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Molecular Endocrinology, doi:10.1210/me.2005-0437
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Molecular Endocrinology 20 (9): 2036-2051
Copyright © 2006 by The Endocrine Society

The N-Terminal A/B Domain of the Thyroid Hormone Receptor-ß2 Isoform Influences Ligand-Dependent Recruitment of Coactivators to the Ligand-Binding Domain

Henghe Tian, Muktar A. Mahajan, Chun Tung Wong, Ioanis Habeos and Herbert H. Samuels

Departments of Pharmacology and Medicine, New York University School of Medicine, New York, New York 10016

Address all correspondence and requests for reprints to: Herbert H. Samuels, Departments of Pharmacology and Medicine, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: herbert. samuels{at}med.nyu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (TRs), expressed as TR{alpha}1, TRß1, and TRß2 isoforms, are members of the steroid hormone nuclear receptor gene superfamily, which comprises ligand-dependent transcription factors. The TR isoforms differ primarily in their N-terminal (A/B) domains, suggesting that the A/B regions mediate distinct transcriptional activation functions in a cell type-dependent or promoter-specific fashion. The nuclear receptor ligand-binding domain (LBD) undergoes a conformational change upon ligand binding that results in the recruitment of coactivators to the LBD. For glucocorticoid receptor and estrogen receptor-{alpha}, the same coactivator can contact both the LBD and A/B domains, thus leading to enhanced transcriptional activation. Very little is known regarding the role of the A/B domains of the TR isoforms. The A/B domain of TRß2 exhibits higher ligand-independent transcriptional activity than the A/B regions of TR{alpha}1 or TRß1. Thus, we examined the role of the A/B domain and the LBD of rat TRß2 in integrating the transcriptional activation function of the A/B and LBD domains by different coactivators. Both domains are essential for a productive functional interaction with cAMP response element-binding protein (CREB)-binding protein (CBP), and we found that CBP binds to the A/B domain of TRß2 in vitro. In contrast, steroid receptor coactivator-1a (SRC-1a) interacts strongly with the LBD but not the A/B domain. The coactivator NRC (nuclear receptor coactivator) interacts primarily with the LBD, although a weak interaction with the A/B domain further enhances ligand-dependent binding with TRß2. Our studies document the interplay between the A/B domain and the LBD of TRß2 in recruiting different coactivators to the receptor. Because NRC and SRC-1a bind CBP, and CBP enhances ligand-dependent activity, our studies suggest a model in which coactivator recruitment of NRC (or SRC-1a) occurs primarily through the LBD whereas the complex is further stabilized through an interaction of CBP with the N terminus of TRß2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THYROID HORMONE RECEPTORS (TRs) are ligand-regulated transcription factors that mediate the biological effects of the thyroid hormones (T3 and T4) (for review see Refs. 1 and 2). Two different genes, TR{alpha} and TRß, encode four isoforms (TR{alpha}1, {alpha}2, ß1, and ß2), which are known to be expressed at the protein level in vivo (1). TR{alpha}2 does not bind T3, and may act to inhibit the function of other TRs on selective genes (1). Alternative 5'-exon splicing and/or separate promoters generate two TRß isoforms, ß1 and ß2, which have unique N-terminal regions (1). TR{alpha}1 and TRß1/ß2, respectively, share a high degree of homology and identity (~85%) except for their N termini, suggesting that the N-terminal region is responsible for modulating isoform-specific activity/function of the TRs.

The TRs belong to a superfamily of nuclear receptors (NRs) that includes receptors for steroid hormones, retinoic acids, 1,25-dihydroxyvitamin D3, and other hormones, as well as orphan receptors for which ligands, if any, have not yet been identified (2). All NRs share a similar modular structure: a variable N-terminal A/B region [which in some NRs encode activation function-1 (AF-1)], followed by the C-region, which contains the DNA-binding domain (DBD), a linker D region, and the C-terminal E/F region. The DEF region contains the ligand-binding domain (LBD) and a ligand-dependent transcriptional activation domain, denoted as activation function-2 (AF-2). Ligand binding to the LBD leads to a conformational change in the receptor, which recruits coactivators to the DNA-bound receptor through their LxxLL motifs (NR boxes). For some nonsteroid NRs [e.g. TRs and retinoic acid receptors (RARs)], unliganded receptor interacts with corepressors, including N-CoR (nuclear receptor corepressor) (3) and SMRT (silencing mediator of retinoid and thyroid hormone receptors) (4), to mediate transcriptional repression. Corepressors and coactivators are thought to modulate transcriptional activity through covalent modification of the chromatin/nucleosome template and by direct interactions with components of the general transcriptional machinery (5).

Several families of coactivators have been isolated and characterized. The most extensively studied is the p160/steroid receptor coactivator (SRC) family, which includes SRC-1/nuclear receptor coactivator-1 (6, 7), transcriptional intermediary factor 2 (TIF-2) glucocorticoid-interacting protein-1 (GRIP-1) (8, 9), and p300/CBP-interacting protein/activator for thyroid hormone receptor (ACTR)/TR activator molecule 1 (TRAM-1)/receptor-associated coactivator 3 (RAC-3) (10, 11, 12, 13, 14). The SRC family interacts with the NR LBD via three centrally located NR boxes containing three highly conserved {alpha}-helical LxxLL motifs (15, 16). One of the isoforms of SRC-1 (SRC-1a) contains a fourth NR box at its extreme C terminus (17). Another well-studied family of coactivators is the cAMP response element-binding protein (CREB)-binding protein (CBP)/p300 family, which interacts with a large variety of transcription factors including the NRs (7, 18, 19). CBP/p300 also interacts with TATA-binding protein, transcription factor IIB, or RNA polymerase II holoenzyme and might serve to link NRs to the basal transcriptional machinery (19). The interaction between CBP and liganded NRs maps to an N-terminal LxxLL motif of CBP (7, 18), although this interaction is much weaker than with the LxxLL motifs of SRC-1/nuclear receptor coactivator-1 (20). Multiprotein complexes referred to as TR-associated protein (TRAP)/vitamin D receptor-interacting protein (DRIP) have also been identified using biochemical approaches (21, 22). TRAP/DRIP complexes are recruited to the LBD of NRs in response to ligand binding through a single subunit TRAP220/DRIP205. Other coactivators that have been reported to interact with TRs include PPAR{gamma} coactivator-1(23), nuclear receptor coactivator (NRC) (24, 25, 26), also referred to as activating signal cointegrator 2/receptor-associated protein 250/PPAR{gamma}-interacting protein/TR-binding protein (27, 28, 29, 30), and NR-interacting factor 3 (31, 32). Among them, NRC was found to associate with CBP with high affinity in vivo (24).

The N-terminal AF-1 function of NRs exhibits ligand-independent constitutive activity when fused to a heterologous DBD (e.g. Gal4) (33). However, in the context of certain full-length NRs, the activity of AF-1 appears to be ligand dependent because ligand is essential for steroid receptor binding to DNA and for the release of corepressors from DNA-bound receptors such as the TRs and RARs. In contrast with the ligand-dependent AF-2 activity, the mechanism(s) underlying AF-1 activity is less well understood. However, recent evidence suggests that some AF-2 coactivators are also capable of interacting with the N-terminal A/B domain of several receptors and mediating their AF-1 function. For example, SRC-1, TIF-2, CBP, and p300 are thought to mediate the AF-1 function of estrogen receptor-{alpha} (ER{alpha}) and/or ERß (34, 35, 36, 37, 38); SRC-1 and TIF-2 interact with the N terminus of AR (39); SRC-1 enhances the AF-1 activity of progesterone receptor (PR) (40); TIF-2 transactivates the AF-1 function of RAR{alpha} (41); and CBP associates with the A/B domain of PPAR{gamma} to stimulate its transcription (42). By interacting with both the N terminus and the C terminus, these coactivators bridge functions of AF-1 and AF-2. Thus, the concomitant interactions of certain coactivators with both AFs result in synergistic activation of transcription (35, 37, 41).

Several studies have investigated the role of the N-terminal A/B domain in regulating the activity of the TRs. Although the 52-amino acid A/B region of TR{alpha}1 does not exhibit AF-1 activity when fused to the Gal4 DBD, it acts to enhance the activity of the receptor through its interaction with transcription factor IIB (43, 44). In contrast with TR{alpha}1, TRß2 exhibits moderate activation of target gene expression in the absence of ligand, which is further enhanced by T3 (45). Unlike TR{alpha}1, the A/B domain of TRß2 is transcriptionally active when fused to a heterologous DBD (46). The N-terminal AF was mapped to two tyrosine-rich regions in chicken TRß2 (cTRß2) (46). In addition, cTRß2 has been reported to regulate transcription through a novel antirepression mechanism by which the LBD recruits SMRT but the A/B domain interacts with the repression domain of SMRT and prevents the subsequent assembly of a functional corepressor complex (45). Although the above results provide mechanistic insights into the basis of the AF-1 function of TRs, it is still not completely clear how the overall transcriptional activity of wild-type TRß2 is generated from its individual AFs.

The AF-1 function of TRß2 could also be mediated by binding of known coactivators to the N terminus of the receptor. Therefore, the present study was undertaken to determine the interaction of the A/B domain of rat TRß2 (rTRß2) with known coactivators. We first established that the A/B domain of rTRß2 has an intrinsic AF. We found that both CBP and NRC (but not SRC-1a) enhance the AF-1 activity of rTRß2 and the N-terminal A/B domain of rTRß2 binds to CBP in the absence of hormone. Functional studies indicate that AF-1 contributes to the overall ligand-dependent activity of wild-type rTRß2, and CBP and NRC integrate the activities of AF-1 and AF-2. Our findings provide evidence for a mechanism by which, in addition to the liganded LBD, the A/B domain of TRß2 provides a second platform for interaction with CBP and NRC (but not SRC-1), which stabilizes the interaction of these factors with the receptor complex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To understand the contribution of the A/B domain of rTRß2 to the overall transcriptional activation of this receptor isoform, mutations were introduced into the A/B domain by PCR-mediated random mutagenesis. The PCR products were cloned into a mammalian expression vector (pEX-LexA) so as to expresses the rTRß2 A/B domain as a LexA-DBD fusion protein. The pEX-LexA-A/B plasmids were sequenced, and a number of mutants that displayed lower activity than the wild-type A/B domain were identified. Two mutants, m199 (Q61R) and m70 (E49D and S72R), with differing activity compared with the wild-type A/B domain, were selected for study. To generate full-length mutant receptors, the A/B domains containing these mutations were exchanged for the wild-type A/B domain in the context of full-length TRß2 in a pEX vector. Figure 1AGo illustrates the T3-mediated transcriptional activity of these TRß2 mutants compared with wild-type TRß2 [TRß2(wt)] and TRß2 lacking the A/B domain ({Delta}A/B). The TRß2(wt) exhibited about 10-fold more T3-dependent transcriptional activation than TRß2({Delta}A/B). The TRß2 mutants exhibited lower levels of T3-dependent stimulation; with TRß2(m199) showing half the activity of TRß2(wt) and TRß2(m70) showing an activity similar to TRß2({Delta}A/B). The inset Western blot shows similar levels of expression of these TRß2 proteins. These results suggest that although the TRß LBD contains an AF-2, the A/B domain of rTRß2 contributes significantly to the overall activity of full-length TRß2. To assess whether the A/B domain of rTRß2 contains an intrinsic AF-1 activity, an RSVT vector expressing LexA, LexA-A/B(wt), LexA-A/B(m199), and LexA-A/B(m70) was expressed in HeLa cells and its intrinsic activity assessed using a LexA-chloramphenicol acetyltransferase (CAT) reporter plasmid (Fig. 1BGo). As previously found with the chicken and human (h) TRß2 A/B domains (46, 47), the results in Fig. 1BGo indicate that the wild-type A/B domain of rTRß2 contains an intrinsic AF. In contrast, LexA-A/B(m70) exhibited no activity compared with LexA alone whereas LexA-A/B(m199) exhibited low but detectable activity. The inset Western blot shows similar levels of expression of these LexA-A/B domain proteins.


Figure 1
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  		Fig. 1. Mutations in the A/B Domain of rTRß2 Decrease the Activity of the Receptor in Mammalian Cells

A, Mutant m199 and m70 A/B domains of rTRß2 were cloned into full-length TRß2 and cotransfected with the reporter plasmid, TRE-DR4A-CAT, as described in Materials and Methods. The T3-dependent activity of these receptors was examined in HeLa cells. Unless otherwise indicated, each sample was analyzed in duplicate, and the experiment was repeated at least two times with similar results. CAT activity was then determined and quantified as described in Materials and Methods. The inset in panel A is a Western blot probed with monoclonal C4 antibody against TRß2-LBD. The bands 1, 2, 3, and 4 are TRß2 {Delta}A/B, TRß2(wt), TRß2(m199), and TRß2(m70), respectively, as described in Material and Methods. B, Differential transactivation by the A/B domains of rTRß2 wt, m199, and m70 was assessed in HeLa cells transfected with RSVT-LexA alone or with the LexA fusion of the A/B domain of rTRß2(wt), m199, or m70 along with a LexA-responsive CAT reporter (3,4,5-LexA- CAT). The concentrations of the various plasmids used are given in Materials and Methods. The inset in panel B is a Western blot probed with polyclonal antibody against LexA. The bands 1, 2, 3, and 4 are LexA, and LexA fusions of the A/B domain of rTRß2(wt), or the A/B domains of m199 or m70 of rTRß2, respectively, as described in Materials and Methods.

 
To examine the contribution of known coactivators to the AF-1 function of rTRß2, we studied SRC-1a, CBP, and NRC as representative coactivators from different coactivator groups. In HeLa cells, the activity of LexA-A/B was examined alone and with coexpression of the different coactivators (Fig. 2Go). Interestingly, coexpression of either NRC or CBP resulted in a 3- to 4-fold increase in the activity of LexA-A/B(wt), whereas SRC-1a was without effect. These results suggest that CBP and NRC contribute to the AF-1 activity of rTRß2 whereas SRC-1a does not. Because NRC (and likely CBP) also contributes to the AF-2 activity of TR, these findings suggest that NRC and/or CBP may modulate both AF-1 and AF-2 through multiple protein-protein contacts with the receptor. In contrast, SRC-1a appears to act primarily through the AF-2 domain of rTRß2.


Figure 2
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  		Fig. 2. NRC and CBP, But Not SRC-1a, Enhance the AF-1 Activity of the A/B Domain of rTRß2 in Mammalian Cells

An RSVT vector expressing LexA or the LexA-A/B domain fusion of rTRß2 was cotransfected into HeLa cells with or without the indicated coactivators (CoAs) using 3,4,5-LexA-CAT as a reporter gene. Refer to Materials and Methods for the concentration of the various plasmids used. CAT activity was then determined as described earlier (Fig. 1Go).

 
To further explore AF-1 and AF-2 interactions of these coactivators, we examined the interaction of full-length rTRß2(wt) and rTRß2 mutants (m199, m70, and {Delta}A/B) using a yeast two-hybrid assay. rTRß2(wt) and mutants were expressed as LexA fusions in yeast along with full-length CBP, NRC, or SRC-1a conditionally expressed from the plasmid pJG4–5. Before studying the interaction of these coactivators with these rTRß2 proteins, we examined the level of expression of rTRß2(wt) and mutants in the presence and absence of T3. Figure 3AGo illustrates a Western blot of yeast extracts expressing full-length LexA-rTRß2 wt, m199, m70, and {Delta}A/B. rTRß2(m199) is expressed at slightly higher levels than rTRß2(wt) whereas rTRß2({Delta}A/B) is expressed at somewhat higher levels than rTRß2(m199). Unlike HeLa cells (Fig. 1Go), rTRß2(m70) is expressed at much lower levels than rTRß2(wt) and the other mutants in yeast. In each case, incubation with T3 slightly increased the expression levels of the receptors. Because the level of expression of rTRß2(m70) was much lower than the other mutants, we limited our coactivator interaction studies in yeast to rTRß2(wt), rTRß2(m199), and rTRß2({Delta}A/B). Figure 3BGo illustrates the effect of T3 on the interaction of CBP with rTRß2(wt), rTRß2(m199), and rTRß2({Delta}A/B) in yeast. Surprisingly, CBP only appeared to interact with rTRß2(wt). Virtually no interaction was found with rTRß2(m199), whereas a low level of interaction was detected with rTRß2({Delta}A/B). The difference in the extent of interaction of CBP with rTRß2({Delta}A/B) and rTRß2(m199) may reflect the higher level of expression of rTRß2({Delta}A/B) in the presence of T3 (Fig. 3AGo). These studies suggest that CBP displays a weak AF-2-dependent association with the rTRß2 LBD and that stable interaction of CBP with rTRß2 requires the receptor A/B domain.


Figure 3
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  		Fig. 3. Ligand-Dependent Interactions of Receptor with CBP in Yeast and Expression Levels of rTRß2 LexA Fusions

A, rTRß2(wt), the A/B domain deleted mutant ({Delta}A/B), m199, and m70 were expressed in yeast strain EGY48 as LexA fusions from pEG{Delta}PL. At least duplicate transformant colonies from each sample were randomly selected and then grown in liquid culture overnight with and without T3. Yeast cell lysates (equal amounts) were then electrophoresed in SDS gels followed by Western blotting using anti-LexA antibody to compare the expression levels. B, The B42 fusion of CBP was tested in yeast two-hybrid assays against rTRß2(wt), m199, and {Delta}A/B rTRß2 as LexA fusion proteins. The interaction was quantified by determining the activity of ß-galactosidase with or without T3 as described in Materials and Methods.

 
Figure 4Go shows a similar experiment comparing the interaction of rTRß2(wt) and rTRß2({Delta}A/B) with CBP, NRC, and SRC-1a. Again, the interaction of CBP with rTRß2(wt) in the presence of T3 is highly dependent on the A/B domain of rTRß2, and a significantly lower interaction was found with rTRß2({Delta}A/B). NRC showed a T3-dependent interaction with rTRß2(wt) similar to that found for CBP. However, unlike CBP, NRC interacted with rTRß2({Delta}A/B) in a ligand-dependent fashion, although less efficiently than with rTRß2(wt) (~2-fold). In contrast with CBP and NRC, SRC-1a appears to exhibit a greater interaction with rTRß2({Delta}A/B) than with rTRß2(wt) in the presence of T3. This apparent enhanced interaction with rTRß2({Delta}A/B), however, parallels the higher expression levels of rTRß2({Delta}A/B) found in the presence of T3 (Fig. 3AGo). These findings suggest that the T3-dependent interaction of CBP with rTRß2(wt) is determined by an interaction of CBP with the A/B domain of rTRß2. Unlike CBP, the A/B domain is not essential for the T3-dependent interaction of NRC with rTRß2, although the A/B domain enhances the interaction. Studies using rTRß2(m199) and NRC were similar to that found with rTRß2({Delta}A/B) and NRC (data not shown). In contrast, the results of Fig. 4Go indicate that maximal T3-dependent interaction of SRC-1a with rTRß2 does not require the rTRß2 A/B domain.


Figure 4
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  		Fig. 4. Interactions of Different Coactivators with Wild-Type and the A/B Domain Deletion Mutant ({Delta}A/B) of rTRß2 in Yeast

Yeast two-hybrid assay was performed in EGY48 using a LexA-LacZ reporter. Full-length coactivators CBP, NRC, and SRC-1a were tested against LexA fusions of the wild-type receptor or A/B-deleted rTRß2. Open columns in each set represent interactions in the presence of T3.

 
To compare the differential coactivation of CBP, NRC, and SRC-1a on rTRß2(wt) and rTRß2({Delta}A/B), dose-dependent effects by these factors in HeLa cells were carried out in the presence of T3. As shown in Fig. 5AGo, in the presence of T3 the enhanced transactivation of rTRß2(wt) by CBP was greater than with rTRß2({Delta}A/B) at all dose points. NRC exhibits a similar pattern as CBP (Fig. 5BGo). In contrast with CBP and NRC, the effect of SRC-1a on the transactivation of rTRß2(wt) is less than that for rTRß2({Delta}A/B) (Fig. 5CGo) at all dose points. The greater transactivation activity of rTRß2({Delta}A/B) than rTRß2(wt) with SRC-1a likely reflects a higher level of expression of rTRß2({Delta}A/B) in mammalian cells as we have observed in yeast. These findings suggest that the A/B domain of rTRß2 contributes to the enhanced transactivation of wild-type rTRß2 mediated by CBP and NRC. In contrast, the A/B domain does not appear to be required for the transactivation of rTRß2(wt) by SRC-1a. This result and the results shown in Fig. 4Go provide evidence for a role of CBP and NRC in mediating both the AF-1 and AF-2 activity of rTRß2. In contrast, SRC-1a appears to affect only the AF-2 activity of rTRß2.


Figure 5
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  		Fig. 5. T3-Dependent Enhancement of rTRß2(wt) and rTRß2({Delta}A/B) Activities by CBP, NRC, and SRC-1a in Mammalian Cells

Increasing amounts of vector expressing CBP (A), NRC (B), or SRC-1a (C) were cotransfected with a fixed amount of rTRß2(wt) or rTRß2({Delta}A/B) in HeLa cells in the presence of T3. The fold enhancement with T3 was measured relative to the control empty vector transfection. Open squares depict the results for rTRß2(wt), and the open circles depict the results for rTRß2({Delta}A/B).

 
CBP is known to function as a transcriptional integrator (7, 19) with an intrinsic histone acetyl transferase domain (19, 48, 49) and other distinct domains, which interact with various components of transcriptional apparatus and have been suggested to contribute to CBP function. Figure 6AGo shows the different regions of CBP used in our study. The most N-terminal region of CBP has been reported to contain a receptor interaction domain (RID) that interacts with the LBD of nuclear receptors through a single LxxLL motif (amino acids 68–72) (20). Figure 6BGo shows the T3-dependent interaction of six separate regions of CBP with rTRß2(wt) and rTRß2({Delta}A/B). Only CBP(1–451) containing the LxxLL receptor interacting domain associates with rTRß2(wt) in the presence of T3. In contrast with rTRß2(wt), the T3-dependent interaction of CBP(1–451) with rTRß2({Delta}A/B) is approximately 5-fold less (Fig. 6BGo) even though rTRß2({Delta}A/B) is expressed at higher levels than rTRß2(wt) (Fig. 3AGo). The expression of the different regions of CBP in yeast was found to be similar as compared by Western blotting (data not shown).


Figure 6
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  		Fig. 6. The N-Terminal Region of CBP and the A/B Domain of rTRß2 Are Necessary for the Receptor-CBP Interaction

A, Schematic representation of different regions of CBP studied as B42 fusions in a yeast two-hybrid assay. B, LexA-rTRß2(wt) and the LexA-A/B domain-deleted mutant (designated as A/B– in the figure) were expressed with full-length B42-CBP and various deletions of CBP as B42 fusions labeled as 1–6 in panel A. The interaction with or without T3 was quantified by determining ß-galactosidase activity as described in Materials and Methods.

 
A similar study was carried out with different regions of NRC. Figure 7AGo illustrates the different regions of NRC examined. NRC is a 2063-amino acid coactivator that interacts in a ligand-dependent fashion with all nuclear hormone receptors. NRC contains a potent N-terminal activation domain (indicated as AD1), a receptor interaction domain containing an LxxLL motif (LxxLL-1), a second activating region (AD2), and a C-terminal region containing a second LxxLL motif (LxxLL-2) (24). LxxLL-1 interacts with all steroid hormone and thyroid hormone/retinoid receptors (24). LxxLL-2 has been reported to interact with liver X receptor (27). ER{alpha} also interacts with LxxLL-2, although with somewhat lower affinity than with the LxxLL-1 region (24). We compared the T3-dependent interaction of rTRß2(wt) and rTRß2({Delta}A/B) with various regions of NRC shown in Fig. 7BGo. No interaction of NRC(1–849) was found with either rTRß2(wt) or rTRß2({Delta}A/B) whereas NRC(849–2063) and NRC(849–1153) interacted with rTRß2 with the extent of interaction greater for rTRß2(wt) than rTRß2({Delta}A/B). The interaction of rTRß2(wt) and rTRß2({Delta}A/B) with NRC(849–2063) is similar to NRC(849–1153), each of which contains the LxxLL-1 region. This finding is consistent with our previous study in yeast using full-length cTR{alpha} or its LBD (24). In that study, we also found that cTR{alpha} showed no detectable interaction with the NRC(849–1153) LxxLL-1 mutant in which the leucines were changed to alanines. Figure 7BGo also compares the interaction of NRC(849–1153) and the NRC(849–1153) LxxLL-1 mutant with rTRß2({Delta}A/B) and rTRß2(wt). Results with rTRß2({Delta}A/B) were similar to that found with cTR{alpha}, indicating no interaction with the NRC(849–1153) LxxLL-1 mutant. In contrast, rTRß2(wt) showed a moderate interaction with the NRC(849–1153) LxxLL-1 mutant which was T3 dependent. The expression levels of the different regions of NRC and the NRC(849–1153) LxxLL mutant were comparable in yeast (data not shown).


Figure 7
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  		Fig. 7. The N-Terminal A/B Domain of rTRß2 Influences the Receptor-NRC Interaction

A, Schematic representation of different regions of NRC studied as B42 fusions in a yeast two-hybrid assay are as indicated. B, Same as Fig. 6BGo, except that the interaction of full-length rTRß2(wt) and the A/B domain-deleted mutant (A/B–) was studied against the different regions of NRC as indicated in panel A.

 
The yeast interaction studies suggest that CBP may associate with the A/B domain of rTRß2 with higher affinity than NRC. This notion is supported by the results of Fig. 8Go, A and B, which examined the binding of the A/B domain of rTRß2, labeled in vitro with [35S]L-methionine in rabbit reticulocyte lysates, with glutathione-S-transferase (GST) fusions of the regions of CBP (see Fig. 6AGo) and with GST-NRC(849–1153). Equal amounts of GST fusion proteins (500 ng) were used in the binding studies. The 35S-labeled A/B domain bound to GST-CBP(1–451) but not to the other GST-CBP proteins or NRC(849–1153). CBP(1460–1891) was not included in the binding study because it did not express efficiently in bacteria. We also studied the binding of the A/B domain of rTRß2 to SRC-1a (amino acids 977-1441) expressed in bacteria as a GST fusion protein. This region of SRC-1a contains not only an LxxLL motif for binding with the LBD of NRs, it also contains a Q-rich area (977–1172) previously suggested as the binding site for the A/B domain of cTRß2 (50). Consistent with our findings in yeast (Fig. 4Go), the interaction of SRC-1a with rTRß2 is A/B domain independent; SRC-1a(977–1441) did not bind with the rTRß2 A/B region (Fig. 8BGo).


Figure 8
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  		Fig. 8. The A/B Domain of rTRß2 Binds to CBP in Vitro, But Not Detectably to NRC or SRC-1a

A, The A/B domain of rTRß2, labeled with [35S]L-methionine by in vitro transcription-translation using rabbit reticulocyte lysates was incubated with bacterial expressed and purified GST fusions of CBP shown in Fig. 6Go. The samples were electrophoresed in SDS gels, and the amount of 35S-labeled A/B domain that bound GST-protein was visualized using a PhosphorImager. B, Same as panel A except that the in vitro binding of the [35S]rTRß2-A/B domain was also tested with GST fusions of rNRC(849–1153) and SRC-1a(977–1441).

 
To further examine the contribution of the A/B domain of TRß2 in the binding of full-length receptor with coactivators, we examined the T3-dependent association of [35S]rTRß2(wt) and [35S]rTRß2({Delta}A/B) with GST-CBP(1–451), GST-NRC(849–1153), and GST-SRC-1a(977–1441). The 977-1441 region of SRC1a was used because it had been previously reported to interact with the A/B domain of cTRß2 (50). Figure 9Go illustrates these results, which were also quantitated (as a percent of total input) using PhosphorImager Storm A20 software (Molecular Dynamics, Inc., Sunnyvale, CA). The binding of [35S]rTRß2(wt) to GST-CBP(1–451) (Fig. 9AGo) and GST-NRC(849–1153) (Fig. 9BGo) was about 3- to 5-fold greater than that for [35S]rTRß2({Delta}A/B) both in the presence and absence of T3. In contrast, GST-SRC-1a(977–1441) (Fig. 9CGo) showed no significant difference in the binding to [35S]rTRß2(wt) or [35S]rTRß2({Delta}A/B) in the presence or absence of T3.


Figure 9
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  		Fig. 9. Ligand-Dependent Binding of rTRß2(wt) and ({Delta}A/B) to CBP(1–451), rNRC(849–1153, and SRC-1a(977–1441) in Vitro GST and the GST-fusion proteins were incubated with 35S-labeled rTRß2(wt) or ({Delta}A/B) in the presence or absence of T3 as described in Materials and Methods. Panel A depicts the results with bacterial expressed GST-CBP(1–451), panel B shows the results with bacterial expressed GST-rNRC(849–1153), and panel C shows the result with GST-SRC-1a(977–1441). The bar diagrams represent the percent of binding as a function of input as quantitated using PhosphorImager Storm A20 software (Molecular Dynamics; Amersham Pharmacia Biotech).

 
The results of Figs. 4Go and 6Go and 9AGo suggest that the LBD and A/B domain both contribute to maximal ligand-dependent interaction of rTRß2 with CBP. To further explore this, we examined the role of the N-terminal LxxLL CBP RID in the rTRß2-CBP interaction. Figure 10AGo shows that mutation of the leucines of the LxxLL to alanines has no effect on the in vitro binding of 35S-labeled A/B domain to CBP(1–451). In addition, a yeast interaction assay (Fig. 10AGo) shows that the CBP LxxLL mutant does not affect the interaction of CBP with the A/B domain, which is consistent with the in vitro binding assays. In contrast, mutation of the LxxLL in CBP(1–451) completely abrogates the ligand-dependent interaction of CBP with wild-type rTRß2 (Fig. 10BGo). Thus, although the N terminus of rTRß2 is important for T3-dependent interaction with CBP, association of CBP with the LBD is required for the formation of a CBP-rTRß2 complex.


Figure 10
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  		Fig. 10. The LxxLL Motif in the N Terminus of CBP is Not Necessary for Interaction with the rTRß2 A/B Domain but Is Necessary for Ligand-Dependent Interaction with Full-Length Receptor

A, The autoradiogram shows the binding of the 35S-labeled A/B domain of rTRß2 to GST-CBP(1–451) wt and to a GST-CBP(1–451) mutant (the leucines of the LxxLL motif were replaced by alanines). The lower bar graphs show a yeast two-hybrid assay for interaction between a LexA fusion of the A/B domain of rTRß2 and B42 fusions of CBP(1–451) wt and the CBP(1–451) LxxLL mutant. B, The autoradiogram shows the expression of the B42 fusions of the CBP(1–451) wt and the CBP(1–451) LxxLL mutant induced by galactose in yeast. The lower bar graphs show the T3-dependent interactions of these B42-CBP proteins (wt and mut) with the wild-type LexA-rTRß2.

 
The A/B domain of cTRß2 was reported to interact with SRC-1 and GRIP-1 (50), whereas the A/B domain of hTRß2 was reported to interact with SRC-1, pCIP, and CBP (47). However, we could not identity a similar association of SRC-1 with the A/B domain of rTRß2 in studies in mammalian cells, yeast, or by binding studies in vitro. The A/B domain of rTRß2 is longer than that of cTRß2 or hTRß2, containing an additional 39 amino acids at the most N-terminus of rTRß2. Thus, it is possible that these 39 amino acids of the A/B domain of rTRß2 may conformationally hinder the association of SRC-1 with the A/B region, accounting for the difference in interaction of cTRß2 and/or hTRß2 for SRC-1 compared with rTRß2. The following describes studies to explore this possibility (data not shown). A 39-amino acid deletion of the rTRß2 A/B domain [rTRß2 (40–159)] was cloned in pEG202 and expressed as a LexA fusion. The interaction of SRC-1a with LexA rTRß2 (40–159) was examined in a yeast two-hybrid assay using the wild-type A/B domain as a control. rTRß2 (40–159) was found to be as active in yeast as the wild-type A/B domain. No further increase in the activity of rTRß2 (40–159) or the wild-type A/B domain was found when SRC-1a was conditionally expressed. The same 39 amino acids were deleted from full-length rTRß2. This N-terminal deletion [rTRß2 (40–538)] was tested for its ability to interact with SRC-1a in yeast in the presence of T3. Again, no difference was noted compared with wild-type rTRß2. Thus, amino acids 1–39 in rTRß2 do not appear to block interaction of SRC-1 with the A/B domain of rTRß2. This suggests that other sequence differences in the A/B domain may be responsible for the differential effects of SRC-1 among chicken, human, and rat TRß2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Studies with TRß2 knockout mice suggest that TRß2 is involved in mediating hypothalamic-pituitary-thyroid feedback control involving TRH and TSH as well as visual and auditory development (51, 52, 53). The TRß isoforms differ only in their A/B domains, suggesting that their N-terminal regions may be involved in the differential regulation of target genes. The chicken and human TRß2 N termini possess a ligand-independent AF that is distinct from the other TR isoforms. Our findings, shown in Figs. 1Go and 2Go, identified that the A/B domain of rTRß2 also contains an intrinsic AF. The AF-1 activity in cTRß2 and hTRß2 has been previously mapped to a 20- to 30-amino acid region in the N terminus (46, 47). Interestingly, m199 and m70 in rTRß2, which exhibit a marked decrease in AF-1 activity (Fig. 1BGo), contain A/B domain mutations in the same 20- to 30-amino acid region as identified in cTRß2 and hTRß2. We investigated the mechanism of AF-1 activity of rTRß2 by examining the interactions of known coactivators with its A/B domain. We sought to clarify how cofactor(s) might mediate cooperativity of the two AFs in the context of wild-type receptor.

Our results indicate that the N terminus of rTRß2 is able to functionally and physically interact with CBP. The contact site of CBP with the TRß2 A/B domain is located within the first 451 amino acids of CBP, which contains the previously reported LxxLL RID (20). However, mutation studies (Fig. 10Go) indicate that the LxxLL motif of CBP does not mediate the direct interaction of CBP with the rTRß2 A/B domain. CBP, as well as NRC, enhances not only the AF-1 activity of the rTRß2 but also the interaction of AF-1 and AF-2 in the context of full-length rTRß2. This functional interaction is particularly notable for the ligand-dependent interaction of CBP with rTRß2 where this interaction is highly dependent on the rTRß2 A/B domain (Fig. 10Go). Mutation of the CBP LxxLL to AxxAA does not alter interaction with the TRß2 A/B domain. However, it completely abrogates ligand-dependent interaction in the context of full-length TRß2 (Fig. 10Go), indicating that both the A/B domain and LBD are important for a functional TRß2-CBP interaction. Given the finding that CBP and NRC associate as a complex in vivo (24), our results suggest that a multiprotein complex involving CBP and NRC could bridge the AF-1 and the AF-2 of rTRß2 to modulate the transcriptional activity of the full-length receptor (Fig. 11Go). Although SRC-1 is also known to associate with CBP/p300 (2, 54), SRC-1 did not affect the AF-1 function of rTRß2 as determined by comparing its effect on rTRß2(wt) and rTRß2({Delta}A/B) in mammalian cells and yeast. Therefore, our results suggest that CBP, NRC, or a complex involving CBP and NRC may mediate cooperativity between the N- and C-terminal regions of rTRß2, which leads to optimal activity (Fig. 11Go).


Figure 11
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  		Fig. 11. Integrating the Activity of AF-1 and AF-2 of rTRß2 by CBP and NRC

The left diagram (A) depicts CBP binding to the N-terminal A/B domain and with T3 via its LxxLL motif to the receptor LBD. Binding to only one of the domains is relatively weak, but association of CBP with the LBD of rTRß2 stabilizes the CBP-rTRß2 interaction. The right panel (B) depicts how a CBP-NRC complex could bind to the wild-type receptor. CBP primarily contacts the A/B domain, and NRC interacts with the LBD. Such a proposal takes into account that the CBP LxxLL motif is a low-affinity motif (20 ) whereas the NRC LxxLL-1 motif is a high-affinity motif that binds the LBD of rTRß2 (24 ). Unlike NRC, CBP has a higher affinity for the A/B domain of rTRß2. Similar to a complex of NRC and CBP, a CBP-SRC-1 complex could also act to integrate AF-1 and AF-2 of rTRß2. For simplicity, the retinoid X receptor (heterodimeric partner of TR) is not included.

 
Some nuclear receptors including TR have been shown to directly interact with CBP in a ligand-dependent manner through their LBDs (7, 18). It has been shown, however, that the affinity of the CBP RID for the LBD of NRs is very low (~50-fold) compared with the RIDs of SRC-1 (20) and NRC (24). Transcriptional activation by RAR-retinoid X receptor heterodimers appears to require the concerted actions of the N-terminal CBP RID and the CBP-interaction domains of SRC-1 (55), suggesting that p160 coactivators might be required for the recruitment of CBP/p300 to NRs. Our results demonstrate that, in addition to a weak ligand-dependent interaction with the TRß-LBD, CBP interacts directly with the A/B domain of rTRß2. CBP enhances the AF-1 activity of the rTRß2 A/B domain as well as the full-length receptor, whereas the effect of SRC-1 appears to primarily affect the AF-2 activity of rTRß2.

Several studies have examined the AF-1 activity of the chicken and human TRß2 isoforms (45, 47, 50, 56). cTRß2 displays less repression activity than the cTRß1 isoform, suggesting that cTRß2 interacts less efficiently with corepressors and/or the AF-1 region of cTRß2 overcomes the extent of repression. Although the LBD of cTRß2 binds SMRT or N-CoR in the absence of ligand, the A/B domain of cTRß2 also appears to contact the silencing domains of SMRT or N-CoR, abrogating its repression function. Such interactions with SMRT/N-CoR do not occur with the A/B domains of other TR isoforms. These findings have led to the proposal of an antirepression model to account for the enhanced activity of cTRß2 (45). Such a mechanism, coupled with the simultaneous ability of the A/B domain of TRß2 to interact with coactivators, would lead to enhanced levels of basal expression, which might be further increased in the presence of T3. The antirepression model may also explain the high levels of TSH and TRH expression in the absence of thyroid hormone. TSH and TRH are negatively regulated by thyroid hormone (1, 57), and this negative regulation appears to require the formation of a coactivator-binding pocket on the surface of the LBD (47). Whether known coactivators paradoxically mediate this negative regulation (58) or if an LxxLL containing repressor molecule found in the pituitary and hypothalamus which might function similar to RIP140 in adipose tissue (59) has not yet been fully clarified.

Although the A/B domains of cTRß2 and hTRß2 appear to interact with SRC-1 in vitro (47, 50), we could not identity a similar association of SRC-1 with the A/B domain of rTRß2 in mammalian cells, in yeast, or in binding studies in vitro. Furthermore, we found that transactivation of the rTRß2 by SRC-1a is A/B domain independent in mammalian cells. rTRß2 was first cloned from a rat pituitary cell cDNA library (60). Subsequently, TRß2 cDNAs from chicken (61) and human (62) were cloned. The rTRß2 A/B domain is longer (161 amino acids) than the A/B domain of the chicken and human TRß2 clones (each 122 amino acids). This difference reflects a 39-amino acid novel sequence at the beginning of the N terminus of rTRß2. However, we found that these 39 amino acids do not account for the differential effect of SRC-1a on the A/B domain of TRß2 from the different species. In addition to a 39-amino acid longer N terminus in rTRß2, the sequence similarity of the A/B domain is greater between human and chicken than the comparable region of the rTRß2 A/B domain. Thus, sequence differences beginning at amino acid 39 of TRß2 to the end of A/B domain may account for the differential coactivator binding activity of the rTRß2 A/B domain compared with cTRß2 and hTRß2.

Hormone-independent transactivation domains have also been found in the variable N-terminal regions of some NRs including glucocorticoid receptor (GR) (63), ER (36), PR (40), and androgen receptor (AR) (64), as well as in RARß (65), retinoid-related orphan receptor-{alpha} (66), and PPAR{alpha} (67). In addition, studies support the notion that the transcriptional activity of certain full-length receptors after ligand binding is mediated through a synergistic effect of AF-1 and AF-2. This synergy has been shown for ER{alpha} (35, 68), ERß (37), RAR{alpha} (41), and AR (39, 69). In the case of ER{alpha}, this is functionally achieved by bridging coactivators, such as CBP/p300 (37), and members of p160 subfamily, such as SRC-1 (34, 68) and TIF-2 (35). These coactivators have been shown to be recruited by both the AF-1 and the AF-2 regions. Recent studies also suggest a direct interaction between the A/B domain and the LBD of AR, PR-B, and ER{alpha}, which may further stabilize coactivator recruitment (39, 68, 70, 71). We examined for such an interaction for rTRß2 with an in vitro binding assay using the 35S-labeled A/B domain of rTRß2 and GST-rTRß2({Delta}A/B). No interaction was found in the presence or absence of T3 (Tian, H., and H. H. Samuels, unpublished observation).

Our results indicate that CBP and NRC require both AF-1 and AF-2 of rTRß2 for maximal transactivation. It is interesting that the A/B and the LBDs of rTRß2 are mutually dependent in leading to a strong interaction with CBP. Although we have not mapped the exact N-terminal sequence in CBP that interacts with the A/B domain of TRß2, the LxxLL motif in the N terminus of CBP does not appear to play a critical role in the interaction with the A/B domain.

Recent studies indicate that protein domains contain amino acid sequences that do not automatically fold into their fully condensed, functional structure (72). The N-terminal A/B domains of many NRs appear to be in a natively unfolded state when expressed as independent proteins (73, 74, 75). For AF-1 function, the A/B domain may adopt a more ordered conformation. Several models for AF-1 folding have been proposed and reviewed by Kumar and Thompson (76). One model predicts induction of AF-1 folding by protein-protein interaction. The unfolded or partially folded A/B region of NRs folds into its active conformation when the protein interacts with a functional binding partner(s). Both structural and biochemical studies on ER{alpha}, AR, and GR have shown that their N termini undergo a transition to a folded state upon interaction with either a component(s) of general transcriptional machinery or with coactivators.

The structure of the A/B domain of the TRs has not been solved. However, the A/B domain of rTRß2 is predicted to be in a random coil-coiled configuration when expressed as a free protein. CBP and NRC both enhance the intrinsic transactivation function of the A/B domain in vivo (Fig. 2Go). Therefore, it remains possible that CBP and/or NRC induces/selects the folding of the A/B domain into a new secondary or tertiary conformation. In addition to their effects on enhancing the AF-1 activity of the TRß2 A/B domain, CBP and/or NRC require the A/B domain of rTRß2 for full ligand-dependent transcriptional activity. The N-terminal region of CBP associates with liganded rTRß2 through both the A/B domain and LBD of the receptor, which acts to stabilize the TRß2-CBP interaction. In contrast with CBP, NRC exhibits a higher affinity for the LBD of rTRß2, and the A/B domain plays a vital but less important role in the rTRß2-NRC interaction (see Figs. 4Go and 9Go). Although CBP or NRC can each bind and enhance the activation of wild-type rTRß2 by T3 (Fig. 11AGo), they may also act as a complex because CBP interacts strongly with NRC in vivo (Fig. 11BGo) (24). Action through a CBP-NRC complex could greatly enhance the activity of rTRß2 through a mechanism by which the A/B domain interacts with CBP while the LBD interacts with NRC (Fig. 11BGo). A similar mechanism might account for the interaction of CBP-SRC-1a complexes with TRß2. In summary, our study provides compelling evidence that for certain coactivators, the N-terminal A/B domain of TRß2 plays an important role in determining the maximal ligand-dependent transactivation potential of the receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
PCR-Based Mutagenesis
The A/B domain of rTRß2 (rTRß2-A/B) was cloned by PCR using full-length rTRß2 as the template. The PCR product was cloned into the BamHI site of pEG{Delta}PL, which was derived from pEG202 (24). Mutations were introduced into the A/B domain of rTRß2 using PCR conditions that randomly introduce about 1 nucleotide change per 200 nucleotides (77). The PCR-generated A/B domain was isolated by gel electrophoresis digested with BglII and then cloned into pEG{Delta}PL to express a LexA-A/B domain fusion in yeast. The LexA fusion with the A/B domain of wild-type rTRß2 is transcriptionally active in yeast. We screened more than 400,000 independent clones of these LexA-A/B domain chimeras to identify mutants that no longer, or very weakly, activated the LacZ reporter. pEG{Delta}PL clones from yeast expressing the expected sized LexA-A/B mutants, as determined by Western blotting, were isolated and sequenced. We sequenced 12 mutants, which displayed activities that varied from 15% to 1% of the wild-type activity. Two mutants were selected for the present study: mutant 199 with 61(Q->R) and mutant 70 with 49(E->D) and 72(S->R). Based on the solution ß-galactosidase assay, mutant 199 retained 10% of the activity of the wild-type A/B domain, whereas m70 exhibited only 1% of the activity.

Plasmids
Full-length wild-type rTRß2 in pBluescript was a gift from Howard Towle (60). rTRß2 was released as an NcoI fragment and cloned into a mammalian pEX vector (78) and the yeast vector, pEG{Delta}PL. To introduce m199 and m70 into full-length TRß2 as a LexA fusion, pEG{Delta}PL containing the m199 and m70 A/B domains was isolated after cutting with XbaI and ligated with the XbaI fragment of TRß2 containing the DBD and LBD to generate the full-length mutants in pEG{Delta}PL. For cell transfection studies, the LexA-A/B component of pEG{Delta}PL was cloned into the RSVT mammalian expression vector (43). The wild-type LexA-A/B region was released from the pEG{Delta}PL-A/B by HindIII and KpnI and then ligated to RSVT. RSVT-LexA-A/B m199 and m70 was constructed the same way but using HindIII and XhoI. The LexA-DBD alone from pEG{Delta}PL vector was also cloned into RSVT to construct the control vector (RSVT-LexA). For in vitro translation, the A/B domain of rTRß2 was transferred from RSVT-LexA-A/B vector to pEX using NcoI and XhoI to construct pEX-rTRß2-A/B. An A/B domain deletion of rTRß2 (rTRß2{Delta}A/B) was constructed by PCR and cloned into pEX using NcoI and Acc65I. The resulting pEX-rTRß2{Delta}A/B was confirmed by sequence analysis and expression studies. To construct pEG{Delta}PL-rTRß2{Delta}A/B the cDNA was released from pEX-rTRß2{Delta}A/B by NcoI and then ligated to NcoI site of pEG{Delta}PL.

All NRC clones are derived from human NRC cDNA unless designated otherwise. pJG4–5{Delta}PL-NRC, pJG4–5-NRC(1–849), pJG4–5-NRC(849–2063), pJG4–5-NRC(849–1153) and its LxxLL-1 mutant, pEX-NRC, and GST-rNRC(849–1153) (rNRC.1) have been described previously (24). All CBP clones were derived from mouse CBP cDNA unless designated otherwise. Rc-RSV-CBP used in transfection studies was a gift from Richard Goodman (79). pJG4–5-CBP was constructed by ligating the full-length CBP fragment derived from Rc-RSV-CBP into the BamHI site of pJG4–5{Delta}PL. All the partial clones of CBP (see Figs. 6Go and 8Go) in pGEX were generously provided by Ralf Janknecht (80). All these CBP partial clones were derived from mouse CBP cDNA except for the 1–451 clone, which was derived from human CBP cDNA. The regions of CBP were released by EcoRI and then cloned into pJG4–5. All SRC-1a cDNAs are human clones. pCR3.1-SRC-1a used in transfection studies was a gift from Bert O’Malley (40). To construct pJG4–5{Delta}PL-SRC-1a, full-length SRC-1a was released as a BspHI-XbaI fragment from pCR3.1-SRC-1a, and the blunt-ended fragment was inserted into the blunt-ended BamHI site of pJG4–5{Delta}PL. All the plasmids generated were confirmed by sequencing analysis. pGEX-SRC-1a(977–1441) was a generous gift from Martin Privalsky (50).

Yeast Two-Hybrid Assay
The yeast strain EGY48, harboring the LacZ reporter plasmid (pSH18–34) (81), was transformed with appropriate bait and prey plasmids. For each transformation, 10 transformants were randomly selected and analyzed on appropriate Xgal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) plates for preliminary evaluation. Typical colonies were then selected for quantitative ß-galactosidase assays as described previously (31). ß-Galactosidase units are expressed as (OD at 420 nm x 1000)/(minutes of incubation x OD at 600 nm of yeast suspension). All ß-galactosidase assays were performed at least two times in duplicate or triplicate.

Cell Culture and Transfections
HeLa cells were cultured as previously described (82). The {Delta}MTV-TRE-DR4A-CAT reporter has been described previously (83). The 3,4,5-LexA-CAT reporter containing LexA binding sites was a gift from Michael Garabedian and Keith Yamamoto (84). HeLa cells were transfected by calcium phosphate coprecipitation as previously described (43, 85). For studies with full-length rTRß2, HeLa cells were cotransfected with 400 ng of wild-type or A/B mutant receptor vector and 1 µg of TRE-DR4A-CAT (TRE-CAT), and the cells were incubated with or without T3 (1 µM). Typically, 5 µg of vector expressing LexA-A/B wild-type or mutant and 5 µg of the reporter were used in transfection studies. LexA-A/B domain plasmids were used at 2 µg to study the effect of different coactivators on the activity of the A/B domain. Vectors expressing CBP, NRC, and SRC-1a coactivators were used at 2–3 µg, and equivalent molar amounts of their corresponding vectors were cotransfected with 5 µg of 3,4,5-LexA-CAT (84). After transfection, cells were incubated at 37 C for about 45 h before being harvested. Typically, CAT assays were carried out using 30–50 µg of cell lysate protein incubated at 37 C for 6–14 h. All transfections were performed in duplicate or triplicate, and the variation in CAT activity of the samples was less than 10%. Each experiment was repeated at least two times.

In Vitro Binding Studies
The glutathione-S-transferase (GST) control, GST-rNRC(849–1153), GST-CBP partial clones, and GST-SRC-1a(977–1441) were expressed in Escherichia coli SG1117 and affinity purified with glutathione-agarose beads (43). 35S-labeled full-length rTRß2, rTBß2-{Delta}A/B, and the A/B domain of rTRß2 were generated by in vitro transcription and translation using rabbit reticulocyte lysates (Promega Corp., Madison, WI). Typically approximately 500 ng of GST protein bound to glutathione-agarose was used per assay. Binding was performed as previously described (43) in 20 mM HEPES (pH 7.9), 1 mM MgCl2, 1 mM dithiothreitol, 10% glycerol, 0.05% Triton X-100, 1 µM ZnCl2 and 200 mM KCl. T3 (1 µM) was added into the binding reaction mixture as indicated in the figure legends. After the binding reaction, the beads were washed three times with the same incubation buffer, and the labeled receptors bound to the beads were examined by sodium dodecyl sulfate (SDS) gel electrophoresis. Five percent of the 35S-labeled receptor input was also electrophoresed in the same gel. The extent of binding was determined by autoradiography and/or quantitation of the percent of binding as a function of input using a PhosphorImager and quantitation using PhosphorImager Storm A20 software (Molecular Dynamics).

Western Blotting Studies
For each transformed yeast strain, the amount of yeast cells used was determined by OD at 600 nm after overnight culture. The protein extracts were then prepared following a CLONTECH protocol using glass beads (425–600 µm; Sigma Chemical Co., St. Louis, MO) and cracking buffer: 8 M urea, 5% SDS, 40 mM Tris-HCl (pH 6.8), 0.4 mg/ml bromophenol blue, a protease inhibitor cocktail (Pierce) and 2 mM phenylmethylsulfonylfluoride. The samples were electrophoresed in SDS gels, transferred to nitrocellulose, followed by Western blotting using an enhanced chemiluminescence detection system (Pierce) as described previously (86). Rat polyclonal anti-LexA antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and monoclonal antibody C4 against an epitope in the C terminus of TRs (87) was generously provided by Sheue-Yann Cheng at the National Institutes of Health. For Western blotting studies in mammalian cells, the various TRß2 or LexA-A/B expression vectors were transfected into HeLa cells, and Western blotting was performed on cell extracts as described previously (88).


   ACKNOWLEDGMENTS
 
We thank Howard Towle for providing us with the rTRß2 clone, Bert O’Malley for the SRC-1a, expression vector, Richard Goodman for the Rc-RSV-mCBP expression vector, Martin Privalsky for pGEX-SRC-1a(977–1441), Ralf Janknecht for the pGEX-CBP clones, and Michael Garabedian and Keith Yammamoto for the 3,4,5, LexA-CAT reporter plasmid. We thank Sheue-Yann Cheng for providing us with monoclonal antibody against TR-LBD. We also thank Evelyn Kono for help with Western blots.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK16636 (to H.H.S.). H.H.S is a member of the New York University Medical Center Cancer Center (CA 016087-23).

First Published Online April 27, 2006

Abbreviations: AF-1, Activation function 1; AR, androgen receptor; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; DBD, DNA-binding domain; DRIP, vitamin D receptor-interacting domain; ER, estrogen receptor; GR, glucocorticoid receptor; GST, glutathione-S-transferase; LBD, ligand-binding domain; N-CoR, nuclear receptor corepressor; NR, nuclear receptor; NRC, nuclear receptor coactivator; PPAR, peroxisomal proliferator-activated receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RID, receptor interaction domain; SDS, sodium dodecyl sulfate; SMRT, silencing mediator of retinoid and thyroid hormone receptors; SRC, steroid receptor coactivator; TIF-2, transcriptional intermediary factor 2; TR, thyroid hormone receptor; TRAP, TR-associated protein; TRß2({Delta}A/B), TRß2 lacking the A/B domain; TRß2(wt), wild-type TRß2.

Received for publication October 28, 2005. Accepted for publication April 7, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiol Rev 81:1097–1142[Abstract/Free Full Text]
  2. Aranda A, Pascual 2001 A Nuclear hormone receptors and gene expression. Physiol Rev 81:1269–1304[Abstract/Free Full Text]
  3. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamil Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  4. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  5. Smith CL, O’Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71[Abstract/Free Full Text]
  6. Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator of the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  7. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414[CrossRef][Medline]
  8. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, 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]
  9. 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[Medline]
  10. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[CrossRef][Medline]
  11. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677–684[CrossRef][Medline]
  12. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  13. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2. Proc Natl Acad Sci USA 94:8479–8484[Abstract/Free Full Text]
  14. Takeshita A, Cardona GR, Koibuchi N, Suen CS, Chin WW 1997 TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1. J Biol Chem 272:27629–27634[Abstract/Free Full Text]
  15. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  16. McInerney EM, Rose DW, Flynn SE, Westin S, Mullen TM, Krones A, Inostroza J, Torchia J, Nolte RT, Assa-Munt N, Milburn MV, Glass CK, Rosenfeld MG 1998 Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev 12:3357–3368[Abstract/Free Full Text]
  17. Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO J 17:232–243[CrossRef][Medline]
  18. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signalling. Nature 383:99–103[CrossRef][Medline]
  19. Goodman RH, Smolik S 2000 CBP/p300 in cell growth, transformation, and development. Genes Dev 14:1553–1577[Free Full Text]
  20. Heery D, Hoare S, Hussain S, Parker M, Sheppard H 2001 Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors. J Biol Chem 276:6695–6702[Abstract/Free Full Text]
  21. Fondell JD, Ge H, Roeder RG 1996 Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex. Proc Natl Acad Sci USA 93:8329–8333[Abstract/Free Full Text]
  22. Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828[CrossRef][Medline]
  23. Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O’Malley B, Spiegelman BM 1999 Activation of PPAR{gamma} coactivator-1 through transcription factor docking. Science 286:1368–1371[Abstract/Free Full Text]
  24. Mahajan M, Samuels H 2000 A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein. Mol Cell Biol 20:5048–5063[Abstract/Free Full Text]
  25. Mahajan MA, Samuels HH 2004 Nuclear hormone receptor co-regulator (NRC): role in hormone action, metabolism, growth, and development. Endocr Rev 26:583–597[Medline]
  26. Mahajan MA, Das S, Zhu H, Tomic-Canic M, Samuels HH 2004 The nuclear hormone receptor coactivator NRC is a pleiotropic modulator affecting growth, development, apoptosis, reproduction, and wound repair. Mol Cell Biol 24:4994–5004[Abstract/Free Full Text]
  27. Lee SK, Anzick SL, Choi JE, Bubendorf L, Guan XY, Jung YK, Kallioniemi OP, Kononen L, Trent JM, Azorsa D, Jhun BH, Cheong JH, Lee YC, Meltzer PS, Lee JW 1999 A nuclear factor, ASC-2, is a cancer-amplified transcriptional coactivator essential for ligand-dependent transactivation by nuclear receptors in vivo. J Biol Chem 274:34283–34293[Abstract/Free Full Text]
  28. Caira F, Antonson P, Pelto-Huikko M, Treuter E, Gustafsson JA 2000 Cloning and characterization of RAP250, a novel nuclear receptor coactivator. J Biol Chem 275:5308–5317[Abstract/Free Full Text]
  29. Ko L, Cardona GR, Chin WW 2000 Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator. Proc Natl Acad Sci USA 97:6212–6217[Abstract/Free Full Text]
  30. Zhu Y, Kan L, Qi C, Kanwar YS, Yeldandi AV, Rao MS, Reddy JK 2000 Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR. J Biol Chem 275:13510–13516[Abstract/Free Full Text]
  31. Li D, Desai-Yajnik V, Lo E, Schapira M, Abagyan R, Samuels HH 1999 NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors. Mol Cell Biol 19:7191–7202[Abstract/Free Full Text]
  32. Li D, Wang F, Samuels H 2001 Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation. Mol Cell Biol 21:8371–8384[Abstract/Free Full Text]
  33. Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. J Biol Chem 270:9535–9542[Abstract/Free Full Text]
  34. McInerney EM, Tsai MJ, O’Malley BW, Katzenellenbogen BS 1996 Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator. Proc Natl Acad Sci USA 93:10069–10073[Abstract/Free Full Text]
  35. Benecke A, Chambon P, Gronemeyer H 2000 Synergy between estrogen receptor {alpha} activation functions AF1 and AF2 mediated by transcription intermediary factor TIF2. EMBO Rep 1:151–157[Medline]
  36. Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP, Chen D, Huang SM, Subramanian S, McKinerney E, Katzenellenbogen BS, Stallcup MR, Kushner PJ 1998 Estrogen receptor activation function 1 works by binding p160 coactivator proteins. Mol Endocrinol 12:1605–1618[Abstract/Free Full Text]
  37. Kobayashi Y, Kitamoto T, Masuhiro Y, Watanabe M, Kase T, Metzger D, Yanagisawa J, Kato S 2000 p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor {alpha} and ß by interacting directly with the N-terminal A/B domains. J Biol Chem 275:15645–15651[Abstract/Free Full Text]
  38. Tremblay A, Tremblay G, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  39. He B, Kemppainen JA, Voegel JJ, Gronemeyer H, Wilson EM 1999 Activation function 2 in the human androgen receptor ligand binding domain mediates interdomain communication with the NH(2)-terminal domain. J Biol Chem 274:37219–37225[Abstract/Free Full Text]
  40. Onate SA, Boonyaratanakornkit V, Spencer TE, Tsai SY, Tsai MJ, Edwards DP, O’Malley BW 1998 The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem 273:12101–12108[Abstract/Free Full Text]
  41. Bommer M, Benecke A, Gronemeyer H, Rochette-Egly C 2002 TIF2 mediates the synergy between RAR{alpha}1 activation functions AF-1 and AF-2. J Biol Chem 277:37961–37966[Abstract/Free Full Text]
  42. Gelman L, Zhou G, Fajas L, Raspe E, Fruchart J, Auwerx J 1999 p300 interacts with the N- and C-terminal part of PPAR{gamma}2 in a ligand-independent and -dependent manner, respectively. J Biol Chem 274:7681–7688[Abstract/Free Full Text]
  43. Hadzic E, Desai-Yajnik V, Helmer E, Guo S, Wu S, Koudinova N, Casanova J, Raaka BM, Samuels HH 1995 A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor {alpha} is essential for transcriptional activation and interaction with the general transcription factor TFIIB. Mol Cell Biol 15:4507–4517[Abstract]
  44. Hadzic E, Habeos I, Raaka BM, Samuels HH 1998 A novel multifunctional motif in the N-terminal A/B domain of T3R{alpha} modulates DNA-binding and receptor dimerization. J Biol Chem 273:10270–10278[Abstract/Free Full Text]
  45. Yang Z, Hong SH, Privalsky ML 1999 Transcriptional anti-repression. Thyroid hormone receptor ß-2 recruits SMRT corepressor but interferes with subsequent assembly of a functional corepressor complex. J Biol Chem 274:37131–37138[Abstract/Free Full Text]
  46. Sjoberg M, Vennstrom B 1995 Ligand-dependent and -independent transactivation by thyroid hormone receptor ß 2 is determined by the structure of the hormone response element. Mol Cell Biol 15:4718–4726[Abstract]
  47. Oberste-Berghaus C, Zanger K, Hashimoto K, Cohen RN, Hollenberg AN, Wondisford FE 2000 Thyroid hormone-independent interaction between the thyroid hormone receptor ß2 amino terminus and coactivators. J Biol Chem 275:1787–1792[Abstract/Free Full Text]
  48. Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641–643[CrossRef][Medline]
  49. Chan HM, La Thangue NB 2001 p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 114:2363–2373[Abstract/Free Full Text]
  50. Yang Z, Privalsky ML 2001 Isoform-specific transcriptional regulation by thyroid hormone receptors: hormone-independent activation operates through a steroid receptor mode of co-activator interaction. Mol Endocrinol 15:1170–1185[Abstract/Free Full Text]
  51. Abel ED, Boers ME, Pazos-Moura C, Moura E, Kaulbach H, Zakaria M, Lowell B, Radovick S, Liberman MC, Wondisford F 1999 Divergent roles for thyroid hormone receptor ß isoforms in the endocrine axis and auditory system. J Clin Invest 104:291–300[Medline]
  52. Ng L, Hurley JB, Dierks B, Srinivas M, Salto C, Vennstrom B, Reh TA, Forrest D 2001 A thyroid hormone receptor that is required for the development of green cone photoreceptors. Nat Genet 27:94–98[Medline]
  53. Abel ED, Kaulbach HC, Campos-Barros A, Ahima RS, Boers ME, Hashimoto K, Forrest D, Wondisford FE 1999 Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin. J Clin Invest 103:271–279[Medline]
  54. Li J, O’Malley BW, Wong J 2000 p300 requires its histone acetyltransferase activity and SRC-1 interaction domain to facilitate thyroid hormone receptor activation in chromatin. Mol Cell Biol 20:2031–2042[Abstract/Free Full Text]
  55. Westin S, Kurokawa R, Nolte R, Wisely G, McInerney E, Rose D, Milburn M, Rosenfeld M, Glass C 1998 Interactions controlling the assembly of nuclear-receptor heterodimers and co-activators. Nature 395:199–202[CrossRef][Medline]
  56. Langlois MF, Zanger K, Monden T, Safer JD, Hollenberg AN, Wondisford FE 1997 A unique role of the ß-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. Mapping of a novel amino-terminal domain important for ligand-independent activation. J Biol Chem 272:24927–24933[Abstract/Free Full Text]
  57. Abel ED, Ahima RS, Boers ME, Elmquist JK, Wondisford FE 2001 Critical role for thyroid hormone receptor ß2 in the regulation of paraventricular thyrotropin-releasing hormone neurons. J Clin Invest 107:1017–1023[Medline]
  58. Tagami T, Park Y, Jameson JL 1999 Mechanisms that mediate negative regulation of the thyroid-stimulating hormone {alpha} gene by the thyroid hormone receptor. J Biol Chem 274:22345–22353[Abstract/Free Full Text]
  59. Christian M, Kiskinis E, Debevec D, Leonardsson G, White R, Parker MG 2005 RIP140-targeted repression of gene expression in adipocytes. Mol Cell Biol 25:9383–9391[Abstract/Free Full Text]
  60. Murray MB, Zilz ND, McCreary NL, MacDonald M, Towle HC 1988 Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors. J Biol Chem 263:12770–12777[Abstract/Free Full Text]
  61. Sjoberg M, Vennstrom B, Forrest D 1992 Thyroid hormone receptors in chick retinal development: differential expression of mRNAs for {alpha} and N-terminal variant ß receptors. Development 114:39–47[Abstract]
  62. Weinberger C, Giguere V, Hollenberg S, Rosenfeld M, Evans R 1986 Human steroid receptors and erbA proto-oncogene products: members of a new superfamily of enhancer binding proteins. Cold Spring Harb Symp Quant Biol 51:759–772[Abstract/Free Full Text]
  63. Iniguez-Lluhi J, Lou D, Yamamoto K 1997 Three amino acid substitutions selectively disrupt the activation but not the repression function of the glucocorticoid receptor N-terminus. J Biol Chem 272:4149–4156[Abstract/Free Full Text]
  64. Alen P, Claessens F, Verhoeven G, Rombauts W, Peeters B 1999 The androgen receptor amino-terminal domain plays a key role in p160 coactivator-stimulated gene transcription. Mol Cell Biol 19:6085–6097[Abstract/Free Full Text]
  65. Folkers GE, van der Leede BM, van der Saag PT 1993 The retinoic acid receptor-ß2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand binding domain. Mol Endocrinol 7:616–627[Abstract/Free Full Text]
  66. Giguere V, Tini M, Flock G, Ong E, Evans RM, Otulakowski G 1994 Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR{alpha}, a novel family of orphan hormone nuclear receptors. Genes Dev 8:538–553[Abstract/Free Full Text]
  67. Juge-Aubry C, Kuenzli S, Sanchez J, Hochstrasser D, Meier C 2001 Peroxisomal bifunctional enzyme binds and activates the activation function-1 region of the peroxisome proliferator-activated receptor {alpha}. Biochem J 353:253–258[CrossRef][Medline]
  68. Metivier R, Penot G, Flouriot G, Pakdel F 2001 Synergism between ER{alpha} transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: requirement for the AF-1 {alpha}-helical core and for a direct interaction between the N- and C-terminal domains. Mol Endocrinol 15:1953–1970[Abstract/Free Full Text]
  69. Ikonen T, Palvimo JJ, Janne OA 1997 Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J Biol Chem 272:29821–29828[Abstract/Free Full Text]
  70. Tetel M, Giangrande P, Leonhardt S, McDonnell D, Edwards D 1999 Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo. Mol Endocrinol 13:910–924[Abstract/Free Full Text]
  71. He B, Kemppainen JA, Wilson EM 2000 FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:22986–22994[Abstract/Free Full Text]
  72. Uversky V, Gillespie J, Fink 2000 A Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 41:415–427[CrossRef][Medline]
  73. Baskakov I, Kumar R, Srinivasan G, Ji Y, Bolen D, Thompson E 1999 Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor. J Biol Chem 274:10693–10696[Abstract/Free Full Text]
  74. Bain D, Franden M, McManaman J, Takimoto G, Horwitz K 2000 The N-terminal region of the human progesterone A-receptor. Structural analysis and the influence of the DNA binding domain. J Biol Chem 275:7313–7320[Abstract/Free Full Text]
  75. Reid J, Kelly S, Watt K, Price N, McEwan I 2002 Conformational analysis of the androgen receptor amino-terminal domain involved in transactivation. Influence of structure-stabilizing solutes and protein-protein interactions. J Biol Chem 277:20079–20086[Abstract/Free Full Text]
  76. Kumar R, Thompson EB 2003 Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions. Mol Endocrinol 17:1–10[Abstract/Free Full Text]
  77. Yajnik V, Blaikie P, Bork P, Margolis B 1996 Identification of residues within the SHC phosphotyrosine binding/phosphotyrosine interaction domain crucial for phosphopeptide interaction. J Biol Chem 271:1813–1816[Abstract/Free Full Text]
  78. Forman BM, Casanova J, Raaka BM, Ghysdael J, Samuels HH 1992 Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers. Mol Endocrinol 6:429–442[Abstract/Free Full Text]
  79. Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  80. Janknecht R, A. N 1996 MAP kinase-dependent transcriptional coactivation by Elk-1 and its cofactor CBP. Biochem Biophys Res Commun 228:831–837[CrossRef][Medline]
  81. Gyuris J, Golemis E, Chertkov H, Brent R 1993 Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803[CrossRef][Medline]
  82. 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 and retinoic acid receptors from an inhibitory cellular factor. Mol Cell Biol 14:5756–5765[Abstract/Free Full Text]
  83. Nelson CC, Hendy SC, Faris JS, Romaniuk PJ 1994 The effects of P-box substitutions in thyroid hormone receptor on DNA binding specificity. Mol Endocrinol 8:829–840[Abstract/Free Full Text]
  84. Godowski PJ, Picard D, Yamamoto KR 1988 Signal transduction and transcriptional regulation by glucocorticoid receptor-LexA fusion proteins. Science 241:812–816[Abstract/Free Full Text]
  85. Mahajan MA, Murray A, Samuels HH 2002 NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor coactivator NRC. Mol Cell Biol 22:6883–6894[Abstract/Free Full Text]
  86. Qi J-S, Desai-Yajnik V, Yuan Y, Samuels HH 1997 Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression. Mol Cell Biol 17:7195–7207[Abstract]
  87. Bhat MK, McPhie P, Ting YT, Zhu XG, Cheng SY 1995 Structure of the carboxy-terminal region of thyroid hormone nuclear receptors and its possible role in hormone-dependent intermolecular interactions. Biochemistry 34:10591–10599[CrossRef][Medline]
  88. Muller S, Berger M, Lehembre F, Seeler JS, Haupt Y, Dejean 2000 A c-Jun and p53 activity is modulated by SUMO-1 modification. J Biol Chem 275:13321–13329[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   TRα  |  TRβ
Coregulators:   CBP  |  SRC-1  |  ASC-2
Ligands:   Thyroid hormone



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