Isoform-Specific Transcriptional Regulation by Thyroid Hormone Receptors: Hormone-Independent Activation Operates through a Steroid Receptor Mode of Coactivator Interaction

Zhihong Yang and Martin L. Privalsky

Section of Microbiology Division of Biological Chemistry University of California at Davis Davis, California 95616


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (T3Rs) are hormone-regulated transcription factors that play important roles in vertebrate homeostasis, differentiation, and development. T3Rs are synthesized as multiple isoforms that display tissue-specific expression patterns and distinct transcriptional properties. Most T3R isoforms associate with coactivator proteins and mediate transcriptional activation only in the presence of thyroid hormone. The pituitary-specific T3Rß-2 isoform departs from this general rule and is able to interact with p160 coactivators, and to mediate transcriptional activation in both the absence and presence of hormone. We report here that this hormone-independent activation is mediated by contacts between the unique N terminus of T3Rß-2 and an internal interaction domain in the SRC-1 (steroid receptor coactivator-1) and GRIP-1 (glucocorticoid receptor interacting protein 1) coactivators. These hormone-independent contacts between T3Rß-2 and the p160 coactivators are distinct in sequence and function from the LXXLL motifs that mediate hormone-dependent transcriptional activation and resemble instead a mode of coactivator recruitment previously observed only for the steroid hormone receptors and only in the presence of steroid hormone. Our results suggest that the transcriptional properties of the different T3R isoforms represent a combinatorial mixture of repression, antirepression, and hormone-independent and hormone-dependent activation functions that operate in conjunction to determine the ultimate transcriptional outcome.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear hormone receptors are transcription factors that mediate cellular responses to a variety of small lipophilic ligands, including the steroids, vitamin D3, retinoids, and thyroid hormones (1, 2, 3, 4). As such, nuclear hormone receptors play essential roles in metazoan reproduction, development, differentiation, and homeostasis. Nuclear hormone receptors function at the molecular level by binding to specific DNA sequences, denoted response elements, and regulating the expression of adjacent target genes. Intriguingly, many nuclear hormone receptors display bimodal transcriptional properties, with a given receptor able to either repress or activate expression of its target genes under different conditions (5, 6, 7, 8, 9, 10).

Best characterized of this class of bimodal nuclear receptors are the thyroid hormone receptors (T3Rs). T3Rs are expressed in virtually all vertebrate tissues and are involved in regulating such diverse physiological processes as general metabolic rate, thermogenesis, central nervous system development, and glucose utilization in response to T3 and T4 (11, 12, 13, 14). The {alpha}- and ß-loci, in turn, can be expressed through differential splicing to generate three primary T3R protein isoforms: T3R{alpha}-1, T3Rß-0 (in birds, analogous to ß-1 in mammals), and T3Rß-2 (Fig. 1AGo). T3R{alpha}-1 expression begins in early embryonic development and continues into the adult, where it is found in most tissue and cell types; the onset of T3Rß-0/1 and ß-2 expression occurs later in embryonic development and generally parallels the appearance of circulatory T3 and T4 hormone (11, 12, 13, 14). Whereas T3Rß-0/1 is expressed in an assortment of different tissue types, the T3Rß-2 isoform is restricted in its expression primarily to the adult pituitary and hypothalamus (11, 12, 13, 14). Genetic disruption experiments confirm that the T3R{alpha} and T3Rß isoforms play different, if overlapping, roles in normal physiology (12, 14).



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Figure 1. Transcriptional Activation by T3Rß-2 in the Absence of Hormone

A, Schematic of different T3R isoforms. The different T3R isoforms described in the text are represented from N to C terminus, with the locations of the DNA binding and hormone binding domains indicated. The amino acid numbering system for T3Rß-2 is shown beneath the corresponding schematic. B, Transcriptional regulation by full-length T3R isoforms. CV-1 cells (left panel) or JEG-3 cells (right panel) were transfected with a TRE-tk-luciferase reporter, a pCH110-lac Z (CV-1) or pCMV-lac Z (JEG-3) internal control reporter, and with a pSG5-expression vector encoding the avian T3R isoforms indicated beneath the panel. The cells were incubated in the absence (hatched bars) or presence (solid bars) of 100 nM T3 hormone and were subsequently harvested, and the relative luciferase activity was determined as described in Materials and Methods. The averages of two or more experiments and the standard deviations are shown. C, Transcriptional regulation by the abstracted N-terminal domains of different T3R isoforms. CV-1 cells were transfected with a 5 X Gal 17-mer luciferase reporter, a pCMV-lac Z internal control reporter, and a pSG5-GAL4DBD expression plasmid containing the N-terminal 50 amino acids of T3R{alpha}, the N-terminal 70 amino acids of T3Rß-2, or the N-terminal 107 amino acids of T3Rß-2, as indicated below the panel. The cells were incubated in the absence of hormone and were subsequently harvested, and the relative luciferase activity was determined as described in Materials and Methods. The averages of two or more experiments and the SD values are shown.

 
The transcriptional properties of the T3Rs reflect the ability of these receptors to associate with auxiliary protein complexes, denoted corepressors and coactivators (5, 6, 7, 8, 9, 10). Once tethered to the nuclear receptor, the corepressors and coactivators mediate the molecular events that confer the ultimate transcriptional outcome, negative or positive. Known corepressor proteins include SMRT (silencing mediator for retinoid and thyroid hormone receptors) and its paralog N-CoR (nuclear receptor corepressor), mSin3, histone deacetylases, and an assortment of additional polypeptides, such as ski, SAP-18 and SAP-30 (5, 6, 7, 8, 9, 10). Coactivator proteins include the p160 polypeptide coactivator family [such as SRC-1 (steroid receptor coactivator) and GRIP-1 (glucocorticoid receptor interacting protein 1)], the CBP (CREB-binding protein)/p300 proteins, and the DRIP/TRAP/SMCC/ARC complex. Corepressors and coactivators appear to modulate transcription both by covalent modification of the chromatin/nucleosome template and by direct interactions with components of the general transcriptional machinery (5, 6, 7, 8, 9, 10).

T3Rs typically repress transcription in the absence of hormone and activate transcription in the presence of cognate T3 hormone (e.g. Refs. 15, 16, 17, 18). In the absence of hormone, a hydrophobic groove on the surface of the nuclear hormone receptor is believed to bind to an {alpha}-helical I/LXXII motif on the surface of the SMRT/N-CoR corepressor, thereby tethering the corepressor complex to the receptor (19, 20, 21). Conversely, the binding of hormone by the nuclear hormone receptor is thought to reorient the C-terminal helix 12 of the receptor so as to occlude the binding site for corepressor, and to form a new binding site for LXXLL protein interaction motifs that are present in many coactivators (22, 23, 24, 25). The interaction surface on the nuclear hormone receptor that binds coactivator in this hormone-dependent fashion maps to helices 3, 4, 5, and 12 within the hormone-binding domain and has been denoted the "activation function-2" (AF-2) domain (22, 23, 24, 25).

The T3R{alpha}-1 and ß-0/1 isoforms conform to this generic model and operate as transcriptional repressors in the absence of T3 and as activators in its presence (15, 16, 18, 26, 27, 28). The T3Rß-2 isoform, however, is a notable exception and fails to repress in the absence of hormone (18, 28). Instead, in the absence of hormone the T3Rß-2 isoform either is neutral in its effects or exhibits a moderate activation of target gene expression that is further enhanced by the presence of hormone (18, 28, 29, 30). We have reported that the inability of the T3Rß-2 to repress transcription is due to an antirepression mechanism by which the unliganded T3Rß-2 recruits the SMRT/N-CoR protein in an inactive form and prevents the subsequent assembly of a functional corepressor complex (31). This mechanism accounts for the failure of T3Rß-2 to repress, but does not fully explain the hormone-independent T3Rß-2 transcriptional activation that is observed with certain promoters or in certain cell contexts.

Here, we report that there is an isoform-specific transcriptional activation domain in the N terminus of T3Rß-2 that is able to recruit the p160 family of coactivators in the absence of hormone. This N-terminal AF-1 activation domain in T3Rß-2 interacts with an internal glutamine-rich (Q-rich) p160 domain that is distinct in sequence and function from the LXXLL motifs that contact the hormone-dependent AF-2 domain. Therefore, the T3Rß-2 isoform makes at least two distinct contacts with the p160 coactivators: the AF-1/Q-rich p160 domain interaction that prevails in the absence of hormone and the AF-2/LXXLL interaction that dominates in the presence of hormone. This hormone-independent mode of coactivator recruitment by T3Rß-2 is reminiscent of the dual contact mode of coactivator interaction reported for androgen receptor (AR), although for the AR these dual contacts serve to enhance hormone-dependent activation, rather than to mediate hormone-independent transcriptional regulation (32, 33, 34, 35, 36). Unlike T3Rß-2, the T3R{alpha}-1 and ß-0 isoforms lack detectable N-terminal coactivator interaction domains, and interact with p160 coactivator by a single set of contacts in a strictly hormone-dependent manner. Taken as a whole, our results indicate that the transcriptional properties of the different T3R isoforms represent an admixture of repression, antirepression, and hormone-independent and hormone-dependent activation functions that operate in conjunction to determine the ultimate transcriptional outcome.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T3Rß-2 Fails to Repress Transcription and Displays Hormone-Independent Activation in Certain Cell Contexts
We first employed a thymidine kinase promoter-luciferase reporter construct bearing a direct repeat (DR)-4 response element to characterize the transcriptional properties of the different T3R isoforms in CV-1 cells. CV-1 cells express very low levels of endogenous T3Rs and exhibit little or no reporter gene responsiveness in the absence of exogenously introduced receptor (Fig. 1BGo, left panel). As previously noted (31), introduction of the avian T3R{alpha}-1 or T3Rß-0 isoforms repressed reporter gene expression in the absence of hormone, resulting in lower luciferase expression than that observed without receptor (Fig. 1BGo, left panel). Conversely, addition of T3 hormone to the cells transfected with T3R{alpha}-1 or T3Rß-0 resulted in a strong stimulation of reporter gene expression above the levels observed in the absence of receptor (Fig. 1BGo). Introduction of the avian T3Rß-2 isoform resulted in an analogous activation of reporter gene expression in the presence of hormone, but in contrast to the other two isoforms, T3Rß-2 failed to repress in the absence of hormone (Fig. 1BGo, left panel).

Rather than being truly neutral in the absence of hormone, the unliganded T3Rß-2 isoform often induced a weak increase in reporter gene expression above that observed with an empty vector (Fig. 1BGo, left panel). This hormone-independent activation by T3Rß-2 could be observed over a range of expression and reporter vector concentrations (data not shown). Other researchers have also described this phenomenon and have noted that the magnitude of this hormone-independent activation differs with different promoters and in different cell types (18, 28, 29). Consistent with these studies, the hormone-independent activation function of the T3Rß-2 isoform was stronger in JEG-3 cells than in CV-1 cells (Fig. 1BGo, right panel). We conclude that T3Rß-2 not only fails to repress, but can actually activate, gene expression in the absence of hormone.

The N Terminus of T3Rß-2 Possesses a Hormone-Independent Activation Function
The T3Rß-0 and ß-2 isoforms are expressed by alternative mRNA splicing and differ only in that the latter contains an extra N-terminal A/B domain (Fig. 1AGo) (18, 37). We therefore examined whether the N terminus of T3Rß-2 possessed an inherent activation function that might contribute to the hormone-independent activation properties of this isoform. An empty GAL4 DNA binding domain (DBD) construct exhibited little or no ability to modulate the expression of a reporter gene containing GAL4 17-mer binding motifs (Fig. 1CGo). In contrast, GAL4DBD-fusions bearing the N-terminal 1–70, or 1–107 amino acids of T3Rß-2 strongly activated expression of the GAL4 17-mer reporter and, as expected from the nature of the fusion, activation by the T3Rß-2 N-terminus did not require T3 hormone (Fig. 1CGo). The N-terminal A/B domain from the T3R{alpha}-1 isoform, when tested as an analogous GAL4DBD fusion, failed to activate transcription from the GAL4 17-mer reporter (Fig. 1CGo), whereas the T3Rß-0 isoform lacks an A/B domain of significant length to test. We conclude that the T3Rß-2 isoform possesses an autonomous, hormone-independent transcriptional activation function within its N terminus that is not present in T3R{alpha}-1 or ß-0.

T3Rß-2 Displays a Hormone-Independent Interaction with the SRC-1 and GRIP-1 Members of the p160 Coactivator Family
SRC-1 and GRIP-1 are members of the p160 class of transcriptional coactivators and play important roles in hormone-dependent transcriptional activation by a variety of nuclear hormone receptors (38, 39). We therefore examined whether T3R{alpha}-1, ß-0, and ß-2 displayed distinctive interactions with these p160 coactivators that paralleled the transcriptional activation properties of these different isoforms. We first tested whether glutathione S-transferase (GST)-T3R constructs, immobilized on glutathione-agarose, were able to bind to the various full-length p160 coactivators, synthesized and radiolabeled by in vitro transcription and translation. Coactivator bound by the immobilized receptors was eluted from the agarose matrix, was resolved by SDS-PAGE, and was visualized by phosphorimager analysis (Fig. 2Go). Only low, background levels of binding of either SRC-1 or GRIP-1 to GST-T3R{alpha}-1 or ß-0 were observed in the absence of hormone (Fig. 2AGo, lanes 4 and 6, and quantified in Fig. 2Go, B and C). In contrast, both p160 coactivators bound at much higher levels to GST-T3R{alpha}-1 and to GST-T3Rß-0 in the presence of 100 nM T3 (Fig. 2AGo, lanes 5 and 7, and quantified in Fig. 2Go, B and C). Neither coactivator exhibited appreciable binding to nonrecombinant GST protein, used as a negative control, in either the absence or the presence of T3 (Fig. 2AGo, lanes 2 and 3, and quantified in Fig. 2Go, B and C). We conclude that the interaction of T3R{alpha}-1 and T3Rß-0 with SRC-1 or with GRIP-1 is strongly dependent on the presence of hormone.



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Figure 2. Binding of the SRC-1a and GRIP-1 Coactivators to Different GST-T3R Fusion Proteins in Vitro

A, Binding of radiolabeled GRIP-1 and SRC-1a to immobilized GST-T3R protein constructs. Radiolabeled GRIP-1 or SRC-1a was incubated with nonrecombinant GST protein, or with GST-fusion proteins representing the full-length open reading frames of T3R{alpha}-1, ß-0, or ß-2, in the absence or presence of 100 nM T3 hormone as indicated above the panels. The GST proteins, immobilized to a glutathione-agarose matrix, were then extensively washed and any proteins remaining bound to the glutathione-agarose matrix were eluted with soluble glutathione and were resolved by SDS-PAGE. Aliquots of the SRC1a and GRIP-1 preparations employed in the binding assays were analyzed in separate lanes for comparison ("10% input"). A phosphorimager scan of the resulting electrophoretograms is presented. B, Quantification of the SRC-1a results. The results from the experiments depicted in panel A were quantified by phosphorimager analysis; the data are expressed as the percentage of the input coactivator protein that remained bound to the different GST fusion proteins after washing. Different scales are used for the (-) and (+) hormone conditions. C, Quantification of the GRIP-1 results. The same protocol as in panel B was employed.

 
In contrast to the GST-T3R{alpha}-1 or ß-0 isoforms, binding of the GST-T3Rß-2 protein to the p160 coactivators displayed both a hormone-independent and a hormone-dependent component (Fig. 2AGo, lanes 8 and 9). Approximately 8.4% of the SRC-1 input bound to GST-T3Rß-2 in the absence of hormone, and this increased to 30.3% in the presence of 100 nM T3 hormone (Fig. 2BGo). Although modest, this interaction of SRC-1 with T3Rß-2 in the absence of hormone was highly reproducible, was not seen with the nonrecombinant GST control, and was approximately 6-fold above that seen with the other receptor isoforms. An analogous, hormone-independent interaction of GST-T3Rß-2 was also observed with the GRIP-1 coactivator; approximately 4% of the input GRIP-1 protein bound in the absence of hormone and this increased to 67% in the presence of 100 nM T3 hormone (Fig. 2B).

The N Terminus of T3Rß-2 Is Responsible for the Hormone-Independent Recruitment of p160 Coactivators
T3Rß-0 and ß-2 differ only due to the presence of an extended N-terminal domain in the latter (Fig. 1AGo), suggesting that the T3Rß-2 N terminus was likely responsible for the hormone-independent interaction of this isoform with the SRC-1 and GRIP-1 coactivators. To test whether the N terminus of T3Rß-2 alone was sufficient for this interaction, we examined the ability of a GST-fusion restricted to the N-terminal 1–107 amino acids of T3Rß2 to bind to the full-length, radiolabeled p160 coactivators. Both SRC-1 and GRIP-1 were able to bind to the abstracted GST-T3Rß-2 N terminus in vitro, whereas little or no coactivator bound to a nonrecombinant GST control or to a corresponding N-terminal domain of T3R{alpha}-1 (Fig. 3Go). In fact, the hormone-independent interaction of T3Rß-2 with the p160 coactivators was more readily detected in this context than when the GST-full-length receptor fusion was employed; approximately 30% of the input SRC-1 and 24% of the input GRIP-1 could bind to the abstracted T3Rß-2 N terminus under these conditions, whereas binding to the nonrecombinant GST control was less than 0.1% of input (Fig. 3Go, bottom panels).



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Figure 3. Binding of the SRC-1a and GRIP-1 Coactivators to a GST-Construct Limited to the N Terminus of T3Rß-2

An experiment similar to that in Fig. 2Go utilizing radiolabeled GRIP-1 and SRC-1a was performed, but the coactivator proteins were incubated with nonrecombinant GST, with a GST-fusion polypeptide representing the N-terminal 107 amino acids of T3R{alpha}-1, or with a GST-fusion polypeptide representing the N-terminal 107 amino acids of T3Rß-2; no hormone was employed. Coactivator proteins remaining bound to the immobilized GST or GST fusion proteins after washing were eluted, were resolved by SDS-PAGE, and were visualized and quantified by phosphorimager analysis. Both a phosphorimager scan and the quantified results (expressed as the percentage of the input coactivator protein that remained bound to the different GST fusion proteins after washing) are presented.

 
The T3Rß-2 N Terminus Interacts with a Glutamine-Rich p160 Coactivator Domain That Maps Outside of the LXXLL Motifs Implicated in Hormone-Dependent Activation
We next examined the sites of contact of the T3Rß-2 N terminus within the SRC-1 and GRIP-1 coactivators. We employed GST fusions representing different subdomains of the p160 coactivators and determined the ability of these GST constructs, isolated from recombinant Escherichia coli, to bind to the different T3R isoforms, synthesized as full-length proteins by in vitro translation (Fig. 4Go). A phosphorimager scan from a representative experiment utilizing a series of GST-SRC-1 constructs is presented (Fig. 4AGo); this experiment, together with others, was also quantified (Fig. 4Go, B and C). Both full-length T3Rß-0 and full-length T3Rß-2 bound in a hormone-enhanced manner to a GST-SRC-1 fusion construct containing the three internal, clustered LXXLL repeats, whereas little or no binding of T3Rß-0 was observed in the absence of hormone (Fig. 4Go, A and B). This was also true of a derivative of SRC-1 limited to the single LXXLL motif at the extreme C terminus, indicating that this single LXXLL motif alone, which is not found in the GRIP-1 paralog, is sufficient for a hormone-dependent recruitment of this coactivator by the T3Rs (Fig. 4Go, A and B). As expected, little or no binding of T3Rß-0 to any of the SRC-1 derivatives was observed in the absence of hormone (Fig. 4Go, A and B, crosshatched bars).



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Figure 4. Mapping of Interaction Domains within the p160 Coactivators

A, Binding of radiolabeled T3Rß-0 and T3Rß-2 to GST fusion proteins representing different domains of the p160 coactivators. Radiolabeled T3Rß-0 (upper panel) or T3Rß-2 (lower panel) protein preparations were incubated with immobilized GST fusion proteins containing different domains of the SRC-1 protein, as indicated above the panels. The incubations were performed in the absence (-) or presence (+) of T3 hormone. The immobilized GST-protein was then washed extensively (maintaining hormone in the washes where appropriate), and the proteins remaining bound to the agarose matrix were eluted with glutathione and were resolved by SDS-PAGE. Aliquots of the T3Rß-0 and ß-2 preparations employed in the binding assays were analyzed in separate lanes for comparison ("10% input"). The locations of the radiolabeled T3Rs were visualized by phosphorimager analysis. As noted in the text, the T3Rß-2 translation products include a polypeptide, initiated at an internal AUG, that lacks the native T3Rß-2 N terminus; this truncated protein is denoted T3Rß-2t. B, Quantification of the SRC-1a results. The results from the experiments depicted in panel A were quantified by phosphorimager analysis and were combined with similar quantifications of experiments employing additional GST-SRC1a fusion constructs. Schematic representations of the different GST and GST-SRC-1a proteins are depicted on the left, and the quantified data, expressed as the percentage of each T3R input preparation that remained bound to the different GST-fusion proteins, is shown on the right. The schematics indicate the locations of the LXXLL motifs, and of the Q-rich internal domain described in the text. C, Quantification of the GRIP-1 results. A similar protocol as in panel B was employed.

 
Conversely, the hormone-independent interaction of T3Rß-2 with GST-SRC-1 did not require the LXXLL motifs of the coactivator, but was dependent instead on a distinct p160 domain mapping between amino acids 977-1172 of SRC-1 (Fig. 4Go, A and B). Analogous results were obtained when the GST pull-down experiments were performed in a reciprocal manner, using the T3R isoform as the GST fusion and the p160 coactivator as the in vitro translation product (data not shown). Removal of this central, glutamine-rich (Q-rich) interaction domain of SRC-1 greatly reduced the ability of T3Rß-2 to bind to coactivator in the absence of hormone, whereas GST-p160 constructs that retained this central domain retained the hormone-independent interaction with T3Rß-2 (Fig. 4Go, A and B). Very similar results were obtained when GST-GRIP-1 fusions were employed in these assays; the hormone-independent interaction of T3Rß-2 with these GST-GRIP-1 coactivator constructs depended on the presence of amino acids 1121–1304 of GRIP-1, which encompasses a Q-rich region closely analogous to that found in the SRC-1 paralog (Fig. 4CGo). The C-terminal region of GRIP-1 lacks the solitary LXXLL motif found in this region of SRC-1 and therefore did not mediate the hormone-dependent interaction with T3Rß-0 and ß-2 that was observed for the otherwise equivalent region of SRC1a (Fig. 4CGo).

Of note, our in vitro translation reactions produce both full-length T3Rß-2 and an artificially truncated T3Rß-2 derivative that lacks the native N terminus [denoted T3Rß-2t and produced by a translational initiation on an internal AUG; Fig. 4AGo (31)]. The T3Rß-2t derivative retains the C-terminal AF-2 domain and displays the expected, hormone-dependent interaction with GST-p160 fusions that contain one or more LXXLL motifs (Fig. 4Go, A and B). Unlike the full-length T3Rß-2 present in the same assays, however, T3Rß-2t did not exhibit significant binding to any GST-p160 derivative in the absence of hormone (Fig. 4Go, A–C). Conversely, the T3Rß-2 N-terminal domain alone was sufficient for binding to GST-p160 derivatives containing the Q-rich interaction domain, but exhibited greatly reduced or no binding to GST coactivator derivatives lacking this domain (Fig. 5Go). These results further support our assignment of the hormone-independent interaction domain of T3Rß-2 to the receptor’s N terminus and confirm that the receptor N terminus interacts primarily with the central Q-rich domain of the p160 coactivators.



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Figure 5. Interaction of the Abstracted N Terminus of T3Rß-2 with the Q-Rich Domain of the p160 Coactivators

A similar experiment as in Fig. 4AGo was performed, but employing a radiolabeled polypeptide limited to the N-terminal 107 amino acids of T3Rß-2. Both the phosphorimager scans and the quantified results (expressed as the percentage of each T3R input preparation that remained bound to the different GST-fusion proteins) are presented. A, Interaction of the T3Rß-2 N terminus with the GST-SRC-1a constructs. B, Interaction of the T3Rß-2 N terminus with the GST-GRIP-1 constructs.

 
The Central Interaction Domain of the p160 Coactivator Can Function Together with, or Independent of, the LXXLL Motifs to Confer Interaction with T3Rß-2
Our global deletion studies suggested the existence of two interaction sites within the p160 coactivators: the LXXLL motifs, which confer a hormone-dependent interaction with all the T3R isoforms tested, and the central Q-rich domain, which confers a hormone- independent interaction with T3Rß-2. To confirm this hypothesis, we created two, more precisely defined GRIP-1 mutants: a small, in-frame deletion of the central Q-rich domain (denoted {Delta}Q-rich), and a mutant GRIP-1 in which all three LXXLL motifs were changed to LXXAA motifs (denoted LXXAA) (Fig. 6AGo). As expected, the T3Rß-0 isoform displayed little or no interaction with wild-type GRIP-1, or with either GRIP-1 mutant, in the absence of hormone (Fig. 6BGo). In the presence of hormone, T3Rß-0 interacted strongly with wild-type GRIP-1 (Fig. 6CGo); this hormone-dependent interaction was severely inhibited by the LXXLL to LXXAA GRIP-1 mutation, but not by deletion of the Q-rich coactivator interaction domain (Fig. 6CGo). Conversely, the hormone-independent interaction of T3Rß-2 with GRIP-1 was eliminated by deletion of the internal Q-rich interaction domain of the coactivator, but was retained in the LXXLL to LXXAA GRIP-1 mutant (Fig. 6BGo). Intriguingly, the LXXLL to LXXAA mutation of GRIP-1 impaired, but did not fully eliminate, the hormone-dependent component of the interaction of T3Rß-2 with this coactivator (Fig. 6CGo). We suggest that the LXXAA substitution mutant of GRIP-1 retains a residual ability to interact with all T3R isoforms in the presence of hormone, as can be observed for T3Rß-0, and that this very modest hormone-dependent interaction is further stabilized for T3Rß-2 due to the additional interaction surface conferred by the N terminus of this isoform. Consistent with this proposal, GRIP-1 mutants that delete, rather than modify, the LXXLL motifs, or that lack both the LXXLL and central interaction domains, do not display this residual, hormone-dependent interaction with T3Rß-2 (e.g. Fig. 4Go and data not shown).



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Figure 6. Interaction of the T3Rß-0 and T3Rß-2 Isoforms with Specific p160 Coactivator Mutants

A, Schematic of the GRIP-1 mutants employed. A schematic representation of the GRIP-1 coactivator is presented from N to C termini. The locations of the LXXLL to LXXAA and the {Delta}Q-rich mutations, described in the text, are shown. B and C, Binding of radiolabeled mutant and wild-type GRIP-1 proteins to different, immobilized GST-T3R fusions. Radiolabeled wild-type or mutant GRIP-1 proteins (as indicated below the panels) were incubated with nonrecombinant GST protein, or with GST-fusion proteins representing the full-length open reading frames of T3Rß-0 or T3Rß-2 (as indicated within each panel). The incubations and washes were performed either in the absence (panel B) or presence (panel C) of 100 nM T3 hormone. The amount of each GRIP-1 protein bound to the immobilized GST-T3R polypeptides after washing was determined as described in Fig. 2Go.

 
The same central, Q-rich region of the p160 coactivators identified here as the interaction site for the T3Rß-2 N terminus has also been reported to be an interaction site for the AF-1 domains present in a number of steroid receptors, such as the AR (32, 33, 34, 35, 36, 40). Unlike T3Rs, however, the unliganded steroid receptors typically are cytoplasmic and do not associate with DNA or activate transcription except on addition of hormone. The AF-1 domain in the native AR, for example, does not function in the absence of androgen, but serves instead in the presence of hormone to stabilize an otherwise weak interaction of the AR AF-2 domain with the coactivator’s LXXLL motifs (32, 33, 34, 35, 36). We investigated whether there was a detectable structural relatedness between the T3Rß-2 and AR N termini. Indeed, several regions of apparent amino acid relatedness could be discerned (Fig. 7Go). The highest level of relatedness was observed in comparisons of amino acids 1–107 of T3Rß-2 with amino acids 360–556 of AR; these two domains shared a global amino acid identity of 21% and a global similarity of 44% in a four-way comparison of avian and human T3Rß-2 to human and mouse AR (Fig. 7Go).



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Figure 7. Comparison of the N-Terminal Domain of T3Rß-2 to That of the AR

The amino acid sequences of the N termini of human (h) and chicken (c) T3Rß-2 s are aligned with a portion of the N termini of the mouse (m) and human (h) ARs. Gaps (dashes) have been introduced to maximize matches between the two sequences. Double dots indicate identical amino acids in equivalent positions in all four sequences; single dots indicate related amino acids in equivalent positions in the four sequences. The amino acid numbering systems are depicted to the left of the corresponding sequences.

 
The Interaction of the N Terminus of T3Rß-2 with p160 Coactivators in Vitro Correlates with Hormone-Independent Transcriptional Activation in Vivo
We asked whether the interactions we observed between the T3Rß-2 N terminus and p160 coactivators in vitro correlated with hormone-independent transcriptional activation in vivo. We first examined whether mutations in the T3Rß-2 N terminus that disrupt p160 coactivator interaction in vitro also impair T3Rß-2 mediated transcriptional activation in transfected cells. A T3Rß-2 bearing an N-terminal deletion of amino acids 6–20 retained a hormone-independent interaction with coactivator in vitro and retained the ability to activate transcription as a GAL4DBD fusion in vivo (Fig. 8Go, A and B). A T3Rß-2 mutant bearing a larger N-terminal deletion, removing amino acids 6–40, exhibited a significant reduction in both the ability to interact with p160 coactivator in vitro, and in the ability to activate GAL4 17-mer reporter gene expression in vivo (Fig. 8Go, A and B). Still larger deletions within the T3Rß-2 N terminus also reduced both coactivator binding in vitro and transcriptional activation in transfected cells, although there is some suggestion of an additional, residual, and relatively weak activation domain mapping C-terminal to amino acid 70 (Fig. 8Go, A and B). A parallel phenomenon was observed in the native T3Rß-2 context, with a deletion of amino acids 6–20 of the T3Rß-2 N terminus having a slightly stimulatory effect on reporter gene expression in the absence of hormone, whereas deletions removing amino acids 6–40 of T3Rß-2 abolished transcriptional activation in this background (Fig. 8CGo). More extreme deletions of the native T3Rß-2 N terminus, removing amino acids 6–60 or 6–70, impinge on the antirepression domain previously described, and not only abolished hormone-independent activation, but converted the unliganded T3Rß-2 into a transcriptional repressor (Fig. 8CGo).



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Figure 8. Mapping of the p160 Interaction and Transcriptional Activation Domains within the T3Rß-2 N Terminus

A, The effect of different deletion mutations on the ability of the T3Rß-2 to interact with SRC1a in the absence of hormone. The ability of different wild-type and mutant T3Rß constructs, expressed as radiolabeled in vitro translation products, to bind to diffferent GST or GST-SRC1a fusions, indicated below the panel, was determined as in Fig. 4Go. B, The effect of different deletion mutations on the ability of the T3Rß-2 N terminus to activate transcription when fused to a GAL4DBD. An experiment similar to that described for Fig. 1C was performed, but utilizing a series of deletions of the T3Rß-2 (1–107 amino acid) N terminus fused to the GAL4DBD. Each mutant construct, as indicated below the panel, was tested for the ability to activate expression of a 5 X GAL 17-mer luciferase reporter when transfected into CV-1 cells. An empty GAL4DBD construct ("empty") and a GAL4DBD fused to the intact 1–107 amino acid domain of T3Rß-2 ("Full") were also tested in parallel for comparison. The average and SD values of two or more repeat experiments are displayed. C, The effect of different deletion mutations on the ability of the native T3Rß-2 to regulate transcription from a hormone response element-reporter construct. An experiment similar to that described for Fig. 1BGo was performed, but utilizing a series of otherwise full-length T3Rß-2 constructs bearing different N-terminal deletion mutations. CV-1 cells were transfected with a TRE-tk-luciferase reporter, a pCH110-lac Z reporter (employed as an internal control), and with a pSG5-expression vector encoding the T3Rß-2 deletion mutants indicated beneath the panel. The cells were incubated in the absence of T3 hormone and were subsequently harvested, and the relative luciferase activity was determined as described in Materials and Methods. The averages of two or more experiments and the SD values are shown.

 
We next tested the effect of ectopic p160 expression on the ability of the T3Rß-2 N terminus to activate transcription when fused to a GAL4DBD. As noted previously, GAL4DBD-fusions bearing the first 1–70, or 1–107 amino acids of T3Rß-2 were able to activate expression of a GAL4 17-mer reporter in transfected CV-1 cells (compare panels B and C in Fig. 9Go with panel A). Cointroduction of ectopic GRIP-1 further enhanced this activation in a dose-dependent manner (Fig. 9Go, B and C). In contrast, cointroduction of GRIP-1 had no significant effect on transcriptional activation by an empty GAL4DBD, by a GAL4DBD-T3R{alpha}-1 N terminus construct, or by a constitutive promoter/ß-galactosidase reporter used as an internal control (Fig. 9Go, A and D, and data not shown). These results suggest that a functional, as well as a physical, interaction can occur between the T3Rß-2 N terminus and the p160 coactivators. We next tested how mutations in the p160 coactivator affected the ability of the coactivator to function with the T3Rß-2 N terminus. A GRIP-1 construct containing the central interaction domain, but lacking the three LXXLL motifs, retained the ability to enhance transcriptional activation by the T3Rß-2 N terminus, whereas a GRIP-1 construct that lacked the internal interaction domain but contained the LXXLL motifs did not significantly enhance reporter expression by the T3Rß-2 N terminus (Fig. 9Go, B and C). We conclude that the ability of the T3Rß-2 N terminus to interact with the internal domain of the p160 coactivators in vitro correlates closely with the ability of the T3Rß-2 N terminus to activate transcription in vivo.



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Figure 9. Functional Interactions between the T3Rß-2 N Terminus and the p160 Coactivators

GAL4DBD constructs either lacking additional sequences (panel A), or fused to the N-terminal 1–70 amino acids of T3Rß-2 (panel B), N-terminal 1–107 amino acids of T3Rß-2 (panel C), or N-terminal 1–50 amino acids of T3R{alpha}-1 (panel D), were tested. These constructs were introduced into CV-1 cells together with a 5 X GAL 17-mer reporter, a pCMV-lac Z reporter (used as an internal control), and varying amounts of a expression vector for wild-type GRIP-1, for the {Delta}Q-rich GRIP-1 mutant, or for the LXXLL to LXXAA GRIP-1 mutant, as indicated below the panels. The cells were incubated in the absence of T3 hormone and harvested, and the relative luciferase activity was determined as described in Materials and Methods.

 
The T3Rß-2 N Terminus Can Interact Simultaneously with p160 Coactivators and with SMRT Corepressor
We have noted previously that the N terminus of the T3Rß-2 isoform makes contacts with the silencing domain of SMRT corepressor, and that these contacts interfere with assembly of a functional corepressor complex (31). We have demonstrated here that an adjacent region of the same T3Rß-2 N-terminal domain can make contact with the Q-rich region of the p160 coactivators. Can these interactions occur simultaneously, such that, as suggested by our model, the N terminus of T3Rß-2 both prevents repression and mediates activation in the absence of hormone? To test this hypothesis, we examined the ability of the abstracted T3Rß-2 N terminus, expressed and purified as a maltose-binding protein (MBP) fusion, to serve as a bridge to tether a radiolabeled, soluble GRIP-1 protein to an immobilized GST-SMRT construct. Little or no binding of the radiolabeled GRIP-1 protein was observed to a nonrecombinant GST construct employed as a negative control (Fig. 10Go, top panel). Addition of either the MBP-T3Rß-2 (1–107) fusion protein, or a native MBP protein to the binding reaction failed to alter this lack of interaction between GRIP-1 and nonrecombinant GST (Fig. 10Go, top panel). Similarly, GRIP-1 did not bind to a GST-SMRT silencing domain fusion either alone or when tested in the presence of the native MBP (Fig. 10Go, bottom panel). However, addition of increasing amounts of the MBP-T3Rß-2 (1–107) fusion protein resulted in a parallel increase in the ability of the radiolabeled GRIP-1 to bind to the GST-SMRT silencing domain fusion (Fig. 10Go, bottom panel). We conclude that the T3Rß-2 N terminus has the capacity to interact concurrently with both SMRT corepressor and p160 coactivators, consistent with a model wherein both antirepression and hormone-independent activation occur simultaneously.



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Figure 10. Simultaneous Interaction of the T3Rß-2 N Terminus with SMRT Corepressor and GRIP-1 Coactivator

The ability of a radiolabeled GRIP-1 protein, synthesized by in vitro transcription and translation, to bind to an immobilized nonrecombinant GST protein (top panel) or to an immobilized GST-SMRT (566–680) construct (representing a portion of the SMRT corepressor silencing domain) was tested by the same general protocol as described for GST-coactivators in Fig. 4Go. The binding reactions were carried out with no further additions (None), in the presence of the indicated amounts of a native MBP, or in the presence of the indicated amounts of a MBP fusion protein representing the N-terminal 1–107 amino acids of T3Rß-2 [MBP-T3Rß-2 (NOREF>1–107)]. Radiolabeled GRIP-1 remaining bound to the GST constructs after repeated washings was eluted and was visualized by SDS-PAGE and phosphorimager analysis.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The N Terminus of T3Rß-2 Contributes to Transcriptional Activation in the Absence of Hormone by Interacting with a Central Domain of the p160 Coactivators
Unlike the T3R{alpha}-1 and T3Rß-0/1 isoforms, the T3Rß-2 isoform is expressed in a highly restrictive tissue pattern limited principally to the hypothalamus and pituitary (12, 13, 14, 41). The T3Rß-2 isoform is also unique in its transcriptional regulatory properties. In contrast to the other T3R isoforms, T3Rß-2 fails to repress transcription in the absence of hormone. Instead, the T3Rß-2 isoform activates transcription in the absence of hormone, and this activation is further stimulated in the presence of hormone (18, 28, 29, 31, 42). We have previously reported that the inability of the T3Rß-2 isoform to repress is not due to a failure to recruit the SMRT or N-CoR corepressors; in common with T3R{alpha}-1 and T3Rß-0/1, the T3Rß-2 isoform interacts strongly with SMRT and with N-CoR in the absence of hormone (31). However, the T3Rß-2 isoform makes additional contacts with the silencing domains of SMRT and N-CoR that are not observed with the T3R isoforms that do repress (31). These additional contacts of T3Rß-2 with SMRT and N-CoR interfere with the subsequent assembly of a larger, functional corepressor complex, a phenomenon that we have denoted antirepression (31). Therefore, the unliganded T3Rß-2 interacts with corepressor, but in an abortive fashion that appears to preclude repression.

Although accounting for the lack of repression by T3Rß-2, this antirepression model does not explain the additional, hormone-independent activation observed for this isoform in many cell lines and promoter contexts. We report here that hormone-independent transcriptional activation by T3Rß-2 maps to the N-terminal domain of this receptor, and that the isolated T3Rß-2 N-terminal domain, when fused to an ectopic GAL4DBD, is capable of autonomous, hormone-independent transcriptional activation in transfected cells. Our results map these transcriptional activation properties to the T3Rß-2 N terminus, with the bulk of this activity requiring amino acids 20 to 40. Our experiments also suggest the existence of a more minor, transcriptional activation domain mapping between amino acids 70 and 107 that is only observed on deletion of more N-terminal sequences. Significantly, both of these activation domains are outside of the region of the T3Rß-2 N terminus that we have shown to mediate antirepression, indicating that activation and antirepression are distinct phenomena. The presence of hormone-independent transcriptional activation domains within the T3Rß-2 N terminus has been previously noted, although there has been disagreement as to the precise location of these activities (18, 28, 30). Our own mapping studies, reported here, generally agree with and help reconcile these prior reports by confirming that there are at least two distinct transcriptional activation domains within the T3Rß-2 N terminus.

How might the hormone-independent T3Rß-2 N-terminal transactivation domains function? The ability of the nuclear hormone receptors to activate transcription in the presence of hormone is dependent on a conformational change in the receptor on binding of hormone agonist, leading to a reorientation of the C-terminal helix 12 domain (19, 20, 21, 22, 24, 25). The reoriented helix 12, together with portions of helix 3, 4, and 5 of the hormone binding domain of the receptor (denoted the AF-2 domain), forms a charge-clamped groove on the surface of the receptor that can interact with LXXLL motifs present in many of the coactivator polypeptides (22, 24, 25). Thus, for most of the nuclear hormone receptors characterized, acquisition of coactivator is strongly dependent on hormone binding. The same mechanism appears to be operative for all the isoforms of T3R tested here, including T3Rß-2. However, in addition to these hormone-dependent interactions of the C-terminal AF-2 domain, the N terminus of T3Rß-2 displays an additional, hormone-independent interaction with the p160 coactivators that is not observed for T3R{alpha}-1 or ß-0. This hormone-independent interaction of the T3Rß-2 N terminus with the SRC-1 and GRIP-1 coactivators can be observed in the absence of the receptor AF-2 domain.

The interaction of the T3Rß-2 N terminus with the SRC-1 and GRIP coactivators closely correlates with the hormone-independent transcriptional activation mediated by T3Rß-2 in transfected cells. For example, the ability of the T3Rß-2 N terminus to activate transcription is significantly enhanced by the cointroduction of ectopic GRIP-1. Conversely, mutations of the T3Rß-2 N terminus that impair its interaction with SRC-1 or GRIP-1 also impair hormone-independent activation without affecting hormone-dependent activation. These results are indicative of a functional, as well as a physical, interaction between the T3Rß-2 N terminus and the p160 coactivators.

As detailed above, hormone-dependent transcriptional activation by nuclear hormone receptors is thought to be primarily mediated by an agonist- induced formation of a docking surface within the AF-2 domain of the receptor that can then interact with the LXXLL amino acid motifs present in many coactivators (22, 24, 25). Indeed, in our experiments the hormone-dependent interaction of the T3R{alpha}-1 and ß-0 isoforms with SRC-1 or GRIP-1 required the presence of at least one LXXLL motif within the p160 coactivators. In contrast to these LXXLL motifs, which mediate hormone-dependent p160 interaction, the hormone-independent interaction of T3Rß-2 with these coactivators was conferred primarily by a distinct set of contacts between the receptor N terminus and a central, glutamine-rich domain of SRC-1 and GRIP-1. Deletion of this glutamine-rich coactivator domain, corresponding to amino acids 1121–1305 of GRIP-1 and equivalent to amino acids 977-1172 of SRC-1, abolished the hormone-independent interaction of native T3Rß-2 with these coactivators without impairing the hormone-dependent interaction of T3Rß-2, T3R{alpha}-1, or T3Rß-0. Consistent with this central coactivator domain being responsible for the hormone-independent activation mediated by T3Rß-2 in vivo, a mutant GRIP-1 lacking the central interaction domain did not enhance transcriptional activation by the T3Rß-2 N terminus, whereas wild-type GRIP-1, or a GRIP-1 mutant retaining the internal interaction domain but lacking the LXXLL motifs, did significantly enhance transcriptional activation by the T3Rß-2 N terminus.

The central glutamine-rich interaction domain of GRIP-1 and SRC-1, implicated here in hormone-independent activation by T3Rß-2, does not display detectable sequence relatedness to the LXXLL coactivator motifs that mediate hormone-dependent activation, nor does the N-terminal region of T3Rß-2 display significant sequence relatedness to the hormone-dependent AF-2 region of this receptor. Intriguingly, however, the N terminus of T3Rß-2 does display detectable sequence relatedness to an N-terminal domain within the ARs that also interacts with the central domain of the p160 coactivators (32, 33, 34, 35, 36). The sequence relatedness between the T3Rß-2 and the AR N termini is modest overall, but includes several subregions exhibiting near 25% identity and more than 50% similarity; notably the regions of highest interrelatedness correspond to the receptor regions necessary for interaction with the p160 coactivators in our genetic analysis. These sequence similarities between T3Rß-2 and AR suggest that a common structural motif, perhaps reflected within the three-dimensional structure of these N-terminal domains, may be responsible for the contacts of these receptors with the p160 central interaction domain.

It is interesting that two nuclear receptors as diverse as T3Rß-2 and AR share a similar, dual contact mode of interaction with the p160 coactivators, particularly given that these shared coactivator contacts appear to be used for different purposes by the two different receptors. The ARs are cytoplasmic in the absence of hormone, and neither directly activate nor directly repress transcription in the unliganded state. Instead, the N-terminal domain of the native ARs is believed to function primarily in the presence of hormone, operating to stabilize an otherwise weak, hormone-dependent interaction of the AR AF-2 domain with the LXXLL coactivator motifs (32, 33, 34, 35, 36, 43). Thus, in AR, the N terminus bolsters the function of the AF-2 domain. In contrast, in T3Rß-2 the N terminus appears able to operate independently of the AF-2 domain to confer transcriptional activation in the absence of hormone. However, our experiments do not preclude an additional role for the T3Rß-2 N terminus in the presence of hormone. In fact, we observed that, even in the presence of hormone, the interaction of T3Rß-2 with mutant coactivators can be stabilized by the interaction of the T3Rß-2 N terminus with the central coactivator domain.

Transcriptional Regulation by Different T3R Isoforms Represents a Mix of Repression, Antirepression, Hormone-Dependent Activation, and Hormone-Independent Activation Functions
The hormone binding domain of the nuclear hormone receptors serves as a primary site of receptor interaction with both corepressors and coactivators. Within the hormone binding domain, portions of helices 3, 5/6, and 11 form overlapping binding sites for corepressors and coactivators, with a hormone-mediated reorientation of helix 12 adjudicating the recruitment of one or the other cofactor. However, our own work, together with that of others, indicates that the N-terminal domain of many of these nuclear receptors makes important additional contacts with corepressors and coactivators, and that these additional contacts can play a pivotal role in regulating transcription. The T3Rß-2 N terminus is a particularly interesting case in point. The T3Rß-2 N terminus makes contacts with both corepressors and with coactivators (31, 44). These N-terminal-mediated contacts act in addition to the corepressor and coactivator contacts conferred by the hormone binding domain and serve both to prevent assembly of a functional corepressor complex and to recruit coactivator in the absence of hormone. In this manner, the intrinsic transcriptional regulatory properties defined by the hormone binding domain are overridden, and the T3Rß-2 is able to activate transcription in both the absence and presence of hormone. Conversely, the absence of these N-terminal modulatory domains in the otherwise identical T3Rß-0 isoform permit the unfettered dominance of the actions of the hormone binding domain, which manifest as repression in the unliganded state and as activation in the presence of hormone.

The use of alternative mRNA splicing to generate both hormone-independent and hormone-dependent forms of transcriptional activator from the same locus appears to be unique to the T3R class of nuclear hormone receptors. However, many other nuclear hormone receptors also encode transcriptional regulatory sequences within their N termini, and a number of these N-terminal sequences appear to operate by recruiting coactivators of various genres (e.g. Refs. 32, 33, 34, 35, 36, 40, 43). These N-terminal coactivator interaction surfaces can confer transcriptional activation in the absence of hormone, can stabilize an otherwise weak coactivator interaction in the presence of hormone, or can diversify the transcriptional response by recruiting coactivators of more than one class to a single nuclear receptor molecule. Taken as a whole, it appears that the nuclear hormone receptors are modular structures that can incorporate a variety of hormone-dependent and independent interaction surfaces for transcriptional cofactors. The contributions of these distinct interaction surfaces can vary for different receptors and receptor isoforms, and these differences result in different modes of coactivator and corepressor recruitment. The particular transcriptional properties of a particular nuclear hormone receptor therefore are a combinatorial mixture of activation and repression, resulting in a transcriptional output that can be carefully customized through evolution to the most physiologically appropriate phenotype.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
The wild-type pSG5-avian T3R{alpha}-1, pSG5-human T3Rß-1, and pSG5-avian T3Rß-2 constructs were described previously (18, 45, 46). The pSG5 avian T3Rß-0 construct was created using PCR and appropriate oligonucleotide primers to add a Kozak consensus/translational start sequence and the T3Rß-0 N-terminal amino acid sequence to the C-terminal portion of the T3Rß open reading frame (47). The pSG5-T3Rß-2 (amino acids 1–107) construct was created by PCR amplification using appropriate primers and standard recombinant DNA techniques (47). The generation of the pSG5-Gal4DBD-T3Rß-1 (amino acids 1–101) construct and of the in-frame, N-terminal deletion mutants of T3Rß-2 ({Delta}6–20, {Delta}6–40, {Delta}6–60, and {Delta}6–70) were previously described (31). The pSG5-GAL4DBD-T3R{alpha}-1 (amino acids 1–50) and the pSG5-Gal4DBD-T3Rß-2 (amino acids 1–70 or amino acids 1–107) constructs were created by standard recombinant DNA approaches, using PCR amplification to introduce appropriate restriction sites where necessary. The pSG5-Gal4DBD-T3Rß-2 N-terminal deletions were constructed by a similar approach, but utilizing the pSG5-T3Rß-2 deletion mutants, described above, as templates.

The different T3R isoforms were cloned into the pGEX-2T and pGEX-KG vectors as full-length constructs by use of PCR amplification, employing oligonucleotide primers to insert appropriate restriction sites, followed by standard recombinant DNA techniques to reconstitute the intact open reading frames (48). The pGEX-KG-avian T3Rß2 (amino acids 1–107) plasmid was constructed by releasing a EcoRI to BamHI fragment from the pSG5-Gal4DBD-T3Rß2 (amino acids 1–107) construct. The pGEX-KG vector was then cleaved with XbaI. Both the linearized vector and the fragment were treated with T4 DNA polymerase, and the resulting blunt-ended DNA fragments were ligated together; the resulting clones were screened for the correct insert orientation.

The pSG5-GRIP-1 and pCR3.1-SRC1a expression plasmids were previously described (38, 39). The GRIP-1 (LXXAA) and GRIP-1 ({Delta}Q-rich) mutants were created by a QuikChange site-directed mutagenesis method, using the protocol recommended by the manufacturer (Stratagene, La Jolla, CA). The latter represents a deletion of amino acids 1185–1260 of GRIP-1. The identity of all mutations was subsequently confirmed by DNA sequence analysis.

The pCR3.1 clones representing subdomains of SRC-1a or GRIP-1 were constructed by PCR amplification using appropriate oligonucleotide primers so as to introduce flanking EcoRI and BamHI restriction sites (for SRC-1a) or EcoRI and BglII restriction sites (for GRIP-1), as well as appropriate upstream Kozak translational start sequences; the resulting restriction fragments were inserted into the EcoRI and BamHI sites in the pCR3.1 vector. The subclones created in this fashion included pCR3.1-SRC-1a (amino acids 977-1441), pCR3.1-SRC-1a (amino acids 1172–1441), pCR3.1-GRIP-1 (a.a.1122–1462), and pCR3.1-GRIP-1 (amino acids 1305–1462). The corresponding pGEX-KG versions of these subclones were constructed by PCR amplification using pairs of primers similar to those described above; the PCR products were subsequently cleaved and ligated into the EcoRI and BamHI sites in the pGEX-KG vector. The pGEX-KG-SRC-1a (560–1136) plasmid was described previously (49).

Protein-Protein Interaction Assays
GST fusion proteins were isolated from Escherichia coli (DH5{alpha}-strain) transformed by the corresponding pGEX-KG or pGEX-2T vectors, and the resulting GST fusion proteins were purified and immobilized by binding to glutathione-conjugated agarose beads as previously described (48). The pSG5- or pCR3.1-based plasmids were transcribed and translated into 35S-radiolabeled proteins in vitro by use of a T7 RNA polymerase-coupled TnT kit (Promega Corp., Madison WI). The 35S-labeled proteins were subsequently incubated for 2 h at 4 C with 25 µl of a 50% slurry of the appropriate immobilized GST fusion protein in 300 µl of HEMG buffer (40 mM HEPES, pH 7.8, 50 mM KCl, 0.2 mM EDTA, 5 mM MgCl2, 0.1% Triton X-100, 10% glycerol, and 1.5 mM dithiothreitol) containing 1x Complete Proteinase Inhibitor (Roche Molecular Biochemicals, Indianapolis, IN) and 10 mg/ml of BSA. After incubation, the immobilized GST-proteins, and any polypeptides bound to them, were washed four times with 500 µl of HEMG buffer. Proteins remaining bound to the glutathione-agarose matrix were eluted with free glutathione and were resolved by SDS-PAGE. The electrophoretograms were visualized and quantified by PhosphorImage analysis (Storm System, Molecular Dynamics, Inc., Sunnyvale, CA).

Transient Transfections
CV-1 cells were maintained at 37 C in DMEM containing 10% FBS in a 5% CO2/bicarbonate-buffering system. Cells were transfected by a Lipofectin-mediated method following the recommendations of the manufacturer (Life Technologies, Gaithersburg, MD). For assays of the function of the full-length T3Rs, approximately 5 x 106 cells were transfected with 20 ng of a pSG5 -T3R{alpha}, -T3Rß-0, -T3Rß-1, -T3Rß-2, or -T3Rß-2 mutant plasmid, 50 ng of a pCH110-lac Z plasmid (employed as an internal transfection control), and 100 ng of a ptk-DR4-luciferase reporter containing a DR-4 thyroid hormone response element (31, 49). pUC18 plasmid was employed to normalize the total transfected DNA per sample to 500 ng. The transfected cells were subsequently propagated in DMEM containing 10% (hormone-depleted) FBS in the presence of either 100 nM of T3-thyronine, or an equivalent amount of ethanol carrier, and were harvested at 48 h post transfection. The luciferase activity was measured and was normalized relative to ß-galactosidase activity as previously described (31, 49). Transcriptional activation by the pSG5-GAL4DBD fusions was assayed by a similar Lipofectin protocol, but employing 25 ng of a pSG5-GAL4DBD vector (either empty or containing a GAL4DBD-N-terminal T3R fusion), 25 ng of a pCMV-lac Z plasmid as an internal control, and 100 ng of a 5 x GAL 17-mer luciferase reporter construct (50). Additional pBluescript plasmid was used to bring the total amount of transfected DNA per assay to 500 ng. The cells were harvested at 48 h post transfection and assayed for luciferase and ß-galactosidase activity as described above. In certain experiments, a pSG5-GRIP-1, pSG5-GRIP-1(LXXLL to LXXAA), or pSG5-GRIP-1({Delta}Q-rich) expression plasmid was also included in the transfections, as indicated in the appropriate figure legends.

JEG-3 cells were maintained in DMEM containing 10% FBS. Transfections of JEG-3 cells were performed using Effectene and the protocol suggested by the manufacturer (QIAGEN, Valencia, CA). Approximately 1.5 x 105 cells were seeded 24 h in advance. The cells were then transfected with 20 ng of the pSG5-T3Rß-2 plasmid, 25 ng of the pCMV-lacZ plasmid, and 100 ng of the ptk-DR4-luciferase reporter plasmid. Additional pUC18 plasmid was used to bring the total amount of transfected DNA per assay to 500 ng. Transfected cells were washed after 6 h and were placed in fresh medium in the presence of 100 nM of T3 thyronine, or an equivalent amount of ethanol carrier. The cells were harvested 24 h post transfection. The luciferase activity was measured and was normalized to ß-galactosidase activity as previously described (31, 49).

Pairwise Sequence Alignment
The N-terminal 107 amino acid of T3Rß-2 was compared by a pairwise sequence alignment to amino acids 360 to 556 of the human AR, using the GeneStream sequence alignment program of BCM Search Launcher (The Baylor College of Medicine Search Launcher).


    ACKNOWLEDGMENTS
 
We thank R. Evans, B. O’Malley, and M. Stallcup for their generosity in providing molecular clones and Valentina Taryanik for dedicated technical assistance.

This work was supported by Public Health Services/NIH Grants R37 CA-53394 and R01 DK-53528.


    FOOTNOTES
 
Address requests for reprints to: Martin L. Privalsky, Section of Microbiology, Division of Biological Chemistry, One Shields Avenue, University of California at Davis, Davis, California 95616. E-mail: mlprivalsky{at}ucdavis.edu

Received for publication December 27, 2000. Revision received March 7, 2001. Accepted for publication March 12, 2001.


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 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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