Folding Requirements of the Ligand-Binding Domain of the Human Mineralocorticoid Receptor

Brigitte Couette1, Stéphan Jalaguier1, Chantal Hellal-Levy, Brigitte Lupo, Jérôme Fagart, Gilles Auzou and Marie-Edith Rafestin-Oblin

INSERM U478 (B.C., C.H-L., J.F., M-E.R-O.) Faculté de Médecine Xavier Bichat Institut Fédératif de Recherche 02 BP 416, 75870 Paris Cédex 18, France
INSERM U300 (S.J., B.L., G.A.) Faculté de Pharmacie 34060 Montpellier, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a series of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus-truncated hMRs showed that the ligand-binding domain (LBD: amino acids 734–984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (Kd) 2.9 vs. 0.47 nM] and does not interact with hsp90. Addition of the five- amino acid sequence (729–733) upstream from the LBD is necessary for interaction with hsp90, but a larger region is needed for high aldosterone affinity. Deletions at the C-terminal end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids reduced the affinity for aldosterone to 1/20 that of the entire protein, and deletion of the last four amino acids completely abolished binding, although the interaction with hsp90 was not affected. These effects can be explained by misfolding of the receptor, since limited proteolysis assays showed that deletions at the C-terminal end of hMR affect the accessibility of the cleavage sites within the DNA-binding domain and the N-terminal part of the hinge region to trypsin. Thus, our results support the idea that a short sequence upstream of the LBD is essential for the interaction of hMR with hsp90 and that the C terminus of hMR and hsp90 are both essential for folding of the receptor in a high-affinity hormone-binding state.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mineralocorticoid receptor (MR) belongs to the steroid/thyroid hormone receptor superfamily of ligand-regulated transcription factors. These factors exert their effects by binding to cis-acting DNA elements (hormone-response elements) in the regulatory region of target genes (1). The nuclear receptors are structurally and functionally related, and specific properties have been assigned to distinct domains (2, 3, 4, 5, 6). The amino-terminal A/B domain has a ligand-independent transactivation function. The central C-domain [DNA-binding domain (DBD)] is highly conserved and composed of two zinc fingers involved in DNA binding and receptor dimerization. A hydrophilic region forms a hinge between the DBD and C-terminal ligand-binding domain (LBD). The LBD consists of approximately 250 amino acids with a complex tertiary structure composed of {alpha}-helices and ß-sheets (7, 8, 9). The LBD mediates complex, overlapping functions including ligand binding, interaction with heat-shock proteins, dimerization, nuclear targeting, and hormone-dependent transactivation.

Aldosterone-dependent activation of gene transcription is thought to involve a series of steps. Aldosterone binds to the receptor and changes the receptor conformation in the LBD (10, 11). This change is believed to lead to the dissociation of the associated proteins, leaving the receptor in a suitable conformation for productive interaction with the transcriptional machinery.

On the basis of sequence homology among nuclear receptors, the LBD of human MR (hMR) has been defined as the sequence from positions 734–984 (1). However, this sequence has not been shown to be the minimum requirement for aldosterone-binding capacity. The deletions from the N-terminal region of the MR have extended only to amino acid 729, just before the LBD (12, 13, 14). These mutant hMRs bind aldosterone with characteristics similar to those of the entire MR, but they are all produced in Escherichia coli as recombinant fusion proteins, and the role of the linked proteins in aldosterone binding is unknown. The role of the C-terminal part of the MR LBD in ligand binding is also unknown. It has been reported that deletion at the C terminus of nuclear receptors abolishes their agonist binding (15, 16, 17, 18, 19, 20, 21, 22, 23). The C-terminal tail of the progesterone receptor (PR) also appears to be crucial for discriminating between agonists and antagonists. Deletion of a small region of the C terminus of the PR greatly reduces progesterone binding, but has a minimal effect on the receptor’s affinity for an antagonist, the RU486 (17). A monoclonal antibody raised against the last 14 amino acids of PR inhibits progesterone binding but not the binding of RU486 (24). Proteolysis assays have also demonstrated differences in the conformations of progesterone, estrogen, and glucocorticoid receptors at the extreme C-terminal end of the protein after the binding of agonists and antagonists (25, 26).

This study was carried out to determine the region of hMR required for interaction with agonists and antagonists, taking into account the cross-talk of ligand and hsp90 binding (27, 28). Mutant hMRs produced in vitro with deletions in either the N- or C-terminal region were used to identify a region upstream from the LBD of hMR that is important for the interaction of hsp90 with the receptor and to show that the extreme C-terminal part of hMR is involved in the folding of the receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Determination of the Core of the hMR LBD
DNA constructs encoding mutant hMRs truncated at either the amino- or carboxy-terminal end of the receptor were produced and expressed in rabbit reticulocyte lysate to identify the minimal region of the hMR with ligand-binding capacity. The resulting proteins (see Fig. 1Go) were tested for their ability to bind aldosterone. The binding of aldosterone to the LBD [MR(734–984)] showed that both the affinity and the maximum number of sites were lower than those of the entire receptor (see Fig. 2Go and Table 1Go), although both proteins were produced at similar levels (data not shown). The hMR sequence (729–984), produced in Escherichia coli as a fusion protein with the maltose-binding protein, has been previously reported to bind aldosterone with the same affinity as the entire MR, when incubated with rabbit reticulocyte lysate (14). It was therefore important to determine whether the five amino acids upstream from the LBD were involved in aldosterone binding. MR(729–984) was produced in rabbit reticulocyte lysate and found to have the same aldosterone-binding capacity as the entire hMR, but its affinity was only one sixth that of the entire hMR (see Fig. 2Go and Table 1Go). MR{Delta}(A/B), a mutant with a larger region upstream from the LBD, had the same affinity as the entire hMR (data not shown). Thus, the five amino acids at positions 729–733 are necessary for aldosterone binding, and a larger sequence is required for aldosterone to be bound with a high affinity.



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Figure 1. Diagramatic Representation of the Entire hMR and the N and C Terminus-Truncated hMRs Used in This Study

The numbers indicate the amino acid positions. The structural domains of hMR are delimited: A/B domain (white bar), DNA binding domain (stippled bar), hinge region (black bar), and ligand binding domain (hatched bar).

 


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Figure 2. Fig. 2. Scatchard Plot of [3H]Aldosterone Binding to the Entire and N Terminus-Truncated hMR

The entire and truncated hMRs were synthesized in vitro in rabbit reticulocyte lysate. The lysate was diluted 2-fold with TEGWD buffer and incubated with increasing concentrations of [3H]aldosterone (0.1 to 100 nM) for 4 h at 4 C. Bound (B) and free (F) steroids were separated by the dextran-charcoal method.

 

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Table 1. Affinity of Aldosterone and Maximum Number of Aldosterone-Binding Sites for Entire and Mutant hMRs

 
The binding of ligands to receptors truncated at their carboxy terminus was tested. Preliminary experiments showed that neither class of ligands bound to mutants lacking the last 40 or the last 20 amino acids (data not shown). A series of mutant hMRs with deletions at the C terminus was therefore prepared to determine the minimal region required to maintain the receptor in a hormone-competent state. The mutant hMRs, MR{Delta}(C1), MR{Delta}(C2), MR{Delta}(C3), and MR{Delta}(C4), were lacking the last one, two, three, or four amino acids of hMR (see Fig. 1Go). The 3H-labeled steroid-binding data showed that deletion of the last one or two amino acids decreased aldosterone binding by 30% (Fig. 3Go). Deletion of the last three amino acids [MR{Delta}(C3)] resulted in a 70% decrease in aldosterone binding, and deletion of the last four amino acids abolished aldosterone binding. Deletion at the C-terminal part of the receptor also led to a decrease in the binding of cortisol (a mineralocorticoid agonist) and RU26752 (a mineralocorticoid antagonist) (Fig. 3Go). The binding of aldosterone to MR{Delta}(C3) was further characterized by Scatchard analysis, and the dissociation kinetics of aldosterone from this mutant hMR were investigated. The affinity of aldosterone for MR{Delta}(C3) was 20-fold lower (Kd 9.03 nM) than that of the full-length receptor (Kd 0.47 nM), and aldosterone dissociated more rapidly from MR{Delta}(C3) (t1/2 13 min) than from the entire receptor (t1/2 105 min) (Fig. 4Go). Thus, the lower affinity of aldosterone for mutated receptors was accompanied by an increase in the off rate of the ligand from the receptor, which also occurs with MR(729–984), a mutant receptor truncated in the amino-terminal region that has an affinity for aldosterone of 2.93 nM (Table 1Go) and a t1/2 of 40 min (Fig. 4Go). Phenylalanine 981 was changed to alanine (F981A) in the full-length molecule to determine whether the fourth amino acid from the C-terminal end of hMR was crucial to the ligand-binding capacity of the receptor. The amount of aldosterone bound to F981A was 30% that of aldosterone bound to the entire receptor (Fig. 3Go), and the affinity was 20-fold lower (Kd 10.95 vs. 0.47 nM). This indicates that the extreme C terminus of the receptor is required for high-affinity ligand binding.



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Figure 3. Binding of Agonists and Antagonists to the Entire and Mutant hMRs

The entire and mutant hMRs were synthesized by translation in vitro. The lysate was diluted 2-fold with TEGWD buffer and incubated with 10 nM [3H]aldosterone (open bars), [3H]cortisol (gray bars) or [3H]RU26752 (hatched bars) for 30 min at 20 C. Bound and free steroids were separated by the dextran-charcoal method. Nonspecific binding was measured in a parallel experiment with a 100-fold excess of the corresponding unlabeled steroid. Results are expressed as specific 3H-labeled steroid binding.

 


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Figure 4. Dissociation Kinetics of [3H]Aldosterone from Entire hMR, MR(729–984), and MR{Delta}(C3)

Mutants and entire hMRs were produced by translation in vitro. The lysate was diluted 2-fold with TEGWD buffer and incubated with 10 nM [3H]aldosterone for 30 min at 20 C. The end of this incubation period was time zero for kinetic analysis. The lysate was then divided into two. One sample was kept at 20 C to measure the stability of steroid-binding sites, and the other was incubated with 1 µM unlabeled aldosterone. Bound and free steroids were separated by the dextran-charcoal method. Nonspecific binding was measured in parallel incubations for each incubation time. Results are corrected for receptor stability and are expressed as a percentage of the binding measured at zero time.

 
Role of hsp90 in Ligand Binding to hMR
It has been reported that hsp90 is required for ligand binding to MR (12, 13, 14). We therefore investigated whether the loss of binding capacity of the N-and C terminus-truncated receptors could be due to the absence of hsp90. The presence of hsp90 in the hMR complex was studied by using 7C10, a mouse monoclonal antibody that recognizes rabbit hsp90 (29). This work examined the oligomeric structure of the entire and mutated hMRs in the absence (Fig. 5Go) or presence (Fig. 6Go) of aldosterone. The entire unliganded hMR sedimented at 10S on a sucrose gradient equilibrated in tungstate-containing buffer (Fig. 5Go). The sedimentation coefficient of the receptor incubated with 7C10 was 12.3S, indicating that hMR is associated with hsp90. MR(729–984) sedimented as a large peak in the 10S region, comprising a hsp90-free entity (4–5S) in addition to the hsp90-associated state of MR(729–984) (9–10S), which was partially displaced by 7C10. This result suggests that the hsp90-MR(729–984) interaction is unstable. The LBD [MR(734–984)] sedimented as a 5.5S entity that was not displaced by 7C10, indicating that hsp90 does not interact with MR(734–984) (Fig. 5Go). All the C terminus-truncated mutant hMRs were recovered associated with hsp90. This is illustrated in Fig. 5Go for MR{Delta}(C4). The mutant hMR sedimented as a 9.4S peak, which was shifted to 12.7S after incubation with 7C10, suggesting its association with hsp90. We further examined the loose interaction between the mutant MR(729–984) and hsp90. As aldosterone stabilizes the hsp90-receptor interaction (30), MR(729–984) was incubated with aldosterone before it was layered on the sucrose gradient (see Fig. 6Go). Under these conditions, we observed a narrow peak at 9.4S that was displaced to 11.4S after incubation with the anti-hsp90 antibody, a sedimentation profile similar to that of the ligand-bound entire hMR (Fig. 6Go). Thus, the interaction between hsp90 and MR(729–984) was less tight than the interaction between hsp90 and the entire hMR, and aldosterone binding to the MR(729–984) stabilized its interaction with hsp90. Altogether, these results indicate that truncation at the C-terminal end of the receptor does not prevent the receptor-hsp90 interaction, whereas a sequence lying upstream of the LBD seems to be crucial for the interaction of hMR with hsp90.



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Figure 5. Sedimentation Gradient Analyses of Entire, N-, and C Terminus- Truncated Mutant hMRs in Ligand-Free Conditions

35S-Labeled MR, MR(729–984), MR(734–984), and MR{Delta}(C4), synthesized in vitro (0.1 ml), were incubated for 1 h at 20 C with 5 µl undiluted control ascites (•) or anti-hsp90 antibody 7C10 ({triangleup}). Samples (100 µl) were layered onto a 5–20% sucrose gradient equilibrated in TEGWD buffer. Gradients were centrifuged for 105 min at 365,000 x g in a VTi 65.2 rotor at 4 C. Collected fractions were analyzed by electrophoresis on a 7.5% (wt/vol) polyacrylamide gel for MR and MR{Delta}(C4) and on a 12.5% (wt/vol) polyacrylamide gel for MR(729–984) and MR(734–984). The band corresponding to the entire or mutant protein was scanned and quantified. The signal intensity was plotted against the relative migration coefficient (Rf). The sedimentation markers were aldolase (A, 7.9S) and BSA (4.6S).

 


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Figure 6. Sedimentation Gradient Analyses of Aldosterone-Bound Entire MR and MR(729–984)

Entire MR and MR(729–984) synthesized in vitro (0.1 ml) were incubated for 10 min at 20 C with 100 nM [3H]aldosterone before 1 h incubation at 20 C with 5 µl undiluted control ascites (•) or anti-hsp90 antibody 7C10 ({triangleup}). Aliquots (100 µl) were treated with dextran-charcoal and layered onto a 5–20% sucrose gradient equilibrated in TEGWD buffer. Gradients were centrifuged for 105 min at 365,000 x g in a VTi 65.2 rotor at 4 C. The radioactivity of each fraction was plotted against the relative migration coefficient (Rf). The sedimentation markers were aldolase (A, 7.9S) and BSA (4.6S).

 
Role of the C-Terminal Amino Acids in Receptor Conformation
We investigated whether the lack of ligand binding to the carboxy-terminus-truncated mutant hMRs could be due to an inappropriate conformation of the receptor. The sensitivity of MR{Delta}(C4) to trypsin was assessed and compared with that of the full-length receptor. The 35S-labeled hMR and MR{Delta}(C4) were incubated in ligand-free conditions with 20 µg/ml trypsin for various periods, and the digestion products were analyzed by SDS-PAGE. Two major fragments of 41 and 30 kDa were generated from the entire hMR and from MR{Delta}(C4) (Fig. 7Go), but the abundance of the two fragments differed. For the entire hMR, the 41-kDa fragment was the most abundant, representing 62% of the two fragments after 5 min of trypsin treatment (Fig. 7Go, lane 2). Under the same conditions, the 41-kDa fragment generated from MR{Delta}(C4) accounted for only 27% of the two fragments (Fig. 7Go, lane 7). These findings show that deletion of the last four amino acids makes the 41-kDa fragment more sensitive to trypsin, suggesting a change in the hMR conformation. We determined whether these fragments contained the LBD by incubating the entire hMR with or without aldosterone and digesting the resulting complexes with increasing concentrations of trypsin for 15 min (Fig. 8AGo). When the entire hMR was incubated with aldosterone before trypsin treatment, the 41- and 30-kDa fragments were resistant to 100 µg/ml of trypsin, whereas they were completely digested in the absence of aldosterone (Fig. 8AGo, compare lane 12 to lane 6). Thus, the binding of aldosterone to hMR modifies the compaction of the receptor so that the 41- and 30-kDa fragments are more resistant to trypsin, which may indicate that these fragments encompass the LBD. We have attempted to define the limits of the 41- and 30-kDa fragments by analyzing the fragments generated by digesting mutant hMRs truncated either at the N- or C-terminal end of the receptor with trypsin (20 µg/ml) for 10 min (Fig. 8BGo). MR{Delta}(A/B), which extends from residue 603 to 984, has a predicted molecular mass of about 41 kDa and presents an electrophoretic tryptic pattern similar to that of the entire hMR (Fig. 8BGo, lanes 1–2). MR{Delta}(C40) generated two fragments of 36 and 26 kDa (Fig. 8BGo, lane 3). MR{Delta}(C20) produced two fragments of 38 and 28 kDa (Fig. 8BGo, lane 4), whereas LBD{Delta}(C20) generated only a 28-kDa fragment (Fig. 8BGo, lane 5). Since truncation at the C-terminal end of the receptor reduced the size of both the 41-kDa and the 30-kDa fragments, the two trypsin-generated fragments may well have the same C-terminal end as the entire receptor. The N-terminal cleavage site compatible with the generation of a 30-kDa fragment is likely to be residue Lys 715. In the hinge region, downstream of a nonclassical sequence of eight proline residues, there is a sequence of amino acid residues that is highly exposed to proteolysis. In this region, the Tyr 710 is very sensitive to chymotrypsin, generating a 30-kDa fragment (Ile711-Lys984) (11). We believe that the tryptic 30-kDa fragment produced in the present study is (Glu716-Lys984). The molecular mass of the 41-kDa fragment together with the trypsin cleavage sites suggest that the predicted amino terminus of the 41-kDa fragment could be one of the three amino acids, Ser591, Pro599, or Ile602, lying just before the DBD. The electrophoretic pattern of MR{Delta}(A/B) supported the relevance of the putative amino terminus limit of the 41-kDa fragment. As shown in Fig. 8BGo (lanes 3–8), all the 26- to 30-kDa fragments generated from the C terminus-truncated mutant hMRs and from F981A were more abundant than the 36- to 41-kDa fragments. This contrasts with the result observed for the entire hMR and MR{Delta}(A/B), for which the 41-kDa fragment was more abundant than the 30-kDa fragment (Fig. 8BGo, lanes 1–2). We therefore conclude that deletions in the C-terminal part of the receptor change the compaction of the receptor by rendering some proteolytic cleavage sites in the DBD and the N-terminal part of the hinge region more accessible to trypsin.



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Figure 7. Proteolysis Kinetics of Entire hMR and MR{Delta}(C4)

35S-labeled MR and MR{Delta}(C4) were produced by translation in vitro and digested at 20 C for various periods of time, with 20 µg/ml trypsin. An equal volume of water was added for the undigested controls (lanes 1 and 6). The digestion products were analyzed by electrophoresis in a 12.5% (wt/vol) polyacrylamide gel and autoradiographed as described in Materials and Methods. The molecular mass markers are indicated on the left-hand side of the figure.

 


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Figure 8. Electrophoretic Analyses of Entire and Mutant hMRs

A, The 35S-labeled hMR was produced by translation in vitro, incubated at 20 C without (lanes 1–6) or with (lanes 7–12) 100 nM aldosterone for 10 min before digestion for 15 min with the indicated concentrations of trypsin. An equal volume of water was added for the undigested controls (lanes 1 and 7). B, The entire MR and a series of mutant hMRs were synthesized by translation in vitro and digested for 10 min with 20 µg/ml trypsin at 20 C. The digestion products were analyzed by electrophoresis in a 12.5% (wt/vol) polyacrylamide gel and autoradiographed as described in Materials and Methods. The molecular mass markers are indicated on the left-hand side of the figure.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this paper we show that a short amino acid sequence upstream the LBD of hMR is necessary for the receptor to interact with the chaperone protein hsp90. We also show that the C-terminal end of the hMR is needed, in addition to hsp90, for the receptor to fold into a conformation able to bind agonists and antagonists.

There is some evidence that hsp90 binds to the LBD of glucocorticoid, progesterone, and estrogen receptors, and that more than one site within the LBD is involved in the hsp90-receptor interaction (31, 32, 33). It has also been shown that a region at the C-terminal end of the DBD is needed for the estrogen receptor to form a heterooligomeric 8S complex (31). The present study shows that a short sequence of five amino acids (729–733) upstream of the LBD of hMR is necessary for its interaction with hsp90. However, this short sequence is not sufficient to stabilize the MR-hsp90 interaction, as a larger region upstream from the LBD appears to be necessary for high-affinity ligand binding. The 30-kDa fragment generated by chymotrypsin, which encompasses the entire LBD and the C-terminal part of the hinge region (Ile711-Lys984), binds aldosterone with an affinity that is only one third that of the full-length receptor (11), whereas MR{Delta}(A/B) has the same affinity as the full-length receptor. A high-affinity receptor can also be generated by fusing a nonreceptor sequence, such as that of the maltose-binding protein, upstream from the MR(729–984) sequence (14). This suggests that the unrelated protein stabilizes the receptor-hsp90 complex. Xu et al (34) also reported a sequence outside the LBD of the glucocorticoid receptor that is necessary for the production of a stable, active LBD and whose effects could be mimicked by nonreceptor proteins.

This paper also provides evidence that truncation at the C terminus of the receptor results in the loss of the MR capacity to bind hormone. This was not due to a reduction in the amount of MR protein or to the absence of interaction with hsp90, but to misfolding of the receptor, as demonstrated by the limited proteolysis assays. All the mutant hMRs truncated at their C terminus generated a fragment of about 41 kDa that was more sensitive to trypsin than that issued from the entire hMR. These fragments probably start just before the DBD, at residue Ser591, Pro599, or Ile602, and encompass the 30-kDa fragment (Glu716-Lys984). Thus, truncating the receptor at the C terminus alters the compaction of the receptor by rendering some proteolytic cleavage sites lying within the DBD and the N-terminal part of the hinge region more accessible to trypsin.

The recently published crystal structures of the LBD of the human retinoid X receptors (7) and of the rat thyroid hormone receptor (8) have shown that the LBD consists of a single structural domain, packed in three layers, composed of 12 {alpha}-helices and four short ß-strands forming a mixed ß-sheet. This general structure may be common to the LBDs of all steroid receptors (9). Helix 12, at the C terminus of the receptor, seems to be crucial for receptor activation because it moved toward the core of the receptor, upon ligand binding, facilitating contact with helices 3 and 4 located at the amino terminus of the LBD. However, the structure of the end of the nuclear receptors downstream from helix 12 is unknown. We have now shown that the third and fourth amino acids from the C-terminal end of the hMR are crucial for the folding of the receptor into a hormone-competent entity. This may also be a feature of other steroid receptors, for which a loss of ligand binding occurs after deletion of the end of the molecule (15, 16, 17, 18, 19, 20, 21, 22, 23). It has been reported that the C-terminal end of PR is involved in discriminating between agonists and antagonists (17, 35). Mutant PRs lacking the C-terminal 42 or 54 amino acids are specifically activated by RU486, a progesterone antagonist, although they are unable to bind progesterone (17). However, this does not seem to hold for all antagonists, because a mutant androgen receptor lacking the 12 C-terminal amino acids still binds RU486, but not other antiandrogens (20). We now find that antimineralocorticoids and aldosterone agonists do not bind to mutant MRs lacking the C terminus of the receptor. A short repressor region in the C terminus of the PR has been identified between amino acids 917 and 928, just after helix 12 (36). This region is the site of interaction with an inhibitory cofactor. Deletion of this region creates a mutant PR that responds positively to RU486. Sequence alignment shows that this repressor region corresponds to amino acids 968–979 in the hMR sequence. In this work, we did not restore antagonist binding to the receptor after deletion of the last 40 amino acids. Thus, if such a repressor region is present in MR, it is not located at the C terminus of the molecule.

It is still not clear why the misfolded C terminus-truncated hMRs do not bind ligands. It could be due to a change in the exposure of one or more of the contact points needed for full steroid-receptor interaction. Aldosterone dissociates more rapidly from the misfolded receptor MR{Delta}(C3). However, it is also possible that, even if hsp90 is associated with the C terminus-truncated hMR proteins, the complex of associated proteins may be destabilized. Indeed, other proteins, in addition to hsp90, have been shown to associate with steroid receptor complexes, such as hsp70 and hsp56, or a variety of other proteins (for review see Refs. 28 and 37), including immunophilins (FKBP54 and cyP-40) (38, 39) and phosphoproteins (p23 and p60) (40, 41). Further studies of the proteins associated with hMR and analysis of the crystal structures of the ligand-free and ligand-bound LBD are required to advance our understanding of the activation of the MR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
[1,2-3H]aldosterone (40–60 Ci/mmol), L-[35S]methionine (1000 Ci/mmol), and 14C-labeled low-range protein molecular mass markers were purchased from Amersham (Les Ulis, France). Nonradioactive aldosterone, cortisol, chymotrypsin, and trypsin were obtained from Sigma (St Louis, MO). Unlabeled and [3H]RU26752 (50–60 Ci/mmol) were provided by Roussel-Uclaf Laboratories (Romainville, France). [1,2-3H]cortisol (50–60 Ci/mmol) and Entensify were obtained from Du Pont-New England Nuclear (Boston, MA). The TNT T7-coupled rabbit reticulocyte lysate system was purchased from Promega (Charbonnières, France). Purified oligonucleotides were purchased from Eurogentec (Seraing, Belgium) or Oligo Express (Paris, France). To avoid steroid adsorption, steroid solutions prepared in ethanol were dried, and the steroids were resuspended in 50% polyethylene glycol 300 (PEG 300) prepared in TEG, to give a final concentration of 5% in the lysate.

Buffers
TEG buffer contained 20 mM Tris-HCl, 1 mM EDTA, and 10% glycerol. TEGW was TEG containing 20 mM sodium tungstate, and TEGWD was TEGW containing 1 mM dithiothreitol. Buffers were adjusted to pH 7.4 at 25 C.

Antibody
The monoclonal anti-hsp90 antibody 7C10, a mouse IgG1 immunoglobulin, was used as diluted ascites fluid (29).

DNA Constructs
The receptor fragment MR{Delta}(A/B) was obtained by PCR amplification, using phMR3750 (1) as a matrix, with the following primers, introducing HindIII sites (underlined) at both ends:

5'-CCCAAGCTTATGTGTTTGGTGTGTGGGGAT-3'. (forward primer); 5'-CCCAAGCTTTCACTTCCGGTGGAAGTAGAG-3' (reverse primer, which introduces a stop codon). The amplified product was purified and ligated into the HindIII site of pGEM11 vector (Promega, France).

The C-terminal truncated MR mutant genes were obtained by PCR amplification using phMR3750 as a matrix. The same forward primer was used for all mutants: 5'-CCCAAGCTTATGGAGACCAAAGGCTACCAC-3' which introduces a HindIII site at the 5' end (underlined). Reverse primers, introducing a HindIII site and a stop codon at the 3' end, were as follows:

MR{Delta}(C1): 5'-CCCAAGCTTTCACCGGTGGAAGTAGAGCGG-3'

MR{Delta}(C2): 5'-CCCAAGCTTTCAGTGGAAGTAGAGCGGCTT-3'

MR{Delta}(C3): 5'-CCCAAGCTTTCAGAAGTAGAGCGGCTTGGC-3'

MR{Delta}(C4): 5'-CCCAAGCTTTCAGTAGAGCGGCTTGGCGTT-3'

MR{Delta}(C20): 5'-CCCAAGCTTTCAGATGATCTCCACCAGC-3'

MR{Delta}(C40): 5'-CCCAAGCTTTCAGAAGCAGAATTCCAGC-3'.

The mutant cDNAs were digested with HindIII, and the mutated cDNAs were purified by agarose gel electrophoresis and electroelution and ligated into HindIII sites of phMR3750.

The MR(729–984) fragment was obtained by PCR amplification from the matrix phMR3750 with the following primers, which introduced a XhoI site at the 5'-end and a SalI site at the 3'-end: 5'-CCCCTCGAGATGACAATC TCACGAGCGCTCACACC-3' (forward primer) 5'-CGGCTGTCGACTCACTT CCGGTGGAAGTAGAG-3' (reverse primer, which introduces a stop codon). The amplified PCR fragment was digested with XhoI and SalI and purified by agarose gel electrophoresis and electroelution. It was then ligated into the XhoI and SalI sites of the pCI vector (Promega, Madison, WI).

The same protocol was used to create the [LBD: MR(734–984)] fragment using the same reverse primer as for MR(729–984) and the following forward primer, which introduced a MluI site at the 5'-end instead of the XhoI site: 5'-TGGACGCGTATGCTCACACCTTCCCCC-3' (forward primer).

LBD{Delta}(C20) was constructed as above using the same forward primer as for MR(734–984), introducing an MluI site. The reverse primer was that used as for the C terminus-truncated hMR.

The F981A mutant was constructed by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit and Pfu DNA polymerase from Stratagene (La Jolla, CA). Mutagenic oligonucleotide primers that generated specific nucleotide changes in the hMR cDNA, coding for amino acid substitutions, were: 5'-AACGCCAAGCCGCTCTACGCCC-ACCGGAAGT GACTGCCC-3' [engineered mutations (underlined) resulted in a change from phenylalanine-981 to alanine]. The reverse primer contained the desired mutation and was annealed to the corresponding sequence on the opposite strand of the plasmid. Mutation-containing DNA was selected by digestion with DpnI, which specifically digests the methylated nonmutated parental DNA template. Mutated DNA was used to transform electrocompetent JM109 cells. All PCR amplifications were performed using Pfu DNA polymerase (Stratagene). In all cases, the presence of the specific mutation and the lack of random mutations was checked by DNA sequence analysis (Genome Express, Grenoble, France).

Coupled Cell-Free Transcription and Translation
Plasmids (1 µg) containing cDNA coding for the full-length hMR (1) or mutant hMRs were transcribed using T7 RNA polymerase, and the mRNA was simultaneously translated in rabbit reticulocyte lysate for 1 h at 30 C according to the manufacturer’s instructions. The reactions were conducted with unlabeled or [35S]methionine in the translation mixture, depending on the experiment.

Hormone-Binding Assay
After translation of the full-length or mutant hMRs, the lysate was diluted 2-fold with TEGWD buffer and incubated for 30 min at 20 C with 10 nM [3H]aldosterone, [3H]cortisol, or [3H]RU26752. Nonspecific binding was measured in a parallel experiment with 100-fold excess of the corresponding unlabeled steroid. Bound and free steroids were separated by the dextran-charcoal method: 25 µl lysate were stirred for 5 min with 50 µl 4% Norit A, 0.4% Dextran-T70 in TEG buffer, and centrifuged at 4500 x g for 5 min at 4 C.

Steroid-Binding Characteristics at Equilibrium
After translation of the full-length or mutant hMRs, the lysate was diluted 2-fold with ice-cold TEGWD buffer and incubated for 4 h at 4 C with [3H]aldosterone (0.1–100 nM). Bound (B) and free (F) steroids were separated by the dextran-charcoal method. Bound steroid was measured by counting the radioactivity of the supernatant. The evolution of B as a function of F was analyzed as previously described (42), and the dissociation constant Kd was calculated.

Kinetic Experiments
After translation of the full-length or mutant hMRs, the lysate was diluted 2-fold with TEGWD buffer and incubated with 10 nM [3H]aldosterone for 30 min at 20 C. The lysate was then divided in two. One half was kept at 20 C to measure the stability of the [3H]aldosterone-MR complexes, and the other was incubated with 1 µM aldosterone for various periods. The bound and free steroids were separated by the charcoal dextran method. Parallel incubations containing [3H]aldosterone plus 100-fold excess of aldosterone were used to calculate the nonspecific binding. The half-lives of the steroid-receptor complexes (t1/2) were calculated from the equation B(t) = B(0) e-(k-1t) where B(0) and B(t) represent the specific steroid binding at times 0 and t of the dissociation period. B(t) was corrected for the stability of steroid binding at each dissociation time.

Sucrose Gradient Centrifugation
Samples were layered onto a 5–20% sucrose gradient prepared in TEGW buffer, and the gradients were centrifuged in a VTi 65.2 rotor at 4 C for 1 h, 45 min at 365,000 x g. Three-drop fractions were collected by piercing the bottom of each tube. Each collected fraction was analyzed by electrophoresis. Aldolase (A, 7.9S) and BSA (4.6S) were used as external sedimentation markers.

Limited Proteolytic Digestion of Translated hMR
Trypsin (diluted in water) was added to 5 µl 35S-labeled translation mix incubated with or without unlabeled steroid for 10 min at 20 C. Aliquots of the digestion product (1 µl) were removed and mixed with 20 µl protein loading buffer, boiled for 5 min, immediately loaded onto a 12.5% SDS-polyacrylamide gel, and subjected to electrophoresis. The gels were then fixed for 30 min in methanol-acetic acid-distilled water (30:10:60), treated with Entensify, and dried. They were autoradiographed at -80 C overnight.

Analysis of Autoradiographs by Scanning Densitometry
Autoradiographs were scanned by image analysis (Optilab, Graftek, France). Results are given as optical density, expressed in arbitrary units.

Miscellaneous
The protein concentration in the lysate was determined by the Bradford method, using BSA as standard (43). The protein concentration of the rabbit reticulocyte lysate was about 50 mg/ml. Radioactivity was measured in a LKB liquid scintillation spectrometer (LKB Instruments, Gaithersburg, MD) after addition of 5 ml of OptiPhase HiSafe (counting efficiency,~50%).


    ACKNOWLEDGMENTS
 
We thank J. L. Arriza and R. Evans for providing phMR3750, C. Radanyi and M. Lombès for the anti-hsp90 antibody (7C10), and Roussel Uclaf for providing RU26752. We are grateful to A. Vandewalle and N. Courtois-Coutry for helpful discussions and D. Mesnier for technical assistance. We thank J. Knight and C. Tritscher for editorial assistance.


    FOOTNOTES
 
Address requests for reprints to: M.E. Rafestin-Oblin, INSERM U478, Faculté de médecine Xavier Bichat, 16 rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France.

1 Considered jointly as first authors. Back

Received for publication September 23, 1997. Revision received November 19, 1997. Accepted for publication February 18, 1998.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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