Dynamic Shuttling and Intranuclear Mobility of Nuclear
Hormone Receptors*
Padma
Maruvada
,
Christopher T.
Baumann§,
Gordon L.
Hager§, and
Paul M.
Yen
¶
From the
Molecular Regulation and Neuroendocrinology
Section, Clinical Endocrinology Branch, NIDDK and
§ Laboratory of Receptor Biology and Gene Expression, NCI,
National Institutes of Health, Bethesda, Maryland 20892
Received for publication, March 21, 2002, and in revised form, December 3, 2002
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ABSTRACT |
We expressed green fluorescent protein
(GFP) chimeras of estrogen, retinoic acid, and thyroid hormone
receptors (ERs, RARs, and TRs, respectively) in HeLa cells to examine
nucleocytoplasmic shuttling and intranuclear mobility of nuclear
hormone receptors (NRs) by confocal microscopy. These receptors were
predominantly in the nucleus and, interestingly, underwent
intranuclear reorganization after ligand treatment.
Nucleocytoplasmic shuttling was demonstrated by
heterokaryon experiments and energy-dependent
blockade of nuclear import and leptomycin-dependent
blockade of nuclear export. Ligand addition decreased shuttling by
GFP-ER, whereas heterodimerization with retinoid X receptor helped
maintain TR and RAR within the nucleus. Intranuclear mobility of the
GFP-NRs was studied by fluorescence recovery after
photo-bleaching ± cognate ligands. Both GFP-TR and GFP-RAR
moved rapidly in the nucleus, and ligand binding did not significantly
affect their mobility. In contrast, estrogen binding decreased the
mobility of GFP-ER and also increased the fraction of GFP-ER that was
unable to diffuse. These effects were even more pronounced with
tamoxifen. Co-transfection of the co-activator, SRC-1, further
slowed the mobility of liganded GFP-ER. Our findings suggest estradiol
and tamoxifen exert differential effects on the intranuclear mobility
of GFP-ER. They also show that ligand-binding and
protein-protein interactions can affect the intracellular mobility of
some NRs and thereby may contribute to their biological activity.
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INTRODUCTION |
Nuclear hormone receptors
(NRs)1 comprise a large
family of ligand-activated transcription factors that bind to hormone
response elements of target genes and regulate their transcription (1, 2). Members of the NR superfamily include the steroid receptors, thyroid hormone, retinoic acid, and vitamin D receptors (TRs, RARs, and
VDRs, respectively) and orphan receptors whose cognate ligands are not
known. NRs are structurally characterized by three distinct domains, an
N-terminal domain, a central DNA-binding domain, and a C-terminal
ligand-binding domain.
Steroid receptors bind to their response elements as homodimers and are
referred to as Type I receptors, whereas RARs, TRs, and VDRs bind to
their response elements and are called Type II receptors as
heterodimers with retinoid X receptors (RXRs). Both Type I and Type II
receptors can recruit co-activators in the presence of ligand, whereas
Type II receptors, notably TRs and RARs, can recruit co-repressors such
as silencing mediator for retinoid and thyroid hormone receptors and
nuclear co-repressor (NCoR) in the absence of ligand and repress the
transcription of target genes (1, 3). The formation of NR/co-activator complexes is associated with histone acetylation (1, 4) of the promoter
region whereas NR/co-repressor complexes recruit histone deacetylases.
Although various protein-protein interactions and enzymatic activities
seem to play important roles in mediating transcriptional activity by
NRs, it is possible that other mechanisms, such as the subcellular
distribution and intranuclear dynamics of NRs, may contribute to
hormonal responses mediated by these receptors.
Recently, several groups have shown that progesterone and
glucocorticoid receptors (PRs and GRs), VDRs, and TRs continuously shuttle between the cytoplasm and nucleus (5-8). We previously used
green fluorescent protein (GFP) fusions of TR
to show that entry
into the nucleus is an energy-mediated process whereas export out of
the nucleus is a passive one (9). Furthermore, our results showed that
ablation of the DNA-binding capacity of TR did not prevent its nuclear
accumulation. Using TR mutants that were defective in their interaction
with various co-regulators, we showed that heterodimerization with RXR
and interaction with NCoR help maintain unliganded TR within the nucleus.
We and others (10-12) have shown that GFP chimeras of ER, GR, and TR
form a punctate pattern in the nucleus after ligand addition, suggesting that intranuclear diffusion and reorganization of NRs may be
important regulatory processes. Recently, Mancini and co-workers (13)
have shown that unliganded ER exhibits high mobility whereas estrogen-bound ER has reduced mobility in the nucleus. Additionally, we
have shown that GR rapidly exchanges with regulatory DNA sequences and
is not statically bound to hormone response elements as thought previously (14). In this paper, we have used GFP chimeras of ER, RAR,
and TR as examples of Type I and II receptors to examine and compare
nucleocytoplasmic shuttling and intranuclear diffusion of NRs in more
detail. In particular, we have used energy depletion studies,
heterokaryon experiments, and fluorescence after photo-bleaching to
examine the movement of these receptors in the absence or presence of
ligand and co-regulators. Our studies suggest that ligand-binding and
protein-protein interactions can significantly influence the mobility
of these NRs and thereby contribute to their biological activity.
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MATERIALS AND METHODS |
Plasmids--
Expression vectors for human ER
, RAR
, and
TR
were described previously (15). GFP fusions were constructed into
the multiple cloning site of pEGFP-C1 (Clontech,
Palo Alto, CA) vector by PCR amplification at XhoI and
EcoRI sites at the C-terminal end of the coding sequence of
NRs. Molecular weight of the receptors was confirmed by Western
blotting using GFP monoclonal antibodies (Clontech,
Palo Alto, CA), and the integrity of all the constructs was confirmed
by DNA sequencing (Veritas, Rockville, MD). Functional characterization
was done by co-transfecting 0.1 µg of the cDNA with 1.7 µg of
reporter thyroid hormone response element (F2) and 1 µg of
-galactosidase into HeLa cells in 35-mm dishes, and luciferase activity was determined in the lysates using a Berthold lumat-LB 9507 luminometer.
Cell Culture and DNA Transfection--
HeLa (human cervical
carcinoma line; ATCC, Manassas, VA) cells were maintained regularly in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
antibiotics (100 units/ml penicillin and streptomycin, 0.5 mg/ml
gentamycin; Invitrogen) and 2 mM
L-glutamine in 5% CO2 incubator at 37 °C.
Various NRs were transiently expressed in HeLa cells grown on
coverslips (1 × 105 cells/well) by the calcium
phosphate (Invitrogen) method (16).
Microscopic Studies--
The cells expressing various GFP-NRs
were viewed under a Leica TCS SP laser scanning confocal
microscope mounted on a DMIRBE inverted epifluorescent
microscope equipped with a ×63 1.4-numerical aperture oil
immersion lens (Heidelberg, Germany). The GFP fluorescence was excited
with a 488-nm laser line from an air-cooled fiber-coupled argon laser
(Coherent Inc., Santa Clara, CA). Typical laser output was less than
10% of its maximal power. DAPI fluorescence was excited by a 385-nm
laser line from a water-cooled argon laser at 25% power (Coherent
Inc., Santa Clara, CA). GFP emission was monitored between 505 and 590 nm, and DAPI emission was followed between 405 and 490 nm. Both GFP and
DAPI were visualized with a pinhole of 1.0 (Airy units) and
detected. Quantitative analysis of receptor distribution was done as
described previously (9).
ATP Depletion Experiments--
ATP depletion experiments were
performed as described previously (9). Cells were incubated for 2 h with 10 mM sodium azide (Merck, Darmstadt, Germany) in
the presence of 6 mM 2-deoxyglucose prior to imaging.
Heterokaryon Assays--
The shuttling nature of NRs was studied
by interspecies heterokaryon assay as described previously (17).
Briefly, 2 × 105 mouse (NIH3T3) cells were plated
onto glass coverslips in 6-well multidishes on day one. Subsequently
the cells were transfected with 0.1 µg of GFP-NR along with carrier
DNA by the calcium phosphate method. After 48 h 1 × 106 human (HeLa) cells were plated onto the coverslips.
After 3 h cells were washed thoroughly with Dulbecco's
phosphate-buffered saline, 100 µl of pre-warmed
polyethylene glycol1500 were added, and the cells were incubated
at 37 °C for 2 min. The cells were washed again with
Dulbecco's phosphate-buffered saline and incubated with Dulbecco's
modified Eagle's medium with 50 µg/ml of cycloheximide incubated for
another 3 h and fixed with 4% paraformaldehyde and mounted on the
glass slides after counter-staining with DAPI for microscopy.
Fluorescence Recovery after Photo-bleaching (FRAP)--
These
studies were carried out as described previously with minor
modifications (14). Briefly, HeLa cells were grown on coverslips in
live cell chambers and maintained in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum at 37 °C. They were transfected
with GFP-NR vectors and treated with ligands. FRAP experiments were
carried out using a Leica TCS SP laser scanning confocal microscope. A
full power beam of 488- and 514-nm laser lines was focused on a defined
region for 0.5 s to bleach the region and then followed the
recovery of fluorescence that region was monitored over time.
Fluorescence intensities of regions of interest were obtained using
Leica TCS NT software and analyzed by Microsoft Excel. The
images were imported as TIFF files, and final images were generated
using Corel software, version 9.0.
Immunofluorescence Studies--
Immunofluorescence was performed
on GFP-tagged ER-transfected HeLa cells on coverslips using
anti-CRM-1 antibodies (1:1000) (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) and visualized under a Leica-Fish microscope, and
images were collected using Metamorph software. The images were
processed using Corel software, version 9.0.
 |
RESULTS |
To study the intracellular distribution of NRs in living cells, we
created vectors expressing GFP chimeras fused to the N-terminal ends of
TR
, ER
, and RAR
(Fig.
1A). Western blot analysis of extracts obtained from transfected cells showed that all the fusion constructs encoded full-length proteins of correct size (data not
shown). The transcriptional activities of the GFP-NRs were assayed in a
transient transfection system, and the levels of ligand-dependent transactivation were similar to unfused
receptors (Fig. 1B) (data not shown).

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Fig. 1.
Characterization of GFP-NR constructs.
A, schematic of the pGFP-NR constructs. Locations of
the cytomegalovirus (CMV) promoter, EGFP coding sequence, NR
coding region, termination codon, and SV40 poly(A) signals are
indicated. B, ligand-dependent transactivation
by GFP-NRs with cognate response element reporters. 0.1 µg of the
pGFP-NR expression vector with 1.7 µg of thyroid hormone response
element (F2), or RARE or ERE reporter, and 1 µg of
-galactosidase control vector were co-transfected into HeLa cells in
the absence or presence of respective ligands (T3, RA, or
E2). Cells were harvested after 48 h, and luciferase
activity of lysates was measured as described under "Materials and
Methods."
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We examined the intracellular distribution of GFP-ER and GFP-RAR in
live cells and found that these receptors localized mostly in the
nucleus, similar to previous observations for TR
and ER
(9, 18)
(see Fig. 2A and Fig.
3A). To quantitate the percentage of NRs present within the
nucleus and cytoplasm, we measured the area-corrected intensity of
GFP-NR fluorescence in both the nucleus and cytoplasm. From these
analyses, we observed that only about 10-15% of unliganded GFP-ER
(Fig. 2A) was present in the cytoplasm, and ER effectively
translocated into the nucleus after estradiol (E2;
10
6 M) or tamoxifen (Txn; 10
8
M) treatment for 1 h (Fig. 2, B and
C). Similar to GFP-ER, ~20% of unliganded GFP-RAR (Fig.
3B) was present in the
cytoplasm, and GFP-RAR almost entirely translocated into the nucleus
after retinoic acid (RA; 10
6 M) treatment
(Fig. 3B). These changes in the nucleocytoplasmic distribution of ER and RAR after ligand addition contrast with our
previous observations for TR
, which maintained ~10% of receptor in the cytoplasm regardless of whether ligand was present (9). The
observed nuclear/cytoplasmic distribution was independent of the amount
of expression vector transfected, as it remained constant when varying
concentrations of GFP-ER and GFP-RAR vectors were transfected into HeLa
cells (data not shown). In these experiments, we also observed that
ligand induced intranuclear reorganization of GFP-ER and GFP-RAR (see
Fig. 2, A-C and Fig. 3, A and
B) similar to previous observations for GR, ER, and, TR
(9-11). In the absence of ligand, GFP-ER and GFP-RAR displayed a
diffuse, reticular pattern whereas in the presence of their cognate
ligands, these receptors exhibited a discrete, punctate pattern. These
effects were rapid with equilibrium patterns occurring within 10 min
after ligand addition (data not shown).

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Fig. 2.
Effect of ligands on the intranuclear
distribution and nucleocytoplasmic shuttling of GFP-ER.
HeLa cells were transfected with 0.1 µg of pEGFP-ER and
subsequently depleted of intracellular ATP levels by treating with
sodium azide (10 mM) in the presence of deoxyglucose (6 mM) and imaged after 2 h. Numbers at the
bottom of each image represent the average nuclear
localization (determined from percent nuclear to total cellular
fluorescence) of at least 30 cells as described under "Materials and
Methods." Shown are GFP-ER-expressing cells with no treatment
(A), with E2 (10 6 M)
(B), or with tamoxifen (10 8 M)
(C) for 1 h and GFP-ER-expressing cells treated with
sodium azide (10 mM) alone to deplete ATP levels
(D) or cells treated with E2 (10 6
M) (E) or tamoxifen (10 8
M) (F) for 1 h before sodium azide
treatment.
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Fig. 3.
Effect of ligand binding and
heterodimerization on the intranuclear distribution and
nucleocytoplasmic shuttling of GFP-RAR. HeLa cells were
transfected with 0.1 µg of pEGFP-RAR ± 0.3 µg of
pcDNA-RXR and were subsequently depleted of intracellular ATP
levels as described in the legend for Fig. 2. Numbers at the
bottom of each image represent the average nuclear
localization (determined from percent nuclear to total cellular
fluorescence) of at least 30 cells as described under "Materials and
Methods." Shown are GFP-RAR-expressing cells with no treatment
(A) or RA (10 6 M) for 1 h
(B) and GFP-RAR co-expressed with pcDNA-RXR
(C), GFP-RAR-expressing cells treated with sodium azide as
mentioned above with no treatment (D) or treated with RA
(10 6 M) for 1 h prior to sodium azide
(E), or GFP-RAR co-expressed with pcDNA-RXR and then
treated with sodium azide (F).
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To further delineate the nucleocytoplasmic shuttling of these
receptors, the intracellular distribution of these receptors was
studied after treatment with sodium azide, which depletes intracellular
stores of ATP and blocks the active transport of proteins across the
nuclear membrane (see Figs. 2 and 3). We showed previously (9) that
nuclear import of GFP-TR
is an energy-mediated process and that its
export is a passive one. We now observed similar findings for both
GFP-ER and GFP-RAR. After a 2-h treatment with sodium azide, more than
20% of GFP-ER redistributed to the cytoplasm indicating that a
subpopulation of GFP-ER shuttles between the nucleus and cytoplasm
(Fig. 2D). Interestingly, treatment with either
E2 or tamoxifen prior to sodium azide treatment prevented cytoplasmic accumulation (Fig. 2, E and F)
indicating that ligand binding to GFP-ER decreases shuttling out of the
nucleus. Similarly, a significant amount of GFP-RAR was found to
undergo nucleocytoplasmic shuttling (Fig. 3D). This
shuttling out of the nucleus was prevented by either ligand treatment
or co-expression of unfused RXR (Fig. 3, E and F)
and suggests that ligand binding and heterodimerization help retain
GFP-RAR in the nucleus.
We then used the heterokaryon system to further study the
nucleocytoplasmic shuttling by GFP-ER. In these studies, NIH3T3 cells
expressing GFP-ER were fused with nonexpressing HeLa cells, in both the
presence and absence of ligands. We observed that GFP-ER translocated
from the NIH3T3 nucleus to the HeLa nucleus, indicating that GFP-ER
rapidly shuttles between the nucleus and cytoplasm (Fig.
4, A and B).
However, in the presence of either E2 or tamoxifen,
translocation of GFP-ER into the HeLa nucleus was reduced (Fig. 4,
C-F) indicating decreased nucleocytoplasmic shuttling.

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Fig. 4.
Heterokaryon experiments to study GFP-ER
nucleocytoplasmic shuttling. GFP-ER-expressing NIH3T3 cells were
fused with HeLa cells using polyethylene glycol1500 for 2 min as
described under "Materials and Methods." The cells were treated
with E2 (10 6 M) or tamoxifen
(10 8 M). The identity of each nucleus was
determined by DAPI staining of the nuclei (right panel).
NIH3T3 nucleus can be differentiated from the HeLa nucleus by the
relatively smaller size and/or the presence of heterochromatin.
A, GFP-ER distribution after fusion; B, DAPI
staining of GFP-ER-expressing nuclei; C, GFP-ER distribution
after fusion and E2 (10 6 M)
treatment; D, DAPI staining of GFP-ER-expressing nuclei upon
E2 treatment; E, GFP-ER distribution after
fusion and tamoxifen treatment (10 8 M);
F, DAPI staining of GFP-ER-expressing nuclei upon tamoxifen
treatment. Cell membrane borders of transfected fused cells are
highlighted.
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To characterize the mechanism of shuttling, we studied ER diffusion in
the heterokaryon system, in the presence of leptomycin B, an inhibitor
of CRM-1 (involved in nucleocytoplasmic export) (Fig.
5). GFP-ER was retained in the HeLa cell
nuclei in the absence of ligand, E2, and tamoxifen
suggesting that ER export occurs by CRM-1 regardless of its
ligand-binding state. Similar results also were observed for GFP-TR
and GFP-RAR (data not shown).

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Fig. 5.
Effect of leptomycin B on GFP-ER shuttling in
the heterokaryon system. GFP-ER-expressing NIH3T3 cells were fused
with HeLa cells. Cells were treated with 10 9
M leptomycin B for 3 h in either the absence or
presence of E2 (10 6 M) or Txn
(10 8 M). Shown is GFP-ER distribution between
heterokaryons treated with no ligand (A), in the presence of
E2 (B), or Txn (C). DNA
counter-staining with DAPI of these treatments is shown in panels
D, E, and F. GFP-ER distribution between
heterokaryons in the presence of leptomyin B was as follows: GFP-ER
treated with no ligand (G), E2 (H),
or Txn (I). DNA counter-staining with DAPI of these
treatments is shown in panels J-L.
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These foregoing findings suggest that GFP-ER and GFP-RAR shuttle
between the nuclear and cytoplasmic compartments. Additionally, our
findings in Figs. 2 and 3 suggest that these receptors reorganize within the nucleus upon ligand binding. To better understand this latter phenomenon, we used FRAP to study the intranuclear
mobility of GFP-ER, GFP-RAR, and GFP-TR in transfected cells in the
presence and absence of ligand. In these studies, a full power laser
beam was focused for 0.5 s on the nuclei of GFP-NR-expressing
cells, causing fluorescence loss in a defined nuclear zone. The
mobility of GFP-NR molecules was then measured as a function of
fluorescence recovery over time in the bleached region.
Under these conditions, we determined the recovery rate of a mobile
fraction of GFP-NRs in which fluorescence recovery could be measured
(t1/2) and observed an immobile fraction in which no
recovery occurred even after 3 min (original fluorescence (100%)
% of recovery after 3 min). The calculated data of ten individual
cells from six different experiments are shown in Table
I. The effect of ligands on the mobility
of GFP-ER was studied using the FRAP technique (see Fig.
6 and Fig. 7A). In
cells transfected with GFP-ER vector,
bleaching for 0.5 s caused only a 55% reduction in fluorescence
within the nuclear zone, indicating that unliganded GFP-ER was
extremely mobile and rapidly moved back to the bleached zone, even
during the time between the bleaching process and first measurement
(see Fig. 6A and Fig. 7A). Rapid recovery was
observed (t1/2 = 1.6 s) as more than 90% of
fluorescence recovery occurred within 3 s, and maximal recovery
occurred in less than 12 s. The immobile fraction was 14%. To
observe the effect of ligands on the mobility of GFP-ER, we incubated
transfected cells with either E2 or tamoxifen for 1 h
and then subjected the cells to FRAP (see Fig. 6, B and C and Fig. 7A). In these studies, we found that
cells treated with E2 had more than 70% loss in
fluorescence initially, and t1/2 = 5.8 s, with
an immobile fraction of 44% (see Fig. 7A and Table I).
These findings demonstrated that E2 reduced the rate at
which GFP-ER returns to the bleached zone and increased the amount of
ER that was unable to diffuse readily. Interestingly, treatment with
the ER antagonist, tamoxifen, resulted in almost total loss in
fluorescence, with little fluorescence recovery even after 3 min
because of a large immobile fraction (see Fig. 6C, Fig.
7A, and Table I) (data not shown). In all these experiments,
the recovered fluorescence was normalized to unbleached control cells
in the same experiments. All the experiments were repeated more than
six times with similar findings.

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Fig. 6.
FRAP of nuclear GFP-ER. A small region
in the nuclei of GFP-ER-expressing cells was bleached with a full power
laser beam for 0.5 s and imaged continuously for the recovery of
fluorescence to measure intranuclear mobility of GFP-ER
(highlighted circle). A, FRAP of
GFP-ER-expressing cells; B, FRAP of GFP-ER-expressing cells
in the presence of E2 (10 6 M);
C, FRAP of GFP-ER-expressing cells in the presence of
tamoxifen (10 8 M).
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Fig. 7.
Time course of FRAP of nuclear GFP-ER in the
presence of ligands and SRC-1 co-activator. A small region in the
nuclei of GFP-ER-expressing cells was bleached with a full power laser
beam for 0.5 s and imaged continuously for the recovery of
fluorescence as a function of movement of GFP-ER. A, FRAP of
GFP-ER-expressing cells in the presence or absence of ligand. FRAP of
GFP-ER-expressing cells (circles), FRAP of GFP-ER-expressing
cells in the presence of E2 (10 6
M) (triangles), and FRAP of GFP-ER-expressing
cells in the presence of Txn (10 8 M)
(squares) are shown. B, FRAP of GFP-ER cells
co-expressed with SRC-1. FRAP of GFP-ER-expressing cells
(circles), FRAP of GFP-ER-expressing cells co-expressed with
SRC-1 (open circles), FRAP of GFP-ER-expressing cells in the
presence of presence of E2 (triangles), and FRAP
of GFP-ER-expressing cells co-expressed with SRC-1 in the presence of
E2 (10 6 M) (open
triangles) are shown.
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Previous studies have shown that p160 co-activators such as SRC-1
interact with ER in a ligand-dependent manner and enhance its transcriptional activity (1). To study the effect of SRC-1 on the
intranuclear mobility of GFP-ER, we performed FRAP on cells co-expressing both GFP-ER and unfused SRC-1, in both the absence and
presence of E2 (Fig. 6B). Interestingly, there
was delayed recovery of fluorescence of unliganded GFP-ER in the
presence of SRC-1 (t1/2 = 3.6 s
versus 1.6 s, respectively) with a maximal recovery
occurring by ~20 s (Fig. 7B). These findings suggest a
possible association between SRC-1 and unliganded GFP-ER that may
decrease the mobility of the latter. In the presence of E2,
more than 80% of fluorescence was bleached in cells transfected with
GFP-ER and SRC-1, compared with 70% bleaching in cells transfected
with only GFP-ER. Additionally, recovery time was slower in cells
transfected with GFP-ER and SRC-1 than GFP-ER alone
(t1/2 = 6.9 s versus 5.8 s,
respectively). These findings suggest that SRC-1 may reduce the
intranuclear mobility of E2-bound ER.
To further investigate the effect of ligands on the mobility of nuclear
hormone receptors, we performed FRAP on cells expressing GFP-RAR in the
presence and absence of RA (Fig.
8A). GFP-RAR was highly mobile
in the absence of ligand as there was only a 40% loss in fluorescence
after photo-bleaching, and maximal recovery occurred by 10 s (Fig.
8A). In the presence of RA, there was little change in the
intranuclear mobility of GFP-RAR (t1/2 = 2.3 s
versus 2.0 s). FRAP studies on unliganded GFP-TR
showed very similar recovery kinetics as GFP-RAR as there was only a 40% loss in fluorescence after photo-bleaching and almost total fluorescence recovery after 10 s (Fig. 8B). In the
presence of T3, there was little change in mobility
of GFP-TR as maximal fluorescence recovery occurred by 5 s, and
t1/2 remained unchanged (1.8 s). In contrast to the
effect of SRC-1 on ER mobility, co-transfection of SRC-1, as well as
NCoR and HDAC-1, did not affect the intranuclear mobility of GFP-TR
(data not shown). We observed previously that nuclear export of ER, RAR, and TR was mediated by CRM-1; thus, we examined whether CRM-1 may
be associated with GFP-ER and modulate its intranuclear mobility (Fig.
9). However, we did not observe
co-localization of CRM-1 and GFP-ER in the absence or presence of
ligand, suggesting that exportin does not modulate intranuclear
mobility of ER.

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Fig. 8.
Time course of FRAP of nuclear GFP-RAR and
GFP-TR in the presence of their respective ligands. The nuclei of
GFP-RAR-expressing cells were bleached with a full power laser beam and
imaged continuously as for Fig. 6. A, FRAP of GFP-RAR. FRAP
of GFP-RAR-expressing cells (circles) and FRAP of
GFP-RAR-expressing cells in the presence of RA (10 6
M) (triangles) are shown. B, FRAP of
GFP-TR. FRAP of GFP-TR-expressing cells (circles) and FRAP
of GFP-TR-expressing cells in the presence of T3
(10 6 M) (triangles) are
shown.
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Fig. 9.
Lack of co-localization of CRM-1 with
GFP-ER. Immunofluorescence was performed on GFP-ER-expressing
cells treated with ligands, using anti-CRM-1 antibody (1:1000) and
visualized by fluorescence microscopy. Shown is GFP-ER expression after
treatment with no ligand (A), E2 (B),
or Txn (C) and CRM-1 immunofluorescence after treatment with
no ligand (D), E2 (E), or Txn
(F). G-I show overlay images for each
treatment. J-L show DNA counter-staining by DAPI
for the same treatments.
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DISCUSSION |
We have used GFP chimeras of ER, RAR, and TR to examine the
cellular localization of several prototypical Type I and Type II NRs in
living cells. In the absence of ligand, all the NRs studied displayed
predominantly nuclear distribution. In contrast, previous studies of
some steroid hormone receptors, particularly GR and PR, showed mostly
cytoplasmic distribution in the absence of ligand (12, 19). This
distribution is likely maintained by the formation of cytoplasmic
complexes that include chaperone proteins such as hsp 70 and hsp 90 (20). The location of unliganded ER is still controversial as earlier
biochemical studies suggested ER was mostly cytoplasmic, but later cell
fractionation and immunohistochemical studies suggested it was
predominantly nuclear (21-24). Recent studies with GFP-ER have not
addressed directly the issue of unliganded ER localization (10, 11).
Here, we show that a small pool of unliganded ER exists in the
cytoplasm, and it constantly shuttles between the cytoplasm and nucleus
in live cells. Moreover, the cellular distribution and shuttling of ER
is markedly affected by estrogen treatment. Interestingly, the estrogen
antagonist, tamoxifen, also has similar effects as estrogen on receptor
cycling and retention, suggesting it does not interfere with these
particular receptor roles. We also showed that the nuclear import of ER
is an energy-dependent process similar to our present
findings for RAR and previous studies for TR and PR (5, 9). We also
demonstrated that nuclear export of ER, RAR, and TR is mediated via the
exportin, CRM-1 (25). Previous studies suggested that VDR, but not PR and GR, were blocked by leptomycin B (26-28). Thus, it is possible there may be more than one mechanism for nuclear export of nuclear hormone receptors.
The mechanism for the nuclear retention of ER is not known; however, it
is possible that ligand binding may facilitate interactions with
nuclear proteins or components. For instance, it is known that liganded
ERs can form multimeric transcription complexes containing
co-activators (1). Recent studies also have shown that
E2-bound ER and T3-bound TR can bind to the
nuclear matrix component (9, 11), which in turn may affect intranuclear mobility and nuclear retention of these receptors.
Our studies also show that unliganded GFP-RAR and GFP-TR shuttle
between the cytoplasmic and nuclear compartments, suggesting that such
shuttling might be a general phenomenon among NRs. In contrast to ER,
ligand binding did not promote major changes in RAR or TR cellular
distribution. Instead, GFP-RAR exhibited reduced shuttling in the
presence of RXR suggesting that heterodimerization plays a major role
in maintaining the normal cellular distribution of RAR. We previously
used GFP chimeras of TR mutants that were defective in homodimerization
or nuclear localization to demonstrate that heterodimerization with RXR
helped maintain TR in the nucleus. Similar results also have been
observed for TR
in an oocyte system and GFP-VDR (7, 8). Our previous
studies also showed that unliganded TR
interaction with the
co-repressor, NCoR, helped maintain nuclear localization (9).
Furthermore, we showed previously that DNA binding by TR did not
significantly contribute to nuclear retention. Taken together, these
findings with the other GFP-NRs suggest that ligand-binding or
protein-protein interactions are key processes that may be
differentially employed by NRs for nuclear localization.
Previous studies have indicated that some NRs undergo intranuclear
reorganization in response to ligand treatment (7, 9-11, 18).
Similarly, all the NRs studied here underwent intranuclear reorganization after hormone treatment. To better understand this phenomenon, we used FRAP to study the intranuclear mobility of the NRs
and the effect of various co-regulators on their mobility. Our studies
demonstrate that NRs move rapidly within the nucleus. Additionally, the
mobility of NRs can be modulated by ligand binding as both
E2 and tamoxifen reduce the mobility of GFP-ER. However, E2 treatment results in partial fluorescence recovery
whereas tamoxifen treatment causes minimal or no fluorescence recovery as it generated a large immobile fraction of ER. These findings suggest
that anti-estrogens can have profound effects on the intranuclear mobility of ER, and this process may be involved in antagonist activity. Of note, our data differ from a study published recently (11)
in which ICI decreased ER intranuclear mobility whereas tamoxifen had minimal effect. Additionally, these studies are consistent with recent findings that showed steroid hormone receptors are dynamic within the nucleus and rapidly exchange between various nuclear components including chromatin (14).
In contrast to ER, ligand did not significantly affect the mobility of
Type II receptors such as GFP-RAR and GFP-TR. Thus, ligands can exert
differential effects on the intranuclear mobility of NRs. We also
studied the effects of various transcriptional co-factors on the
intranuclear mobility of NRs. GFP-ER had decreased mobility when
co-expressed with SRC-1 perhaps by forming of an NR/co-activator
complex or by stabilizing NR within larger multiprotein complexes. In
this connection, nuclear hormone receptors have been shown to
co-localize with co-activators and interact with proteins in the
proteasome complex (29-31). This may potentially be a mechanism for
down-regulating transcriptional activity by NRs. It also is interesting
to note that SRC-1 decreased the mobility of both unliganded and
E2-bound ER, suggesting that SRC-1 itself may be in a
dynamic equilibrium with ER, even when the latter is in an unliganded state.
In summary, we have shown that several representative NRs undergo
nucleocytoplasmic shuttling in an energy-dependent manner. These NRs also have rapid intranuclear mobilities, which, in the case
of ER, can be modulated by ligand binding. Estrogen and tamoxifen have
differential effects on ER mobility, and the marked effect of tamoxifen
on ER mobility may contribute to its anti-estrogenic properties. When
our results of GFP-ER, GFP-RAR, and GFP-TR are taken together, they
suggest that ligand binding and interaction of NRs with transcriptional
co-factors may exert differential effects on the nucleocytoplasmic
shuttling and intranuclear mobility of NRs. These, in turn, may
represent novel regulatory processes, which affect the biological
activity of NRs.
 |
ACKNOWLEDGEMENTS |
We thank Drs. James McNally and Tatiana
Karpova, Imaging Core Facility, Laboratory of Receptor Biology and
Gene Expression, NCI, National Institutes of Health for technical
advice on analyzing FRAP kinetic data.
 |
FOOTNOTES |
*
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: Molecular
Regulation and Neuroendocrinology Section, Clinical Endocrinology
Branch, National Institutes of Health, Bldg. 10, Rm. 8D12, Bethesda, MD 20892. Tel.: 301-594-6797; Fax: 301-402-4136; E-mail:
pauly@intra.niddk.nih.gov.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M202752200
 |
ABBREVIATIONS |
The abbreviations used are:
NR, nuclear hormone
receptor;
TR, thyroid hormone receptor;
RAR, retinoic acid receptor;
VDR, vitamin D receptor;
RXR, retinoid X receptor;
NCoR, nuclear
co-repressor;
PR, progesterone receptor;
GR, glucocorticoid receptor;
GFP, green fluorescent protein;
ER, estrogen receptor;
EGFP, enhanced
GFP;
DAPI, 4',6-diamidino-2-phenylindole;
FRAP, fluorescence
recovery after photo-bleaching;
E2, estradiol;
Txn, tamoxifen;
RA, retinoic acid;
SRC-1-steroid receptor coactivator-1, T3, triiodithyronine.
 |
REFERENCES |
1.
|
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344[Abstract/Free Full Text]
|
2.
|
Yen, P. M.
(2001)
Physiol. Rev.
81,
1097-1142[Abstract/Free Full Text]
|
3.
|
Xu, L.,
Glass, C. K.,
and Rosenfeld, M. G.
(1999)
Curr. Opin. Genet. Dev.
9,
140-147[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Yao, T.-P.,
Ku, G.,
Zhou, N.,
Scully, R.,
and Livingston, D. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10626-10631[Abstract/Free Full Text]
|
5.
|
Guiochon-Mantel, A.,
Lescop, P.,
Christin-Maitre, S.,
Loosfelt, H.,
Perrot-Applanat, M.,
and Milgrom, E.
(1991)
EMBO J.
10,
3851-3859[Abstract]
|
6.
|
Hache, R. J.,
Tse, R.,
Reich, T.,
Savory, J. G.,
and Lefebvre, Y. A.
(1999)
J. Biol. Chem.
274,
1432-1439[Abstract/Free Full Text]
|
7.
|
Prufer, K.,
Racz, A.,
Lin, G. C.,
and Barsony, J.
(2000)
J. Biol. Chem.
275,
41114-41123[Abstract/Free Full Text]
|
8.
|
Bunn, C. F.,
Neidig, J. A.,
Freidinger, K. E.,
Stankiewicz, T. A.,
Weaver, B. S.,
McGrew, J.,
and Allison, L. A.
(2001)
Mol. Endocrinol.
15,
512-533[Abstract/Free Full Text]
|
9.
|
Baumann, C. T.,
Maruvada, P.,
Hager, G. L.,
and Yen, P. M.
(2001)
J. Biol. Chem.
276,
11237-11245[Abstract/Free Full Text]
|
10.
|
Htun, H.,
Holth, L. T.,
Walker, D.,
Davie, J. R.,
and Hager, G. L.
(1999)
Mol. Biol. Cell
10,
471-486[Abstract/Free Full Text]
|
11.
|
Stenoien, D. L.,
Mancini, M. G.,
Patel, K.,
Allegretto, E. A.,
Smith, C. L.,
and Mancini, M. A.
(2000)
Mol. Endocrinol.
14,
518-534[Abstract/Free Full Text]
|
12.
|
Lim, C. S.,
Baumann, C. T.,
Htun, H.,
Xian, W.,
Irie, M.,
Smith, C. L.,
and Hager, G. L.
(1999)
Mol. Endocrinol.
13,
366-375[Abstract/Free Full Text]
|
13.
|
Stenoien, D. L.,
Patel, K.,
Mancini, M. G.,
Dutertre, M.,
Smith, C. L.,
O'Malley, B. W.,
and Mancini, M. A.
(2001)
Nat. Cell Biol.
3,
15-23[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
McNally, J. G.,
Muller, W. G.,
Walker, D.,
Wolford, R.,
and Hager, G. L.
(2000)
Science
287,
1262-1265[Abstract/Free Full Text]
|
15.
|
Liu, Y.,
Takeshita, A.,
Nagaya, T.,
Baniahmad, A.,
Chin, W. W.,
and Yen, P. M.
(1998)
Mol. Endocrinol.
12,
34-44[Abstract/Free Full Text]
|
16.
|
Sambrook, J.,
Fritxch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
17.
|
Wu, J.,
Zhou, L.,
Tonissen, K.,
Tee, R.,
and Artzt, K.
(1999)
J. Biol. Chem.
274,
29202-29210[Abstract/Free Full Text]
|
18.
|
Zhu, X. G.,
Hanover, J. A.,
Hager, G. L.,
and Cheng, S. Y.
(1998)
J. Biol. Chem.
273,
27058-27063[Abstract/Free Full Text]
|
19.
|
Htun, H.,
Barsony, J.,
Renyi, I.,
Gould, D. L.,
and Hager, G. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4845-4850[Abstract/Free Full Text]
|
20.
|
Pratt, W. B.,
and Toft, D. O.
(1997)
Endocrinol. Rev.
18,
306-360[Abstract/Free Full Text]
|
21.
|
King, W. J.,
and Greene, G. L.
(1984)
Nature
307,
745-747[Medline]
[Order article via Infotrieve]
|
22.
|
Raam, S.,
Lauretano, A. M.,
Vrabel, D. M.,
Pappas, C. A.,
and Tamura, H.
(1988)
Steroids
51,
425-439[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Welshons, W. V.,
Lieberman, M. E.,
and Gorski, J.
(1984)
Nature
307,
747-749[Medline]
[Order article via Infotrieve]
|
24.
|
Dauvois, S.,
White, R.,
and Parker, M. G.
(1993)
J. Cell Sci.
106,
1377-1388[Abstract/Free Full Text]
|
25.
|
Fukuda, M.,
Asano, S.,
Nakamura, T.,
Adachi, M.,
Yoshida, M.,
Yanagida, M.,
and Nishida, E.
(1997)
Nature
390,
308-311[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Prufer, K.,
and Barsony, J.
(2002)
Mol. Endocrinol.
16,
1738-1751[Abstract/Free Full Text]
|
27.
|
Tyagi, R. K.,
Amazit, L.,
Lescop, P.,
Milgrom, E.,
and Guiochon-Mantel, A.
(1998)
Mol. Endocrinol.
12,
1684-1695[Abstract/Free Full Text]
|
28.
|
Liu, J.,
and DeFranco, D. B.
(2000)
Mol. Endocrinol.
14,
40-51[Abstract/Free Full Text]
|
29.
|
Baumann, C. T.,
Ma, H.,
Wolford, R.,
Reyes, J. C.,
Maruvada, P.,
Lim, C.,
Yen, P. M.,
Stallcup, M. R.,
and Hager, G. L.
(2001)
Mol. Endocrinol.
15,
485-500[Abstract/Free Full Text]
|
30.
|
Hauser, S.,
Adelmant, G.,
Sarraf, P.,
Wright, H. M.,
Mueller, E.,
and Spiegelman, B. M.
(2000)
J. Biol. Chem.
275,
18527-18533[Abstract/Free Full Text]
|
31.
|
Lonard, D. M.,
Nawaz, Z.,
Smith, C. L.,
and O'Malley, B. W.
(2000)
Mol. Cell
5,
939-948[Medline]
[Order article via Infotrieve]
|
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