GGMM
The 5' end was amplified from the GMGG template using the primers GR1 and MR828R. The 3' end was amplified from the GGMM template using the primers MR828F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (804–844)-GGMM
The 5' end was amplified from the GMGG template using the primers GR1 and MR844R. The 3' end was amplified from the GGMM template using the primers MR844F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (834–874)-GGMM
The 5' end was amplified from the pRShGR template using the primers GR1 and MR834R. The 3' end was amplified from the GMMM template using the primers MR834F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (847–874)-GGMM
The 5' end was amplified from the pRShGR template using the primers GR1 and MR847R. The 3' end was amplified from the GMMM template using the primers MR847F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (820–844)-GGMM
The 5' end was amplified from the pRShGR template using the primers GR1 and MR820–844R. The 3' end was amplified from the MR (808–846) chimera template using the primers MR820–844F and MR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and MR2.

MR (804–844)-GR
The 5' end was amplified from the GMGG template using the primers GR1 and MR844R. The 3' end was amplified from the pRShGR template using the primers MR844F and GR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and GR2.

MR (820–844)-GR
The 5' end was amplified from the pRShGR template using the primers GR1 and MR820–844R. The 3' end was amplified from the MR (808–844)-GR chimera template using the primers MR820–844F and GR2. The two PCR products were joined together by overlap extension PCR using the primers GR1 and GR2.

Construction of Point Mutations
Single, double, and multiple point mutations were made to incorporate combinations of amino acids from MR (820–844) into the GGMM chimera. These mutations were created using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), as per the manufacturer’s instructions. The template used for mutagenesis was GGMM subcloned into pBluescript (Stratagene). Successful mutagenesis was confirmed by direct sequencing in pBluescript, and the LBD was subcloned back into the pRShGR vector. The primers used for PCR are presented in the supplemental data. The primers were named after the MR residues that they incorporate into the GGMM sequence. Mutations to incorporate amino acid changes are underlined.

Subsequent combinations (see Table 3Go) used the same primers on the new templates where the mutagenesis primers did not overlap the existing mutations. In the case of the Phe (826) and Tyr (828) double mutation combined with Thr (822)-Gln (825), the primers 5'-TCA AGT GCA AAC TTT CTG TAT TTT GCT CCT GAT CTG-3' (forward) and 5'-CAG ATC AGG AGC AAA ATA CAG AAA GTT TGC ACT TGA-3' (reverse) were used on a template containing the Thr (822)-Gln (825) mutation. To create the combined Ser (824), Tyr (828), Phe (835), and Ser (843) mutations in GGMM, three additional primer sets were used sequentially.

Tissue Culture and Transactivation Assay
CV-1 African green monkey cells were grown at 37 C in DMEM [supplemented with 0.075% sodium bicarbonate, 10 mM HEPES, 1 mM glutamine, nonessential amino acids and penicillin (10 U/liter)-streptomycin (10 µg/liter)-fungazone (0.025 µg/liter)] + 10% fetal bovine serum (FBS). The cells were trypsinized and replated in 48-well plates at a density of 2 x 104 cells/well in DMEM + 5% FBS. After 20–24 h the cells were transfected with 1 µg of the chimera expression plasmid, 1 µg of mouse mammary tumor virus-luciferase (MMTV-LUC) reporter plasmid, and 0.25 µg of pRSV-ßGAL control plasmid using the calcium phosphate precipitation method. For the studies presented in Table 4Go, the pRL-tk plasmid containing the Renilla luciferase gene (Promega) was used as the transfection control. The cells were then incubated at 37 C in DMEM + 10% charcoal-stripped FBS for 20–24 h. Fresh DMEM + 10% charcoal-stripped FBS was added to the cells before addition of steroid. The cells were incubated with steroid for 24 h at 37 C and then harvested for assay. Measurement of transactivation was performed using the Dual-Light kit (Tropix, Bedford, MA) or the Renilla Luciferase Assay System (Promega), as per the manufacturers’ instructions. The cells were incubated with 100 µl lysis buffer for 30 min at room temperature. A 10-µl aliquot was removed for assay. Steroid-dependent luciferase activity was measured first, the tubes were incubated for 1 h at room temperature to allow the luciferase protein to degrade, and then ß-galactosidase or Renilla luciferase activity, which was included as a control for transfection efficiency, was measured. All measurements were performed with two independent points in two separate experiments.

Ligand-Binding Assay
Ligand-binding assays were performed in COS-1 cells. Cells were grown at 37 C in DMEM + 10% FBS. The cells were trypsinized and replated in 12-well plates at a density of 8 x 104 cells/well. After 20–24 h the cells were transfected with the expression plasmids using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) as per the manufacturer’s instructions, and then incubated overnight in DMEM + 10% FBS. Before assay the medium was replaced with DMEM (no FBS) and the cells incubated for one hour at 37 C. They were then washed three times with ice-cold PBS. The cells were incubated with radioligand in DMEM (no FBS) at 37 C for 1 h. They were then washed three times with ice-cold PBS and then lysed using 1 M NaOH. The suspension was added to scintillant and radioactivity was measured in a Packard 2500 TR liquid scintillation counter (Packard Instrument Co., Meriden, CT).

Scatchard Analysis
Scatchard analysis of [3H]aldosterone binding was performed to determine the affinities (Kd) of the constructs for the ligand. For the MR LBD and MR:GR chimeras, the concentrations of radioligand used were 25, 10, 4, 1.6, 0.64, and 0.26 nM. To examine [3H]aldosterone binding to the GR, the concentrations of radioligand used were 50, 25, 12.5, 6.25, and 3.2 nM. For each concentration of radioligand, nonspecific binding was determined by coincubation with a 500-fold excess of unlabeled aldosterone. Results were analyzed using the EBDA-Ligand program (36). Cortisol binding to the chimeras was assessed by incubating 20 nM [3H]dexamethasone with 0, 0.2, 1, and 10 µM cortisol. Comparison of the Kd values was performed using Student’s two-tailed t tests and the GB-STAT software (Dynamic Microsystems Inc., Houston, TX).


   ACKNOWLEDGMENTS
 
We thank Professors R. M. Evans, S. Nordeen, G. Ooi, and W. Tilley for the generous gifts of the plasmids pRShGRNX, pRShMRNX, MMTV-LUC, pRen-LUC, and pRSV-ß-GAL. We thank Francine Brennan for her work to conclude this study. We also thank Claudette Thiedeman and Sue Panckridge for help in preparation of the manuscript.


   FOOTNOTES
 
This work was supported by the National Health & Medical Research Council through a Project Grant and a Career Fellowship (to P.J.F.) (no. 122200) and also by a generous donation from Mrs. Eva and Mr. Les Erdi.

Current address for F.M.R.: Royal Children’s Hospital, Parkville 3050, Victoria, Australia.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 6, 2007

Abbreviations: AR, Androgen receptor; DBD, DNA-binding domain; FBS, fetal bovine serum; GR, glucocorticoid receptor; LBD, ligand-binding domain; LUC, luciferase; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; PR, progesterone receptor; TA, triamcinolone acetonide.

Received for publication June 13, 2006. Accepted for publication February 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Fuller PJ, Young MJ 2005 Mechanisms of mineralocorticoid action. Hypertension 46:1227–1235[Abstract/Free Full Text]
  2. Gomez-Sanchez EP, Zhou M, Gomez-Sanchez CE 1996 Mineralocorticoids, salt and high blood pressure. Steroids 61:184–188[CrossRef][Medline]
  3. Young M, Funder JW 2000 Aldosterone and the heart. Trends Endocrinol Metab 11:224–226[CrossRef][Medline]
  4. Pitt B 2004 Effect of aldosterone blockade in patients with systolic left ventricular dysfunction: implications of the RALES and EPHESUS studies. Mol Cell Endocrinol 217:53–58[CrossRef][Medline]
  5. Lifton RP, Gharavi AG, Geller DS 2001 Molecular mechanisms of human hypertension. Cell 104:545–556[CrossRef][Medline]
  6. Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai FTF, Sigler PB, Lifton RP 2000 Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289:119–123[Abstract/Free Full Text]
  7. Gronemeyer H, Gustafsson JÅ, Laudet V 2004 Principles for modulation of the nuclear receptor superfamily. Nat Rev 3:950–964
  8. Arriza JL, Weinberger C, Cerell, G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268–275[Abstract/Free Full Text]
  9. Pascual-Le Tallec L, Lombès M 2005 The mineralocorticoid receptor: a journey exploring its diversity and specificity of action. Mol Endocrinol 19:2211–2221[Abstract/Free Full Text]
  10. Rogerson FM, Fuller PJ 2000 Mineralocorticoid action. Steroids 65:61–73[CrossRef][Medline]
  11. Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306–360[Abstract/Free Full Text]
  12. Rupprecht R, Reul JMHM, van Steensel B, Spengler D, Söder M, Berning B, Holsboer F, Damm K 1993 Pharmacological and functional characterization of human mineralocorticoid and glucocorticoid receptor ligands. Eur J Pharmacol 247:145–154[CrossRef][Medline]
  13. Arriza JL 1991 Aldosterone action: perspectives from the cloning of the mineralocorticoid receptor. In: Bonvalet JP, Farman N, Lombès M, eds. Aldosterone: fundamental aspects. Paris: Colloque INSERM/John Libbey Eurotext; 13–21
  14. Rogerson FM, Dimopoulos N, Sluka P, Chu S, Curtis AJ, Fuller PJ 1999 Structural determinants of aldosterone binding selectivity in the mineralocorticoid receptor. J Biol Chem 274:36305–36311[Abstract/Free Full Text]
  15. Fagart J, Huyet J, Pinon GM, Mayer C, Rafestin-Oblin M-E 2005 Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension. Nat Struct Mol Biol 12:554–555[CrossRef][Medline]
  16. Bledsoe RK, Madauss KP, Holt JA, Apolito CJ, Lambert MH, Pearce KH, Stanley TB, Stewart EL, Trump RP, Willson TM, Williams SP 2005 A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor. J Biol Chem 280:31283–31293[Abstract/Free Full Text]
  17. Li Y, Suino K, Daugherty J, Xu HE 2005 Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor. Mol Cell 19:367–380[CrossRef][Medline]
  18. Vivat V, Gofflo D, Wurtz J-M, Bourguet W, Philibert D, Gronemeyer H 1997 Sequences in the ligand-binding domains of the human androgen and progesterone receptors which determine their distinct ligand identities. J Mol Endocrinol 18:147–160[Abstract/Free Full Text]
  19. Robin-Jagerschmidt C, Wurtz J-M, Guillot B, Gofflo D, Benhamou B, Vergezac A, Ossart C, Moras D, Philibert D 2000 Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and nodulate the receptor transactivation capacity. Mol Endocrinol 14:1028–1037[Abstract/Free Full Text]
  20. Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nature Struct Biol 3:206[CrossRef][Medline]
  21. Bruns CM, Hubatsch I, Ridderström M, Mannervik B, Tainer JA 1999 Human glutathione transferase A4–4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products. J Mol Biol 288:427–439[CrossRef][Medline]
  22. Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD, Consler TG, Parks DJ, Stewart EL, Willson TM, Lambert MH, Moore JT, Pearce KH, Xu HE 2002 Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110:93–105[CrossRef][Medline]
  23. Kauppi B, Jakob C, Farnegardh M, Yang J, Ahola H, Alarcon M, Calles K, Engstrom O, Harlan J, Muchmore S, Ramqvist AK, Thorell S, Ohman L, Greer J, Gustafsson JA, Carlstedt-Duke J, Carlquist M 2003 The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism. J Biol Chem 278:22748–22754[Abstract/Free Full Text]
  24. White PC, Mune T, Agarwal AK 1997 11ß-Hhydroxysteroid dehydrogenase and the syndrome of apparent mineralocorticoid excess. Endocr Rev 18:135–156[Abstract/Free Full Text]
  25. De Kloet ER, Vreugdenhil E, Oitzl MS, Joëls M 1998 Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269–301[Abstract/Free Full Text]
  26. Baker ME 1997 Steroid receptor phylogeny and vertebrate origins. Mol Cell Endocrinol 135:101–107[CrossRef][Medline]
  27. Sloman KA, Desforges PR, Gilmour KM 2001 Evidence for a mineralocorticoid-like receptor linked to branchial chloride cell proliferation in freshwater rainbow trout. J Exp Biol 204:3953–3961
  28. Rogerson FM, Yao Y-Z, Smith BJ, Dimopoulos N, Fuller PJ 2003 Determinants of spironolactone binding specificity in the mineralocorticoid receptor. J Mol Endocrinol 31:573–582[Abstract]
  29. Rogerson FM, Yao Y, Smith BJ, Fuller PJ 2004 Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor. Clin Exp Pharmacol Physiol 31:704–709[CrossRef][Medline]
  30. Martinez ED, Pattabiraman N, Danielsen M 2005 Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination. Exp Cell Res 308:320–333[CrossRef][Medline]
  31. Lind U, Greenidge P, Gustafsson J-Å, Wright APH, Carlstedt-Duke J 1999 Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids. J Biol Chem 274:18515–18523[Abstract/Free Full Text]
  32. Carlsson P, Koehler KF, Nilsson L 2005 Glucocorticoid receptor point mutation V571M facilitates coactivator and ligand binding by structural rearrangement and stabilization. Mol Endocrinol 19:1960–1977[Abstract/Free Full Text]
  33. Rogerson FM, Brennan FE, Fuller PJ 2003 Dissecting mineralocorticoid receptor structure and function. J Steroid Biochem Mol Biol 85:389–396[CrossRef][Medline]
  34. Fuller PJ, Smith BJ, Rogerson FM 2004 Cortisol resistance in the New World revisited. Trends Endocrinol Metab 15:296–299[CrossRef][Medline]
  35. Nettles KW, Sun J, Radek JT, Sheng S, Rodriguez AL, Katzenellenbogen JA, Katzenellenbogen BS, Greene GL 2004 Allosteric control of ligand selectivity between estrogen receptors {alpha} and ß: implications for other nuclear receptors. Mol Cell 13:317–327[CrossRef][Medline]
  36. McPherson GA, Molenaar P, Raper C, Malta E 1983 Analysis of dose-response curves and calculation of agonist dissociation constants using a weighted nonlinear curve fitting program. J Pharmacol Methods 10:231–241[CrossRef][Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   GR  |  MR
Ligands:   Dexamethasone  |  Spironolactone  |  Aldosterone  |  Progesterone




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rogerson, F. M.
Right arrow Articles by Fuller, P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rogerson, F. M.
Right arrow Articles by Fuller, P. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals