Nuclear Cytoplasmic Shuttling by Thyroid Hormone Receptors

MULTIPLE PROTEIN INTERACTIONS ARE REQUIRED FOR NUCLEAR RETENTION*

Christopher T. BaumannDagger §, Padma Maruvada§, Gordon L. HagerDagger ||, and Paul M. Yen

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-beta (TRbeta ) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TRbeta was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TRbeta . By mutating defined regions of TRbeta , we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TRbeta mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TRbeta ; thus, site-specific DNA binding is not essential for maintaining TRbeta within the nucleus. Both ATP depletion studies and heterokaryon analysis demonstrated that TRbeta rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TRbeta within the nucleus. In summary, our findings demonstrate that TRbeta rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TRbeta with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TRbeta 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 TRbeta . Conversely, an earlier study with a GFP chimera of TRbeta 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 TRbeta , we have examined the intracellular distribution of wild-type TRbeta 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 TRbeta . In particular, TRbeta mutants that cannot bind N-CoR have markedly decreased nuclear localization in the absence of hormone. Furthermore, ablation of the DNA-binding capacity of TRbeta does not prevent its nuclear accumulation. Whereas a significant fraction of wild-type TRbeta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Expression vectors for wild-type TRbeta , the TRbeta mutants (24), RXR (25), and N-CoR (26) were described previously. GFP fusion vectors of wild-type TRbeta (pEGFP-TRbeta ) and the TRbeta mutants (pEGFP-TRbeta -mutant) were constructed as follows. The TRbeta 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 TRbeta -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 TRbeta . pEGFP-RXR was constructed as described above for pEGFP-TRbeta . 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-TRbeta 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-TRbeta 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-TRbeta 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-TRbeta 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-TRbeta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To study the intracellular distribution of TRbeta in living cells, we generated a GFP fusion to the N terminus of wild-type TRbeta (GFP-TRbeta ; Fig. 1A). In addition, GFP fusions were made to a series of TRbeta mutants (Fig. 1B). GFP-TRbeta -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-TRbeta -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-TRbeta -429 (R429Q) and GFP-TRbeta -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-TRbeta constructs used in this study. A, schematic of the pEGFP-TRbeta . Locations of the cytomegalovirus (CMV) promoter, enhanced GFP (EGFP) coding sequence, TRbeta coding region, termination codon, and SV40 poly(A) signal are all indicated. B, domain structure of wild-type (WT) TRbeta 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.

Analysis of the intracellular distribution of GFP-TRbeta in living cells demonstrated that the majority of TRbeta localized within the nucleus in both the absence and presence of T3 (Fig. 2, A and B). To quantitate the percentage of GFP-TRbeta present within the nucleus, the area-corrected intensity of GFP-TRbeta fluorescence in both the nucleus and the cytoplasm was calculated (see "Experimental Procedures"). From these analyses, we determined that 85-90% of GFP-TRbeta was localized within the nucleus in both the absence and presence of T3 (Fig. 2, A and B, and Table I). In addition, GFP-TRbeta 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-TRbeta (data not shown). We found a somewhat lower cytoplasmic concentration of TRbeta 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-TRbeta in HeLa cells. pEGFP-TRbeta (100 ng) was transfected into HeLa cells, and the intracellular distribution of GFP-TRbeta was determined by laser-scanning confocal microscopy. Representative image of EGFP-TRbeta 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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Table I
Intracellular distribution of GFP-TRbeta and GFP-TRbeta mutants

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-TRbeta occurred. In the absence of T3, GFP-TRbeta was organized in a diffuse, reticular pattern (Fig. 2A). Upon addition of T3, GFP-TRbeta redistributed into a discrete punctate pattern (Fig. 2B). To quantitate the intranuclear distribution of GFP-TRbeta in the absence and presence of T3, we adopted the method of Htun et al. (27). Briefly, the average intensity of GFP-TRbeta 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-TRbeta 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-TRbeta was 0.11 ± 0.01, whereas in the presence of T3, the coefficient of variation increased to 0.25 ± 0.07. Therefore, GFP-TRbeta undergoes a quantitative intranuclear redistribution upon the addition of T3.

We then examined the intracellular distribution of the TRbeta mutants described above. GFP-TRbeta -345, which binds ligand poorly, exhibited a similar intracellular distribution as GFP-TRbeta 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-TRbeta -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-TRbeta -127 was found within the nucleus (Fig. 3, C and D, and Table I). Even though both liganded and unliganded TRbeta 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 TRbeta .



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Fig. 3.   Intracellular distribution of GFP-TRbeta -345 and GFP-TRbeta -127 in HeLa cells. pEGFP-TRbeta -345 (A and B) or pEGFP-TRbeta -127 (C and D) (100 ng each) was transfected into HeLa cells, and the intracellular distribution of the each GFP-TRbeta 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."

Surprisingly, GFP-TRbeta -AHT, which does not interact with N-CoR, was significantly more cytoplasmic in the absence of ligand than GFP-TRbeta (Fig. 4A and Tables I and II). Quantitative analyses of the intracellular distribution of this mutant demonstrated that less than 50% of the GFP-TRbeta -AHT was localized to the nucleus (Tables I and II). However, after addition of T3, a substantial portion of GFP-TRbeta -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 TRbeta . However, in the presence of ligand, N-CoR interactions are dispensable for maintaining TRbeta within the nucleus.



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Fig. 4.   Intracellular distribution of GFP-TRbeta -AHT in HeLa cells. pEGFP-TRbeta -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-TRbeta -AHT in the absence of 10-6 M T3. B, representative example of GFP-TRbeta -AHT in the presence of T3. C, effect of RXR on the intracellular distribution of GFP-TRbeta -AHT. RXR and GFP-TRbeta -AHT were coexpressed in HeLa cells, and the intracellular distribution of GFP-TRbeta -AHT was followed as described above. D, effect of N-CoR on the intracellular distribution of GFP-TRbeta -AHT. N-CoR and GFP-TRbeta -AHT were coexpressed in HeLa cells, and the intracellular distribution of GFP-TRbeta -AHT was followed as described above. E, effect of wild-type TRbeta on the intracellular distribution of GFP-TRbeta -AHT. Wild-type TRbeta and GFP-TRbeta -AHT were coexpressed in HeLa cells, and the intracellular distribution of GFP-TRbeta -AHT was followed as described above. Values represent the average nuclear intensity (as percent total) of GFP-TRbeta -AHT as described under "Experimental Procedures."


                              
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Table II
Effect of associated proteins on the intracellular distribution of GFP-TRbeta -AHT

To investigate if additional factors were required for retaining TRbeta within the nucleus, the intracellular distribution of GFP-TRbeta -429 was studied. GFP-TRbeta -429 selectively forms heterodimers with the retinoid X receptor (RXR) and is defective in homodimerization. In the absence of ligand, GFP-TRbeta -429 was ~75% nuclear (Fig. 5A and Table I), similar to that seen with GFP-TRbeta -127 (Fig. 3C and Table I). However, in the presence of T3, GFP-TRbeta -429 translocated to the nucleus and adopted an intracellular distribution that was indistinguishable from wild-type TRbeta (Fig. 5B). These results demonstrate that homodimerization has only a minor role in maintaining unliganded TRbeta within the nucleus and is not required for the nuclear localization of liganded TRbeta .



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Fig. 5.   Intracellular distribution of GFP-TRbeta -429 and GFP-RXR in HeLa cells. pEGFP-TRbeta -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."

To investigate further whether heterodimerization played a role in the nuclear localization of the unliganded TRbeta , we expressed unfused RXR in the presence of GFP-TRbeta -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-TRbeta -AHT. Fig. 4C demonstrates that this was indeed the case as GFP-TRbeta -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-TRbeta -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-TRbeta -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-TRbeta -AHT to interact with N-CoR. In addition, coexpressing unfused TRbeta had no effect on the intracellular distribution of GFP-TRbeta -AHT (Fig. 4E and Table II), consistent with the results seen with the heterodimer-specific GFP-TRbeta -429. Taken together, these findings further underscore the role of heterodimerization on nuclear localization of the unliganded TRbeta .

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-TRbeta -AHT and RXR suggest that at least a portion of the intracellular TRbeta may also shuttle between the nucleus and the cytoplasm. To confirm this hypothesis, the intracellular distribution of GFP-TRbeta 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-TRbeta redistributed to the cytoplasm (Fig. 6, A and B, and Table III), demonstrating that a subpopulation of GFP-TRbeta 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-TRbeta (Fig. 6, C and D, and Table III) demonstrating that the population of GFP-TRbeta that shuttles was unaffected by ligand binding. This observation was confirmed by heterokaryon analysis (Fig. 6G). Here, GFP-TRbeta -expressing NIH3T3 cells were fused to nonexpressing HeLa cells, and GFP-TRbeta was found to translocate from the NIH3T3 nuclei to the HeLa nuclei, again confirming that GFP-TRbeta can rapidly shuttle between the nuclear and the cytoplasmic compartments.



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Fig. 6.   Nuclear cytoplasmic shuttling of GFP-TRbeta in HeLa cells. HeLa cells were transfected with 100 ng of pEGFP-TRbeta 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-TRbeta prior to treatment with sodium azide. B, GFP-TRbeta -expressing cells treated with sodium azide for 2 h as described under "Experimental Procedures." C, GFP-TRbeta -expressing cells treated with sodium azide for 1 h prior to the addition of 10-6 M T3. D, GFP-TRbeta 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-TRbeta 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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Table III
Effect of sodium azide on the intracellular distribution of GFP-TRbeta and GFP-TRbeta -AHT

These findings suggested that nuclear import of both the unliganded and liganded TRbeta was an energy-dependent process. In further support of this point, the effect of sodium azide on GFP-TRbeta -AHT was studied. In the presence of sodium azide, a significant redistribution of this receptor was observed, with ~50% of the nuclear-localized GFP-TRbeta -AHT redistributing to the cytoplasm (Table III). As shown above, addition of ligand to GFP-TRbeta -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 TRbeta 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 TRbeta . To confirm these observations further, cells were cotransfected with GFP-TRbeta 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-TRbeta and demonstrated again the importance of both RXR and N-CoR in maintaining TRbeta 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 TRbeta . In both the absence (Fig. 7A) and presence (Fig. 7B) of ligand, high salt and detergent extractions of GFP-TRbeta -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-TRbeta was completely lost from the nucleus, whereas a portion of the liganded GFP-TRbeta 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 TRbeta but also increases the association of the receptor with nonchromatin components of the nucleus.



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Fig. 7.   Ligand-bound GFP-TRbeta but not unliganded GFP-TRbeta can associate with an insoluble nuclear com ponent. HeLa cells were transfected with 100 ng of pEGFP-TRbeta 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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 TRbeta (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 TRbeta in living cells. In the absence of T3, ~10-15% of the intracellular GFP-TRbeta 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 TRbeta association with heat shock proteins (22). However, this study was performed in vitro, and a potential association between TRbeta and the growing family of chaperones has not been exhaustively investigated. Currently it is not known whether the cytoplasmic pool of TRbeta exists as monomers or homodimers (our data show RXR is exclusively nuclear, thus eliminating the possibility of cytoplasmic TRbeta -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 TRbeta located within the cytoplasm may be responsible for some of the observed nongenomic effects of thyroid hormones.

To understand the properties of TRbeta responsible for its observed intracellular distribution, several TRbeta mutants were studied. Mutations that disrupted site-specific DNA binding (GFP-TRbeta -127), ligand binding (GFP-TRbeta -345), and homodimerization (GFP-TRbeta -429) have minimal effects on the intracellular distribution of TRbeta , 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 TRbeta . First, GFP-TRbeta -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-TRbeta -AHT redistributed to the cytoplasm as compared with ~10% of wild-type TRbeta . This is likely due to the fact that a larger percentage of GFP-TRbeta -AHT shuttles between the cytoplasm and the nucleus as compared with GFP-TRbeta (due to the inability to interact with N-CoR). Furthermore, coexpression of N-CoR with GFP-TRbeta 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 TRbeta -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 TRbeta . Although the major function of N-CoR interaction with TRbeta may be to mediate basal repression of transcription in positively regulated target genes, our data show that nuclear retention of TRbeta may be another role. Previous cotransfection studies have shown that TRbeta -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 TRbeta within the nucleus. Coexpression of RXR enhances the nuclear localization of unliganded GFP-TRbeta -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-TRbeta 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 TRbeta or TRbeta -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-TRbeta , indicating that RXR itself does not readily shuttle between the nucleus and cytoplasm. Lazar and co-workers (43) have observed that RXR heterodimerization with TRbeta -AHT restores basal repression by binding to N-CoR. Thus, TRbeta /RXR/N-CoR may form a complex that maintains TRbeta in the nucleus and increases basal repression (Fig. 8).



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Fig. 8.   Model for intracellular distribution of the unliganded TRbeta . A, wild-type TRbeta (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 TRbeta 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, TRbeta -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), TRbeta -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 TRbeta . TRbeta -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 TRbeta 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-TRbeta 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 TRbeta , decrease the rate of nuclear export, and alter the equilibrium of the receptor. Since GFP-TRbeta -AHT cannot interact with N-CoR, the rate of nuclear export increases, and the observed intracellular distribution of GFP-TRbeta -AHT is more cytoplasmic. Coexpression of RXR with GFP-TRbeta -AHT increases the localization of this mutant within the nucleus, presumably by forming GFP-TRbeta -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-TRbeta -AHT is exported from the nucleus. In the presence of sodium azide, nuclear import is blocked, and the subpopulation of shuttling GFP-TRbeta accumulates in the cytoplasm (nuclear export is unaffected by sodium azide). However, in the presence of either RXR or N-CoR, GFP-TRbeta 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 TRbeta will reside. Additionally, it is possible that other cytoplasmic factor(s) may help compartmentalize a subset of unliganded TRbeta s in the cytoplasm (i.e. decrease the rate of nuclear import). Post-translational modification of TRbeta is yet another potential mechanism for regulating the intracellular distribution of TRbeta . TRbeta 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 TRbeta within the nucleus are less clear since all of the TRbeta 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 TRbeta 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-TRbeta -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 TRbeta may also be rapidly exchanging between chromatin and the nucleoplasm. If so, then DNA binding would not be a critical factor in maintaining TRbeta within the nucleus.

A central tenet of the current TRbeta 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 TRbeta 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 TRbeta can interact with nuclear components other than chromatin, whereas unliganded TRbeta 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 TRbeta 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 TRbeta in living cells using GFP technology. We also have observed that T3 promotes nuclear redistribution of TRbeta . Additionally, we have shown that TRbeta 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 TRbeta 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; TRbeta , thyroid hormone receptor-beta ; RXR, retinoid X receptor; N-CoR, nuclear corepressor; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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