Nuclear Cytoplasmic Shuttling by Thyroid Hormone Receptors
MULTIPLE PROTEIN INTERACTIONS ARE REQUIRED FOR NUCLEAR
RETENTION*
Christopher T.
Baumann
§,
Padma
Maruvada§¶,
Gordon L.
Hager
, and
Paul M.
Yen¶
From the
Laboratory of Receptor Biology and Gene
Expression, NCI and ¶ Molecular Regulation and Neuroendocrinology
Section, Clinical Endocrinology Branch, NIDDKD, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, December 11, 2000
 |
ABSTRACT |
In this report, we have studied the intracellular
dynamics and distribution of the thyroid hormone receptor-
(TR
)
in living cells, utilizing fusions to the green fluorescent protein.
Wild-type TR
was mostly nuclear in both the absence and presence of
triiodothyronine; however, triiodothyronine induced a
nuclear reorganization of TR
. By mutating defined regions of TR
,
we found that both nuclear corepressor and retinoid X receptor are
involved in maintaining the unliganded receptor within the nucleus. A
TR
mutant defective in DNA binding had only a slightly altered
nuclear/cytoplasmic distribution compared with wild-type TR
; thus,
site-specific DNA binding is not essential for maintaining TR
within
the nucleus. Both ATP depletion studies and heterokaryon analysis
demonstrated that TR
rapidly shuttles between the nuclear and the
cytoplasmic compartments. Cotransfection of nuclear corepressor and
retinoid X receptor markedly decreased the shuttling by maintaining
unliganded TR
within the nucleus. In summary, our findings
demonstrate that TR
rapidly shuttles between the nucleus and the
cytoplasm and that protein-protein interactions of TR
with various
cofactors, rather than specific DNA interactions, play the predominant
role in determining the intracellular distribution of the receptor.
 |
INTRODUCTION |
Thyroid hormone receptors
(TRs)1 are nuclear hormone
receptors, which can directly regulate transcription by binding to
thyroid hormone response elements of target genes. In the absence of
T3, TRs repress basal transcription of positively regulated
genes, whereas in the presence of T3, TRs activate
transcription (1, 2). In basal repression, TRs associate with
corepressors such as N-CoR and SMRT, forming a corepressor complex (3,
4). This complex includes other components, such as histone
deacetylases, which, in turn, can alter the chromatin structure of
target genes. During transcriptional activation, liganded TRs associate
with coactivators such as SRC-1 and form coactivator complexes,
including components such as the CREB-binding protein, p300, and P/CAF
(5). Several of these factors have histone acetyltransferase activity (6, 7); thus, chromatin remodeling is thought to be a component of both
activation and repression by nuclear receptors. Although these
protein-protein interactions are important for transcriptional activity, it is possible that other mechanisms, such as the subcellular distribution of TRs in the absence or presence of ligand, may contribute to regulation of TR transcriptional activity. In this connection, steroid hormone receptors, such as the glucocorticoid (8-10), progesterone (11, 12), and androgen receptors (13), are at
least partially cytoplasmic in the absence of hormone and translocate
into the nucleus in the presence of hormone. In their unliganded
states, these receptors are believed to be associated with a complex of
chaperone proteins in the cytoplasm, including heat shock proteins 70 and 90 (14-18). It is thought that these interactions are critical in
maintaining the cytoplasmic distribution of the unliganded receptor.
The intracellular distribution of TR
has been characterized both by
cell fractionation and indirect immunofluorescence approaches. By
using these techniques, the receptor is found completely in the
nucleus, both in the presence and absence of ligand (19-21). Since the
receptor will bind specific DNA recognition sites in the presence and
absence of ligand (22), the prevailing model holds that the
constitutive association with the nuclear compartment is due to DNA
binding by TR
. Conversely, an earlier study with a GFP chimera of
TR
identified a significant pool of unliganded receptor in the
cytoplasm (23). To examine the potential roles of various receptor
functions on the intracellular distribution of TR
, we have examined
the intracellular distribution of wild-type TR
and a battery of
receptor mutants, which are defective in specific receptor functions,
including ligand binding, DNA binding, homodimerization, and nuclear
corepressor (N-CoR) interaction. These studies have been performed in
living cells using GFP chimeras. Our findings suggest that
heterodimerization with RXR and interaction with N-CoR both play
important roles in the ligand-independent nuclear retention of TR
.
In particular, TR
mutants that cannot bind N-CoR have markedly
decreased nuclear localization in the absence of hormone.
Furthermore, ablation of the DNA-binding capacity of TR
does not
prevent its nuclear accumulation. Whereas a significant fraction of
wild-type TR
is likely bound to DNA in the absence of hormone, we
conclude that the formation of multifactor complexes with heterodimer
partners and corepressors is primarily responsible for nuclear
accumulation of the unliganded thyroid receptor.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
Expression vectors for wild-type TR
, the TR
mutants (24), RXR (25), and N-CoR (26) were described previously. GFP fusion vectors of wild-type TR
(pEGFP-TR
) and the TR
mutants (pEGFP-TR
-mutant) were constructed as follows. The TR
coding region was amplified by the polymerase chain reaction with primers that
inserted an XhoI site at the 5'-end and an EcoRI
site at the 3'-end of the TR
-coding sequence. Each was then cloned
in frame into XhoI/EcoRI-cut pEGFP-C1
(CLONTECH, Palo Alto CA) resulting in a fusion
protein where GFP was at the N terminus of TR
. pEGFP-RXR was
constructed as described above for pEGFP-TR
. The integrity of all
GFP fusions were confirmed by DNA sequencing.
Cell Culture and DNA Transfection--
HeLa cells (human
cervical carcinoma line, ATCC, Manassas, VA) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, 0.5 mg/ml gentamicin, and 2 mM L-glutamine in 5% CO2 at 37 °C. 100 ng of the indicated GFP-TR
fusion
vector were transfected into HeLa cells grown on 1.5 glass coverslips
(1 × 105 cells/well) with calcium phosphate
(Invitrogen, Carlsbad CA) for 18 h. Cells were then washed twice
with PBS, fed with fresh media, and imaged as described below.
Microscopic Studies--
Cells expressing GFP fusion
proteins were imaged on a Leica TCS SP laser-scanning confocal
microscope mounted on a DMIRBE inverted epifluorescence microscope
equipped with a 63× 1.4 N.A. oil immersion lens. GFP fluorescence was
excited by a 488 nm laser line from an air-cooled
fiber-coupled argon laser (Coherent Laser, 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 Laser, 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 on an 8-bit PMT. All images obtained represent the
average of four sequentially obtained images.
Quantitative Analysis of Receptor Distributions--
The
percentage of GFP-TR
within the nucleus was determined as follows
using the analysis features built into the Leica TCS SP software
package. First, the nucleus was encircled (using the polygon tool) and
the total fluorescence intensity, and the total area of the nucleus was
determined. This was then done for the entire cell to yield the total
fluorescence intensity and the total area of the entire cell.
Background fluorescence was determined by measuring the total
fluorescence of a random region within the field of view and dividing
that value by the total area of that region. This yields the total
background per unit area (BA) within the field of view. The BA was then
multiplied by the total area of both the nucleus and the entire cell to
give the total background within the nucleus (BN) and within the entire
cell, respectively (BC). The BN was then subtracted from the total
fluorescence intensity of the nucleus and BC from the total
fluorescence intensity of entire cell to yield the background-corrected
intensity of both the nucleus (FN) and of the entire cell (FC).
Finally, FN was divided by FC to give the percentage of total
fluorescence found within the nucleus. For each condition, this
analysis was done on a minimum of 15 cells from at least two
independent experiments.
Measurements of the coefficient of variation of the nuclear localized
GFP-TR
were done essentially as described previously (27) with the
following modifications. Data analysis was performed on a Gateway
E-5200 with Scion Image (Scioncorp, Frederick, MD). For each cell, a
minimum of 7 line profiles were generated, and for each condition, a
minimum of 10 cells was analyzed from two independent experiments.
Heterokaryon Analysis--
Heterokaryon analysis was performed
as described previously (28). Briefly, 2 × 105 mouse
cells (NIH3T3) were plated on glass coverslips in a 6-well dish. The
following day, the cells were transfected with 1 µg of pEGFP-TR
as
described above. After 48 h, 1 × 106 human cells
(HeLa) were plated on the same coverslips. Following a 3-h incubation,
the cells were washed thoroughly with PBS and treated with 100 µl of
warmed PEG1500. 2 h later the cells were washed again with PBS and
incubated with Dulbecco's modified Eagle's medium with 50 µ g/ml
cycloheximide for 3 h, fixed with 4% paraformaldehyde, stained
with DAPI (to visualize chromatin), and mounted on glass slides. Cells
were imaged by confocal microscopy as described above.
ATP Depletion Experiments--
ATP depletion studies were
performed as described previously (29). Cells were incubated for 2 h in the presence of sodium azide prior to imaging.
Cell Extraction--
Cell extraction procedures were carried out
according to the methods described previously (30, 31). Briefly,
GFP-TR
-expressing cells were grown to 70% confluency and treated
with or without 10
6 M
T3. Cells were washed once with PBS and then treated with
100 mM NaCl and 0.5% Triton X-100 for 3 min to remove the
soluble components of the cell followed by DNase treatment for 50 min at 32 °C to remove DNA. Cells were then treated with 1 M
ammonium sulfate to remove chromatin. Finally cells were washed with
PBS, stained with DAPI (to visualize DNA), and fixed with 4%
paraformaldehyde. GFP fluorescence was visualized after each extraction
step as described above.
 |
RESULTS |
To study the intracellular distribution of TR
in living cells,
we generated a GFP fusion to the N terminus of wild-type TR
(GFP-TR
; Fig. 1A). In
addition, GFP fusions were made to a series of TR
mutants (Fig.
1B). GFP-TR
-AHT contains three point mutations in the
hinge region (A223G, H224G, and T227G) that abrogate its interaction
with the nuclear corepressor N-CoR (32). GFP-TR
-127 contains a
mutation within the DNA-binding domain (C127A) that destroys the
integrity of the first zinc finger and prevents the receptor from
binding DNA in vitro (33). GFP-TR
-429 (R429Q) and
GFP-TR
-345 (G345R) are natural mutants from patients with resistance
to thyroid hormone and are deficient in homodimerization and ligand
binding, respectively (33). Western blot analysis indicated that all
GFP fusion constructs generated proteins of the predicted size (data
not shown). In addition, the transcriptional activity of each GFP
fusion was assayed in transient cotransfection studies and was found to
be similar to that reported previously for the unfused receptors (data
not shown).

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Fig. 1.
Schematic of GFP-TR
constructs used in this study. A, schematic of
the pEGFP-TR . Locations of the cytomegalovirus (CMV)
promoter, enhanced GFP (EGFP) coding sequence, TR coding
region, termination codon, and SV40 poly(A) signal are all indicated.
B, domain structure of wild-type (WT) TR and
each of the four mutants used in this study. Location of the
DNA-binding domain (DBD), hinge region, ligand binding
domain (LBD), and activation function 2 (AF2) are
all shown. * represents the location of the indicated mutations. The
ability of each mutant to perform various receptor functions is
indicated by a + or . n.d., not determined.
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Analysis of the intracellular distribution of GFP-TR
in living cells
demonstrated that the majority of TR
localized within the nucleus in
both the absence and presence of T3 (Fig.
2, A and B). To
quantitate the percentage of GFP-TR
present within the nucleus, the
area-corrected intensity of GFP-TR
fluorescence in both the nucleus
and the cytoplasm was calculated (see "Experimental Procedures").
From these analyses, we determined that 85-90% of GFP-TR
was
localized within the nucleus in both the absence and presence of
T3 (Fig. 2, A and B, and Table
I). In addition, GFP-TR
was excluded
from the nucleoli, as has been reported for other nuclear receptors
(Fig. 2, A and B (8, 11, 13, 27)). The observed nuclear/cytoplasmic ratio was independent of the amount of DNA transfected, indicating that protein expression levels did not impact
the observed intracellular distribution of GFP-TR
(data not shown).
We found a somewhat lower cytoplasmic concentration of TR
in
hormone-free cells than that reported by Zhu et al. (23)
(nuclear/cytoplasmic = 1.5 or ~60% nuclear); in the presence of
ligand, our results are in complete accord. The quantitative difference
in the amount of unliganded receptor in the cytoplasm may be due to
differences in experimental conditions between our studies and that of
Zhu et al. (23).

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Fig. 2.
Intracellular distribution of
wild-type GFP-TR in HeLa cells.
pEGFP-TR (100 ng) was transfected into HeLa cells, and the
intracellular distribution of GFP-TR was determined by
laser-scanning confocal microscopy. Representative image of EGFP-TR
in HeLa cells in the absence (A) and presence (B)
of 10 6 M T3.
Arrows indicate nucleoli. Numbers at
bottom of each image represents the average nuclear
localization (in percent total) as described under "Experimental
Procedures." C and D, representative line
graphs displaying the variation of pixel intensities across a nuclei in
the absence (C) and presence (D) of
10 6 M T3. Values
represent the mean pixel intensity across the graph. s.d. is
the S.D.
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In previous studies on the intranuclear distribution of other
nuclear receptors, such as the estrogen receptor, addition of ligand
led to an intranuclear rearrangement of the receptor (27, 34).
Similarly, upon T3 addition, an intranuclear rearrangement of GFP-TR
occurred. In the absence of T3, GFP-TR
was
organized in a diffuse, reticular pattern (Fig. 2A). Upon
addition of T3, GFP-TR
redistributed into a discrete
punctate pattern (Fig. 2B). To quantitate the intranuclear
distribution of GFP-TR
in the absence and presence of
T3, we adopted the method of Htun et al. (27).
Briefly, the average intensity of GFP-TR
along a random linear line
was determined. Dividing the S.D. of the average intensity by the
average intensity yielded the coefficient of variation, an intensity
corrected value representing the range of GFP-TR
intensities along a
defined line. A low coefficient of variation, indicative of a more
diffuse distribution, would be represented by a relatively smooth line
profile, as shown in Fig. 2C. A high coefficient of
variation would indicate a more focal distribution and is represented
by uneven line profile (Fig. 2D). In the absence of ligand,
the coefficient of variation for GFP-TR
was 0.11 ± 0.01, whereas in the presence of T3, the coefficient of variation increased to 0.25 ± 0.07. Therefore, GFP-TR
undergoes a
quantitative intranuclear redistribution upon the addition of
T3.
We then examined the intracellular distribution of the TR
mutants described above. GFP-TR
-345, which binds ligand poorly, exhibited a similar intracellular distribution as GFP-TR
in the absence and presence of T3 (Fig.
3, A and B, and
Table I) with no change in the intranuclear distribution of this
receptor (Fig. 3, A and B, and data not shown).
Next, the intracellular distribution of GFP-TR
-127 was studied. This
receptor contains a disrupted zinc finger within the DNA-binding domain
and is unable to bind DNA. In both the absence and presence of
T3, ~80% of GFP-TR
-127 was found within the nucleus
(Fig. 3, C and D, and Table I). Even though both
liganded and unliganded TR
are believed to be associated with DNA,
these results suggest that site-specific DNA binding plays only a minor
role in maintaining the nuclear localization of wild-type TR
.

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Fig. 3.
Intracellular distribution of
GFP-TR -345 and
GFP-TR -127 in HeLa cells. pEGFP-TR -345
(A and B) or pEGFP-TR -127 (C and
D) (100 ng each) was transfected into HeLa cells, and the
intracellular distribution of the each GFP-TR mutant was determined
by laser-scanning confocal microscopy. Representative image in the
absence (A and C) and presence (B and
D) of 10 6 M
T3. Numbers at bottom of each image
represents the average nuclear localization (as percent total) as
described under "Experimental Procedures."
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Surprisingly, GFP-TR
-AHT, which does not
interact with N-CoR, was significantly
more cytoplasmic in the absence of ligand than GFP-TR
(Fig.
4A and Tables I and
II). Quantitative analyses of the
intracellular distribution of this mutant demonstrated that less than
50% of the GFP-TR
-AHT was localized to the nucleus (Tables I and
II). However, after addition of T3, a substantial portion
of GFP-TR
-AHT translocated into the nucleus, resulting in ~85% of
the receptor localizing within the nucleus (Fig. 4B and
Tables I and II). These results suggest that, in the absence of
T3, N-CoR interactions likely play an important role in
maintaining the nuclear localization observed with wild-type TR
.
However, in the presence of ligand, N-CoR interactions are dispensable for maintaining TR
within the nucleus.

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Fig. 4.
Intracellular distribution of
GFP-TR -AHT in HeLa cells. pEGFP-TR -AHT
(100 ng) was transfected into HeLa cells, and the intracellular
distribution of the receptor was followed by laser-scanning confocal
microscopy. A, representative example of GFP-TR -AHT in
the absence of 10 6 M
T3. B, representative example of GFP-TR -AHT
in the presence of T3. C, effect of RXR on the
intracellular distribution of GFP-TR -AHT. RXR and GFP-TR -AHT were
coexpressed in HeLa cells, and the intracellular distribution of
GFP-TR -AHT was followed as described above. D, effect of
N-CoR on the intracellular distribution of GFP-TR -AHT. N-CoR and
GFP-TR -AHT were coexpressed in HeLa cells, and the intracellular
distribution of GFP-TR -AHT was followed as described above.
E, effect of wild-type TR on the intracellular
distribution of GFP-TR -AHT. Wild-type TR and GFP-TR -AHT were
coexpressed in HeLa cells, and the intracellular distribution of
GFP-TR -AHT was followed as described above. Values represent the
average nuclear intensity (as percent total) of GFP-TR -AHT as
described under "Experimental Procedures."
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To investigate if additional factors were required for retaining TR
within the nucleus, the intracellular distribution of GFP-TR
-429 was
studied. GFP-TR
-429 selectively forms heterodimers with the retinoid
X receptor (RXR) and is defective in homodimerization. In the absence
of ligand, GFP-TR
-429 was ~75% nuclear (Fig.
5A and Table I), similar to
that seen with GFP-TR
-127 (Fig. 3C and Table I). However,
in the presence of T3, GFP-TR
-429 translocated to the
nucleus and adopted an intracellular distribution that was
indistinguishable from wild-type TR
(Fig. 5B). These
results demonstrate that homodimerization has only a minor role in
maintaining unliganded TR
within the nucleus and is not required for
the nuclear localization of liganded TR
.

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Fig. 5.
Intracellular distribution of
GFP-TR -429 and GFP-RXR in HeLa cells.
pEGFP-TR -429 (A and B) or pEGFP-RXR
(C and D) (100 ng each) were transfected into
HeLa cells, and the intracellular distribution of the each GFP fusion
was determined by laser-scanning confocal microscopy. Representative
image in the absence (A) and presence (B) of
10 6 M T3 or the
absence (C) and presence(D) of
10 6 M 9-cis-retinoic
acid. Numbers at bottom of each image represents
the average nuclear localization (as percent total) as described under
"Experimental Procedures."
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To investigate further whether heterodimerization played a role in the
nuclear localization of the unliganded TR
, we expressed unfused RXR
in the presence of GFP-TR
-AHT, which was mostly cytoplasmic in the
absence of ligand. If RXR was involved in the nuclear retention of the
receptor, we predicted that coexpression of RXR should increase the
nuclear localization of the predominantly cytoplasmic GFP-TR
-AHT.
Fig. 4C demonstrates that this was indeed the case as
GFP-TR
-AHT localized to the nucleus when cotransfected with RXR. In
addition, as the amount of RXR expression vector transfected into the
cell was increased, the amount of GFP-TR
-AHT retained within the
nucleus also increased (Table II). As expected, GFP-RXR was found
almost completely in the nucleus in both the absence and presence of
its ligand, 9-cis-retinoic acid (Fig. 5, C and D). Coexpression of N-CoR with GFP-TR
-AHT had no effect
on the intracellular distribution of this mutant (Fig. 4D
and Table II) as would be predicted by the inability of GFP-TR
-AHT
to interact with N-CoR. In addition, coexpressing unfused TR
had no
effect on the intracellular distribution of GFP-TR
-AHT (Fig.
4E and Table II), consistent with the results seen with the
heterodimer-specific GFP-TR
-429. Taken together, these findings
further underscore the role of heterodimerization on nuclear
localization of the unliganded TR
.
Previous studies by Milgrom and co-workers (29) have shown that both
the estrogen and progesterone receptors are actively transported into
the nucleus and return to the cytoplasm by passive diffusion. Our
studies with GFP-TR
-AHT and RXR suggest that at least a portion of
the intracellular TR
may also shuttle between the nucleus and the
cytoplasm. To confirm this hypothesis, the intracellular distribution
of GFP-TR
was studied after treatment with sodium azide, which
depletes intracellular stores of ATP and previously has been shown to
block the ATP-dependent uptake of both the estrogen and
progesterone receptors into the nucleus (29). After a 2-h treatment
with sodium azide, ~10% of the nuclear localized GFP-TR
redistributed to the cytoplasm (Fig. 6,
A and B, and Table
III), demonstrating that a subpopulation
of GFP-TR
shuttles between the nucleus and cytoplasm. In addition,
treatment of cells with T3, either before or after the
addition of sodium azide, had no effect on the intracellular
redistribution of GFP-TR
(Fig. 6, C and D, and
Table III) demonstrating that the population of GFP-TR
that shuttles
was unaffected by ligand binding. This observation was confirmed by
heterokaryon analysis (Fig. 6G). Here, GFP-TR
-expressing
NIH3T3 cells were fused to nonexpressing HeLa cells, and GFP-TR
was
found to translocate from the NIH3T3 nuclei to the HeLa nuclei, again
confirming that GFP-TR
can rapidly shuttle between the nuclear and
the cytoplasmic compartments.

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Fig. 6.
Nuclear cytoplasmic shuttling of
GFP-TR in HeLa cells. HeLa cells were
transfected with 100 ng of pEGFP-TR and subsequently depleted of ATP
by sodium azide treatment as described under "Experimental
Procedures." Numbers at bottom of each image
represent the average nuclear localization (as percent total) as
described under "Experimental Procedures." A, GFP-TR
prior to treatment with sodium azide. B,
GFP-TR -expressing cells treated with sodium azide for 2 h as
described under "Experimental Procedures." C,
GFP-TR -expressing cells treated with sodium azide for 1 h
prior to the addition of 10 6 M
T3. D, GFP-TR expressing cells treated with
sodium azide for 2 h after the addition of
10 6 M T3 for 1 h. Coexpression of either N-CoR (E) or RXR (F)
blocks the observed redistribution of GFP-TR after treatment with
sodium azide. G, pEGFP-TRb-expressing NIH3T3 cells were
fused with HeLa cells as described in "Experimental Procedures."
The identity of each nuclei was determined by DAPI staining of the
nuclei (left) and labeled on the figure. The distribution of
the EGFP-TRb after fusion is shown on the right.
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These findings suggested that nuclear import of both the unliganded and
liganded TR
was an energy-dependent process. In further support of this point, the effect of sodium azide on GFP-TR
-AHT was
studied. In the presence of sodium azide, a significant
redistribution of this receptor was observed, with ~50% of the
nuclear-localized GFP-TR
-AHT redistributing to the cytoplasm (Table
III). As shown above, addition of ligand to GFP-TR
-AHT led to a
redistribution of the receptor from the cytoplasm to the nucleus (Fig.
4, A and B, and Table II). However in the
presence of sodium azide, this nuclear translocation was blocked (Table
III), again demonstrating that nuclear import of TR
is an
ATP-dependent process.
As described above (Figs. 4 and 5 and Table II), both N-CoR and RXR
interactions played important roles in maintaining the correct
intracellular distribution of TR
. To confirm these observations further, cells were cotransfected with GFP-TR
and either RXR or
N-CoR and then treated with sodium azide. Coexpression of either N-CoR
(Fig. 6E and Table III) or RXR (Fig. 6F, Table
III) blocked the sodium azide-induced redistribution of GFP-TR
and
demonstrated again the importance of both RXR and N-CoR in maintaining
TR
within the nucleus.
Previous experiments with the estrogen receptor found that the
ligand-bound estrogen receptor is in a tighter association with the
nucleus than the unliganded receptor (27, 34). We have found very
similar results with TR
. In both the absence (Fig.
7A) and presence (Fig.
7B) of ligand, high salt and detergent extractions of
GFP-TR
-expressing cells failed to remove all of the receptor from
the nuclei, indicating that the receptor was tightly associated with
one or more factors within the nucleus. However, after DNase and
ammonium sulfate treatment to remove the chromatin, the unliganded
GFP-TR
was completely lost from the nucleus, whereas a portion of
the liganded GFP-TR
remained associated with an extraction-resistant
component of the nucleus (Fig. 7B, bottom panel, data not
shown). Therefore, ligand binding not only induces a change in the
intranuclear distribution of TR
but also increases the association
of the receptor with nonchromatin components of the nucleus.

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Fig. 7.
Ligand-bound GFP-TR
but not unliganded GFP-TR can associate
with an insoluble nuclear com ponent. HeLa cells were
transfected with 100 ng of pEGFP-TR and sequentially extracted as
described under "Experimental Procedures" both before
(A) and after (B) treatment with
10 6 M T3. GFP
fluorescence was followed by laser-scanning confocal microscopy.
DAPI-stained nuclei (right) are included in each image to
indicated the presence or absence of DNA following each
treatment.
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 |
DISCUSSION |
Nuclear receptors can be divided into three broad categories,
based on their unliganded distributions as follows: those that are
primarily nuclear (estrogen receptor (27, 35, 36) and TR
(23, our
results)); those that are primarily cytoplasmic (glucocorticoid receptor (8, 10, 37) and androgen receptor (13)); and those with a
mixed distribution (mineralocorticoid receptor (38) and progesterone
receptor (11, 12)). However, in all cases, addition of ligand leads to
a near complete nuclear localization of the receptors (39). Thus it is
possible there may be a continuum of cellular/nuclear distributions
among the unliganded nuclear hormone receptors that plays a role in
modulating their activity in both the absence and presence of ligand.
We have used fluorescence microscopy in conjunction with GFP fusion
proteins to examine the contribution of various receptor parameters to
the intracellular distribution of TR
in living cells. In the absence
of T3, ~10-15% of the intracellular GFP-TR
was found
within the cytoplasm. The cytoplasmic distribution seen for several
members of the steroid receptor family, including the glucocorticoid
and progesterone receptors, has been postulated to be stabilized by
interactions with molecular chaperones, primarily hsp70 and hsp90 (40).
A previous study did not demonstrate TR
association with heat shock
proteins (22). However, this study was performed in vitro,
and a potential association between TR
and the growing family of
chaperones has not been exhaustively investigated. Currently it is not
known whether the cytoplasmic pool of TR
exists as monomers or
homodimers (our data show RXR is exclusively nuclear, thus eliminating
the possibility of cytoplasmic TR
-RXR heterodimers). Nongenomic
effects by thyroid hormone and other nuclear hormone receptor ligands
have previously been described (41). It is possible that a population
of TR
located within the cytoplasm may be responsible for some of
the observed nongenomic effects of thyroid hormones.
To understand the properties of TR
responsible for its observed
intracellular distribution, several TR
mutants were studied. Mutations that disrupted site-specific DNA binding (GFP-TR
-127), ligand binding (GFP-TR
-345), and homodimerization (GFP-TR
-429) have minimal effects on the intracellular distribution of TR
, demonstrating that these receptor properties are not major contributors to the intracellular localization of the receptor.
We have demonstrated several lines of evidence that suggest that
interactions with N-CoR play an important role in maintaining the
correct intracellular distribution of TR
. First, GFP-TR
-AHT was
primarily cytoplasmic in the absence of T3; upon addition of T3, this mutant translocated to the nucleus.
Additionally, in the presence of sodium azide, ~50% of the
nuclear-localized GFP-TR
-AHT redistributed to the cytoplasm as
compared with ~10% of wild-type TR
. This is likely due to the
fact that a larger percentage of GFP-TR
-AHT shuttles between the
cytoplasm and the nucleus as compared with GFP-TR
(due to the
inability to interact with N-CoR). Furthermore, coexpression of N-CoR
with GFP-TR
in the presence of sodium azide blocked the
redistribution of the receptor observed without N-CoR. Although the
x-ray crystal structure suggests that the amino acid residues mutated
in TR
-AHT may not be on the surface of the receptor (42), this
mutant has been shown, by several groups, to be deficient in N-CoR
interactions (32, 43, 44). Taken together, our data strongly implicate N-CoR in maintaining the correct intracellular distribution of the
unliganded TR
. Although the major function of N-CoR interaction with
TR
may be to mediate basal repression of transcription in positively
regulated target genes, our data show that nuclear retention of TR
may be another role. Previous cotransfection studies have shown that
TR
-AHT is defective in mediating basal repression (26, 45). It is
possible that the inability to interact with N-CoR may result in an
inability of the AHT mutant to be retained within the nucleus, and
hence contribute to its inability to repress basal transcription. Thus,
compartmentalization of TRs within the cell may be a mechanism to
modulate transcriptional activity.
In addition to N-CoR interactions, our findings demonstrate that RXR
interactions contribute to the retention of TR
within the nucleus.
Coexpression of RXR enhances the nuclear localization of unliganded
GFP-TR
-AHT, suggesting that heterodimerization is important for
maintaining the nuclear localization of the thyroid hormone receptor.
Again, as found in the experiments with N-CoR, coexpression of RXR with
GFP-TR
blocked the azide-dependent redistribution of the
wild-type receptor to the cytoplasm. Of note, GFP-RXR was exclusively
localized to the nucleus even when coexpressed with TR
or TR
-AHT
(data not shown). In addition, sodium azide treatment had no effect on
the distribution of GFP-RXR under conditions that led to cytoplasmic
retention of GFP-TR
, indicating that RXR itself does not readily
shuttle between the nucleus and cytoplasm. Lazar and co-workers (43)
have observed that RXR heterodimerization with TR
-AHT restores basal
repression by binding to N-CoR. Thus, TR
/RXR/N-CoR may form a
complex that maintains TR
in the nucleus and increases basal
repression (Fig. 8).

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|
Fig. 8.
Model for intracellular distribution
of the unliganded TR . A,
wild-type TR (yellow) exists in equilibrium with multiple
protein factors within the cell, including RXR (green) and
N-CoR (orange), and acts to repress transcription from
target genes (red ×). These interactions are proposed to
maintain TR within the nucleus by maintaining the rate of nuclear
import (large arrow) greater than that of nuclear export
(small arrow). For simplicity, only N-CoR and RXR
interactions are shown. B, when unable to interact with
N-CoR, TR -AHT accumulates within the cytoplasm due to an increase in
the rate of nuclear export as compared with nuclear import (right
side of B). However, in the presence of coexpressed RXR
(left half of B), TR -AHT is retained within
the nucleus, either as a direct consequence of RXR interactions or
through the recruitment of N-CoR by RXR to the AHT/RXR heterodimer
(43). C, DNA binding is not essential for the correct
nuclear distribution of TR . TR -127, which cannot bind DNA, is
still retained within the nucleus through interactions with RXR and
N-CoR.
|
|
In conjunction with our preceding data, we propose a model of TR
intracellular distribution in which a subpopulation of the receptor is
continually shuttling between the cytoplasm and nucleus. This continual
shuttling results in a dynamic equilibrium between the rate of
ATP-dependent nuclear import and passive nuclear export (Fig. 8). The observed nuclear localization of GFP-TR
results when the rate of nuclear import exceeds that of nuclear export. Nuclear
proteins, such as RXR and N-CoR, interact with the nuclear localized
TR
, decrease the rate of nuclear export, and alter the equilibrium
of the receptor. Since GFP-TR
-AHT cannot interact with N-CoR, the
rate of nuclear export increases, and the observed intracellular
distribution of GFP-TR
-AHT is more cytoplasmic. Coexpression of RXR
with GFP-TR
-AHT increases the localization of this mutant within the
nucleus, presumably by forming GFP-TR
-AHT/RXR heterodimers (which,
based on the results of Lazar and colleagues (43), can interact with
N-CoR) and thereby decrease the rate in which GFP-TR
-AHT is exported
from the nucleus. In the presence of sodium azide, nuclear import is
blocked, and the subpopulation of shuttling GFP-TR
accumulates in
the cytoplasm (nuclear export is unaffected by sodium azide). However,
in the presence of either RXR or N-CoR, GFP-TR
remains in the
nucleus (i.e. the rate of nuclear export is decreased). In
the cell, it is possible that limiting amounts of these and other
cofactors may help determine which compartment unliganded TR
will
reside. Additionally, it is possible that other cytoplasmic factor(s)
may help compartmentalize a subset of unliganded TR
s in the
cytoplasm (i.e. decrease the rate of nuclear import).
Post-translational modification of TR
is yet another potential
mechanism for regulating the intracellular distribution of TR
. TR
can be phosphorylated in vitro and in vivo, but
the effect(s) of phosphorylation on the cellular distribution of the
receptor currently are not known (46, 47). The factor(s) that maintain
the liganded TR
within the nucleus are less clear since all of the
TR
mutants described here are mostly nuclear in the presence of
ligand. Studies are now underway to address this important issue.
The current model views unliganded TR
as statically bound to
thyroid response elements and actively involved in basal
repression of positively regulated genes through corepressor
interactions (19, 48). Upon addition of T3, the
corepressors are lost and exchanged for coactivator complexes. As a
consequence, DNA binding is thought to be critical for maintaining the
unliganded receptor within the nucleus (22). Therefore, our data
showing the predominantly nuclear distribution of GFP-TR
-127 was
initially surprising. However, a recent study on the intranuclear
dynamics of the glucocorticoid receptor has found that nuclear
receptors may not be statically bound to chromatin (49). Through
direct photobleaching studies, it was observed that the glucocorticoid
receptor is not statically bound to chromatin but instead is
rapidly exchanging between chromatin and the nucleoplasm. Based on
these results, it is reasonable to speculate that the unliganded TR
may also be rapidly exchanging between chromatin and the nucleoplasm.
If so, then DNA binding would not be a critical factor in maintaining
TR
within the nucleus.
A central tenet of the current TR
models is that the receptor
remains bound to the same chromatin response element when ligand is
added, and the differential activity of the receptors with and without
ligand results from the different macromolecular complexes that
interact with the ligand-free and ligand-bound receptors. However, we
have observed an intranuclear reorganization of TR
in the presence
of T3, suggesting that a significant pool of the receptor
is moving from one site to another in response to ligand. Similar
ligand-dependent intranuclear distributions have been observed for other nuclear hormone receptors, including the estrogen receptor, glucocorticoid receptor, mineralocorticoid receptor, and
androgen receptor (8, 34, 50, 51). Therefore, ligand-bound nuclear
receptors may adopt similar intranuclear distributions, although the
exact nature of these nuclear substructures is still a matter of
debate. Our studies with high salt, detergent-extracted and
DNase-treated, nuclei suggest that a significant portion of liganded
TR
can interact with nuclear components other than chromatin, whereas unliganded TR
cannot. Similar results have been reported for
other nuclear receptors including estrogen receptor and glucocorticoid receptor (27, 52). Therefore, the intranuclear redistribution of these
receptors appears to recruit the receptors to an insoluble component of
the nuclear substructure. Why then would the liganded nuclear receptors
be resistant to DNase treatment? One possibility is that in addition to
being bound to DNA, the liganded nuclear receptors interact with other
nuclear substructures, and it is these interactions that instill the
DNase insensitivity to their distribution. A second possibility is that
the DNase-insensitive receptors represent a subpopulation of liganded
receptors that are, for one reason or another, inactive. Previous
observations have found that the ligand-bound forms of several nuclear
receptors, including the estrogen, progesterone, and retinoic acid
receptors are rapidly degraded by the 26 S proteasome (53-57).
Therefore, it is intriguing to speculate that the DNase-resistant forms
of TR
and of the other nuclear receptors may represent a population of receptors that are currently being degraded or are targeted for
degradation by the proteasome.
In summary, we have observed nuclear/cytoplasmic shuttling of TR
in
living cells using GFP technology. We also have observed that
T3 promotes nuclear redistribution of TR
. Additionally, we have shown that TR
can localize in the nucleus even in the absence of ligand or DNA binding. Furthermore, RXR and N-CoR may promote this nuclear localization in the absence of ligand. We speculate that nuclear/cytoplasmic shuttling may be a novel mechanism for modulating transcription by TR
and other nuclear hormone receptors. Understanding how various factors regulate this shuttling should provide new insight into nuclear hormone receptor action.
 |
Note Added in Proof |
While this work was in progress, Prufer
et al. ((2000) J. Biol. Chem. 275;
41114-41123) found that the retinoid X receptor promoted nuclear
localization and ligand-dependent nuclear organization of vitamin D receptors.
 |
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.
§
Both authors contributed equally to this work.
To whom correspondence should be addressed: Laboratory of
Receptor Biology and Gene Expression, NCI, 41 Library Dr., Bldg. 41, Rm. B602, National Institutes of Health, Bethesda, MD 20892-5055. Tel.:
301-496-9867; Fax: 301-496-4951; E-mail:
hagerg@exchange.nih.gov.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M011112200
 |
ABBREVIATIONS |
The abbreviations used are:
TRs, thyroid hormone
receptors;
T3, triiodothyronine;
TR
, thyroid
hormone receptor-
;
RXR, retinoid X receptor;
N-CoR, nuclear
corepressor;
GFP, green fluorescent protein;
DAPI, 4,6-diamidino-2-phenylindole;
PBS, phosphate-buffered saline.
 |
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