Thyroid Hormone-mediated Enhancement of Heterodimer Formation between Thyroid Hormone Receptor beta  and Retinoid X Receptor*

(Received for publication, December 18, 1996, and in revised form, March 5, 1997)

Trevor N. Collingwood Dagger §, Alison Butler §, Yukiko Tone Dagger , Rory J. Clifton-Bligh Dagger , Malcolm G. Parker and V. Krishna K. Chatterjee Dagger par

From the Dagger  Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ and the  Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D3, and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian two-hybrid assay, we have found that recruitment of a VP16-RXR chimera by a Gal4-TRbeta ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T3). This was also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibited hormone-dependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR was also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T3-induced corepressor release. Finally, we have shown that the interaction between RXR and TR is augmented by T3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. We propose that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling.


INTRODUCTION

The thyroid hormone receptor (TR)1 is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. A subset of receptors, including those for estrogen and glucocorticoid, bind to regulatory sequences in target gene promoters as homodimers, with cooperative DNA binding and dimerization mediated by regions within their DNA- and ligand-binding domains (1-7). The thyroid hormone receptor is also capable of binding to thyroid hormone response elements (TREs) as a monomer or homodimer, but the latter complex is known to dissociate in the presence of thyroid hormone (T3) (8-11). More recently, it has been shown that TR as well as receptors for vitamin D3, retinoic acid (RAR), and eicosanoids form heterodimeric complexes with an auxiliary factor, the retinoid X receptor (RXR) (12-22). The TR·RXR heterodimer binds TREs with higher affinity than TR alone and remains stable in the presence of T3 (8-11), suggesting that this complex mediates ligand-dependent transcriptional activation.

The isolated DNA-binding domains (DBDs) of TR and RXR are capable of both cooperative binding to and discrimination of direct repeat TREs (23). The dimerization interface between DBDs was mapped initially by mutational analyses (24) and then elucidated from the crystal structure of TR·RXR DBDs bound to a DR4 TRE (25). A separate dimerization interface in the carboxyl-terminal domains of nuclear receptors was first delineated as a series of nine conserved hydrophobic heptad repeats (26) predicted to form alpha -helices with the potential to mediate a coiled-coil protein interaction. A 40-amino acid region encompassing the ninth heptad has been shown to be critical for heterodimerization (27). This region is almost coincident with helical segments (helices 9 and 10) that constitute the dimer interface in the crystal structure of the ligand-binding domain (LBD) of hRXRalpha (28). In addition, mutation of hydrophobic residues at either end of this heptad in TR or RAR abolishes the interaction with RXR (10, 29, 30).

Several studies have shown a solution interaction between RXR and other nuclear receptors in the absence of DNA or ligand. Thus, RAR·RXR or TR·RXR complexes can be coimmunoprecipitated in the absence of ligand (18-20), and a constitutive interaction between the LBDs of RXR and RAR (31) or TR (10, 32) has been demonstrated in mammalian cells and yeast (33). However, the influence of ligand on dimerization between RXR and TR is unclear. In this study, we have investigated the role of T3 in modulating the interaction between the LBDs of TRbeta and RXRalpha .


MATERIALS AND METHODS

Expression Vectors and Reporter Constructs

The wild-type pSG-TR and pSG-RXR mammalian expression vectors, respectively, contain the LBDs of human TRbeta (amino acids 174-461) and RXRalpha (amino acids 198-462) coupled to the Gal4 DBD (amino acids 1-147) in pSG424. Mutant expression vectors include pSG-TR(L454A), containing a L454A substitution (32), and pSG-TR(AHTm), with the triple substitution A223G/H224G/T227A in pSG-TR. pVP-RXR-(1-462) and pVP-RXR(DE) consist of either full-length or the LBD (amino acids 198-462) of hRXRalpha , respectively, coupled to the activation domain of VP16 in the expression vector pAASV. The reporter plasmid UAStkLUC contains Gal4 recognition sequences and the thymidine kinase promoter in pA3LUC. The internal control plasmid Bos-beta gal contains the promoter of human elongation factor 1alpha linked to beta -galactosidase (32). The yeast expression vectors pMA-TR(wt) and pMA-TR(L454A) contain the LBDs of either wild-type human TRbeta or the L454A mutant described above coupled to the Gal4 DBD in pMA424, respectively, and pMA210 contains full-length Gal4 cDNA. pGAD-RXR consists of residues 198-462 of hRXRalpha coupled to the activation domain of Gal4 in pGAD10. The bacterial expression vector pGEX-RXR contains full-length hRXRalpha linked to glutathione S-transferase (GST) in pGEX2T.

Mammalian Cell Culture and Transfections

JEG-3 human choriocarcinoma cells were cultured in Opti-MEM supplemented with 2% (v/v) fetal bovine serum and 1% penicillin/streptomycin/Fungizone and transferred to Opti-MEM with 1% penicillin/streptomycin/Fungizone prior to transfection by a 4-h exposure to calcium phosphate as described previously (10). Following a 36-h incubation, cells were lysed, and luciferase activity was measured and normalized using beta -galactosidase activity.

Yeast beta -Galactosidase Assays

SFY526 yeast cells were cultured and cotransformed with the appropriate expression vectors and assayed following the protocol detailed by the manufacturer (CLONTECH). beta -Galactosidase activity in liquid culture assays was corrected for cell density. Cells were grown in the absence or presence of 1 µM triiodothyroacetic acid (TRIAC).

Protein-Protein Interaction Assays

GST-RXR fusion protein was expressed in Escherichia coli and purified as described previously (32). [35S]Methionine-labeled Gal4-TRbeta was synthesized by coupled in vitro transcription and translation. Aliquots of GST-RXR immobilized on glutathione-Sepharose were preincubated with 4 mg ml-1 bovine serum albumin in P buffer (50 mM Tris-HCl, 0.1 M KCl, 0.14 M NaCl, 0.5% Nonidet P-40, 10% glycerol, and 2 mM dithiothreitol, pH 8.0) at room temperature prior to 10-fold dilution in P buffer and equilibration at 37 °C. Aliquots of in vitro translated Gal4-TR were diluted in P buffer in the absence or presence of 5 µM T3 and equilibrated at 30 °C for 1 h to allow saturation with ligand. Equal volumes of the GST-RXR and Gal4-TR solutions were then mixed and incubated at 37 °C for the specified duration, followed by washing four times with 20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40, pH 8.0, and analysis by SDS-polyacrylamide gel electrophoresis. Gels were stained with Coomassie Blue to check for equal loading of fusion protein and then exposed to autoradiography.


RESULTS

Gal4-TR and VP16-RXR Chimeras Interact in Vivo

To examine the interaction between the ligand-binding domains of TR and RXR, we used a mammalian two-hybrid assay in which chimeric proteins containing the LBDs of human TRbeta or hRXRalpha or full-length hRXRalpha were fused to either the Gal4 DBD or the VP16 activation domain and coexpressed in JEG-3 cells (Fig. 1). Transfection of either receptor fusion with the Gal4 DBD or VP16 alone resulted in negligible transcriptional activity. Similarly, coexpression of Gal4-TR and VP16-TR or Gal4-RXR and VP16-RXR did not enhance promoter activity. However, cotransfection of TR and RXR chimeras resulted in a significant increase in reporter gene activity, suggesting that a constitutive interaction between the LBDs of these receptors does occur in vivo.


Fig. 1. Constitutive heterodimeric, but not homodimeric, interaction of TR and RXR LBDs in vivo. JEG-3 cells in six-well plates were transfected with 0.5 µg of each of the specified Gal4- and VP16-receptor fusion vectors, along with 5 µg of UAStkLUC reporter and 0.3 µg of Bos-beta gal internal control plasmid. -Fold induction of luciferase activity is relative to Gal4-TR + VP16. Data are the means ± S.E. of three separate experiments.
[View Larger Version of this Image (25K GIF file)]

T3 Enhances the Interaction of Gal4-TR with VP16-RXR

Since transcriptional activation by nuclear receptors is generally dependent on cognate ligand, we next examined the influence of T3 in the above paradigm. As noted previously, there was a significant constitutive interaction between Gal4-TR(wt) and VP16-tagged full-length RXR or the RXR LBD (Fig. 2, bars 2 and 3). The addition of thyroid hormone enhanced reporter gene activity by 25-50-fold. One interpretation of this finding was that the additional activation represented increased recruitment of VP16-RXR to the promoter via its interaction with Gal4-TR. However, T3 also significantly augmented the activity of Gal4-TR alone (Fig. 2, bar 1), presumably by stimulating the known ligand-dependent transcriptional activation function (AF-2) contained within the TR LBD. This raised the possibility that the ligand-dependent increase in reporter activation was due to the AF-2 activity of TR superimposed on activation associated with the VP16 moiety.


Fig. 2. T3 enhances the interaction between TR and RXR in mammalian cells. JEG-3 cells in 24-well plates were cotransfected with 50 ng of pSG-TR(wt) (Gal4-TR(wt)), pSG-TR(L454A) (Gal4-TR(L454A)), pAASV (VP16), pVP-RXR (VP16-RXR-(1-462)), and pVP-RXR(DE) (VP16-RXR(DE)) as specified, along with 500 ng of UAStkLUC reporter and 100 ng of Bos-beta gal internal control. The cells were then incubated in the absence or presence of 0.1 µM T3. -Fold induction of luciferase activity is relative to Gal4-TR(wt) in the absence of ligand. Data are the means ± S.E. of at least three individual experiments. Note the discontinuous scale of the vertical axis.
[View Larger Version of this Image (23K GIF file)]

T3-mediated Enhancement Is Not Due to AF-2 Activity of Gal4-TR

To differentiate between these possibilities, we used a mutant Gal4-TR fusion containing a single amino acid substitution (L454A) in the carboxyl-terminal amphipathic alpha -helix that abolishes the AF-2 activity of TR without altering its ligand binding function (32). The mutant Gal4-TR(L454A) chimera also exhibited constitutive interaction with VP16-RXR (Fig. 2, bars 5 and 6), and again, reporter activity was markedly enhanced with the addition of T3. However, the mutant Gal4-TR(L454A) fusion exhibited no ligand-dependent transcriptional activation when tested alone (Fig. 2, bar 4), suggesting that the T3-dependent increase in reporter activity was not due to AF-2 activity, but rather to an increased recruitment of VP16-RXR.

T3 Enhances Gal4-TR and VP16-RXR Interaction in Yeast

Although full-length TRbeta has been shown to exhibit both constitutive and ligand-dependent activation functions with TRE-containing reporter genes in yeast (34), a chimera consisting of the TRbeta LBD coupled to the DBD of LexA was transcriptionally inactive in Saccharomyces cerevisiae (33). This lack of AF-2 activity in yeast prompted us to examine the influence of ligand on the interaction of TR and RXR LBDs to substantiate our findings in mammalian cells. Wild-type or mutant (L454A) Gal4-TR fusions were cotransformed into yeast with either the activation domain (AD) of Gal4 alone or a chimera containing the LBD of RXR fused to the activation domain (AD-RXR). Recombinant yeast cells were grown either in the absence or presence of TRIAC prior to detection of beta -galactosidase expression (Fig. 3A). Treatment with TRIAC did not induce beta -galactosidase activity with coexpressed Gal4-TR(wt) and AD, but ligand markedly enhanced beta -galactosidase expression with a Gal4-TR(wt) and AD-RXR combination. To exclude the possibility that the coexpressed RXR was simply facilitating the inherent AF-2 activity of Gal4-TR, we tested the Gal4-TR(L454A) mutant and found a comparable TRIAC-dependent enhancement of beta -galactosidase activity with AD-RXR. To quantify the magnitude of the ligand-dependent enhancement, yeast cells containing either wild-type or mutant (L454A) Gal4-TR fusions, together with either AD or AD-RXR, were grown in the absence or presence of TRIAC, and cell extracts were assayed for beta -galactosidase activity (Fig. 3B). The results confirmed that neither the wild-type nor the mutant (L454A) Gal4-TR fusions exhibit AF-2 activity following TRIAC treatment. Both fusions showed a comparable constitutive association with coexpressed AD-RXR that was enhanced 20-25-fold by the addition of TRIAC.


Fig. 3. Interaction between TR and RXR in yeast is enhanced by ligand. A, in a yeast two-hybrid assay, cells were transformed with pMA210 + pGAD10 (GAL4), pMA424 + pGAD-RXR (Gal + AD-RXR), pMA-TR(wt) + pGAD10 (Gal-TR(wt) + AD), pMA-TR(wt) + pGAD-RXR (Gal-TR(wt) + AD-RXR), or pMA-TR(L454A) + pGAD-RXR (Gal-TR(L454A) + AD-RXR) and grown on plates in the absence or presence of 1 µM TRIAC. Colonies were transferred to filters and assayed for beta -galactosidase reporter expression using 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside as substrate. B, yeast cells were transformed with pMA-TR(wt) + pGAD10 (WT), pMA-TR(L454A) + pGAD10 (L454A), pMA-TR(wt) + pGAD-RXR (WT + AD-RXR), or pMA-TR(L454A) + pGAD-RXR (L454A + AD-RXR) and then grown in liquid culture in the absence (open bars) and presence (closed bars) of 1 µM TRIAC. Data are the means ± S.E. of at least four individual experiments. Note the discontinuous vertical axis.
[View Larger Version of this Image (34K GIF file)]

T3 Enhancement Is Not Due to Corepressor Release

Unliganded TR is known to mediate repression of basal gene transcription, and this silencing function has been localized to its LBD (35, 36). Recently, two putative corepressor proteins (N-CoR and SMRT) have been identified that bind TR·RXR heterodimers in the absence of ligand, but that dissociate following ligand binding (37, 38). This raised the possibility that ligand-induced release of corepressor(s) by promoter-bound Gal4-TR·VP16-RXR complexes could account for the T3-dependent increased reporter activity observed in two-hybrid assays. To discount this possibility, we studied another mutant Gal4-TR fusion, AHTm, which contains a triple substitution (A223G/H224G/T227A)2 that has been shown to abolish the interaction of the TR·RXR heterodimer with N-CoR (38). When tested independently (Fig. 4, bar 2), hormone-dependent reporter gene activation by Gal4-TR(AHTm) was preserved, but significantly reduced (15%) compared with Gal4-TR(wt) (bar 1), indicating that this mutant receptor retains ligand binding and activation functions. The Gal4-TR(AHTm) mutant unexpectedly showed negligible constitutive interaction with coexpressed VP16-RXR compared with the wild-type receptor fusion (Fig. 4, bars 3 and 4). Following T3 treatment, there was a significant enhancement of reporter activity with Gal4-TR(wt), as seen previously, but in addition, Gal4-TR(AHTm) mediated a similar response, suggesting that corepressor release did not account for this effect.


Fig. 4. T3-induced TR-RXR interaction is not due to corepressor release. JEG-3 cells were transfected as described in the legend to Fig. 2, but with the inclusion of pSG-TR(AHTm) (Gal-TR(AHTm)). Cells were grown in the absence or presence of 0.1 µM T3. Data are the means ± S.E. of three individual experiments. Note the discontinuous vertical axis.
[View Larger Version of this Image (16K GIF file)]

T3 Enhances TR·RXR Heterodimer Formation in Vitro

To determine whether T3 could enhance dimerization between the TR and RXR LBDs, we utilized an in vitro protein-protein interaction assay in which GST-RXRalpha was incubated with the [35S]methionine-labeled Gal4-TR(wt) chimera in the absence or presence of T3. The results indicated a weak constitutive association between these two proteins in the absence of hormone, which did not change upon extended coincubation (Fig. 5, A and B). However, in the presence of 1 µM T3, the TR-RXR interaction was enhanced by up to 10-fold, reaching a maximum after ~40 min of coincubation (Fig. 5 (A and B) and data not shown).


Fig. 5. T3 enhances the interaction between TR and RXR in vitro. Bacterially expressed GST or GST-RXR fusion protein was incubated with 35S-labeled Gal4-TR for up to 60 min in the absence or presence of 2.5 µM T3. Gal-TR Input represents 10% of the total Gal4-TR used in each incubation. The Gal4-TR doublet is due to initiation of translation from an internal methionine. A, autoradiograph of a representative experiment; B, quantitation of radiolabeled Gal4-TR binding in A. open circle , -T3; bullet , +T3.
[View Larger Version of this Image (35K GIF file)]

9-cis-RA Attenuates the Recruitment of VP16-RXR by Gal4-TR

Finally, we examined the influence of 9-cis-RA, the ligand for RXR, on the interaction of Gal4-TR and VP16-RXR in the mammalian cell two-hybrid system. Either the Gal4 DBD or Gal4-TR(L454A) was coexpressed with VP16-tagged full-length RXR or the RXR LBD. The presence of 1 µM 9-cis-RA, in the absence of T3, attenuated the constitutive interaction of Gal4-TR(L454A) with VP16-RXR, particularly with the full-length VP16-RXR fusion (Fig. 6, bar 9 versus bar 11). The enhanced interaction of Gal4-TR(L454A) with VP16-RXR following T3 was also attenuated by 9-cis-RA (Fig. 6, bar 6 versus bars 8 and 10 versus bar 12).


Fig. 6. 9-cis-RA attenuates the TR-RXR interaction in vivo. JEG-3 cells were transfected as described in the legend to Fig. 2. Cells were then incubated in the absence or presence of 0.1 µM T3 or 0.1 µM 9-cis-RA as indicated. -Fold induction is relative to the activity of Gal4-TR(L454A) + VP16 in the absence of ligand. Data are the means ±S.E. of at least three independent experiments. Note the discontinuous vertical axis.
[View Larger Version of this Image (30K GIF file)]


DISCUSSION

Heterodimerization between TR and RXR is a central mechanism for T3-induced regulation of many target gene promoters. While a number of studies in vitro have suggested that the formation of TR·RXR complexes on DNA is not significantly influenced by ligand, the role of hormone in mediating solution interactions between these receptors has not been elucidated. Here we have shown a constitutive heterodimeric interaction between the LBDs of TR and RXR in the absence of ligand, confirming earlier observations (20, 32, 40), while in addition demonstrating a lack of homodimeric interaction for either LBD. This latter finding suggests that the two receptor LBDs possess complementary dimerization interfaces and, furthermore, that their DBDs and the response element to which they bind also play a major role in determining the nature of the dimeric complex since TR·TR and RXR·RXR homodimers do form readily on DNA (24, 41, 42). We have made the additional observation that the addition of T3 markedly increased reporter gene activity up to 50-fold in cells containing coexpressed Gal4-TR and VP16-RXR fusion proteins, suggesting that T3 enhances the recruitment of VP16-RXR to promoter-associated Gal4-TR. However, alternative explanations were also examined.

The possibility that the enhanced activity represented T3-mediated induction of the ligand-dependent transactivation function (AF-2) present in the TR LBD was discounted since this effect was also observed with the mutant thyroid hormone receptor fusion protein Gal4-TR(L454A), which lacks AF-2 activity, as well as in yeast, where the AF-2 function of the TR LBD is not manifest. The suggestion that the apparent enhanced recruitment of VP16-RXR was due to T3-mediated release of corepressor(s) was eliminated since Gal4-TR(AHTm), which contains mutations shown to abrogate the interaction with the putative transcriptional corepressor N-CoR (38), also showed T3-dependent recruitment of VP16-RXR. Furthermore, while previously reported T3-induced dissociation of TR homodimers may facilitate interaction of the Gal4-TR monomer with RXR (9, 11), the data in Fig. 1 show no evidence of TR LBD homodimer formation. We considered the possibility that the apparent enhanced recruitment of VP16-RXR was not due to augmentation of a direct interaction between TR and RXR LBDs, but the result of ligand-dependent recruitment of additional cofactors, capable of binding to either receptor partner and promoting their interaction (43). Using a two-hybrid assay system in mammalian cells, Kraus et al. (44) have shown a ligand-dependent interaction between the amino- and carboxyl-terminal regions of the estrogen receptor that is markedly attenuated by a mutation in the AF-2 region of the carboxyl-terminal domain. One mechanism proposed by the authors involved ligand-dependent recruitment of an accessory factor to the AF-2 region, thereby facilitating the interaction between the two receptor regions. However, our data do not favor such a mechanism for two reasons. First, we have recently shown that the L454A mutation in the AF-2 region of TR abrogates its ligand-dependent interaction with at least two putative intermediary proteins (SRC-1 and RIP140) (45), yet Gal4-TR(L454A) retained the ability to recruit VP16-RXR in response to ligand; second, we have also shown that T3 can enhance the association of Gal4-TR and GST-RXR by up to 10-fold (Fig. 5, A and B) in vitro, a context in which facilitation by auxiliary factors is unlikely. Furthermore, the in vitro data also rule out the possibility that the augmented reporter gene activity in vivo was mediated by a ligand-dependent alteration in Gal4-TR expression or stability leading to increased VP16-RXR recruitment.

There is precedent for our observations from previous studies with other members of the nuclear receptor family. Ligand-dependent homodimerization between fusion proteins containing the activation or DNA-binding domains of Gal4 linked to the full-length estrogen receptor has been demonstrated using a two-hybrid assay system in yeast (46). The solution interaction of the vitamin D3 receptor with GST-RXR, in the absence of DNA, has been examined using surface plasmon resonance (47). The addition of the ligand 1,25-dihydroxyvitamin D3 enhanced the affinity of the vitamin D3 receptor for RXR by 7-fold as a result of an increase in the association rate constant, and we hypothesize that the T3-mediated enhancement of the TR-RXR interaction might involve a similar mechanism. Another example in which ligand promotes dimerization is provided by the ecdysone receptor-ultraspiracle interaction, where muristerone has been shown to promote formation of ecdysone receptor-ultraspiracle complexes as well as formation of ecdysone receptor-ultraspiracle or ecdysone receptor-RXR heterodimers on DNA (48). Last, a genomic footprinting study has shown that occupancy of the retinoid response element in the RARbeta 2 gene occurs only after treatment of cells with retinoic acid (49). Although it was suggested that this might represent an enhanced affinity of the liganded RAR·RXR heterodimer for DNA, the effect may also be due to a ligand-dependent increase in solution heterodimer formation.

Studies using circular dichroism spectroscopic analysis have provided evidence for a ligand-induced conformational change in TR, while gel shift experiments have shown that the mobility of DNA-bound complexes is increased with T3 (9, 11, 50). Crystallographic studies of the LBD of the alpha  isoform of TR, which is highly homologous to TRbeta (51), also suggest an influence of ligand binding on dimerization. The ninth heptad repeat of TR is at the amino-terminal end of helix 11, on the surface of which are many residues implicated in dimerization. However, residues at the carboxyl-terminal end of helix 11 border the hormone-binding cavity and form critical contacts with ligand. This involvement of helix 11 in both functions is in keeping with the colocalization of ligand binding and dimerization to a similar region in the estrogen receptor (3). Evidence in favor of this relationship is provided by the observation that the effect of a ninth heptad mutation (L421R in human TRbeta , L365R in chick TRalpha , and M377R in human RARalpha ), which abolishes the interaction of a number of receptors with RXR can be reversed by the addition of cognate ligand, suggesting a role for hormone in stabilizing dimerization (10, 30). The Gal4-TR(AHTm) triple mutant used in this study also failed to interact with RXR in the absence of ligand, suggesting that these residues, which lie within helix 1, either constitute part of the dimerization interface or allosterically influence the integrity of dimerization regions. However, with T3, the interaction of Gal4-TR(AHTm) with VP16-RXR was fully restored, emphasizing the central role of ligand binding in stabilizing the integrity of the LBD and thereby promoting the formation of a dimerization interface.

Although RXR enhances DNA binding by TR and subsequent T3-induced receptor signaling, the role of 9-cis-RA in TR·RXR dimerization is unclear. The RXR moiety of DNA-bound receptor heterodimers typically binds to the 5'-half site of a TRE (23, 52, 53), but is unable to bind 9-cis-RA due to the negative allosteric influence of its dimer partner (54, 55). We found that 9-cis-RA inhibited both the constitutive and T3-induced interaction of Gal4-TR with VP16-RXR in the two-hybrid assay. 9-cis-RA-mediated inhibition of the constitutive TR-RXR interaction suggests that this effect is not due to disruption of T3 occupancy of TR by an inhibitory allosteric influence of liganded RXR. An alternative possibility is that 9-cis-RA induces dimerization of the VP16-RXR fusion to itself or endogenous RXR or orphan receptors, titrating it away from Gal4-TR. Indeed, studies in vitro have shown that 9-cis-RA enhanced homodimerization by RXR on DNA (56), while with coexpressed RXRalpha and TRalpha in CV-1 cells, the addition of 9-cis-RA or RXR-specific ligands augmented formation of RXR homodimers, thereby inhibiting T3-dependent activation via TR (57).

We have shown that the solution interaction between the ligand-binding domains of TR and RXR is markedly enhanced by T3. Although TR can clearly form unliganded complexes with RXR on cognate response elements to mediate silencing of basal transcription in many contexts (40), the ability to form TR·RXR heterodimers in solution may confer certain advantages. First, since RXR is a factor common to the signaling pathways of a number of other nuclear receptors, it might not always be expedient for RXR to be sequestered into DNA-bound unliganded receptor complexes, except when in a repressing role. Accordingly, ligand-dependent dimerization in solution would facilitate channeling of RXR to the desired signaling pathway and may also confer a negative regulatory influence on other receptor signaling pathways requiring this factor. We therefore propose that ligand-enhanced heterodimerization prior to DNA binding may constitute an additional early level of control in nuclear receptor-mediated gene expression.


FOOTNOTES

*   This work was supported by the Wellcome Trust, the Medical Research Council (United Kingdom), and the Imperial Cancer Research Fund.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.
§   Equal first authors.
par    Wellcome Senior Clinical Research Fellow. To whom correspondence should be addressed: Dept. of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2QQ, UK. Tel.: 11-44-1223-336842; Fax: 11-44-1223-336846; E-mail: kkc1{at}mole.bio.cam.ac.uk.
1   The abbreviations used are: TR, thyroid hormone receptor; TRE, thyroid hormone response element; T3, thyroid hormone; RAR, retinoic acid receptor; RXR, retinoid X receptor; hRXRalpha , human retinoid X receptor alpha ; DBD, DNA-binding domain; LBD, ligand-binding domain; GST, glutathione S-transferase; TRIAC, triiodothyroacetic acid; AD, activation domain; 9-cis-RA, 9-cis- retinoic acid.
2   The original codon nomenclature has been altered to correspond to the sequence containing 461 residues (39).

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