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INSERM U478 (C.H.-L., A.S., M.-E.R.-O.) Faculté de
médecine Xavier Bichat Institut fédératif de
recherche 02 B.P. 416, 75780 Paris Cédex 18, France
Institut de Génétique et de Biologie
Moléculaire et Cellulaire (J.F., J.-M.W., D.M.) B.P.
163, 67404 Illkirch Cédex CU de Strasbourg, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The precise mechanisms whereby transcription is stimulated remain
unknown. In the absence of hormone, steroid receptors are associated
with an inhibitory multiprotein complex composed of hsp90, hsp70, p59,
and other factors (5). The binding of the steroid to the hMR induces a
change in the receptor conformation that is believed to lead to the
dissociation of the multiprotein complex formed with receptor (6, 7).
Recent studies have identified two related proteins, a nuclear receptor
corepressor (N-CoR) and a silencing mediator for the retinoid and
thyroid-hormone receptors (SMRT), that mediate the repression of
transcription by thyroid hormone receptor (TR) and retinoic acid
receptor (RAR) (8, 9). The nuclear hormone receptors that interact with
N-CoR and SMRT now include other members of the NR superfamily, such as
COUP-TF, and the thiazolidinedione-peroxisome proliferator-activated
receptor 
(PPAR) (10, 11).
Several crystal structures of NR-LBDs have been described: the
ligand-free form (apo receptor) of the retinoid X receptor 
(RXR)
and PPAR, the ligand-bound form (holo receptor) of
all-trans-retinoic acid receptor (RAR), TR, ER,
PR, and PPAR (12, 13, 14, 15, 16, 17, 18). All these NR-LBDs have a common fold with 11
to 12 -helices (numbered H1-H12) and one ß-sheet arranged as an
antiparallel -helical "sandwich" in a three-layer structure
(19). Ligand binding causes the LBD to adopt a more compact structure;
the helix H11 is repositioned in the continuity of H10 and the helix
H12, that comprises the autonomous activating domain (AF-2AD), is
folded back toward the LBD core. The repositioning of the activation
helix H12, together with additional structural changes such as bending
of H3, brings it into a distinct receptor environment, thus creating an
interface suitable for NR coactivator binding (20). Many coactivators
interacting with the nuclear receptors in a ligand-dependent manner
have been identified to date. These include steroid
receptor-coactivator 1 (SRC-1), transcriptional intermediary factor II
(TIF-II)/GRIP-1, and 140-kDa receptor-interacting proteins (RIP-140),
TIF1, and CBP/p300 (10, 11).
Because agonist-induced repositioning of the helix 12 is crucial to bring steroid receptors in a suitable conformation for productive interaction with transcriptional machinery, the question arises whether the extended region connecting the helices H11 and H12 (H11-H12 loop), which is highly conserved among steroid receptors, plays a role in the intramolecular process(es) of MR activation. A detailed mutagenesis analysis of the hMR H11-H12 loop was therefore carried out. The ligand-binding properties and transcriptional activities of the mutant hMRs were measured. Their ability to interact with coactivators was assessed as well as their conformational states. In the light of a three-dimensional model of the hMR-LBD, we propose that H11-H12 loop is required to fold the hMR into a ligand-binding competent state and to stabilize the active receptor conformation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and hPR is presented
in Fig. 1
. The residues of the H111-H12
loop are highly conserved among MRs. hGR has one residue and hER
three residues less than the hMR, whereas the H11-H12 loops in hAR and
hPR have the same length as in the hMRs. Residues of the hMR H11-H12
loop were replaced by alanine and the corresponding mutant receptors
referred to as H950A, L952A, K953A, V954A, E955A, F956A, and P957A.
Wild-type and mutant hMRs were expressed in vitro and tested
for their expression level and ability to bind aldosterone. As observed
in Fig. 2A, similar amounts of the
wild-type and mutant hMRs were synthesized according to the protein
band intensity with a molecular mass of 110 kDa. H950A, L952A and E955A
retained the aldosterone binding capacity of the wild-type receptor
(Fig. 2B). K953A, V954A, and P957A displayed 50–75% of the
aldosterone binding capacity. In contrast F956A was unable to bind
aldosterone (Fig. 2B). Moreover, the mutant hMRs transiently expressed
in COS-7 cells were tested for their ability to bind aldosterone.
L952A, K953A, V954A, and P957A displayed 35–65% of the aldosterone
binding capacity. H950A and E955A retained 100% of the aldosterone
binding capacity whereas F956A has completely lost this function (data
not shown).
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). Aldosterone dissociated from
L952A, K953A, and P957A much more rapidly (t1/2
values of 3–10 min) than from the wild-type receptor (120 min) (Table 1) and the aldosterone-E955A complex was characterized by a half-life
time of 42 min.
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), suggesting
that replacement of Phe956 by an alanine does not prevent the
hsp90-receptor interaction.
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Conformation Analysis of the Mutant hMRs by Limited Proteolysis
Assays
We investigated whether replacement Phe956 by alanine
induced a change in the receptor conformation leading to the lack of
aldosterone binding. The sensitivity of F956A to chymotrypsin was
examined and compared with that of the wild type. The
35S-labeled wild-type hMR and F956A synthesized
in vitro were incubated in ligand-free conditions with 15
µg/ml chymotrypsin for 5–30 min, and the digestion products were
analyzed by SDS-PAGE (Fig. 4). The
wild-type receptor and F956A were both synthesized at the same level
(Fig. 4, lanes 1 and 6). Two major fragments corresponding to molecular
masses of 27 and 30 kDa were generated from both the wild-type hMR and
F956A, but the abundance of the two fragments differed. After 5 min
chymotrypsin treatment, the intensity of the 30-kDa fragment generated
from the wild-type receptor was higher than that of the 27-kDa fragment
(Fig. 4, lane 2), and the two fragments were completely digested after
a 30-min proteolysis (Fig. 4, lane 6). In the case of F956A, the
intensity of the 30-kDa fragment was lower than that of the 27-kDa
fragment (Fig. 4, lane 8), and the 30- and 27-kDa fragments were
completely digested after 15 and 30 min proteolysis, respectively (Fig. 4, lanes 10 and 12). The 30-kDa fragment corresponds to the sequence
Ile711-Lys984, which contains the C-terminal part of the hinge region
and the entire LBD of the receptor (7). The 27-kDa fragment has not
been identified. Proteolysis experiments performed with a truncated
hMR, lacking the A/B domain, generated the 30- and 27-kDa fragments,
suggesting that the 27-kDa fragment did not encompass the A/B region.
Altogether these findings revealed an increase in the receptor
sensitivity to proteolysis upon Phe956 substitution, suggesting a
change in the receptor compaction.
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, lanes 2). When the wild-type and
mutant hMRs (L952A, K953A, V954A, and P957A) were first incubated with
aldosterone and then with chymotrypsin for 10 min treatment, a 30-kDa
fragment was detected (Fig. 5, lanes 3–7). The 30-kDa fragment
generated from the mutant hMRs was partially (L952A) or completely
(K953A, V954A, and P957A) digested after 60 min chymotrypsin treatment,
whereas 30-kDa fragment generated from the wild-type receptor was
highly resistant (Fig. 5, lanes 8–12). These results suggest that
replacement of Leu952, Lys953, Val954, and Pro957 by alanine does not
prevent the aldosterone-induced compaction of the receptor.
Nevertheless, these aldosterone-mutant hMRs complexes are less
resistant to proteolysis than the wild-type receptor associated with
aldosterone, suggesting a different or less stable conformation of
these mutant receptors compared with the wild-type hMR.
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. The
aldosterone induced-activity of H950A and E955A was nearly the same as
that of the wild-type hMR. L952A and P957A displayed approximately 50%
of the wild-type receptor activity; in contrast, K953A, V954A, and
F956A have completely lost the receptor activity (Fig. 6A).
Dose-response curves were also generated by adding increasing
concentrations of aldosterone to transfected cells (Fig. 6B).
Aldosterone increased the luciferase activity of the wild-type hMR in a
dose-dependent manner with an ED50 value of
approximately 10-10 M.
Replacement of His950 by alanine did not modify the aldosterone-induced
transactivation activity of hMR, and replacement of Glu955 by alanine
induced a shift in the dose-response curve of the aldosterone-induced
luciferase activity toward higher concentrations with an
ED5O value of approximately
10-9 M. The aldosterone-
mediated activities of L952A, K953A, V954A, and P957A were low
(Fig. 6B). Progesterone, a potent mineralocorticoid antagonist (23),
was able to inhibit the aldosterone-induced activity of H950A and E955A
by 80%, as it does for the wild-type hMR (data not shown). Thus,
except H950A, which retained the transcriptional activity of the
wild-type receptor, all the other mutant hMRs, within the H11-H12 loop,
displayed a lower transcriptional activity, suggesting that this loop
is involved in the hMR activation process.
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(24, 25, 26). The wild-type hMR did not interact with coactivators
in the absence of ligand (Fig. 7A, lanes
3, 6, and 9) or in the presence of progesterone (Fig. 7A, lanes 5, 8,
and 11). Incubation of hMR with aldos-terone promoted the
interaction of the receptor with coactivators (Fig. 7A, lanes 4, 7, and
10). Our results are consistent with those observed with GRIP1 and
SCR-1 for the rat MR (27, 28) and other NRs indicating that the
receptor-coactivator interaction is agonist dependent (10, 11). As
observed for the wild-type hMR, the ligand-free H950A, K953A, and E955A
did not interact with hTIF1 (Fig. 7B, lanes 1, 3, and 5). In the
presence of aldosterone, H950A interacted with hTIF1 (Fig. 7B, lane
2), and a weak interaction was detected for E955A (Fig. 7B, lane 6).
hTIF1 did not interact with aldosterone-associated K953A (Fig. 7B, lane 4), and no interaction of L952A, V954A, and P957A with hTIF1
was detected (data not shown). The interaction profile of the mutant
hMRs with RIP140 and SRC-1 was the same as that observed with hTIF1
(data not shown). Altogether, these results showed that, with the
exception of H950A and E955A, all the mutant hMRs have lost their
ability to bind coactivators.
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). The polar residues face the solvent,
whereas the hydrophobic ones are buried and point toward the core of
the protein. Phe956 is within the region delimited by the helices H3,
H11, and H12 and forms van der Waal contacts with Asn770 (H3), Phe946
(H11), Ser949 and Val954 (H11-H12 loop), Leu960, Val961 and Ile964
(H12), and the aldosterone C21-hydroxyl group (3, 2 Å) (Fig. 9A). Val954 is deeply buried (<10% of
solvent accessibility) and is in close contact with Glu763, Leu766,
Ser767 and Asn770 (H3), Leu952 and Phe956 (H11-H12 loop) (Fig. 9B).
Leu952 is less well buried (24% accessibility) than Val954 and is
close to Thr945 (H11) and Val954 (H11-H12 loop) (Fig. 9B). The two
charged residues of the H11-H12 loop (Lys953 and Glu955) could form
salt bridges with Glu763 and Arg771, respectively, two residues of H3
(Fig. 9B). As in the hPR crystal structure, the backbone oxygen atom of
Glu955 forms a strong hydrogen bond with the Asn770 amide group (Figs. 8 and 9A). From this model, it appeared that there are numerous
contacts between the H11-H12 loop residues and also between the
residues of this loop and residues of H3, H11, and H12.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Replacement of Phe956 by alanine within the hMR causes a complete loss of ligand binding and transcriptional activity. The hMR homology model, based on the structural data of the progesterone-associated hPR, revealed that the 21-hydroxyl group of aldosterone is in a favorable position to make van der Waals contacts with Phe956. But it is unlikely that the loss of aldosterone binding capacity upon Phe956 substitution is due solely to the absence of this contact, since progesterone, which lacks the 21-hydroxyl group, is also unable to bind F956A (data not shown). The inability of F956A to bind agonist and antagonist ligands is not due to a lack of interaction with hsp90 since F956A sedimented at 8–10S as hsp90-receptor complexes (21). The loss of ligand binding capacity upon Phe956 substitution is due to a change in the receptor conformation, as F956A displays a high sensitivity to chymotrypsin compared with the wild-type receptor. Altogether, these results suggest that the residue Phe956 is essential to fold the hMR into a ligand binding competent state.
Replacement of His950 by alanine does not modify the aldosterone binding and transcriptional activities of the wild-type receptor. In contrast, replacement of the other H11-H12 loop residues decreased (Leu952, Glu955, Pro957) or completely abolished (Lys953, Val954) the receptor activity without notably modifying the affinity of the receptor for aldosterone. The different sensitivities of ligand binding and transcriptional activities to substitution of an H11-H12 loop residue have been reported for other steroid receptors. A naturally occurring mutant androgen receptor, in which Val889 (V954 in hMR) is replaced by a methionine, is nearly completely insensitive to androgens, whereas it has a normal binding affinity for androgens (29). Similarly, a mutant GR, in which Ile747 (Val954 in hMR) is replaced by threonine, can bind dexamethasone like the wild-type receptor, but high dexamethasone concentrations are needed to stimulate the transactivation function (30).
The first step after aldosterone binding to the receptor is a change in the receptor conformation, revealed by an increase in the resistance of the receptor to proteolysis (7). The binding of aldosterone to L952A, K953A, V954A, and P957A increases the resistance of the receptor to chymotrypsin, suggesting that aldosterone bound to these mutant hMRs causes a change in receptor compaction in a way similar to the effect of aldosterone bound to the wild-type receptor. But, as aldosterone dissociates more rapidly from mutant hMRs than from the wild-type receptor, it is less effective in protecting these mutant hMRs against proteolysis than the wild-type receptor. Thus, the ability of aldosterone to protect mutant hMRs against proteolysis depends upon the stability of the aldosterone-receptor complex.
There have been several reports that coactivators interact with NRs
when receptors are transcriptionally active (31, 32, 33, 34). The introduction
of a mutation into the TRß-H12 (35), the vitamin D receptor-H12 (36),
and the ER-H3 (37) causes an alteration in the NR activity together
with an impaired interaction with coactivators. The loss of these two
functions upon mutation of residues within the helices H12 or H3 is not
surprising, given the model proposed from structural data that compares
the ligand-induced receptor transconformation to a mouse trap
mechanism, by which structural changes such as bending of helix H3,
bring the helix H12 into a distinct receptor environment creating the
areas for coactivator binding. Furthermore, analysis of the structure
of agonist-associated PPAR and hER LBD complexed with a peptide
encompassing the coactivator LXXLL region shows that this peptide is
bound, in a -helical conformation, by a hydrophobic groove formed by
residues from helices H3, H4, H5, and H12 (38, 39). The present study
shows that aldosterone-associated-hMR interacts with SRC-1 RIP140 and
hTIF1. In contrast, neither ligand-free nor
antagonist-associated-hMR bind these coactivators, indicating that the
hMR-coactivator interaction is agonist dependent. Those mutant hMRs
that retain the full transcriptional activity interact with
coactivators, as it is observed for H950A. The poor ability of E955A to
recruit coactivators is consistent with the lower sensitivity of this
mutant hMR (ED50 10-9
M) compared with that of the wild- type receptor
(10-10 M). The inability of L952A,
K953A, V954A, and P957A to recruit coactivators, whereas L952A and
P957A display 50% of the wild-type receptor activity, might be due to
the rapid dissociation of aldosterone from these mutant hMRs and/or to
poor receptor-coactivator contacts resulting from a modification of the
aldosterone-associated-hMR conformation.
The homology model of the hMR LBD reveals numerous contacts in the
H11-H12 region that might help to stabilize the aldosterone
associated-hMR conformation. There are van der Waals contacts between
the hydrophobic residues of the H11-H12 loop (Val 954 with Leu952 and
Phe956) and also between residues of the loop and the nearby amino
acids of H3, H11, and H12. In addition, the two polar residues of the
loop (Lys953 and Glu955) are in suitable positions to make salt bridges
with two residues of H3 (Glu763 and Arg771). These contacts may help
anchor the H11-H12 loop to helix H3, as mutation of these residues
reduces (Glu955) or abolishes (Lys953) receptor activity without
preventing the ligand binding and resistance to proteolysis.
Furthermore, the oxygen atom of the Glu955 main chain forms a strong
hydrogen bond with Asn770 in helix 3, a residue that is critical in the
binding of C21-hydroxylated agonists such as aldosterone (40). This
network of contacts is essential for the transition from the
ligand-free inactive to the liganded active receptor state. The
structure of the ligand-free hMR is unknown: it could be similar to
that of the ligand-free RXR (12), in which helix H11 obstructs the
ligand binding cavity, and the H11-H12 loop and H12 point away from the
LBD core. The residues of the H11-H12 loop might be compared with a zip
fastener, in which each residue of the H11-H12 loop is a link, which
helps to promote the precise positioning of H12. It is likely that the
accommodation of the receptor around aldosterone is a rate limiting
step, as proposed by Ribeiro et al. (41) for TRß, which
depends greatly upon the integrity of the H11-H12 loop residues.
The results described here point out the role of the H11-H12 loop in stabilizing the inactive ligand-free and active aldosterone-associated hMR conformation states. With the help of other factors, such as hsp90, the loop H11-H12 maintains the receptor in an open state able to bind agonist and antagonist ligands. It is also essential for establishing the network of contacts required for ordering the activation helix H12 allowing coactivators recruitment. The similarity of the overall organization of NRs and the existence of mutations in other NRs with similar effects on the receptor activation led us to propose that the H11-H12 loop is involved in producing the active state in the other nuclear receptors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression and Reporter Constructs
The plasmid pchMR was constructed by excising a 3.6-kb
HindIII-HindIII fragment containing the entire
coding sequence of the hMR gene and about 270 bp and 400 bp of the 5'-
and 3'-untranslated regions from plasmid 3750 (1). This fragment was
subcloned into the expression vector pcDNA3 (Invitrogen,
NV leek, The Netherlands). pFC31Luc, which contains the MMTV promoter
driving the luciferase gene, was obtained from H. Richard-Foy (LMBE,
Toulouse, France) (42).
Site-Directed Mutagenesis
Site-specific mutagenesis of the hMR was performed by the method
of Nelson and Long (43). The mutant hMR fragments were obtained by PCR
amplification using GeneAmp (Perkin-Elmer Cetus, Norwalk,
CT). Four primers were used: a reverse hybrid primer downstream of an
AflII enzyme restriction site, composed of a nucleotide
sequence complementary to the cDNA reverse strand and a single 5'-20
nucleotide sequence: (5'-GGGGTACTAGTAACCCGGGCACCTCTGCCA GCTCTGCCC-3'),
a forward primer upstream of a BpU1102I enzyme restriction site:
(5'-ACGAAGTGTTTCTACTGGATC-3'), a reverse primer of identical sequence
to the single sequence 5'-20 nucleotide: (5'-GGGGTACTAGTAACCCGGGC-3'),
and a forward mutagenic primer with base mismatch was used as
follows:
H950A:5'-ACCTTCCGAGAGTCCGCTGCGCTGAAG GTA-3',
L952A: 5'-TCCCATGCGGCGAAGGTAGAGTTCCCC-3',
K953A: 5'-TCCCATgCgCTGgCggTAgAgTTCCC C-3',
V954A: 5'-TCCCATgCgCTgAAggCAgAgTTCCCC-3',
E955A: 5'-CTgAAGGTAGCGTTCCCCGCAATGCTG-3',
F956A: 5'-CTGAAGGTAGAGGCCCCCGCAATGCTG-3'
P957A: 5'-CTgAAggTAgAgTTCgCCgCAATgCTg-3').
The amplified products were digested with BpU1102 and AflII, purified by agarose gel electrophoresis, subcloned into the BpU1102 and AflII sites pchMR vector and transformed into electrocompetent JM109 cells. The presence of the specific mutation and the lack of random mutations were checked by DNA sequence analysis (Genome Express, Grenoble, France).
Cell Culture and Transfection
COS-7 cells were cultured in DMEM (Life Technologies, Inc., Cergy Pontoise, France) supplemented with 10%
heat-inactivated FCS, 2 mM glutamine, 100 IU/ml penicillin,
and 100 µg/ml streptomycin in a humidified atmosphere with 5%
CO2. Cells were maintained in the medium
supplemented with 10% charcoal-stripped FCS 4 h before and
thoughout the transfection procedure. Cells were transfected by the
phosphate calcium precipitation method (Promega Corp.
system). The phosphate solution, prepared for six-well trays, contained
5 µg of one of the receptor expression vectors (wild-type or mutant
pchMR), 10 µg pFC31Luc (42) that contained the MMTV promoter driving
the luciferase gene, and 5 µg pSVß, including the gene coding for
ß-galactosidase. The steroids to be tested were added to the cells
12 h after transfection and incubation continued for 24 h.
Cell extracts were then prepared and assayed for luciferase (44) and
ß- galactosidase activities (45). To standardize for transfection
efficiency, the relative light units, obtained in the luciferase assay,
were divided by the optical density obtained in the ß-galactosidase
assay.
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]-labeled methionine in the translation
mixture, depending on the experiment. The protein concentration of the
rabbit reticulocyte lysate, determined by the Bradford method (46) with
BSA as standard, was about 50–70 mg/ml.
In Vitro Hormone-Binding Assay
Reticulocyte lysates containing the wild-type or mutant hMRs
were diluted 2-fold with TEGWD buffer (20 mM sodium
tungstate and 1 mM dithiothreitol in TEG) and incubated for
30 min at 20 C with 10 nM
[3H]aldosterone. Nonspecific binding was
measured in a parallel experiment with a transcription and translation
in the reticulocyte lysate without receptor. 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 4,500 x g for 5 min at 4 C.
Bound steroid was measured by counting the radioactivity of the
supernatant. Radioactivity was measured, in disintegrations per min, in
a liquid scintillation spectrometer (LKB, Rockville, MD)
after adding 5 ml OptiPhase HiSafe (counting efficiency 
50%).
Hormone Binding in Whole Cells
COS-7 cells transiently transfected as described above were
incubated for 30 min at 20 C with 10 nM
[3H]aldosterone. The cells were rinsed twice
with 1 ml ice-cold PBS (pH 7.4), and bound steroids were extracted by
incubating the cells with 0.5 ml ethanol at 20 C for 30 min. The
radioactivity of the ethanol extracts was measured in a
LKB liquid scintillation spectrometer after adding 5 ml
OptiPhase HiSafe (counting efficiency 50%).
Steroid-Binding Characteristics at Equilibrium
Reticulocyte lysates containing the wild-type or mutant hMRs
were 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 unbound (U) steroids were separated by
the dextran-charcoal method. The change in B as a function of U was
analyzed (47) and the dissociation constant at equilibrium
(Kd) calculated.
Kinetic Experiments
Reticulocyte lysates containing the wild-type or mutant hMRs
were diluted 2-fold with ice-cold TEGWD buffer and incubated with 10
nM [3H]aldosterone for 1 h 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 10 µM
aldosterone for various times. After each incubation period, bound and
free steroid were separated by the charcoal dextran method. Parallel
incubations containing [3H]aldosterone plus a
1000-fold excess of unlabeled 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)Ê = ÊB(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) is 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
[35S]-labeled wild-type or mutant hMRs
synthesized in vitro were incubated with or without
unlabeled aldosterone (10-7
M) for 10 min at 20 C. Chymotrypsin (20 µg/ml
or 150 µg/ml) was added to 9 µl
[35S]-labeled translation mix incubated with or
without aldosterone for various times 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 Universal Autoradiography Enhancer,
dried, and autoradiographed at -80 C overnight.
Production of GST Fusion Proteins
The vectors pGEX2TK containing GST, GST fused with the RIP-140
amino acid sequence 683-1158 (GST-RIP-140), and GST fused with the
hTIF1 amino acid sequence 630–854 (GST-hTIF1) were provided by
V. Cavaillès (48). GST-SRC-1 encoding a fusion protein containing
GST and residues 570–780 of hSRC-1 was provided by M. G. Parker
(49). GST and GST fusion proteins were expressed as described by Kaelin
et al. (50). Overnight cultures of Escherichia
coli expressing the recombinant GST plasmids were diluted in
Luria-Bertani medium (LB). Cultures at an absorbance at 600 nm of
0.8–1.2 were induced for 3 h with isopropyl
ß-D-thiogalactoside (0.1
mM final concentration). Bacteria were then
collected by centrifugation, suspended 1:10 in NETN (0.5% Nonidet P-40
(NP40), 1 mM EDTA, 20 mM
Tris-HCl, 100 mM NaCl, pH 8.0) containing
protease inhibitors. The suspension was sonicated and then centrifuged.
Protein concentration was estimated by the Bradford method, and the
bacterial proteins were separated by SDS-PAGE and visualized by
Coomassie blue staining.
GST Pull-Down Assays
An aliquot of crude bacterial extract (1 ml) containing GST fusion
proteins was incubated for 30 min at 4 C with 100 µl
glutathione-Sepharose beads, previously washed three times, in NETN
[final concentration 1:1 (vol/vol)]. The glutathione-Sepharose beads
were then washed three times with NETN. The wild-type and mutant hMRs
were transcribed, translated, and 35S-labeled in
rabbit reticulocyte lysate following the manufacturer’s instructions.
The resulting receptors were incubated without (ethanol: no hormone) or
with 1 µM aldosterone or progesterone for 10 min at 20 C
and then with the fusion proteins on glutathione-Sepharose beads for
1 h at 4 C. The beads were washed, suspended in 20 µl loading
buffer, boiled for 3 min, and analyzed by SDS-PAGE. Signals were
amplified with Entensify, and gels were autoradiographed at -80 C
overnight.
hMR Homology Model
The hMR-LBD homology model and the docking of the ligands were
prepared as previously described (40). Briefly, the hMR-LBD homology
model was generated by the Modeller package (version 4.0) (51) using
the hPR crystal structure as a template (G. Auzou, J. Fagart, A.
Souque, C. Hellal-Levy, J. M. Wurtz, D. Moras, and M. E.
Rafestin-Oblin, manuscript in preparation).
Aldosterone was docked manually in the pocket using the
probe-accessible and van der Waals volumes as guides; these volumes
were generated with VOIDOO (52). The side chains in the vicinity of the
ligand were positioned in favorable orientation using a rotamer library
of the O package (53). The Charmm package (QUANTA/CHARMM package,
Molecular Simulation, Inc., Burlington, MA) was used for all
calculations. The complex was energy minimized in 2000 steps with a
dielectric constant of 2, using the Powell procedure. During the
minimization process, the hydrogen bonds were defined by upper harmonic
distance restraints (60 kcal Å-2 force
constant), and the overall structure of the LBD was maintained by
harmonic position restraints (30 kcal Å-2 force
constant) of the C atoms of residues defining the secondary
structure elements.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was suported by INSERM (APEX 98–34, MERO) and by the Fondation pour la recherche medicale (C.H.L.).
Received for publication January 12, 2000. Revision received April 13, 2000. Accepted for publication April 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. Nature 375:377–382[CrossRef][Medline]
. Nature 395:137–143[CrossRef][Medline]
. Nature 395:137–143
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