Analysis of the Functional Role of Steroid Receptor Coactivator-1 in Ligand-Induced Transactivation by Thyroid Hormone Receptor

M. Jeyakumar, Michael R. Tanen and Milan K. Bagchi

The Population Council and The Rockefeller University, New York, New York 10021


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear hormone receptors belonging to the steroid/thyroid/retinoid receptor superfamily are ligand-inducible transcription factors. These receptors modulate transcription of specific cellular genes, either positively or negatively, by interacting with specific hormone response elements located near the target promoters. Recent studies indicated that the hormone- occupied, DNA-bound receptor acts in concert with a cellular coregulatory factor, termed coactivator, and the basal transcription machinery to mediate gene activation. Consistent with this scenario, a number of nuclear proteins with potential coactivator function have been isolated. In the present study, we demonstrate that steroid receptor coactivator-1 (SRC-1), a recently isolated candidate coactivator, functions as a positive regulator of the thyroid hormone receptor (TR)-mediated transactivation pathway. In transient transfection experiments, coexpression of SRC-1 significantly enhanced ligand-dependent transactivation of a thyroid hormone response element (TRE)-linked promoter by human TRß. Our studies revealed that deletion of six amino acids (451–456) in the extreme COOH-terminal region of TRß resulted in a receptor that retained the ability to bind T3 but failed to be stimulated by SRC-1. These six amino acids are part of an amphipathic helix that is highly conserved among nuclear hormone receptors and contains the core domain of the ligand-dependent transactivation function, AF-2. In agreement with this observation, in vitro protein binding studies showed that SRC-1 interacted with a ligand binding domain peptide (145–456) of TRß in a T3-dependent manner, whereas it failed to interact with a mutant ligand binding domain lacking the amino acids (451–456). We demonstrated that a synthetic peptide containing the COOH-terminal amino acids (437–456) of TRß efficiently blocked the ligand-induced binding of SRC-1 to the receptor. These results suggest that the conserved amphipathic helix that constitutes the AF-2 core domain of TRß is critical for interaction with SRC-1 and thereby plays a central role in coactivator-mediated transactivation. We further observed that a heterodimer of TRß and retinoid X receptor-{alpha} (RXR{alpha}), either in solution or bound to a DR+4 TRE, recruited SRC-1 in a T3-dependent manner. The AF-2 of TR was clearly involved in this process because a TR-RXR heterodimer containing a mutant TRß (1–450) with impaired AF-2 failed to bind to SRC-1. Surprisingly, the RXR-specific ligand 9-cis-retinoic acid induced binding of SRC-1 to the RXR component of the TRE-bound heterodimer. This novel finding suggests that RXR, as a heterodimeric partner of TR, has the potential to play an active role in transcriptional regulation. Our results raise the interesting possibility that a RXR-specific ligand may modulate T3-mediated signaling by inducing additional interactions between TRE-bound TR-RXR heterodimer and the coactivator.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone receptors (TRs), {alpha} and ß, which mediate the physiological actions of thyroid hormones, belong to the nuclear hormone receptor superfamily of ligand-inducible transcription factors that also includes receptors for steroid hormones, retinoids, and vitamin D3 (1, 2, 3, 4). TR interacts with distinct DNA sequences, termed thyroid hormone response elements (TREs), in the target gene promoter and functions either as a transcriptional activator or a repressor depending on the hormonal status of the receptor (5). In the absence of hormone, TR functions as a silencer of basal level transcription from the target promoter (6, 7, 8, 9, 10, 11). Ligand binding to TR releases transcriptional silencing and leads to the activation of target gene expression (6, 7, 8, 9). The precise mechanisms by which TR exerts its transcriptional effects on a TRE-linked promoter are unclear.

Recent studies in several laboratories indicate that nuclear receptors repress or enhance transcription by interacting with multiple cellular coregulatory factors, which function as signaling intermediates between the receptors and the RNA polymerase II transcription machinery (for a review see 12 . We and others have demonstrated that unliganded TR associates with a negative coregulatory factor, termed corepressor (13, 14, 15, 16, 17). The receptor-corepressor complex binds to the TRE and actively represses target gene transcription by impairing the activity of the basal transcription machinery (13, 14, 15, 16, 17). The binding of T3 to TR results in the dissociation of the corepressor from the receptor, leading to the reversal of transcriptional repression (13, 14, 15, 16, 17). The hormone-occupied, DNA-bound receptor is then believed to act in unison with a positive coregulatory factor, termed coactivator, to mediate gene activation (9, 14). The coactivators are envisioned to act as bridging molecules between the activation domain(s) of the receptor and the basal transcription machinery.

Studies by Chambon and co-workers (18, 19) utilizing transcriptional interference or ‘squelching’ between different steroid receptors first suggested that cofactors in addition to the activated hormone receptor and the basal transcription apparatus are necessary for gene activation. We observed recently that ligand-occupied TR, TR{alpha} or TRß, inhibits transactivation of a progesterone-responsive reporter gene by progesterone receptors in CV1 or human breast carcinoma T47D cells (20). The transcriptional interference occurred in the absence of a DNA response element for the ‘interfering receptor’ (TR) in the target promoter. It was also totally dependent on the presence of the hormonal ligand T3. Conceptually, the target protein(s) of an inhibitory cross-talk between two different nuclear receptors is one or more basal transcription factors or coactivator(s). We demonstrated that T3-occupied TR ligand-binding domain (LBD) could bind to and functionally deplete a soluble cofactor(s) that is critical for transactivation of a progesterone-responsive gene in T47D nuclear extracts, while the basal level of trancription from a minimal TATA promoter or activated transcription from a control adenovirus major-late promoter remained unaffected (20). These results provided strong biochemical evidence in favor of the existence of a common, limiting coactivator molecule that mediates the interaction of ligand-bound steroid or TR with the RNA polymerase II transcription machinery to achieve the activated level of the target gene expression.

Consistent with this scenario, a number of putative mediator proteins have been isolated during the past 2 yr by a number of laboratories using yeast two-hybrid assay or far-Western cloning (12, 21, 22, 23, 24, 25, 26, 27, 28, 29). Screening of cDNA libraries employing baits containing the LBDs of nuclear receptors led to the isolation of multiple candidate coactivators such as steroid receptor coactivator-1 (SRC-1) (23), SUG-1 (24), transcriptional intermediary factor-1 (TIF-1) (25), receptor-interacting protein-140 (RIP-140) (26), TIF-2 (27), glucocorticoid receptor-interacting protein 1 (GRIP1) (28), p160 (21, 29), and CREB-binding protein (CBP) (29). It is now clear that SRC-1, p160, TIF-2, and GRIP1 are members of a family of structurally related coregulatory factors, which significantly enhance transactivation by several nuclear receptors in the presence of the cognate ligand (23, 27, 28, 29). It has also been reported that each of these proteins interacts directly with a nuclear receptor in a ligand-dependent manner (23, 24, 25, 26, 27, 28, 29). However, the molecular interactions between a coactivator such as SRC-1 and the transactivation domain(s) of a nuclear receptor have not been explored in detail.

In this study, we analyzed the functional interactions between SRC-1 and a TR-retinoid X receptor (RXR) heterodimer to investigate the mechanisms of action of the coactivator in TR-mediated transactivation. Using transient transfection experiments, we demonstrated that SRC-1 functions as a transcriptional coactivator for hormone-occupied TRß. A conserved COOH-terminal amphipathic {alpha}-helix in the LBD of TRß is essential for ligand-induced interaction with the coactivator. We provide strong evidence that this conserved {alpha}-helix is necessary for interactions with SRC-1. We also observed that both receptor partners within a DNA-bound TR-RXR heterodimer display the ability to recruit SRC-1. Interaction of the coactivator with each partner of the heterodimer depends on the cognate hormonal ligand. These results suggest a plausible mechanism by which a combination of TR- and RXR-specific hormonal signals in a target cell may influence the transcriptional activity of a TRE-linked promoter by simultaneously activating both partners within a TR-RXR heterodimer.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Conserved AF-2 Core Domain of TRß Is Essential for Coactivator Function of SRC-1
Previous studies in our laboratory indicated that a limited pool of cellular coactivator(s) is critical for efficient TR-mediated transactivation (20). An array of putative mediator molecules that interact with nuclear receptors in a ligand-dependent manner have been isolated recently by different laboratories (21, 22, 23, 24, 25, 26, 27, 28, 29). Onate et al. (23) reported that SRC-1, a candidate coactivator, enhanced hormone-induced transactivation by multiple nuclear receptors including TR. We examined the effects of coexpression of SRC-1 on ligand-dependent transcriptional activity of human TRß in CV-1 cells. As shown in Fig. 1Go, T3 induced a 4- to 5-fold TRß-mediated transactivation of a thyroid hormone response element (TRE)-linked reporter gene in the absence of any cotransfected SRC-1 (compare lanes 1 and 2). Cotransfection of an expression vector containing SRC-1 did not affect transcriptional activity of unliganded TR but led to a dramatic enhancement in transactivation by liganded TR (lane 4). The SRC-1-induced enhancement of transactivation was typically about 4-fold over that mediated by TR alone effecting a net 20-fold T3-dependent transactivation of the TRE-linked promoter (compare lanes 1 and 4). Consistent with the earlier report by Onate et al., SRC-1 therefore functions as a coactivator of TRß-mediated transactivation of a TRE-linked promoter in the presence of T3.



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Figure 1. The AF-2 Core Domain of TRß Is Critical for Coactivator Function of SRC-1

CV-1 cells were transiently transfected with pCI-TRß or pCI-TRß{Delta}C6 (2 µg) together with TRE tk-CAT (10 µg) as a T3-responsive reporter plasmid and pSV ß-gal (2 µg) as an internal control. The expression vector pCI-SRC-1 (2.5 µg) was added as indicated. The cells were treated with or without T3 (10 nM) as shown for 18 h before harvesting. CAT assays were performed as described in Materials and Methods. CAT activity was normalized to ß-galactosidase activity. The activity in the absence of hormone and SRC-1, but in the presence of pCI-TRß (lane 1), was taken as 1 and the relative values for test measurements (lanes 2–8) were obtained. Three independent sets of the same experiment were performed, and the results are shown as mean value ± the average deviation from the mean.

 
Conceptually, the coactivator molecule provides the functional link between the transactivation domain(s) of a ligand-bound nuclear receptor and the basal transcription machinery during gene activation. Previous studies localized a ligand-dependent transactivation function in the extreme C-terminal region (440–456) of TRß (9, 30, 31). This region, known as AF-2 core domain, forms an amphipathic {alpha}-helix that contains amino acid residues that are highly conserved among the nuclear hormone receptors and has been shown to be critical for the ligand-dependent transactivation by these receptors (9, 30, 31). Baniahmad et al. (9) previously showed that deletion of the last six carboxy-terminal amino acids (451–456) in TRß resulted in a mutant receptor, TRß{Delta}C6, which retained the ability to bind thyroid hormone but was impaired in AF-2 function.

To determine whether the AF-2 core domain of TR is involved in coactivator function, we employed transient transfection to examine the effects of SRC-1 on transcriptional activity of TRß{Delta}C6. As reported previously, we found that this TR mutant failed to transactivate a TRE-linked promoter in a T3-dependent manner (Fig. 1Go, lanes 5 and 6). Cotransfection of a vector expressing SRC-1, which markedly enhanced transactivation by full-length TRß, did not exhibit any effect on transcriptional activity of the AF-2 mutant either in the presence or in the absence of T3 (lanes 7 and 8). These results clearly implied that conserved amino acids 451–456 within the C-terminal AF-2 core domain of TRß play a critical role in the coactivator function of SRC-1.

AF-2 Core Domain of TRß Is Essential for Ligand-Dependent Interaction with SRC-1
Since SRC-1 functions as a transcriptional coactivator for TR, it is likely to undergo protein-protein interactions with the ligand-bound receptor. We therefore examined the interaction between SRC-1 and TRß in vitro. A recombinant TRß fused to glutathione-S-transferase (GST) was used in this experiment. As shown in Fig. 2AGo, very little binding of 35S-labeled SRC-1 to GST-TRß immobilized on an affinity matrix was detected in the absence of T3 (lane 2). In the presence of T3, however, we observed a remarkable enhancement in the binding of SRC-1 to GST-TRß (lane 3). These results demonstrated that TRß undergoes direct interaction with SRC-1 in a strictly ligand-dependent manner.



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Figure 2. The AF-2 Core Domain of TRß Is Essential for Ligand-Dependent Interaction with SRC-1

Panel A, 35S-labeled SRC-1 was produced by in vitro transcription-coupled translation and incubated with GST (lane 1, 1 µg) or purified GST-TRß (lanes 2 and 3, 1 µg) immobilized on GSH-resin either in the presence or absence of T3 (1 µM). After stringent washings, the resin-bound proteins were eluted with a gel-loading buffer, analyzed by SDS-PAGE, and visualized by fluorography. A 130-kDa polypeptide representing full-length SRC-1 is indicated by an arrow. Smaller 35S-labeled fragments represent truncated forms of SRC-1 generated during in vitro transcription-translation. Three independent sets of the same experiment were performed, and the results of a representative experiment are shown. Panel B (top), linear structures of TRß and its deletion mutants. Panel B (middle), Various deletion mutants of TRß were expressed as GST fusion proteins in E. coli BL21. The numbers indicate amino acid end points of the truncated receptor. Each GST-tagged mutant (1 µg) was immobilized on glutathione-resin and incubated with 35S-labeled SRC-1 produced by in vitro transcription-coupled translation either in the presence (1 µM) or absence of T3. The resin-bound proteins were washed extensively, eluted, and analyzed by SDS-PAGE followed by fluorography. Panel B (bottom), the absorbance values of the SRC-1 signals (indicated by arrow) were determined by densitometry. The plotted values (lanes 4–9) were adjusted by deducting the background value of SRC-1 retained nonspecifically on the GST resin alone (lane 2 or 3). The input lane represents 20% of the total volume of reticulocyte lysate added to each reaction. Three independent sets of the same experiment were performed, and the results of a representative experiment are shown.

 
We next investigated the role of the AF-2 core domain of TRß in ligand-dependent interaction with SRC-1. For this purpose, we analyzed the interaction of SRC-1 with a panel of TRß deletion mutants (Fig. 2BGo, top panel). We observed that the LBD fragment of TRß containing the amino acids (145–456) exhibited remarkable ligand-dependent binding to SRC-1 (middle and bottom panels, lanes 4 and 5). By our estimate, about 20-fold enhancement in SRC-1 binding to GST-TR (145–456) was observed in the presence of T3 (bottom panel, compare lane 4 with lane 5). In contrast, a fragment of TRß containing the N-terminal amino acids (1–260) showed only modest (about 2-fold) ligand-independent binding to SRC-1 (middle and bottom panels, compare lane 8 or 9 with lane 4). Interestingly, a receptor mutant GST-TR (82–450) that lacked the N terminus, as well as the last six COOH-terminal amino acids, but retained hormone binding, failed to exhibit any interaction with SRC-1 either in the presence or in the absence of T3 (lanes 6 and 7). Thus, the deletion of critical amino acids (451–456) within the AF-2 core domain of TRß, which results in the loss of ligand-induced transcriptional activity of this receptor, also disrupts the interaction between the receptor and SRC-1. Taken together, these results prompt us to propose that the conserved AF-2 core domain of TR critically influences the ligand-dependent interaction between SRC-1 and the receptor.

A 20-Amino Acid Synthetic Peptide Containing the AF-2 Core Domain of TRß Blocks the Interaction between the Receptor and SRC-1
Danielian et al. (30) and others (9, 31) have identified a 17-amino acid AF-2 core region containing an amphipathic {alpha}-helix whose main features are well conserved between all known nuclear receptors that transactivate in a hormone-dependent manner. Point mutations within the AF-2 core domain decrease or abolish ligand-dependent activation, even though the ligand binding, DNA binding, and dimerization properties remain unaffected (9, 31). Furthermore, this domain displays an autonomous activation function when fused to a heterologous DNA binding domain (9, 31). Recent crystal structure analyses of the nuclear receptors TR, retinoid acid receptor (RAR), and RXR suggest that induction of AF-2 activity upon ligand binding corresponds to a major conformational change involving the repositioning of the {alpha}-helix containing the AF-2 core domain (32, 33, 34). These observations raised the possibility that this domain is involved in creating the surface that interacts directly with a transcriptional coactivator such as SRC-1. We reasoned that if this postulation is correct, then a peptide containing the AF-2 core domain might be able to disrupt the binding of the full-length receptor to SRC-1 by occluding the coactivator-binding site.

To examine this possibility, we synthesized a peptide containing the last 20 C-terminal amino acids (437–456) containing the AF-2 core domain of TRß. A control peptide containing the same region but harboring mutations in several key amino acids, which are known to be critical for AF-2 activity (9, 31), was also generated (Fig. 3Go, top panel). We then incubated ligand-bound TRß with 35S-labeled SRC-1 in the presence or in the absence of excess AF-2 core or control peptide. As shown in Fig. 3Go (middle panel), incubation with an excess of the control peptide did not significantly affect the ligand-induced binding of SRC-1 to TRß (compare lane 2 with lane 5 or 6). Incubation with a similar excess of the AF-2 core peptide, on the other hand, drastically inhibited the binding of SRC-1 to hormone-occupied TRß (compare lane 2 with lanes 3 or 4). We observed that approximately 50% of [35S]SRC-1 binding to 2 pmol of TRß was suppressed in the presence of 10 nmol of AF-2 core peptide (Fig. 3Go, bottom panel). One can consider two different mechanisms by which the AF-2 core domain may inhibit coactivator binding. It is conceivable that the AF-2 core helix itself might represent a discrete binding site for the coactivator. In this scenario, the AF-2 core peptide competes with the TR LBD for the same binding site on the SRC-1. Alternatively, the AF-2 {alpha}-helix may align with another region(s) of the receptor to generate the proper binding surface for the coactivator. In this latter scenario, the peptide may inhibit SRC-1 binding by disrupting the intramolecular interaction that creates the coactivator binding site. Either way, interference of SRC-1 binding to the full-length receptor by the AF-2 core peptide strongly suggests that the C-terminal amphipathic {alpha}-helix of TRß is critically involved in creating the surface in the activated receptor that interacts with the coactivator.



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Figure 3. A Synthetic Peptide Containing the AF-2 Core Domain Efficiently Blocks SRC-1 Binding by TRß

Top panel, The amino acid sequences of the synthetic AF-2 core peptide and the control peptide are shown. The positions of the point mutations in the control peptide that abolish ligand-induced transactivation by TR are indicated by asterisks. Middle panel, Purified GST-TRß (2 pmol) was immobilized on GSH-resin and treated with T3 (1 µM). The immobilized ligand-occupied TRß was then incubated with 35S-SRC-1 (10 µl) in the absence (lane 2) or in the presence of increasing amounts of a synthetic AF-2 core peptide (lane 3, 50 nmol; lane 4, 100 nmol) or the control peptide (lane 5, 50 nmol; lane 6, 100 nmol). The resin-bound proteins were processed and analyzed by SDS-PAGE as described in Materials and Methods. Three independent sets of the same experiment were performed, and the results of a representative experiment are shown. Bottom panel, The AF-2 core peptide inhibits 35S-SRC-1 binding to TRß in a dose-dependent manner. The value for 35S-SRC-1 bound to GST-TRß in the presence of T3 and in the absence of any added peptide is taken as 100%. Open and closed circles indicate the amounts of 35S-SRC-1 that remained bound to TRß in the presence of the indicated amounts of the control and the AF-2 core peptides, respectively.

 
Both Receptor Partners in a TR-RXR Heterodimer Can Interact with SRC-1 in a Ligand-Dependent Manner
TR forms an obligate heterodimer with RXR, which exists ubiquitously in all tissues (3, 35). These heterodimers are readily generated either in solution or on appropriate DNA response elements. Heterodimerization with RXR markedly enhances binding of TR to its response element (3, 35). It is thus likely that TR-RXR heterodimers may represent the functional form of TR in vivo. We therefore investigated how each receptor partner within a TR-RXR heterodimer interacts with SRC-1 in response to its cognate hormone. In this experiment, described in Fig. 4Go, we used bacterially expressed GST-tagged RXR and hexahistidine-tagged TRß. The GST-RXR fusion protein was first immobilized on glutathione affinity resin. The TR-RXR heterodimers were generated in the absence of DNA by incubating the immobilized RXR with excess TRß. The formation of heterodimers in solution was ascertained by isolating the immobilized complexes, followed by analysis of the protein components by SDS-PAGE and by protein staining. Equivalent intensities of TR- and RXR-specific bands representing equivalent molar amounts of the receptors were visualized in the isolated complexes confirming efficient heterodimer formation under these conditions (Fig. 4AGo). The heterodimerization was not significantly affected by the presence of either a combination of T3 (1 µM) and 9-cis-retinoic acid (RA) (1 µM) (compare lanes 2 and 3 with lanes 6 and 7) or excess direct repeat (DR)+4 TRE (compare lane 2 with lane 3, lane 6 with lane 7). The resin complexed to TR-RXR heterodimers was isolated by brief centrifugation and washed repeatedly. The resin-bound receptors were then treated with either T3 or 9-cis-RA or solvent follwed by incubation with 35S-labeled SRC-1.



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Figure 4. Both Partners of a TR-RXR Heterodimer Display Ligand-Dependent Binding to SRC-1

Panel A, Purified GST-RXR (10 pmol) was immobilized on GSH-resin and incubated with (lanes 2, 3, 6, and 7) or without (lanes 1 and 5) excess purified His6-TRß (40 pmol). In lanes 4 and 8, GSH-resin was incubated with His6-TRß (40 pmol) alone. DR+4 TRE (1 µg) and ligands T3 (1 µM) and 9-cis-RA (1 µM) were added during incubation as indicated. Binding was allowed to proceed for 1 h at 4 C in a buffer containing 20 mM Tris-HCl (pH 7.4), 60 mM NaCl, 1 mM dithiothreitol, and 15% glycerol. The unbound proteins were removed by washing the resin repeatedly with the same buffer. The bound proteins retained on the resin were then eluted by boiling with a gel-loading buffer containing SDS, analyzed by SDS-PAGE, and visualized by Coomassie blue staining. Note that the GST portion of GST-RXR{alpha} contributes to the slightly better Coomassie blue staining exhibited by this fusion protein compared with His6-TRß. Panel B (upper), GST-RXR (10 pmol) was immobilized on GSH-resin and incubated with excess His6-TRß (lanes 3, 5, and 7, 20 pmol; lanes 4, 6, 8, and 9, 40 pmol) to form heterodimers. The unbound proteins were washed off the resin. The immobilized heterodimers were then treated with T3 or 9-cis-RA (1 µM) as indicated. The resin-bound hormone-receptor complexes were incubated with 10 µl retic lysate containing 35S-SRC-1. The resin-bound proteins were finally processed and analyzed by SDS-PAGE as described in Materials and Methods. Panel B (lower), the absorbance values of the SRC-1 signals (indicated by arrow) were determined by densitometry. The plotted values (lanes 1–9) were adjusted by deducting the background value of SRC-1 retained nonspecifically on the GST resin alone (lane GST). Three independent sets of the same experiment were performed, and the results of a representative experiment are shown.

 
As shown in Fig. 4BGo, resin-bound GST-RXR or GST-RXR-TR did not retain any significant SRC-1 signal in the absence of any hormone (lanes 1, 5, and 6). Addition of the RXR-specific ligand, 9-cis-RA, led to a marked enhancement in the binding of SRC-1 to GST-RXR alone (lower panel, compare lanes 1 and 2). Interestingly, heterodimerization with TR did not affect 9-cis-RA-dependent binding of SRC-1 to RXR (compare lane 2 with lanes 3 or 4). Our results also clearly showed that RXR in a preformed heterodimer could bind to its ligand and recruit SRC-1 in a ligand-dependent manner (lanes 3 or 4). As expected, addition of T3 to the heterodimer also markedly induced interaction of SRC-1 with the TR component (lanes 7 and 8). Taken together, these results demonstrated that in a TR-RXR heterodimer both receptor partners are capable of interacting with a transcriptional coactivator in a strictly ligand-dependent fashion.

Ligand-Dependent Recruitment of SRC-1 by a DNA-Bound TR-RXR Heterodimer
It is conceivable that a DNA response element may modulate interaction of a TR-RXR heterodimer with SRC-1. A recent report indicated that the polarity or relative configuration of half-sites within a retinoid response element dictates the interaction of a RAR-RXR heterodimer with a nuclear receptor corepressor (14). A TR-RXR heterodimer binds to a DNA response element consisting of two direct repeat half-sites (DR+4) of consensus sequence AGGTCA in an asymmetric manner: RXR occupies the upstream half-site and TR occupies the downstream half-site (36, 37). We therefore examined whether the binding of a TRß-RXR{alpha} heterodimer to the DR+4 TRE influences its interaction with SRC-1. For this purpose, we first assembled TR-RXR heterodimers on biotinylated DR+4 TRE oligodeoxynucleotides. The DNA-bound TR-RXR complexes were then isolated by binding to an avidin-linked resin, and the resin-bound heterodimers were incubated with 35S-labeled SRC-1 either in the presence or in the absence of T3 or 9-cis-RA. After stringent washings, the resin-bound proteins were analyzed by SDS-PAGE for the presence of 35S-labeled SRC-1. The results of these experiments are shown in Fig. 5Go, A and B.



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Figure 5. Ligand-Dependent Interactions between SRC-1 and a TR-RXR Heterodimer Bound to a DR+4 Element

Panel A (upper), 1 µg of double-stranded, biotinylated DR+4 oligonucleotide was immobilized on avidin-linked agarose. The resin-bound DNA was initially incubated without (lane 3) or with His6-TRß (lanes 4–9, 20 pmol) and His6-RXR{alpha} (lanes 4–9, 80 pmol). The resin-DNA-protein complexes were then treated with T3 or 9-cis-RA (1 µM) as indicated. The resulting resin-bound complexes were reacted with 10 µl retic lysate containing 35S-SRC-1. The resin-bound proteins were processed and analyzed by SDS-PAGE as described in the Materials and Methods. Panel A (lower), the absorbance values of the SRC-1 signals (indicated by arrow) were determined by densitometry. The plotted values (lanes 3–9) were adjusted by deducting the background value of SRC-1 retained nonspecifically on the resin alone (lane 2). The input lane represents 20% of the total volume of reticulocyte lysate containing labeled SRC-1 that is added to each reaction. Panel B (upper), 1 µg of double-stranded, biotinylated DR+4 oligonucleotide was immobilized on avidin-linked agarose. The resin-bound DNA was initially incubated with His6-TRß (20 pmol) alone (lanes 1–3) or His6-RXR{alpha} (80 pmol) alone (lanes 4–6) or with a combination of both (lanes 7–11). The resin-DNA-protein complexes were then treated with T3 or 9-cis-RA (1 µM) as indicated. In certain control experiments (lanes 1, 2, 4, 5, 7, 8, and 10), the indicated proteins were incubated with avidin-linked agarose containing no bound DNA. The resulting resin-bound complexes were reacted with 10 µl of retic lysate containing 35S-SRC-1. The resin-bound proteins were processed and analyzed by SDS-PAGE as described in Materials and Methods. Panel B (lower), the absorbance values of the SRC-1 signals (indicated by arrow) were determined by densitometry. Panel C (upper), immobilized biotinylated DR+4 oligonucleotides were initially incubated with His6-RXR{alpha} (lanes 1–4, 80 pmol) together with either His6-TRß (lanes 1 and 2, 20 pmol) or His6-TRß{Delta}C6 (lanes 3 and 4, 20 pmol). This was followed by treatment with T3 (1 µM) or 9-cis RA (1 µM) as indicated. Panel C (lower), the absorbance values of the SRC-1 signals (indicated by arrow) were determined by densitometry. The plotted values (lanes 1–6) were adjusted by deducting the background value of SRC-1 retained nonspecifically on the resin alone (data not shown). The input lane represents 20% of the total volume of reticulocyte lysate containing labeled SRC-1 that is added to each reaction.

 
We noted that low levels of SRC-1 were bound nonspecifically to the avidin-linked resin in the absence of DNA (Fig. 5AGo, lane 2). This nonspecific retention of SRC-1 was not significantly affected when the resin was incubated with biotinylated TRE alone (lane 3) or with TR and RXR in the absence of TRE (lanes 8 and 9). The results in Fig. 4AGo suggest that a heterodimer is generated on the DR+4 TRE when one receptor partner is incubated with DNA in the presence of an excess of the other partner. The binding of SRC-1 increased only slightly when resin-bound TRE-TR-RXR complexes were used in the absence of any hormonal ligand (lane 4). A pronounced enhancement in the binding of SRC-1 was observed only when resin-bound TRE-TR-RXR complexes were incubated with the 35S-labeled protein in the presence of either T3 or 9-cis-RA (upper and lower panels, compare lane 4 with lane 5 or 6). These results were in excellent agreement with those described in Fig. 4BGo using TR-RXR heterodimers formed in solution. Coaddition of both ligands did not produce any significant change in SRC-1 binding compared with each ligand added singly (compare lane 5 or 6 with lane 7). The limiting amount of SRC-1 in the binding reaction did not allow us to determine whether the two ligand-occupied receptors in a heterodimer bound to the coactivator in an additive or a synergistic fashion.

We also examined the binding of 35S-labeled-SRC-1 to the DR+4-bound resin when TR or RXR was used singly (Fig. 5BGo). Previous reports indicated that TR alone could bind to DR+4 either as a monomer or homodimer (5, 38, 39). It has been reported that in the presence of T3, TR homodimers are destabilized whereas the binding of TR monomers to DR+4 TRE is favored (38, 39). Our results indicated that TR alone could bind to the TRE under the conditions of our experiment and recruit SRC-1 in a T3-dependent manner (compare lanes 1 and 2). Upon addition of excess RXR to TR, heterodimers formed on DR+4 TRE, and these complexes also bound to SRC-1 in a T3-dependent manner (lanes 7 and 8). Although the binding of TR monomer and homodimer is weaker than the heterodimer, the amount of SRC-1 recruited by TR-RXR was found to be only slightly greater than that recruited by TR alone (compare lanes 2 and 8). This observation was not surprising, however, because the molar concentration of DNA-bound receptor complexes in the binding reaction far exceeded that of SRC-1.

We also observed that RXR by itself failed to bind to DR+4 TRE and, consequently, no significant recruitment of SRC-1 was detected on this DNA element when RXR alone was used (lanes 4 and 5). Interestingly, a marked 9-cis-RA-induced SRC-1 binding to the DNA-bound complex was observed only when a combination of RXR and TR was incubated with DR+4 (lanes 9 and 10). The simplest explanation for this observation is that a TR-RXR heterodimer is the predominant complex on the DR+4 TRE when both receptors are incubated together with this DNA. It is also evident from these studies that each receptor partner within the heterodimer positioned at the TRE binds to its cognate ligand and is able to recruit the coactivator in an entirely ligand-dependent manner.

We next investigated whether T3-induced recruitment of SRC-1 by a TR-RXR heterodimer was dependent on an intact AF-2 of TR. For this purpose, we used the mutant TRß{Delta}C6 with impaired AF-2 activity. As shown in Fig. 5BGo, whereas a DNA-bound heterodimer containing a full-length TR displayed strong T3-dependent binding to SRC-1 (compare lanes 1 and 2), a heterodimer of TRß{Delta}C6 and RXR failed to exhibit any interaction with the coactivator either in the presence or in the absence of T3 (lanes 3 and 4). These results are in agreement with our finding that the ligand-dependent AF-2 of TR plays an essential role in coactivator function. Interestingly, addition of 9-cis-RA elicited binding of SRC-1 to the TRß{Delta}C6-RXR heterodimer (lane 5). The 9-cis-RA-induced binding of SRC-1 to RXR is presumably mediated via the AF-2 of this receptor. Although the deletion of TRß AF-2 did not appear to significantly influence the 9-cis-RA-induced binding of RXR to the coactivator, we cannot rule out allosteric control of the activity of one heterodimeric partner by the other as recently proposed by Schulman et al. (40), especially because the coactivator is limiting in our assays. We nonetheless conclude that, in the presence of the cognate ligand, each receptor component of TR-RXR heterodimer requires the AF-2 core domain for ligand-dependent interactions with SRC-1. This scenario is entirely consistent with the proposed role of SRC-1 as a coactivator in nuclear receptor-mediated transactivation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present study analyzes the molecular interactions underlying the functional role of a newly identified nuclear receptor coactivator, SRC-1, in the TR-mediated transactivation pathway. A coactivator is thought to facilitate hormone-dependent gene activation by serving as a physical link between the transactivation domain of a ligand-occupied receptor and the basal transcription machinery. In agreement with this view, coexpression of SRC-1 in transient transfection experiments markedly enhanced TR- and T3-mediated transactivation of a target promoter. We found that the regulatory effect of SRC-1 is critically dependent on the ligand-induced transactivation function AF-2 of TR. We also observed that SRC-1 is able to interact with both TR and its heterodimeric partner, RXR, in a ligand-specific manner. SRC-1 therefore displays many hallmarks of a genuine coactivator for TR-mediated transactivation.

Our studies reveal that an AF-2 core domain that is conserved among nuclear receptors plays a central role in SRC-1-mediated transactivation. In this study, we demonstrate that a 20-amino acid peptide containing the AF-2 core domain efficiently inhibits the binding of TR LBD to SRC-1. More importantly, mutations of key acidic (Glu 452, Glu 455, Asp 456) and hydrophobic (Phe 454) amino acids, which impair the ligand-induced transactivation by TR (9, 31), also abolish the ability of the peptide to interfere with SRC-1 binding to TR. These results provide compelling evidence that the conserved AF-2 core helix of the receptor is essential for interactions with a nuclear receptor coactivator. Recent crystal structural analysis of the LBDs of TR, RXR, and RAR suggests that the AF-2 core domain, which forms an amphipathic {alpha}-helix, also known as helix 12, undergoes a striking conformational transition upon hormone binding (32, 33, 34). In the unoccupied receptor, the helix 12 projects into the solvent (32). In the hormone-occupied receptor, the helix folds back toward the receptor to form a part of the ligand-binding cavity (33, 34). The helix packs loosely with the hydrophobic residues facing inward toward the ligand-binding pocket and the charged residues extending into the solvent (33, 34). It is conceivable that in the hormone-bound receptor, the helix presents itself as a binding site for the coactivator. As the hydrophobic residues interact with the ligand-binding core to stabilize the domain, certain charged residues might be available for direct interaction with the coactivator. Alternatively, the ligand-induced repositioning of helix 12 may trigger a reorganization in other parts of the receptor, creating an interaction surface for the coactivator. In this scenario, the AF-2 core domain may play a critical role by participating in intramolecular interactions with another region(s) of the LBD to help create the proper coactivator-binding site.

Collingwood et al. (41) have recently reported that a naturally occurring TRß mutant, in which leucine at position 454 in the AF-2 core domain is changed to valine, still retains its ability to respond to SRC-1 in transient transfection experiments. The apparent discrepancy between our results and those of Collingwood et al. could arise from the use of different TRß mutants in these studies. In the case of Collingwood et al. (41), the point mutant L454V retained moderate interactions with SRC-1. Consequently, in transient transfection assays, overexpressed SRC-1 could still potentiate transactivation by this mutant TR, presumably by binding to the altered AF-2 core domain. In contrast, we employed a truncation mutant, TRß{Delta}C6, which lacked several conserved amino acids within the AF-2 helix. This deletion rendered TRß{Delta}C6 completely ineffective in SRC-1 binding and as a result, this mutant receptor failed to respond to the coactivator (Fig. 1Go).

Although the failure of the AF-2 mutant, TRß{Delta}C6, to respond to SRC-1-mediated activation in our transfection experiments may appear to be simply due to a disruption in coactivator binding, more complex scenarios can be considered. Recent studies suggest that AF-2 core domain may influence the interaction of the receptor with corepressor molecules (40). Unliganded TR or RAR associates with a corepressor, which is released from the receptor upon hormone binding (13, 15, 16). Interestingly, addition of hormone did not affect the transcriptional activity of the AF-2 mutant (Fig. 1Go, lane 6). This is presumably due to a failure to release the corepressor. Previous studies have shown that TRß{Delta}C6 binds ligand and undergoes certain conformational changes in response to ligand binding (42) but remains a constitutive transcriptional repressor (9). Therefore, hormone binding is not sufficient to dissociate the corepressor from the AF-2 mutant. Schulman et al. (40) have recently proposed that the AF-2 core domain itself participates in the transition from repressive to active state by an as yet unknown mechanism. One can speculate that the AF-2 helix helps to maintain a receptor conformation that allows release of corepressor upon hormone binding. The loss of this structure in the TRß{Delta}C6 mutant prevents corepressor release. Based on the crystal structure of TR, however, deletion of six amino acids from the AF-2 core helix is not expected to disrupt the compact bound state of the receptor or its hormone-binding cavity, although subtle alterations in the receptor conformation cannot be ruled out (34). Alternatively, one can imagine that the binding of the coactivator to the AF-2 helix of a ligand-bound receptor serves as the switch that triggers a conformation change, which in turn facilitates corepressor release. Recent observations by Glass and co-workers (14) however, imply that interactions with the corepressor are dominant over the recruitment of coactivators. These studies also suggest that the DNA response elements can allosterically control the receptor-corepressor association. Further analyses of the complex interplay between the DNA-bound nuclear receptor and the corepressor or coactivator are therefore needed to understand how these coregulatory molecules modulate the gene-regulatory activity of the receptor.

A surprising finding of the present studies is the observation that the ligand 9-cis-RA induced binding of SRC-1 to RXR in a TR-RXR heterodimer. Previous studies reported that RXR, once heterodimerized, is incapable of binding its ligand (43, 44). It was proposed that RXR in the heterodimer enhances DNA binding by the partner receptor but is a silent partner in transactivation (3, 43, 44). We, however, observed that heterodimerization of RXR with TR did not prevent its binding to 9-cis-RA or its ligand-dependent interaction with SRC-1. RXR, therefore, not only binds to its ligand in the context of the heterodimer, but also maintains its ability to interact with a transcriptional coactivator. Our results, therefore, suggest a potentially active role of RXR in TR-mediated transcription. The reason for the apparent discrepancy between our results and those published previously is not clear. Our results are in good agreement, however, with the work reported previously by Kersten et al. (45) and more recently by Minucci et al. (46). Whereas Kersten et al. (45) using a fluorescence-based method demonstrated that RXR in a RAR-RXR heterodimer can bind to its ligand (45), Minucci et al. employed a differential proteolytic analysis to conclude that RXR in such a heterodimer not only binds its ligand but also functions as a transcriptionally active partner by enhancing retinoid-dependent gene expression (46). However, the role of RXR in transactivation by the TR-RXR heterodimer is yet to be settled unequivocally. In light of the conflicting results from different laboratories, it appears that RXR within a heterodimer may play either a silent or an active role depending on cell, promoter, and ligand contexts.

An interesting issue raised by the present study is the differential transactivation activities displayed by TR and RXR within the heterodimer in response to the cognate ligand. Although both partner receptors can individually bind to the coactivator in a ligand-dependent manner, T3 but not an RXR-specific ligand, when added alone, activated transcription from a TRE-linked promoter in transfection assays (43, 46). Minucci et al. (46) reported that although a RXR-specific ligand, when added singly, failed to activate a retinoid-responsive promoter, it synergistically stimulated transcription when added together with a RAR ligand. Taken together, these studies indicated that a full display of the transcriptional activity of RXR in a TR-RXR or RAR-RXR heterodimer depends on the ligand of its heterodimeric partner. It appears that the transactivation potential of RXR in the heterodimer is somehow masked in the absence of either T3 or the RAR ligand. It is possible that a corepressor, which is complexed with the unliganded TR or RAR in the heterodimer, blocks access of the coactivator to RXR. In this scenario, addition of either T3 or the RAR ligand, which triggers the release of the corepressor from the cognate heterodimeric partner, is obligatory for RXR-dependent transactivation.

In summary, the results presented here demonstrate that a conserved AF-2 core domain of TR plays an essential role in interactions with a transcriptional coactivator, SRC-1, to regulate the activity of a TRE-linked promoter. Our studies further reveal that RXR, the heterodimeric partner of TR, also possesses the potential to modulate T3-mediated signaling by interacting with the coactivator in a 9-cis-RA-dependent manner. These results provide interesting insights into the complex molecular interactions among multiple nuclear receptors and transcriptional coregulatory factors that determine the response of hormone-regulated promoters to diverse ligand signals.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
In vitro transcription and nuclease-treated rabbit reticulocyte lysate translation kits were purchased from Promega Corp. (Madison, WI.) L-[35S]methionine (>1000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL).

Plasmid Construction
The SRC-1 cDNA used in this study was originally isolated by Onate et al. (23). The 3432-nucleotide cDNA extends from positions -62 to + 3361 and contains an open reading frame of 1061 amino acids. The plasmids pCI-TRß and pCI-SRC-1 expressing human TRß and human SRC-1, respectively, were constructed by inserting cDNAs encoding these proteins into vector pCI under the control of human cytomegalovirus immediate-early promoter (Promega). pCI-TRß{Delta}C6 was constructed by incorporating the previously described mutant TRß{Delta}C6 (9) in the pCI vector. The construction of the reporter plasmid TRE-tkCAT has been described previously (20).

Recombinant Nuclear Receptors and Their Mutants
Recombinant nuclear receptors GST-TRß and GST-RXR{alpha} containing GST fused to the amino-terminal sequences of TRß and RXR{alpha}, respectively, were expressed in Escherichia coli BL21. Various deletion mutants of TRß were expressed as GST fusion proteins: GST-TR (145–456), GST-TR (80–450), and GST-TR (1–260). The receptor fragments were generated by PCR and engineered into the bacterial expression vector pGX-2T (Pharmacia, Piscataway, NJ). The construction, expression, and purification of His6-TRß and His6-RXR{alpha} were described previously (11). The protein concentrations of mutant receptors were determined by comparing the intensity of Coomassie-stained band of each receptor fusion protein with that of a known BSA standard in SDS-PAGE.

Transient Transfection Experiments
CV-1 cells were maintained in DMEM (GIBCO BRL, Grand Island, NY) supplemented with 5% FBS (Hyclone Laboratories, Logan, UT). Semiconfluent cells were transiently transfected using the calcium phosphate coprecipitation procedure as described previously (20). Briefly, 5 x 105 cells were plated on 10-cm tissue culture dishes in phenol red-free DMEM medium containing 5% charcoal-stripped serum and after 24–48 h were transfected with plasmid DNAs. Typically, cells received 10 µg chloramphenicol acetyltransferase (CAT) reporter plasmid, 2 µg pCI-TRß or pCI-TRß{Delta}C6, 2.5 µg pCI-SRC-1 or empty pCI vector, and 2 µg of an internal control plasmid pSV-ßgal (Promega), which contains the gene for ß-galactosidase enzyme. After 12–14 h of exposure to the calcium phosphate precipitate, the cells were washed with PBS and incubated in fresh phenol red-free, serum-free medium with 10-8 M T3 or solvent. Cells were harvested after 18 h for determination of ß-galactosidase and CAT activities as described previously (20). The amount of cell extract used per CAT assay was determined after normalization with respect to the ß-galactosidase activity. Quantification of the CAT activities was performed by liquid scintillation analysis of the acetylated [14C]-chloramphenicol product and the remaining unacetylated substrate. Each transient transfection experiment was repeated at least three times.

GST Pull-Down Assay
Each GST fusion protein (0.1–1.0 µg) was immobilized on a glutathione (GSH)-resin (15 µl), and the immobilized protein was treated with or without T3 (1 µM) or 9-cis-RA (1 µM) at room temperature for 15 min. 35S-labeled SRC-1 was generated from an expression vector pBK-CMV-SRC-1 by in vitro transcription-coupled translation in reticulocyte lysates following manufacturer’s instructions. An aliquot (10 µl) of the translation mix containing labeled SRC-1 was then incubated with immobilized GST fusion protein in a binding buffer containing 20 mM Tris-HCl (pH 7.4), 60 mM NaCl, 1 mM dithiothreitol, 15% glycerol, and 0.1% NP-40. Binding was allowed to proceed for 1 h at 4 C. The resin was then washed repeatedly with the binding buffer. The 35S-labeled SRC-1 that was retained on the resin was eluted by boiling with a gel-loading buffer containing SDS, analyzed by SDS-PAGE, and visualized by fluorography.

In peptide competition experiments, GST-TRß (0.1 µg) was first immobilized on a GSH-resin followed by treatment with T3 (1 µM). The resin-bound hormone-receptor complex was then incubated with 35S-labeled SRC-1 (10 µl translation mix) in the presence of increasing molar excess of either AF-2 core or control peptide. The 35S-labeled SRC-1 that was retained on the resin after stringent washings was then eluted by boiling with a gel-loading buffer containing SDS and analyzed by SDS-PAGE. The AF-2 core and control peptides were synthesized by the Rockefeller University Biopolymer Facility (New York, NY).

Biotinylated DNA Pull-Down Assay
For this assay, the protocol of Kurokawa et al. (14) was used with certain modifications. The sequence of the sense strand of the DR+4 TRE was: 5'-GAAGGGGATCCGGGTAAGGTCACAGGAGGTCACGAA-3'. The sense oligodeoxynucleotide was biotinylated at the 5'-end by using a biotin-phosphoramidite during synthesis. Equal amounts of sense and antisense oligonucleotides were annealed to form the double-stranded DR+4 element. One microgram (~35 pmol) of the double-stranded oligonucleotide was coupled to 12 µl avidin-linked agarose for 1 h at room temperature. The immobilized biotinylated TRE was then incubated with purified His6-TRß (20 pmol) or His6-RXR{alpha} (80 pmol) or a combination of both for 30 min at room temperature. The incubation was performed in a binding buffer containing 20 mM Tris-HCl (pH 7.4), 60 mM NaCl, 1 mM dithiothreitol, 15% glycerol, and 0.1% NP-40. The resin was collected by brief centrifugation and washed extensively with the binding buffer. The resin-bound DNA-receptor complexes were then treated with either T3 (1 µM) or 9-cis-RA (1 µM) or solvent for 20 min at room temperature. Ten microliters of translation mix containing 35S-labeled SRC-1 were then added to the receptor-DNA complexes, and incubation was carried out for 30 min at room temperature. The resin was again collected and washed repeatedly with the binding buffer. The bound proteins were eluted with a gel-loading buffer containing SDS and analyzed by SDS-PAGE. 35S-labeled SRC-1 was visualized by fluorography. The intensity of the full-length SRC-1 polypeptide was quantitated by densitometry. The optical densities were corrected by subtracting from the test values the nonspecific binding of SRC-1 to the resin in the absence of DNA and the receptors.


    ACKNOWLEDGMENTS
 
We are grateful to Bert W. O’Malley and Ming-Jer Tsai for the generous gift of human SRC-1 cDNA.


    FOOTNOTES
 
Address requests for reprints to: Milan K. Bagchi, The Population Council and The Rockefeller University, 1230 York Avenue, New York, New York, 10021.

This work was supported by NIH Grant R01DK-50257–02 (to M.K.B).

Received for publication January 31, 1997. Accepted for publication February 28, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tsai M-J, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  3. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers, orphan receptors. Cell 83:841–850[Medline]
  4. Beato M, Herrlich P, Schutz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:851–857[Medline]
  5. Lazar MA 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Medline]
  6. Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erbA functions as a thyroid hormone receptor antagonist. Nature 339:593–597[CrossRef][Medline]
  7. Baniahmad A, Kohne AC, Renkawitz R 1992 A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA oncogene product and in the retinoic acid receptor. EMBO J 11:1015–1023[Abstract]
  8. Casanova J, Helmer E, Selmi-Ruby S, Qi J-S, Au-Fliegner M, Desai-Yajnik V, Koudinova N, Yarm F, Raaka BM, Samuels HH 1994 Functional evidence for ligand-dependent dissociation of thyroid hormone, retinoic acid receptors from an inhibitory cellular factor. Mol Cell Biol 14:5756–5765[Abstract]
  9. Baniahmad A, Leng X, Burris TP, Tsai SY, Tsai M-J, O’Malley BW 1995 The t4 activation domain of the thyroid hormone receptor is required for release of a putative corepressor(s) necessary for transcriptional silencing. Mol Cell Biol 15:76–86[Abstract]
  10. Fondell JD, Roy AL, Roeder RG 1993 Unliganded thyroid hormone receptor inhibits formation of a functional initiation complex: implications for active repression. Genes Dev 7:1400–1410[Abstract]
  11. Tong G-X, Tanen MR, Bagchi MK 1995 Ligand modulates the interaction of thyroid hormone receptor b with the basal transcription machinery. J Biol Chem 270:10601–10611[Abstract/Free Full Text]
  12. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators, corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  13. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–403[CrossRef][Medline]
  14. Kurokawa R, Soderstrom M, Horlein AJ, Halachmi S, Brown M, Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic acid receptors determined by a co-repressor. Nature 377:451–454[CrossRef][Medline]
  15. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  16. Tong G-X, Jeyakumar M, Tanen MR, Bagchi MK 1996 Transcriptional silencing by unliganded thyroid hormone receptor b requires a soluble corepressor that interacts with the ligand-binding domain of the receptor. Mol Cell Biol 16:1909–1920[Abstract]
  17. Sande S, Privalsky ML 1996 Identification of TRACs (T3 receptor-associating cofactors), a family of cofactors that associate with, modulate the activity of, nuclear hormone receptors. Mol Endocrinol 10:813–825[Abstract]
  18. Meyer ME, Gronemeyer H, Turcotte B, Bocquel MT, Tasset D, Chambon P 1989 Steroid hormone receptors compete for factors that mediate their enhancer function. Cell 57:433–442[Medline]
  19. Meyer ME, Qurin-Stricker C, Chambon P, Gronemeyer H 1992 A limiting factor mediates the differential activation of promoters by the human progesterone receptor isoforms. J Biol Chem 267:10882–10887[Abstract/Free Full Text]
  20. Zhang X, Jeyakumar M, Bagchi MK 1996 Ligand-dependent cross-talk between steroid, thyroid hormone receptors. J Biol Chem 271:14825–14833[Abstract/Free Full Text]
  21. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins — possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  22. Lee JW, Choi H-S, Gyuris J, Brent R, Moore DD 1995 Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Mol Endocrinol 9:243–254[Abstract]
  23. Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  24. Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid hormone receptor with a conserved transcriptional mediator. Nature 374:91–94[CrossRef][Medline]
  25. Le Douarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat B, Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T 18. EMBO J 14:2020–2033[Abstract]
  26. Cavailles V, Dauvois S, Horset FL, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP 140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  27. Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Abstract]
  28. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MRG 1996 RIP1, A novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  29. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MGA 1996 CBP integrator complex mediates transcriptional activation, AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  30. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone-dependent transcriptional activation by steroid hormone receptors. EMBO J 11:1025–1033[Abstract]
  31. Barettino D, Vivanco Ruiz MM, Stunnenberg HG 1994 Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor. EMBO J 13:3039–3049[Abstract]
  32. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha} Nature 375:377–382[CrossRef][Medline]
  33. Renaud J-P, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  34. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  35. Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM 1992 Retinoid x receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446–449[CrossRef][Medline]
  36. Kurokawa R, Yu VC, Naar A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG, Glass CK 1993 Differential orientations of the DNA-binding domain, carboxy-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev 7:1423–1435[Abstract]
  37. Perlmann T, Rangarajan PN, Umesono K, Evans RM 1993 Determinants for selective RAR, TR recognition of direct repeat HR Es. Genes Dev 7:1411–1422[Abstract]
  38. Yen PM, Darling DS, Carter RL, Forgione M, Umeda PK, Chin WW 1992 Triiodothyronine (T3) decreases binding to DNA by T3-receptor homodimers but not receptor-auxiliary protein heterodimers. J Biol Chem 267:3565–3568[Abstract/Free Full Text]
  39. Ribeiro RCJ, Kushner PJ, Apriletti JW, West BL, Baxter JD 1992 Thyroid hormone alters in vitro DNA binding of monomers, dimers of thyroid hormone receptors. Mol Endocrinol 6:1142–1152[Abstract]
  40. Schulman IG, Juguilon H, Evans RM 1996 Activation, repression by nuclear hormone receptors: hormone modulates an equilibrium between active, repressive states. Mol Cell Biol 16:3807–3813[Abstract]
  41. Collingwood TN, Rajanayagam O, Adams M, Wagner R, Cavailles V, Kalkhoven E, Matthews C, Nystrom E, Stenlof K, Lindstedt G, Tisell L, Fletterick RJ, Parker MG, Chatterjee VKK 1997 A natural transactivation mutation in the thyroid hormone b receptor: impaired interaction with putative transcriptional mediators. Proc Natl Acad Sci USA 94:248–253[Abstract/Free Full Text]
  42. Leng X, Tsai SY, O’Malley BW, Tsai M-J 1993 Ligand-dependent conformational changes in thyroid hormone, retinoic acid receptors are potentially enhanced by heterodimerization with retinoic x receptor. J Steroid Biochem Mol Biol 46:643–661[CrossRef][Medline]
  43. Forman BM, Umesono K, Chen J, Evans RM 1995 Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 81:541–550[Medline]
  44. Kurokawa R, Direnzo J, Boehm M, Sugarman J, Gloss B, Rosenfeld MG, Heyman RA, Glass CK 1994 Regulation of retinoid signaling by receptor polarity, allosteric control of ligand binding. Nature 371:528–531[CrossRef][Medline]
  45. Kersten S, Dawson MI, Lewis BA, Noy N 1996 Individual subunits of heterodimers comprised of retinoic acid, retinoid x receptors interact with their ligands independently. Biochemistry 35:3816–3824[CrossRef][Medline]
  46. Minucci S, Leid M, Toyama R, Saint-Jeannet J-P, Peterson V, Horn V, Ishmael JE, Bhattacharyya N, Dey A, Dawid IG, Ozato K 1997 Retinoid x receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand, enhances retinoid-dependent gene expression. Mol Cell Biol 17:644–655,[Abstract]