Nucleocytoplasmic Trafficking of Steroid-free Glucocorticoid
Receptor*
Robert J. G.
Haché
§,
Raymond
Tse
,
Terry
Reich
,
Joanne G. A.
Savory¶, and
Yvonne A.
Lefebvre
§
From the Departments of
Medicine and
§ Biochemistry, Microbiology, and Immunology and the
¶ Graduate Program in Biochemistry, University of Ottawa, The Loeb
Health Research Institute at the Ottawa Hospital,
Ottawa, Ontario K1Y 4E9, Canada
 |
ABSTRACT |
Glucocorticoid receptor (GR) recycles between an
inactive form complexed with heat shock proteins (hsps) and localized
to the cytoplasm and a free liganded form that regulates specific gene
transcription in the nucleus. We report here that, contrary to previous
assumptions, association of GR into hsp-containing complexes is not
sufficient to prevent the shuttling or trafficking of the GR across the
nuclear membrane. Following the withdrawal of treatment with cortisol
or the hormone antagonist RU486, GRs recycled rapidly into
hsp-associated, hormone-responsive complexes. However,
cortisol-withdrawn receptors redistributed to the cytoplasm very slowly
(t1/2 = 8-9 h) and RU486-withdrawn receptors not
at all. Persistent localization of these GRs to the nucleus was not due
to a gross defect in export, since in both instances the complexed
nuclear GRs transferred efficiently between heterokaryon nuclei.
Moreover, the addition of a nuclear retention signal to the N terminus
of GR induced the transfer of naive receptor to the nucleus in the
absence of steroid. These results suggest that the localization of GR
to the cytoplasm is determined by fine control of the rates of transfer
of GR across the nuclear membrane and/or by active retention that
occurs independently from the association of GR with hsps.
 |
INTRODUCTION |
In the absence of hormone, steroid receptors are packaged into
similar multiprotein complexes that migrate at 8 S on sucrose gradients
(1, 2). These complexes contain heat shock proteins (hsps),1 most particularly
hsp90 (3). They also contain hsp90-associated proteins, such as hsp70,
p23, p14, and high molecular weight immunophilins, including Cyp40 and
FKBP56 (3). Hormone binding induces dissociation of the active
receptors from the hsp complexes and promotes sequence-specific DNA
binding and transcriptional regulation. Steroid binding is transient,
and the loss of hormone from the receptor leads to a repackaging or
recycling of the receptor into the hsp-containing complex (4). For the
glucocorticoid receptor (GR), association with hsps is required for
steroid binding (5, 6).
When liganded, steroid receptors are shuttling proteins that traffic
continuously between nucleus and cytoplasm (7-9). The primary steroid
receptor nuclear localization signal (NLS), NL1, overlaps with the
C-terminal ends of the receptor DNA binding domains (10-13).
Additionally, the receptor for glucocorticoids (GR) has a second,
ligand-dependent NLS (NL2) in the ligand binding domain
whose sequence has not been delimited (11). Receptor nuclear export
signals, however, remain to be identified. For GR, nuclear localization
also appears to be dependent in large part on nuclear retention
mediated through the binding of the receptors to DNA (14, 15), the
nuclear matrix (16, 17), and other nuclear components (18).
Despite similarities in nuclear-cytoplasmic trafficking in the liganded
state, the similar nature of their NLSs, and a common association into
hsp-containing complexes, steroid receptors are differentially
localized in the cell in the absence of hormone. Unliganded estrogen
(ER) (19) and progesterone receptors (PR) (20) are nuclear, while naive
GR, mineralocorticoid receptor, and possibly androgen receptor are
localized to the cytoplasm (11, 14, 21, 22). The molecular basis for
these differences in localization is not understood. However, the
nuclear localization of ER and PR does not appear to be due to a defect
in nuclear export, since naive PR transfers rapidly between nuclei in
heterokaryon fusion experiments (9).
The usual explanation offered for the cytoplasmic localization of naive
GR is that its association into the hsp-containing complex acts to mask
its NL1 in a way that prevents recognition by the nuclear import
machinery (23-25). This proposal is supported by a study demonstrating
that hsp association of GR prevents the binding of an NL1-specific
antibody (26).
However, other results are inconsistent with this simple model. For
example, the redistribution of GR to the cytoplasm upon hormone
withdrawal occurs far more slowly than could be expected to be
accounted for by the time required for hormone dissociation and hsp
reassociation (14, 27-29). This is true even for GR DNA binding
mutants that never become more than 75% nuclear in the presence of
hormone (14). Second, overexpression of GR in many cell lines results
in the nuclear localization of the hsp-associated receptor, without
discernible change in other properties (30, 31). Third, treatment of
cytoplasmic GRs expressed by transient transfection with the hormone
antagonist RU486 results in a nuclear GR that reacquires steroid
responsiveness following antagonist withdrawal but does not
redistribute to the cytoplasm (14, 32).
In this study, we have directly examined the nucleocytoplasmic
trafficking of hormone-free GR prior to hormone treatment and following
hormone withdrawal. Our results demonstrate that hsp-associated GRs
exchange continuously between nucleus and cytoplasm and suggest that
the cytoplasmic localization of naive receptors occurs through a
process that is more complex than has been previously appreciated.
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MATERIALS AND METHODS |
Cell Culture, Plasmids, and Transfections--
GrH2 cells
(derived from a rat hepatoma cell line stably transfected with rat GR,
expressing approximately 100,00 GR molecules/cell) (33), COS7 cells
(ATCC CRL-1651), or NIH 3T3 (ATCC CRL-6474) cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum.
For chloramphenicol acetyl transferase (CAT) assays, lipofectamine (15 µg) was used to transfect GrH2 cells with plasmids RSV-
gal (1 µg), to monitor transfection efficiency, and pBLMMTVCAT (34) (MMTV
631/+105, 3 µg), as the reporter gene. Sixteen hours after
transfection, the cells were switched to serum-free medium. After
16 h in serum-free medium, hormonal treatments were initiated. RU38486 (RU486) was generously provided by RousselUclaf. Cells were
treated and harvested as indicated in individual experiments. CAT
assays were carried out as we have previously described (14).
-Galactosidase activity was used to correct transfection efficiency, and the results displayed are the average of three experiments performed in duplicate.
The DNA-binding domain of c-Abl, amino acids 862-959 (35), was cloned
as a StuI-SalI fragment into the pTL-GR
expression vector (36) and into a pTLmyc-GR vector with Lys-Asn
mutations at amino acids 513-515.
Indirect Immunofluorescence (IIF)--
To monitor the
subcellular distribution of GR by immunofluorescence, GrH2 or COS7
cells were plated onto poly-L-lysine (500 µg/ml)-coated
glass coverslips and incubated overnight in 35-mm tissue culture plates
in DMEM containing 10% charcoal-stripped fetal calf serum. Cells were
synchronized to G0 by incubation in serum-free DMEM for a
further 21 h prior to the initiation of treatment. Dexamethasone,
cortisol, and RU486 were added to final concentrations of
10
6 M in serum-free medium. To monitor
redistribution of GR to the cytoplasm following hormone withdrawal,
cells pretreated with cortisol (10
6 M) or
RU486 (10
6 M) for 1 h were washed three
times with phosphate-buffered saline and twice in serum-free medium
with 5% bovine serum albumin (BSA). Subsequent incubation was in
serum-free supplemented with BSA to 5%.
For heterokaryon analyses, GrH2 cells were co-plated with NIH 3T3 cells
onto poly-L-lysine-coated coverslips. After attachment, the
cells were cultured in serum-free medium for 16 h, treated with
RU486 (10
6 M) or cortisol (10
6
M) for 1 h, and washed as described above and then
with serum-free medium for 2 h before fusion. Cell fusion was
performed by adding 50% polyethylene glycol 4000 (Life Technologies,
Inc.) for 2 min at 37 °C (37). The cells were then washed five times
with calcium/magnesium-free Hanks' balanced salt solution and
incubated for 4 h in serum-free DMEM before being processed for
IIF. Nuclei from GrH2 cells and NIH 3T3 cells were identified by
staining with the Hoechst dye 33258 at 0.5 µg/ml.
IIF was carried out exactly as described previously (14) with primary
murine BUGR-2 antibody (Affinity BioReagents, Inc.). Quantification was
performed using double-blind encryption. For each time point, at least
400 stained cells were counted in each experiment. Each experiment was
repeated 3-8 times over a period of several months.
Biochemical Fractionation--
To prepare cytoplasmic and
nuclear fractions, GrH2 cells (5 × 105 cells/sample)
were collected, washed, and suspended in hypotonic buffer (10 mM HEPES, pH 7.9, at 4 °C, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol). The
cells were lysed with a Dounce homogenizer (B pestle), and nuclei were
collected by centrifugation (3000 × g) (38). For each
experiment, we confirmed before fractionation that fewer than 5% of
the homogenized cells excluded trypan blue. The supernatant was saved
as the cytosolic fraction. Nuclei were suspended in a low salt buffer
(20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 0.02 M KCl), and proteins were extracted with a high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1.2 M KCl) over 30 min. Prior to fractionation through 10%
SDS-polyacrylamide gels, GRs were concentrated and desalted by
immunoprecipitation from the cytosolic and nuclear fractions as
described previously (14, 39). After electrophoresis, samples were
electrophoretically transferred to Immobilon P (Millipore Corp.,
Bedford, MA), and GR was probed on Western blots with BuGR-2 and sheep
anti-mouse IgG conjugated to horseradish peroxidase (Amersham Pharmacia
Biotech). Detection of GR was by enhanced chemiluminescence (Amersham
Pharmacia Biotech). Chemiluminescent signals were quantified using a CH imaging screen (Bio-Rad) on a Bio-Rad GS-525 Molecular Imager.
Analysis of GR by Sucrose Density Gradient--
Whole cell
extracts of GrH2 cells prepared as described previously (14) were
fractionated through 15-30% sucrose gradients for 16 h at 55,000 rpm at 4 °C (40). 300-µl fractions were collected, immunoprecipitated with BuGR-2, and fractionated through 10%
SDS-polyacrylamide gels. Chemiluminescent signals were quantified using
a CH screen on a Bio-Rad GS-525 Molecular Imager. Markers for the
gradients were aldolase (7.3 S) and BSA (4.6 S).
Steroid Binding Assays--
GrH2 cells were cultured overnight
in serum-free DMEM supplemented with 5% BSA prior to treatment with
cortisol (10
6 M) or RU486 (10
6
M) for 1 h. Cells were withdrawn from ligand treatment
in serum-free media and harvested at the time points indicated, and
cell number was determined before resuspension in phosphate-buffered
saline containing either 3H-cortisol or
3H-cortisol + 50 µM cold cortisol (1000-fold
excess) as described previously (41). After a 30-min incubation, the
cells were washed five times with phosphate-buffered saline and counted
in a liquid scintillation counter. Experiments were performed three
times in duplicate, and values were compared with the initial level of
specific cortisol binding in untreated cells.
 |
RESULTS |
RU486-withdrawn GR Fails to Redistribute to the
Cytoplasm--
Maintenance of GR-expressing cells in culture in the
absence of serum allows for the examination of the subcellular
trafficking of a stable pool of GR without appreciable new synthesis or
degradation of the receptor molecules for periods exceeding 48 h
(14). Further, under these culture conditions, the cells mimic the
usual G0-G1 state of differentiated
glucocorticoid-responsive cells in the majority of mammalian target
tissues (42). Previously, we have observed in transient transfection
experiments in which GRs were overexpressed that redistribution of GR
to the cytoplasm following hormone withdrawal occurred only over a
period of several hours (14). Receptors in RU486-treated cells,
however, failed to redistribute appreciably to the cytoplasm for at
least 48 h following withdrawal of treatment.
To begin a detailed analysis of the exchange of unliganded GRs between
nucleus and cytoplasm that underlies their localization, we sought to
determine whether GRs expressed at more physiological levels
redistributed similarly to overexpressed receptor in response to
hormone agonists and antagonists. In untreated GrH2 cells, which stably
express rat GR and have been used extensively to study glucocorticoid
hormone action (7, 43), the receptor is entirely localized to the
cytoplasm prior to hormone treatment (7). To monitor the time course of
redistribution of GRs in these cells, we employed an indirect
immunofluorescence assay that we and others have previously
demonstrated to allow quantification of the changes in the subcellular
localization of steroid hormone receptors over a period of time (13,
14).
Treatment of GrH2 cells with the nonmetabolizable hormone agonist
dexamethasone induced a rapid and nearly complete nuclear uptake
(t1/2 = 4-5 min, 90-95% nuclear) of GR (Fig.
1). This rate was identical to the one we
previously observed for transiently transfected GRs (14) and the rate
that others have noted for endogenous GR in rat thymus cells measured by biochemical fractionation (27). Similar uptake results were also
obtained with the natural steroid
cortisol.2 Nuclear transfer
of GR upon RU486 treatment was similar but occurred more slowly
(t1/2 = 9-10 min). This difference is believed to
reflect a decreased rate of dissociation of RU486-bound GR from the
heat shock protein complex rather than a difference in nuclear import
(44). Thereafter, in the continuous presence of dexamethasone or
RU486, greater than 90% of cells contained GR that remained
exclusively nuclear for at least 48 h.

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Fig. 1.
Nuclear uptake of GR after treatment with
10 6 M dexamethasone or 10 6
M RU486. Dexamethasone ( ) or RU486 ( ) treatment
of GrH2 cells was initiated 16 h after serum withdrawal.
Subcellular distribution was determined by semiquantitative IIF as has
previously been described (14). Immunofluorescent cells were classified
into five categories ranging from completely nuclear (N) to
completely cytoplasmic. The percentage of cells with exclusively
nuclear distribution is plotted as a function of time after hormone
treatment. S.E. values were calculated from eight experiments performed
in duplicate. Error bars are not visible, since in this presentation
the error was for all samples smaller than the point plotted. Display
of the data with a logarithmic time scale facilitates discrimination of
localization at early times after treatment. Previous studies have
shown that the results obtained with this technique accurately reflect
the subcellular distribution of GR obtained by other methods
(14).
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Upon withdrawal of a 1-h treatment with cortisol, a process that
included thorough washing of the cells to ensure complete washout of
the steroid, GR redistributed to the cytoplasm only very slowly over an
extended period of time (Fig. 2,
t1/2 = 8-9 h). This contrasts with the rapid off
rate (t1/2 = 10 min) of cortisol and RU486 from GR
(27). More strikingly, upon withdrawal of RU486 treatment, GR remained
fully nuclear and did not appreciably redistribute to the cytoplasm.
Further, the persistence of GR in the nucleus upon ligand withdrawal is unlikely to reflect the low residual level of intracellular ligand, since we have previously demonstrated, in transient transfection experiments, that a GR mutant with a 10-fold lower affinity for ligand
responded to the withdrawal of RU486 and cortisol identically to WT GR
(14).

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Fig. 2.
Redistribution of GR from the nucleus of GrH2
cells after withdrawal of cortisol or RU486 from the serum-free tissue
culture medium following a 1-h treatment. Withdrawal from
10 6 M RU486 ( ) or 10 6
M cortisol ( ) was initiated by extensive washing,
followed by incubation in ligand-free medium as described under
"Materials and Methods." To facilitate comparison of the rates of
redistribution of cortisol and RU486-treated GRs from the nucleus upon
withdrawal, values for nuclear occupancy are expressed as a percentage
of the equilibrium nuclear localizations at 1 h after the addition
of ligand (t0). The percentages of
immunofluorescent cells with exclusively nuclear distribution
(N) are displayed. The values represent the average of three
independent experiments performed in duplicate. Again, the S.E. values
are not illustrated, since they were smaller than the size of the
points. The addition of cycloheximide (50 µg/ml) to incubations did
not alter kinetics of export.2
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RU486 and Cortisol-withdrawn GRs Rapidly Reassemble into an 8 S
Complex Able to Bind Ligand and Regulate
Transcription--
Previously, it has been demonstrated that upon
withdrawal of hormone agonist, the subnuclear localization of GR is
altered prior to its redistribution to the cytoplasm (29), but the
association of GR with heat shock proteins during this period has not
been examined. However, this intermediate state is marked by an
increased sensitivity of the GR in the agonist-withdrawn cells to
biochemical extraction.
In GrH2 cells treated with cortisol or RU486 for 1 h, virtually
100% of the GRs remained nuclear upon hypotonic lysis of the cells at
4 °C (Fig. 3A). However,
2 h after withdrawal of cortisol, a time when over 90% of the GRs
were nuclear according to immunofluorescence data, the receptor was
completely lost into the cytosol upon hypotonic cellular lysis. We
observed the same result with RU486-withdrawn cells. Within 2 h of
withdrawal of RU486, essentially all of the receptor fractionated
biochemically into the cytosol upon hypotonic lysis of the cells (Fig.
3A).

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Fig. 3.
RU486-withdrawn receptors rapidly reassociate
into 8 S complexes that are lost from the nucleus upon hypotonic
cellular lysis. A, biochemical fractionation of
RU486-withdrawn receptors. GrH2 cells in serum-free medium were
fractionated by hypotonic cellular lysis into nuclear (N)
and cytoplasmic (C) fractions following treatment with
either 10 6 M dexamethasone or
10 6 M RU486 for 1 h (withdrawal = t0) and after 2 h of withdrawal from the
cortisol/RU486 treatment in serum-free medium (t = 2 h). GRs quantitatively immunoprecipitated from the extracts were
fractionated on a 10% SDS-polyacrylamide gel and identified by Western
blotting with GR antibody BuGR2 (14). The distribution of GRs between
the two fractions was quantified by a phosphor imager. B,
sucrose density analysis of GR withdrawn from RU486 treatment for
2 h. Following a 1-h treatment with 10 6
M RU486 and a 2-h withdrawal period, a whole cell extract
of GrH2 cells was fractionated over linear (15-30%) sucrose density
gradients with aldolase (7.3 S) and BSA (4.6 S) markers. GRs in
individual fractions were immunoprecipitated, resolved on 10%
SDS-polyacrylamide gels, and quantified by phosphor imager analysis of
Western blots. Migration of the RU486-withdrawn GR (solid
line) is compared with that of naive GR extracted from the
cytoplasm of untreated GrH2 cells (dashed line)
and dexamethasone-liganded GRs extracted from the nucleus of cells
treated with 10 6 M dexamethasone for 1 h
(dotted line).
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The loss of nuclear occupancy upon hypotonic cellular lysis following
the withdrawal of hormone was reminiscent of both the facile nuclear
extraction of liganded GRs containing mutations that eliminate DNA
binding (14) and that of untreated nuclear ERs and PRs associated with
hsps (45). Association of GRs with heat shock proteins is reflected on
sucrose gradients by a shift in sedimentation coefficient from 4 to 8 S
(1). To determine whether GR in RU486-treated cells had recycled into
an 8 S complex upon withdrawal of treatment, the cytosol from
hypotonically lysed cells shown in Fig. 3A was fractionated
over sucrose gradients (Fig. 3B). Within 2 h of the
withdrawal of treatment, GRs from the RU486-treated cells appeared to
be completely reassociated with hsps into 8 S complexes.
Association of GR into the hsp complex occurs through a highly ordered,
ATP-dependent process that can be efficiently reconstituted in rabbit reticulocyte lysate at 30 °C (4, 46) but that is not known
to occur in cellular extracts at 4 °C (3). Nonetheless, to discount
the possibility of artifactual reassociation of GR with hsps in our
cellular extracts, we carried out several control experiments. In the
first instance, we assessed the ability of heat-transformed GRs to
reassociate with hsps in cellular extracts at 4 °C. Following heat
transformation in whole cell extract, incubation of the GR-containing
extract for 30 min on ice in the presence of molybdate was completely
ineffective in promoting the reassociation of GR into the 8 S
complex.2 This was also true for heat-transformed in
vitro translated GR mixed with whole cell extract that had been
spared the heat treatment and for cellular GRs in extracts supplemented
with untreated whole cell extracts from the GR
parental
cell line of GrH2 cells.2 Therefore, we conclude that the
rapid reassociation of GRs with heat shock proteins upon withdrawal of
RU486 was not sufficient to initiate the redistribution of GR to the
cytoplasm. Similarly, reassociation of cortisol-withdrawn GRs into 8 S
complexes was complete at 2 h, when only 10% of the receptors had
redistributed to the cytoplasm.2
Ligand binding by GR is strictly dependent upon an ordered assembly of
the receptor with the heat shock proteins that fixes the receptor
ligand binding domain in a conformation competent to bind ligand
(4-6). To determine whether cortisol and RU486-withdrawn GRs also
reacquired ligand binding ability more rapidly than they redistributed
to the cytoplasm, we performed whole cell steroid binding assays on
naive cells and cells withdrawn for increasing times from treatment
with cortisol and RU486 (Fig. 4). The
results indicate that over 75% of high affinity glucocorticoid binding sites were regenerated in GrH2 cells within 1 h of the withdrawal of the cells from either cortisol or RU486. Indeed, approximately 40%
of the binding sites were regenerated within 30 min of withdrawal. Thus, upon withdrawal of steroid treatment, the GRs reassociated into 8 S complexes competent to bind steroid well in advance of their
redistribution to the cytoplasm. Further, it suggests that the failure
of GRs in RU486-treated cells to redistribute to the cytoplasm upon
hormone withdrawal was not due to a defect in the recycling of the
receptor into a hormone-responsive state.

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Fig. 4.
RU486 and cortisol-withdrawn GRs rapidly
recover ligand binding activity. GrH2 cells in serum-free medium
were treated with 10 6 M cortisol or
10 6 M RU486 for 1 h. Following
withdrawal of the stimuli, the cells were withdrawn from ligand in
serum-free media and harvested at the times indicated. Whole cell
binding assays to measure specific GR binding sites were performed in
duplicate in phosphate-buffered saline with 3H-cortisol for
30 min at 0 °C. Binding to the withdrawn cells is expressed as a
percentage of the level of binding in untreated cells (un,
stippled bar), which was approximately 100,000 sites/cell, similar to previous reports (33). Cortisol binding upon
withdrawal from cortisol is shown by the solid
bars, while cortisol binding upon withdrawal from RU486 is
represented by the open bars. The data shown is
the average ± S.E. of three experiments performed in
duplicate.
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To evaluate the effect of recycling of GR from RU486 treatment in the
nucleus on its ability to activate transcription in response to
subsequent hormonal stimuli, we monitored transcription from a mouse
mammary tumor virus (MMTV) reporter gene containing a strong
glucocorticoid-responsive promoter (Fig.
5). RU486 is known to be a partial
glucocorticoid agonist that induces a weak response from the MMTV
promoter. Treatment of GrH2 cells transiently transfected with an MMTV
CAT reporter gene with RU486 for 1 h had only a small effect on
CAT activity produced from the MMTV promoter (lanes
1 and 2). The CAT activity recorded thereafter was the same whether the cells had been withdrawn from RU486 for 1 h or 23 h prior to harvesting (lanes 2 and
4). This indicated that production of the low level of CAT
protein in response to RU486 treatment was complete within 1 h of
withdrawal of the antagonist. Therefore, subsequent
hormone-dependent increases in CAT activity would directly
reflect the consequences of secondary steroidal treatment on
transcription of the reporter gene.

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Fig. 5.
Induction of MMTV transcription by steroid in
RU486-withdrawn GrH2 cells. GrH2 cells transfected with an MMTVCAT
reporter plasmid were placed in serum-free medium for 16 h prior
to the initiation of hormonal treatments, which was taken as the
starting time for experiments. Cells harvested for CAT assay were
treated as follows. Lane 1, control, incubated
2 h in serum-free medium; lane 2, 1 h
with 10 6 M RU486, followed by 1 h of
withdrawal in serum free medium; lane 3, control
incubated an additional 24 h in serum-free medium; lane
4, 1 h with 10 6 M RU486,
followed by 23 h of withdrawal in serum-free medium;
lane 5, 1 h with 10 6
M RU486 followed by withdrawal for 2 h in serum-free
medium and secondary treatment with 10 6 M
dexamethasone for a further 21 h; lane 6,
3-h initial incubation in serum-free medium followed by a 21-h
treatment with 10 6 M dexamethasone. Numerical
values for CAT activities are shown above the
bars and represent the average ± S.E. from three
experiments performed in duplicate.
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This enabled us to compare the steroid hormone induction of MMTV
transcription in naive cells with that in cells subjected to a cycle of
a 1-h RU486 treatment and a 1-h withdrawal. Treatment of naive cells
with dexamethasone for 21 h, at a time after transfection equal to
the completion of the 1-h RU486 withdrawal, led to a strong induction
of CAT activity (lane 6). Secondary treatment of
cells with nuclear, RU486-withdrawn GRs resulted in a similar total
accumulation of CAT activity 21 h after dexamethasone treatment (lane 5). When corrected by subtraction for the
contributions to the total CAT activity made by the hormone-independent
and RU486-dependent accumulation of CAT, the primary and
secondary responses to dexamethasone were almost identical. A similar
result was also obtained when the period of secondary treatment was
initiated 23 h following withdrawal of RU486.2
Together, these results indicated that the nuclear RU486-withdrawn GRs
were not discernibly different from the cytoplasmic GRs in untreated
cells in their association into a high molecular weight complex,
ability to bind steroid hormone, and ability to activate transcription
in response to steroid treatment. However, they did appear to be
permanently localized to the nucleus.
Persistent Nuclear Localization of RU486-treated GRs Is
Reversible--
In order to determine whether the nuclear localization
of RU486-withdrawn GR could be reversed by secondary steroid treatment, the localization of GR in GrH2 cells was monitored by
immunofluorescence following the withdrawal of a secondary cortisol
treatment from cells initially treated with RU486 (Fig.
6). For the first cycle, the cells were
treated with RU486 for 1 h, washed extensively, and incubated for
2 h in hormone-free medium. Secondary stimulation was accomplished
by treatment with cortisol for 1 h. The consequences of withdrawal
from the cortisol were monitored over the subsequent 24 h.

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Fig. 6.
RU486-withdrawn GR recovers ability to
redistribute to the cytoplasm after secondary treatment with steroid
agonist. GrH2 cells treated with 10 6 M
RU486 for 1 h and then withdrawn from RU486 for 2 h were
treated a second time for 1 h with 10 6 M
cortisol, followed by withdrawal from cortisol in serum-free medium in
the presence (dotted line) or absence
(solid line) of 10 µg/ml cycloheximide.
Subcellular distribution of GR was monitored by IIF at the time points
indicated over the course of the experiment beginning at 1 h
following RU486 treatment. The percentage of cells with exclusively
nuclear (N) GRs is shown. The data are the average of four
separate experiments performed in duplicate ± S.E.
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Notably, following withdrawal of this secondary treatment with
cortisol, GRs redistributed to the cytoplasm in a manner that closely
mimicked the redistribution of the receptor following withdrawal of a
primary treatment with cortisol (compare with Fig. 2). These results
indicate that the changes in GR, or elsewhere in the cell, that
occurred in response to RU486 and led to the long term localization of
GR in the nucleus following withdrawal of the antagonist, could be
reversed by a subsequent cycle of binding to steroid agonist. In these
experiments, it was also clear that the redistribution of GR was not
due to the synthesis of new receptor, as maintenance of the cells in
cycloheximide throughout the course of treatment resulted in a closely
overlapping pattern of subcellular distribution of GR compared with
untreated cells.
RU486- and Cortisol-withdrawn, hsp-associated GRs Shuttle between
Nucleus and Cytoplasm--
While the export of GR and other steroid
receptors from the nucleus is poorly understood, it is known that
liganded 4 S GRs traffic continuously or shuttle between nucleus and
cytoplasm (7). It has also been demonstrated that unliganded,
hsp-associated PRs shuttle continuously between nucleus and cytoplasm
despite its apparent nuclear localization (9, 47). One potential explanation for the block in redistribution of GRs to the cytoplasm upon withdrawal of RU486 and for the slow return of GRs in cells withdrawn from steroid treatment was that the export of GRs from the
nucleus upon loss of steroid or RU486 was somehow compromised (7).
To investigate this possibility, we evaluated the ability of nuclear
unliganded, 8 S GRs to be exchanged between heterologous nuclei in cell
fusion experiments (Fig. 7). Following
treatment with cortisol or RU486 for 1 h and withdrawal for 2 h (a time at which GRs treated by each ligand migrated at 8 S on
sucrose gradients and were competent to bind hormone; Figs.
3B and 4), GrH2 cells were fused with NIH 3T3 cells, and the
exchange of GRs between nuclei was assessed by immunofluorescence. Upon
fusion to the NIH 3T3 cells, the unliganded, hsp-associated GRs in the nuclei of RU486-withdrawn cells transferred efficiently to the NIH 3T3
nuclei (Fig. 7, A and B). Similarly, the GRs in
GrH2 cells withdrawn from cortisol also redistributed equally between
the two nuclei of the heterokaryons at this early time following
hormone withdrawal when the GRs were still localized to the nucleus
(Fig. 7, C and D). Thus, these results indicated
that unliganded, hsp-associated GRs in cells withdrawn from steroid or
RU486 treatment shuttled continuously between nucleus and cytoplasm.
For GRs withdrawn from cortisol, this continuous shuttling between
nucleus and cytoplasm was separate from the slow relocalization of GRs
to the cytoplasm observed above. Therefore, the failure of GRs to
redistribute rapidly to the cytoplasm upon withdrawal of cortisol or
RU486 could not be accounted for by loss of the ability of the GRs to be exported from the nucleus.

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|
Fig. 7.
GRs traffic between nuclei of heterokaryons
2 h after the withdrawal of RU486 and cortisol treatment.
GrH2 and NIH 3T3 cells plated together at high density incubated for
16 h in serum-free medium and then were treated with
10 6 M RU486 (A and B)
or 10- 6 M cortisol (C and
D) for 1 h. The cells were then washed and incubated
for a further 2 h in serum-free medium. Cellular fusion was
promoted by treatment with polyethylene glycol. After fusion, the cells
were incubated for a further 4 h in serum-free medium. The cells
were then processed for detection of GR by IIF (A and
C). Hoechst dye was used to distinguish GrH2 nuclei from NIH
3T3 nuclei (B and D). The arrows point
to NIH 3T3 and GrH2 nuclei in the right panels.
Immunofluorescence in both GrH2 and NIH 3T3 cells indicates the
transfer of GR to the NIH 3T3 nuclei. The images displayed are examples
of the results consistently observed in over 400 fused cells viewed
over three independent cell fusion experiments. FITC, fluorescein
isothiocyanate
|
|
Localization of Unliganded GR to the Nucleus by the Addition of a
Nuclear Retention Signal--
Unliganded 8 S GR prior to exposure to
hormone has been proposed to be localized to the cytoplasm through a
masking of the nuclear localization signals by the heat shock protein
complex (23-25). However, to date there has been no direct
demonstration that unliganded cytoplasmic GR does not transiently enter
the nucleus. By contrast, our results indicated that the packaging of
GR into hormone-responsive nuclear 8 S complexes following hormone
withdrawal was not a barrier to the nuclear import of GR. In order to
determine whether the unliganded GRs localized to the cytoplasm of
untreated cells might also possess the ability to transiently shuttle
into the nucleus, we designed an experiment that would allow us to
directly visualize the accumulation of untreated GRs in the nucleus.
Specifically, we reasoned that if unliganded naive GRs shuttled
transiently between nucleus and cytoplasm, then the addition to GR of a
signal demonstrated to promote protein retention in the nucleus might
be expected to shift the equilibrium distribution of GR in untreated
cells toward the nucleus.
The creation of GR fusion proteins by adding sequences to the N
terminus of GR is not known to force redistribution of GR to the
nucleus in the absence of nuclear localization activity within the
fragment. Indeed, fusion of heterologous proteins with the ligand
binding domain of GR has long been used as a tool to regulate the
nuclear entry of protein fragments (48-50).
We have previously reported that the ability to bind DNA promotes the
nuclear retention of liganded, transformed GR, following the entry of
GR into the nucleus (14). c-Abl is a nuclear tyrosine kinase that has
the ability to bind DNA (51). Unlike most other DNA-binding proteins
(10), the c-Abl DNA binding domain is not linked to an NLS-like
sequence (35). Thus the c-Abl DNA binding domain alone is unable to
promote the transport a cytoplasmic protein into the nucleus (35).
Therefore, to test our hypothesis that the unliganded GRs in untreated
cells can exchange transiently between nucleus and cytoplasm, we sought
to determine whether the 97-amino acid-long c-Abl DNA binding domain
expressed fused to the N terminus of GR would alter the localization of
GR in the absence of ligand (Fig. 8).

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Fig. 8.
The addition of a nuclear retention sequence
to GR directs naive, unliganded receptor to the nucleus. IIF of
COS7 cells transiently transfected to express either c-Myc-GR fusion
protein (A and B), GFP-GR fusion protein
(C and D), c-Abl-GR fusion protein with three
Lys-Asn substitutions in the GR NL1 (E and F),
c-Abl-GR fusion protein (G and H). IIF was
performed on cells incubated in serum-free medium, prior to
(A, C, E, and G) and
following a 2-h treatment with 10 6 M
dexamethasone (B, D, F, and
H).
|
|
This experiment was performed by expressing GR fusion proteins by
transient transfection into COS7 cells as we have previously described
(14) and monitoring the localization of the fusion proteins by indirect
immunofluorescence. Initially, several controls were performed to
ensure that any nuclear transfer of the Abl-GR fusion protein would
correlate directly with the ability of the c-Abl DNA binding domain to
interact with DNA. First, to reiterate that the fusion of peptides to
the N terminus of GR do not affect the hormone responsiveness of GR or
nonspecifically promote the transfer of GR to the nucleus by promoting
the exposure of the constitutive GR NLS, we examined the localization
of GRs with N-terminal fusions of a 6× c-Myc epitope tag or green
fluorescent protein (Fig. 8, A-D)
Expression of GR with the 82-amino acid c-Myc epitope tag (Fig. 8,
A and B) or the 240-amino acid GFP sequence (Fig.
8, C and D) resulted in the expression of GR
fusion proteins that were fully cytoplasmic in the absence of hormone
(<3 ± 2% nuclear; Fig. 8, A and C) and
that transferred indistinguishably from WT GR to the nucleus in
response to dexamethasone (94 ± 3% nuclear; Fig. 8, B
and D).
Second, expression of the c-Abl DNA binding domain fused to GR
containing three point mutations that inactivate the basic NL1 motif of
the receptor3 was also
completely localized to the cytoplasm in the absence of hormone
(92 ± 3% cytoplasmic; Fig. 8E). This result confirmed that the c-Abl DNA binding domain has no inherent nuclear localization potential. Upon the addition of hormone, this chimeric receptor transferred partially to the nucleus (21 ± 3% nuclear + nuclear > cytoplasmic, 50 ± 6% nuclear = cytoplasmic;
Fig. 8F). This behavior closely mimics that expected for WT
GR whose entry into the nucleus is dependent solely upon the
hormone-dependent NL2 activity in the receptor ligand
binding domain (52, 53).
By contrast, the fusion protein with the c-Abl DNA binding domain fused
to WT GR was localized mainly to the nucleus prior to any exposure of
the cells to hormone (81 ± 3% nuclear + nuclear > cytoplasmic; Fig. 8G) and became fully nuclear in response
to treatment of the cells with dexamethasone (96 ± 2% nuclear;
Fig. 8H). The localization of c-Abl-GR to the nucleus was
not due to inappropriate dissociation of the heat shock proteins, since
transcriptional responses to the c-Abl-GR fusion protein to
dexamethasone were the same as that observed for WT GR.2
Further, Western analysis showed that all of the GR constructs expressed in this experiment accumulated in the cells to the same level
as wild type GR.2
These data indicate that the GR NL1 was constitutively accessible in
c-Abl-GR fusion protein and mediates the hormone-independent import of
the hsp-associated protein into the nucleus. They also provide the
first evidence that unliganded, hsp-associated, cytoplasmic GRs may
constitutively shuttle between nucleus and cytoplasm. Thus, in both the
liganded and unliganded states, GR appears to exist in a dynamic
equilibrium between the nucleus and cytoplasm.
 |
DISCUSSION |
The cytoplasmic localization of GR prior to the addition of
hormone is one tangible difference in the molecular mechanism of action
of GR compared with ER and PR for which an explanation remains elusive.
In the present work, we have determined that unliganded, hsp-complexed,
hormone-responsive GRs exist in a dynamic equilibrium between nucleus
and cytoplasm and shuttle continuously across the nuclear membrane.
These observations suggest that the nucleocytoplasmic trafficking of
unliganded GR is fundamentally similar to the trafficking of ER and PR.
Further, our results suggest that the localization of unliganded,
hsp-complexed GR to the cytoplasm is accomplished through an active
mechanism that can be differentially affected by the transient binding
of hormone agonists and antagonists.
8 S, hsp-associated GRs were observed to traffic across the nuclear
membrane similarly to the liganded, free receptors. First, following
withdrawal from both RU486 and cortisol, GR recycled rapidly into
hormone-responsive complexes but redistributed at best only very slowly
to the cytoplasm. Nonetheless, despite their prolonged nuclear
occupancy, these 8 S GRs transferred efficiently between nuclei in
heterokaryon experiments. Thus, the persistence of these GRs in the
nucleus following hsp association occurred in the absence of obvious
effects on the trafficking of GR across the nuclear membrane.
Second, DNA binding is hypothesized to facilitate the concentration of
liganded GR in the nucleus by decreasing the pool of GR available for
export (14). That the addition of an ectopic DNA binding domain from
c-Abl could stimulate a very strong, NL1-dependent, accumulation of naive GR in the nucleus is substantive evidence that
cytoplasmic unliganded GRs are also in a dynamic equilibrium between
nucleus and cytoplasm. However, for unliganded WT receptors expressed
at physiological levels, this equilibrium is normally expected to
strongly favor cytoplasmic localization of the receptor population.
Third, additional support for the nuclear-cytoplasmic trafficking of
unliganded, 8 S GRs is found in the reports that overexpression of GR
in some cells promotes the partial transfer of GR to the nucleus in the
absence of hormonal stimulus. (30, 31). In other experiments, we have
recently observed that this shift in the naive GR population toward the
nucleus upon receptor overexpression is also dependent upon
NL1.3
While our results strongly support the rapid transfer of unliganded GR
between cellular compartments, they do not directly address the status
of the GR molecules during transport through the nuclear pore. In
principle, nuclear pores are large enough to accommodate the passage of
molecular complexes considerably larger in size than hsp-complexed
receptors (mRNA-protein complexes, for example) (54).
Alternatively, steroid receptors have been shown to be in a dynamic
equilibrium between hsp-complexed and free forms (55). Therefore, it is
also possible that the transfer of unliganded, hsp-complexed steroid
receptors across the nuclear membrane is dependent upon transient
complex disassembly, followed by reassembly on the other side of the
membrane. We note, however, that in our experiments, the loss of 8 S
GRs from the nucleus upon hypotonic cellular lysis occurred under
conditions that precluded reassociation of 8 S complexes in cytosol.
This would favor the direct export of intact 8 S GRs from the nucleus.
Our results indicate that changes in the localization of the GR
population between cytoplasm and nucleus are superimposed upon a
background of individual receptors shuttling rapidly across the nuclear
membrane. What then determines the localization of hsp-associated
receptors to nucleus or cytoplasm? Two possibilities seem likely.
First, active retention mechanisms may limit the accessibility of 8 S
GR to the nuclear-cytoplasmic transport machinery. For example, it has
recently been shown that following withdrawal of hormonal ligand GRs
become concentrated in a discrete nuclear subcompartment prior to their
relocalization to the cytoplasm (12). Targeting 8 S GR to this
subcompartment could promote the maintenance of the GR population in
the nucleus by decreasing its access to the nuclear export machinery in
a manner similar to that discussed above for DNA binding. However, it
is also possible that localization of naive GR to the cytoplasm
requires active cytoplasmic retention. Active retention in the
cytoplasm has been observed for other proteins (56, 57), and such a
retention mechanism would be more compatible with the observation that
overexpression of GR can lead to nuclear accumulation of the receptor
in some cells (30, 31).
Alternatively, since nuclear import and export are now known to involve
distinct transporters, is it also possible that changes in the
localization of GR reflect changes in the relative activities of the
nuclear localization and nuclear export signals on GR. Both import and
export are complex processes, and knowledge of active mechanisms to
regulate the rates of protein transport across the nuclear membrane
through postranslational modification and association with modifying
proteins are emerging (58-61).
In this respect, it would appear notable that GR is a phosphoprotein
whose phosphorylation state is known to increase and decrease in
response to the addition and withdrawal of hormone in actively growing
cells (62, 63). Thus, it is tempting to speculate that postranslational
modification of the receptor by phosphorylation plays a role in the
persistent nuclear localization of 8 S GR following hormone withdrawal.
However, several results suggest that the phosphorylation sites that
have been identified to date for GR in actively growing cells, which
are localized in the N terminus of the receptor, are unlikely to play a
direct role in localizing 8 S GR in the cell. First, fusion of the GR N
terminus with the ER DNA and ligand binding domains resulted in a
protein that was constitutively localized to the nucleus (64). Similar
analyses of GR-ER and GR-PR chimeras also suggested that cytoplasmic
localization of unliganded GR is determined by its ligand binding
domain (13). Second, substitution of individual and multiple
phosphorylation sites in the N terminus of GR have been reported to
have no effect on the localization of naive receptor to the cytoplasm
of asynchronously growing COS1 cells (65). Last, the phosphorylation at
these sites is highly dependent upon the cell cycle and mitogenic
stimulli with the peak in phosphorylation occurring during
G2 (62, 66-69).
Interestingly, however, GR is known to become differentially
hyperphosphorylated in response to RU486 compared with glucocorticoid agonist (68, 70). Thus, the differential modification of GR remains an
attractive possibility as a mediator of the prolonged but reversible
nuclear localization of GR following the withdrawal of RU486 treatment.
Alternatively, it is also possible that the composition of the hsp-GR
complex may vary in ways that rapidly lead to hormone-responsive complexes following the withdrawal of RU486 and cortisol but limit the
relocalization of the receptor to the cytoplasm. Distinguishing between
these possibilities will require a careful comparison of the
composition and postranslational modification of cytoplasmic and
nuclear 8 S GR complexes that are stably maintained during G0. Characterization of the molecular basis for the
persistent nuclear localization of cortisol- and RU486-withdrawn GRs
could be expected to contribute toward understanding the biological imperative that has dictated the cytoplasmic localization of unliganded GR but allows for the constitutive nuclear localization of the closely
related receptors for estrogens and progestins.
 |
ACKNOWLEDGEMENTS |
We thank our colleague M. Ekker for helpful
discussions and comments on the manuscript. We are grateful to K. Yamamoto and D. DeFranco for providing cell lines and plasmids employed
in this work and R. Van Etten for providing a c-Abl cDNA. We also appreciate the assistance provided by F. Sackey in establishing the
indirect immunofluorescence technique.
 |
FOOTNOTES |
*
This work was funded by a grant from the Medical Research
Council of Canada (to Y. A. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: The Ottawa
Hospital Loeb Research Institute, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada. Tel.: 613-761-5142; Fax: 613-761-5036; E-mail: lefebvre{at}civich.ottawa.on.ca.
The abbreviations used are:
hsp, heat shock
protein; GR, glucocorticoid receptor; ER, estrogen receptor; PR, progesterone receptor; MMTV, mouse mammary tumor virus; IIF, indirect
immunofluorescence; NLS, nuclear localization signal; CAT, chloramphenicol acetyl- transferase; DMEM, Dulbecco's modified
Eagle's medium; BSA, bovine serum albumin; WT, wild type.
2
R. J. G. Haché, R. Tse, T. Reich, J. G. A. Savory, and Y. A. Lefebvre, unpublished observation.
3
Savory, J. G. A., Hsu, B., Laquain,
I. R., Giffin, W., Reich, T., Haché, R. J. G., and
Lefebvre, Y. A. (1999) Mol. Cell. Biol., in press.
 |
REFERENCES |
-
Litwack, G.,
Cake, M. H.,
Filler, R.,
and Taylor, K.
(1978)
Biochem. J.
169,
445-448[Medline]
[Order article via Infotrieve]
-
Traish, A. M.,
Muller, R. E.,
and Wotiz, H. H.
(1984)
Endocrinology
114,
1761-1769[Abstract]
-
Pratt, W. B.,
and Toft, D. O.
(1997)
Endocr. Rev.
18,
306-360[Abstract/Free Full Text]
-
Scherrer, L. C.,
Dalman, F. C.,
Massa, E.,
Meshinchi, S.,
and Pratt, W. B.
(1990)
J. Biol. Chem.
265,
21397-21400[Abstract/Free Full Text]
-
Bresnick, E. H.,
Dalman, F. C.,
Sanchez, E. R.,
and Pratt, W. B.
(1989)
J. Biol. Chem.
264,
4992-4997[Abstract/Free Full Text]
-
Cadepond, F.,
Schweizer-Groyer, G.,
Segard-Maurel, I.,
Jibard, N.,
Hollenberg, S. M.,
Giguere, V.,
Evans, R. M.,
and Baulieu, E. E.
(1991)
J. Biol. Chem.
266,
5834-5841[Abstract/Free Full Text]
-
Madan, A. P.,
and DeFranco, D. B.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
3588-3592[Abstract]
-
Dauvois, S.,
White, R.,
and Parker, M. G.
(1993)
J. Cell Sci.
106,
1377-1388[Abstract/Free Full Text]
-
Guiochon-Mantel, A.,
Lescop, P.,
Christin-Maitre, S.,
Loosfelt, H.,
Perrot-Applanat, M.,
and Milgrom, E.
(1991)
EMBO J.
10,
3851-3859[Abstract]
-
LaCasse, E. C.,
and Lefebvre, Y. A.
(1995)
Nucleic Acids Res.
23,
1647-1656[Medline]
[Order article via Infotrieve]
-
Picard, D.,
and Yamamoto, K. R.
(1987)
EMBO J.
6,
3333-3340[Abstract]
-
Tang, Y.,
Ramakrishnan, C.,
Thomas, J.,
and DeFranco, D. B.
(1997)
Mol. Biol. Cell
8,
795-809[Abstract]
-
Ylikomi, T.,
Bocquel, M. T.,
Berry, M.,
Gronemeyer, H.,
and Chambon, P.
(1992)
EMBO J.
11,
3681-3694[Abstract]
-
Sackey, F. N. A.,
Haché, R. J. G.,
Reich, T.,
Kwast-Welfeld, J.,
and Lefebvre, Y. A.
(1996)
Mol. Endocrinol.
10,
1191-1205[Abstract]
-
Danielsen, M.,
Northrop, J. P.,
and Ringold, G. M.
(1986)
EMBO J.
5,
2513-2522[Abstract]
-
van Steensel, B.,
Jenster, G.,
Damm, K.,
Brinkmann, A. O.,
and van Driel, R.
(1995)
J. Cell. Biochem.
57,
465-478[Medline]
[Order article via Infotrieve]
-
Tang, Y.,
and DeFranco, D. B.
(1996)
Mol. Cell. Biol.
16,
1989-2001[Abstract]
-
van Steensel, B.,
Brink, M.,
van der Meulen, K.,
van Binnendijk, E. P.,
Wansink, D. G.,
de Jong, L.,
de Kloet, E. R.,
and van Driel, R.
(1995)
J. Cell Sci.
108,
3003-3011[Abstract/Free Full Text]
-
Puca, G. A.,
Medici, N.,
Armetta, I.,
Nigro, V.,
Moncharmont, B.,
and Molinari, A. M.
(1986)
Ann. N. Y. Acad. Sci.
464,
168-189[Abstract]
-
Press, M. F.,
and Greene, G. L.
(1988)
Endocrinology
122,
1165-1175[Abstract]
-
Simental, J. A.,
Sar, M.,
Lane, M. V.,
French, F. S.,
and Wilson, E. M.
(1991)
J. Biol. Chem.
266,
510-518[Abstract/Free Full Text]
-
Jenster, G.,
Trapman, J.,
and Brinkmann, A. O.
(1993)
Biochem. J.
293,
761-768[Medline]
[Order article via Infotrieve]
-
Czar, M. J.,
Lyons, R. H.,
Welsh, M. J.,
Renoir, J. M.,
and Pratt, W. B.
(1995)
Mol. Endocrinol.
9,
1549-1560[Abstract]
-
Hutchison, K. A.,
Scherrer, L. C.,
Czar, M. J.,
Stancato, L. F.,
Chow, Y. H.,
Jove, R.,
and Pratt, W. B.
(1993)
Ann. N. Y. Acad. Sci.
684,
35-48[Abstract]
-
Pratt, W. B.
(1993)
J. Biol. Chem.
268,
21455-21458[Free Full Text]
-
Urda, L. A.,
Yen, P. M.,
Stoney Simons, S. J.,
and Harmon, J. M.
(1989)
Mol. Endocrinol.
3,
251-260[Abstract]
-
Munck, A.,
and Holbrook, N. J.
(1984)
J. Biol. Chem.
259,
820-831[Abstract/Free Full Text]
-
Qi, M.,
Hamilton, B. J.,
and DeFranco, D.
(1989)
Mol. Endocrinol.
3,
1279-1288[Abstract]
-
Yang, J.,
Liu, J.,
and DeFranco, D. B.
(1997)
J. Cell Biol.
137,
523-538[Abstract/Free Full Text]
-
Martins, V. R.,
Pratt, W. B.,
Terracio, L.,
Hirst, M. A.,
Ringold, G. M.,
and Housley, P. R.
(1991)
Mol. Endocrinol.
5,
217-225[Abstract]
-
Sanchez, E. R.,
Hirst, M.,
Scherrer, L. C.,
Tang, H. Y.,
Welsh, M. J.,
Harmon, J. M.,
Simons, S. S., Jr.,
Ringold, G. M.,
and Pratt, W. B.
(1990)
J. Biol. Chem.
265,
20123-20130[Abstract/Free Full Text]
-
Qi, M.,
Stasenko, L. J.,
and DeFranco, D. B.
(1990)
Mol. Endocrinol.
4,
455-464[Abstract]
-
Miesfeld, R.,
Rusconi, S.,
Godowski, P. J.,
Maler, B. A.,
Okret, S.,
Wikstrom, A. C.,
Gustafsson, J. A.,
and Yamamoto, K. R.
(1986)
Cell
46,
389-399[Medline]
[Order article via Infotrieve]
-
Cato, A. C.,
and Weinmann, J.
(1988)
J. Cell Biol.
106,
2119-2125[Abstract]
-
Van Etten, R. A.,
Jackson, P.,
and Baltimore, D.
(1989)
Cell
58,
669-678[Medline]
[Order article via Infotrieve]
-
Préfontaine, G. G.,
Lemieux, M. E.,
Schild-Poulter, C.,
Pope, L.,
LaCasse, E.,
Walker, P.,
and Haché, R. J. G.
(1998)
Mol. Cell. Biol.
18,
3416-3430[Abstract/Free Full Text]
-
Borer, R. A.,
Lehner, C. F.,
Eppenberger, H. M.,
and Nigg, E. A.
(1989)
Cell
56,
379-390[Medline]
[Order article via Infotrieve]
-
Weil, P. A.,
Luse, D. S.,
Segall, J.,
and Roeder, R. G.
(1979)
Cell
18,
469-484[Medline]
[Order article via Infotrieve]
-
Giffin, W.,
Torrance, H.,
Rodda, D. J.,
Préfontaine, G. G.,
Pope, L.,
and Haché, R. J.
(1996)
Nature
380,
265-268[CrossRef][Medline]
[Order article via Infotrieve]
-
Thole, H. H.
(1994)
J. Steroid Biochem. Mol. Biol.
48,
463-466[CrossRef][Medline]
[Order article via Infotrieve]
-
Distelhorst, C. W.,
Kullman, L.,
and Wasson, J.
(1987)
J. Steroid Biochem.
26,
59-65[Medline]
[Order article via Infotrieve]
-
Pardee, A. B.
(1989)
Science
246,
603-607[Medline]
[Order article via Infotrieve]
-
Hsu, S.,
and DeFranco, D. B.
(1995)
J. Biol. Chem.
270,
3359-3364[Abstract/Free Full Text]
-
Distelhorst, C. W.,
and Howard, K. J.
(1990)
J. Steroid Biochem. Mol. Biol.
36,
25-31
-
Catelli, M. G.,
Binart, N.,
Jung-Testas, I.,
Renoir, J. M.,
Baulieu, E. E.,
Feramisco, J. R.,
and Welsh, W. J.
(1985)
EMBO J.
4,
3131-3135[Abstract]
-
Hutchison, K. A.,
Czar, M. J.,
Scherrer, L. C.,
and Pratt, W. B.
(1992)
J. Biol. Chem.
267,
14047-14053[Abstract/Free Full Text]
-
Guiochon-Mantel, A.,
Lescop, P.,
Christin-Maitre, S.,
Perrot-Applanat, M.,
and Milgrom, E.
(1992)
Ann. Biol. Clin.
50,
387-392
-
Picard, D.,
Salser, S. J.,
and Yamamoto, K. R.
(1988)
Cell
54,
1073-1080[Medline]
[Order article via Infotrieve]
-
Godowski, P. J.,
Picard, D.,
and Yamamoto, K. R.
(1988)
Science
241,
812-816[Medline]
[Order article via Infotrieve]
-
Jackson, P.,
Baltimore, D.,
and Picard, D.
(1993)
EMBO J.
12,
2809-2819[Abstract]
-
Dikstein, R.,
Heffetz, D.,
Ben-Neriah, Y.,
and Shaul, Y.
(1992)
Cell
69,
751-757[Medline]
[Order article via Infotrieve]
-
Cadepond, F.,
Gasc, J. M.,
Delahaye, F.,
Jibard, N.,
Schweizer-Groyer, G.,
Segard-Maurel, I.,
Evans, R.,
and Baulieu, E. E.
(1992)
Exp. Cell Res.
201,
99-108[Medline]
[Order article via Infotrieve]
-
Jewell, C. M.,
Webster, J. C.,
Burnstein, K. L.,
Sar, M.,
Bodwell, J. E.,
and Cidlowski, J. A.
(1995)
J. Steroid Biochem. Mol. Biol.
55,
135-146[CrossRef][Medline]
[Order article via Infotrieve]
-
Dingwall, C.
(1996)
Nature
384,
210-211[CrossRef][Medline]
[Order article via Infotrieve]
-
Smith, D. F.
(1993)
Mol. Endocrinol.
7,
1418-1429[Abstract]
-
Pines, J.,
and Hunter, T.
(1994)
EMBO J.
13,
3772-3781[Abstract]
-
Li, X.,
Shou, W.,
Kloc, M.,
Reddy, B. A.,
and Etkin, L. D.
(1994)
J. Cell Biol.
124,
7-17[Abstract]
-
Jans, D. A.,
Ackermann, M. J.,
Bischoff, J. R.,
Beach, D. H.,
and Peters, R.
(1991)
J. Cell Biol.
115,
1203-1212[Abstract]
-
Moll, T.,
Tebb, G.,
Surana, U.,
Robitsch, H.,
and Nasmyth, K.
(1991)
Cell
66,
743-758[Medline]
[Order article via Infotrieve]
-
Rihs, H. P.,
Jans, D. A.,
Fan, H.,
and Peters, R.
(1991)
EMBO J.
10,
633-639[Abstract]
-
Sidorova, J.,
and Breeden, L.
(1993)
Mol. Cell. Biol.
13,
1069-1077[Abstract]
-
Orti, E.,
Hu, L. M.,
and Munck, A.
(1993)
J. Biol. Chem.
268,
7779-7784[Abstract/Free Full Text]
-
Hu, L. M.,
Bodwell, J.,
Hu, J. M.,
Orti, E.,
and Munck, A.
(1994)
J. Biol. Chem.
269,
6571-6577[Abstract/Free Full Text]
-
Picard, D.,
Kumar, V.,
Chambon, P.,
and Yamamoto, K. R.
(1990)
Cell Regul.
1,
291-299[Medline]
[Order article via Infotrieve]
-
Webster, J. C.,
Jewell, C. M.,
Bodwell, J. E.,
Munck, A.,
Sar, M.,
and Cidlowski, J. A.
(1997)
J. Biol. Chem.
272,
9287-9293[Abstract/Free Full Text]
-
Hu, J. M.,
Bodwell, J. E.,
and Munck, A.
(1997)
Mol. Endocrinol.
11,
305-311[Abstract/Free Full Text]
-
Krstic, M. D.,
Rogatsky, I.,
Yamamoto, K. R.,
and Garabedian, M. J.
(1997)
Mol. Cell. Biol.
17,
3947-3954[Abstract]
-
Orti, E.,
Mendel, D. B.,
Smith, L. I.,
and Munck, A.
(1989)
J. Biol. Chem.
264,
9728-9731[Abstract/Free Full Text]
-
Rodatsky, I.,
Logan, S. K.,
and Garabedian, M. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2050-2055[Abstract/Free Full Text]
-
Hoeck, W.,
Rusconi, S.,
and Groner, B.
(1989)
J. Biol. Chem.
264,
14396-14402[Abstract/Free Full Text]
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