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
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ABSTRACT
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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 734984) 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 (729733) 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.
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INTRODUCTION
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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
-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 734984
(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 receptors
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.
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RESULTS
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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. 1
) were tested for their ability to bind
aldosterone. The binding of aldosterone to the LBD [MR(734984)]
showed that both the affinity and the maximum number of sites were
lower than those of the entire receptor (see Fig. 2
and Table 1
), although both proteins were
produced at similar levels (data not shown). The hMR sequence
(729984), 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(729984) 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. 2
and Table 1
). MR
(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
729733 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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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
(C1), MR
(C2), MR
(C3), and MR
(C4), were
lacking the last one, two, three, or four amino acids of hMR (see Fig. 1
). The 3H-labeled steroid-binding data showed that
deletion of the last one or two amino acids decreased aldosterone
binding by 30% (Fig. 3
). Deletion of the
last three amino acids [MR
(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. 3
). The binding of aldosterone to MR
(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
(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
(C3) (t1/2 13 min) than from the entire receptor
(t1/2 105 min) (Fig. 4
).
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(729984), a mutant receptor
truncated in the amino-terminal region that has an affinity for
aldosterone of 2.93 nM (Table 1
) and a t1/2 of
40 min (Fig. 4
). 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. 3
), 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(729984), and
MR (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.
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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. 5
) or presence (Fig. 6
) of aldosterone. The entire unliganded
hMR sedimented at 10S on a sucrose gradient equilibrated in
tungstate-containing buffer (Fig. 5
). The sedimentation coefficient of
the receptor incubated with 7C10 was 12.3S, indicating that hMR is
associated with hsp90. MR(729984) sedimented as a large peak in the
10S region, comprising a hsp90-free entity (45S) in addition to the
hsp90-associated state of MR(729984) (910S), which was partially
displaced by 7C10. This result suggests that the hsp90-MR(729984)
interaction is unstable. The LBD [MR(734984)] sedimented as a 5.5S
entity that was not displaced by 7C10, indicating that hsp90 does not
interact with MR(734984) (Fig. 5
). All the C terminus-truncated
mutant hMRs were recovered associated with hsp90. This is illustrated
in Fig. 5
for MR
(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(729984) and hsp90. As aldosterone stabilizes
the hsp90-receptor interaction (30), MR(729984) was incubated with
aldosterone before it was layered on the sucrose gradient (see Fig. 6
).
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. 6
). Thus, the interaction between hsp90 and MR(729984) was less
tight than the interaction between hsp90 and the entire hMR, and
aldosterone binding to the MR(729984) 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 6. Sedimentation Gradient Analyses of
Aldosterone-Bound Entire MR and MR(729984)
Entire MR and MR(729984) 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 ( ).
Aliquots (100 µl) were treated with dextran-charcoal and layered onto
a 520% 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).
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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
(C4) to trypsin
was assessed and compared with that of the full-length receptor. The
35S-labeled hMR and MR
(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
(C4) (Fig. 7
), 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. 7
, lane 2). Under the same conditions, the
41-kDa fragment generated from MR
(C4) accounted for only 27% of the
two fragments (Fig. 7
, 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. 8A
). 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. 8A
, 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. 8B
). MR
(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. 8B
, lanes 12). MR
(C40) generated
two fragments of 36 and 26 kDa (Fig. 8B
, lane 3). MR
(C20) produced
two fragments of 38 and 28 kDa (Fig. 8B
, lane 4), whereas LBD
(C20)
generated only a 28-kDa fragment (Fig. 8B
, 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
(A/B) supported the relevance of the putative amino terminus limit
of the 41-kDa fragment. As shown in Fig. 8B
(lanes 38), 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
(A/B), for which the 41-kDa fragment was more abundant than the
30-kDa fragment (Fig. 8B
, lanes 12). 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 (C4)
35S-labeled MR and MR (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 16) or with
(lanes 712) 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.
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DISCUSSION
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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 (729733) 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
(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(729984) 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
-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 968979 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
(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.
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MATERIALS AND METHODS
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Chemicals
[1,2-3H]aldosterone (4060 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 (5060 Ci/mmol) were
provided by Roussel-Uclaf Laboratories (Romainville, France).
[1,2-3H]cortisol (5060 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
(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
(C1): 5'-CCCAAGCTTTCACCGGTGGAAGTAGAGCGG-3'
MR
(C2): 5'-CCCAAGCTTTCAGTGGAAGTAGAGCGGCTT-3'
MR
(C3): 5'-CCCAAGCTTTCAGAAGTAGAGCGGCTTGGC-3'
MR
(C4): 5'-CCCAAGCTTTCAGTAGAGCGGCTTGGCGTT-3'
MR
(C20): 5'-CCCAAGCTTTCAGATGATCTCCACCAGC-3'
MR
(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(729984) 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(734984)] fragment
using the same reverse primer as for MR(729984) 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
(C20) was constructed as above using the same forward primer as
for MR(734984), 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 manufacturers 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 (0.1100 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 520% 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. 
Received for publication September 23, 1997.
Revision received November 19, 1997.
Accepted for publication February 18, 1998.
 |
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