Pituitary Resistance to Thyroid Hormone Syndrome Is Associated with T3 Receptor Mutants that Selectively Impair ß2 Isoform Function
Wei Wan,
Behnom Farboud and
Martin L. Privalsky
Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California 95616
Address all correspondence and requests for reprints to: Martin L. Privalsky, Section of Microbiology, One Shields Avenue, University of California at Davis, Davis, California 95616. E-mail: mlprivalsky{at}ucdavis.edu.
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ABSTRACT
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Resistance to thyroid hormone (RTH) syndrome is an inherited inability to respond appropriately to T3 hormone. In generalized RTH, the T3 response of both the pituitary and periphery is disrupted. In pituitary (or central) RTH, the ability of the pituitary to sense (and down-regulate) elevated T3 is selectively impaired, whereas the periphery remains relatively T3 responsive, resulting in peripheral thyrotoxicity. Both forms of disease are linked to mutations in thyroid hormone receptor (TR)-ß. TRß is expressed by alternate mRNA splicing as two isoforms: TRß2, found primarily in the pituitary/hypothalamus, and TRß1, expressed broadly in many tissues. We report here that the wild-type TRß2 isoform displays an enhanced T3 response relative to the TRß1 isoform. Mutations associated with generalized RTH (P453S, G345S) impair both TRß2 and TRß1 function proportionally, whereas mutations associated with pituitary-specific RTH (R338L, R338W, R429Q) disproportionately disrupt TRß2 function. We propose that in the normal organism, and in generalized RTH, TRß2 in the pituitary can sense rising T3 levels in advance of TRß1 in the periphery, preventing thyrotoxicity. In contrast, the TRß mutations associated with pituitary RTH disproportionately disrupt the pituitarys ability to sense and suppress elevated T3 levels in advance of the periphery, producing symptoms of thyrotoxicity.
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INTRODUCTION
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THYROID HORMONE (T3 and its prohormone T4) play key roles in vertebrate physiology and development (1, 2, 3, 4, 5). T3 functions by binding to thyroid hormone receptors (TRs), which bind in turn to specific DNA response elements and either repress or activate transcription of adjacent target genes (6, 7, 8, 9, 10). On positive-acting DNA response elements, TRs recruit corepressors and repress target gene transcription in the absence of hormone, but release corepressors, recruit coactivators, and activate transcription in response to T3 (6, 9, 10, 11, 12). Less well understood are negative-acting DNA response elements that are activated by TRs in the absence, but repressed in the presence of T3; although corepressors and coactivators have been implicated in negative response element regulation, their precise roles remain incompletely understood (e.g. Refs. 13, 14, 15, 16). Negative response elements are found in the TSH
, TSHß, and TRH genes, where they participate in an important negative feedback loop through which the pituitary/ hypothalamus/thyroid axis stabilizes circulating T3/T4 levels (17, 18, 19, 20, 21, 22, 23, 24, 25).
TRs are encoded by multiple genetic loci and by alternative mRNA splicing to generate a series of interrelated receptor isoforms: TR
1, TRß1, and TRß2 (10, 26). Both TR
1 and TRß1 are expressed in virtually all adult tissues (2, 27). In contrast, TRß2 is highly restricted in its expression pattern and is most abundant in the pituitary and hypothalamus (2, 27). The different isoforms of TR play distinct, if overlapping, roles in development and homeostasis (1, 2, 10, 26, 27, 28, 29, 30, 31, 32). The TRß2 isoform, for example, although able to activate positive-response genes, plays a particularly important function in the negative feedback suppression of T3/T4 synthesis in the pituitary/hypothalamus/thyroid axis (5, 17, 28, 31, 33, 34, 35, 36).
Inherited mutations in TRß result in resistance to thyroid hormone (RTH) syndrome, a human endocrine disorder that manifests as an inability to respond appropriately to circulating T3/T4 (5, 27, 37, 38, 39, 40, 41, 42, 43). In most kindreds characterized, the mutant TR fails to release corepressor and/or to bind coactivator at physiological T3 levels (16, 44, 45, 46, 47, 48, 49, 50, 51, 52). As a result, RTH-mutant TRs are impaired in their ability to activate positive-acting response elements and to repress negative response elements. Notably, these RTH-mutant TRs function as dominant negatives and can interfere in trans with the actions of wild-type TRs coexpressed in the same cells (i.e. from the TR
locus or from the unaffected TRß allele in a heterozygote).
Two broad categories of RTH syndrome, generalized vs. pituitary, have been defined clinically (5, 27, 37, 38, 39, 40, 41, 42, 43). Generalized resistance (GRTH) presents as a broad failure to respond properly to elevated thyroid hormone, resulting in a loss of negative feedback inhibition in the pituitary-hypothalamus-thyroid axis (leading to unsuppressed TSH, TRH, and T3/T4 levels) and a parallel loss of T3/T4 response in the periphery. This loss of peripheral responsiveness permits GRTH to mimic aspects of hypothyroidism despite the elevated levels of circulating T3/T4. Pituitary resistance (PRTH, also known as centralized resistance) presents instead as a more narrow failure of negative feedback hormone sensing in the pituitary/hypothalamus/thyroid axis, again resulting in unsuppressed TSH, TRH, and T3/T4 levels, but with sufficient retention of a peripheral T3/T4 responsive to induce symptoms of thyrotoxicity (53, 54, 55). Although these categories are useful conceptually and clinically, RTH syndrome is a complex disorder that can present as a continuum of possible symptoms (42, 56, 57, 58, 59, 60, 61).
TRß1 and TRß2 isoforms differ in the ability to bind to coactivators and to activate transcription in the absence of hormone (14, 19, 62, 63, 64). We report here that wild-type TRß2 also displays a higher activation of positive response elements and a stronger repression of negative response elements over a wide range of T3 concentrations compared with wild-type TRß1. As a result, TRß2 mediates the same transcriptional response at low T3 concentrations as does TRß1 at higher T3 concentrations. This is suggestive of a hysteresis mode of regulation by which the pituitary TRß2 isoform can sense (and down-regulate) increases in circulating T3/T4 in advance of the periphery. Furthermore, we found that the effects of a given RTH syndrome mutation can manifest differently when expressed as TRß1 vs. TRß2. Notably, TR mutations associated with GRTH exhibited proportional inhibitory effects when expressed as either the TRß1 or TRß2 isoform, decreasing overall responsiveness to T3 hormone but preserving the ß2 > ß1 response at subsaturating hormone conditions. In contrast, TR mutations associated with PRTH displayed a selective impairment when expressed as the TRß2 isoform, resulting in a ß2 = ß1 response at subsaturating hormone. Our results suggest that TR mutations that result in a disproportionate inhibition of the T3 response of the TRß2 isoform may selectively disrupt the ability of the pituitary and hypothalamus to sense rising T3 levels in advance of the periphery, thereby contributing to the central resistance characteristic of PRTH syndrome.
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RESULTS
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TRß2 Possesses an Enhanced Ability to Activate Positive-Response Elements at All Subsaturating T3 Concentrations
In the absence of hormone, TRß1 represses transcription at positive-response elements, whereas TRß2 activates gene expression under the same conditions (6, 7, 8, 9, 10, 11, 65). We confirmed these results in transient transfections of CV-1 cells, which express little or no endogenous TRs, using a prototypic DR-4 (direct repeat-4) positive-response element in a thymidine kinase (TK) promoter-luciferase reporter. In the absence of T3, introduction of TRß1 into the CV-1 cells repressed expression of the DR-4-TK-luciferase reporter below the basal levels observed without receptor (Fig. 1A
, compare TRß1 luciferase values, triangles, vs. no receptor, circles, at zero hormone). Conversely, introduction of TRß2 into the CV-1 cells activated DR4-TK-luciferase expression to above basal levels under the same conditions (Fig. 1A
, compare TRß2, diamonds, vs. no receptor, circles, at zero hormone). This constitutive activation by unliganded TRß2 has been linked to the ability of this isoform to recruit coactivators through hormone-independent contacts between the TRß2 N terminus and a glutamine-rich domain in the p160 coactivators (14, 19, 62, 63, 64).

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Fig. 1. Differences in Positive Transcriptional Regulation by the TRß1 and ß2 Isoforms at Subsaturating Hormone Concentrations
A, Reporter gene regulation by wild-type TR isoforms. A DR-4-tk-luciferase (positive TRE) reporter was introduced into CV-1 cells together with an empty pSG5 expression vector (no TR), a pSG5 wild-type TRß1 expression vector (wtTRß1) or a pSG5 wild-type TRß2 expression vector (wt TRß2), as described in Materials and Methods. After 24 h at 37 C, T3 hormone (or ethanol carrier alone) was added to the concentrations indicated, the cells were incubated an additional 24 h at 37 C and harvested as noted in Materials and Methods. Luciferase levels were calculated and normalized to the levels of expression of a ß-galactosidase reporter cointroduced as an internal control. The average and SE of two or more experiments are presented. Curves are fitted to a sigmoid dose response with dotted lines represent 95% confidence levels. B, Expression levels of different TR isoforms in transfected cells. Expression of the wild-type and mutant isoforms in transfected CV-1 cells was analyzed using anti-TR antisera in an SDS-PAGE immunoblotting protocol (81 ). TR levels were quantified using an ECL+ substrate and an Alpha-Innotech (San Leandro, CA)/Fluorochem 8900 detector. Average and SE from multiple experiments are presented.
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We next compared the regulatory properties of TRß1 and TRß2 in the presence of varying levels of T3. The wild-type TRß1 isoform was converted from a repressor to an activator of the DR4-TK-luciferase reporter in response to T3, with maximal reporter activation observed at 100 nM hormone and above (Fig. 1A
). The wild-type TRß2 isoform, although able to activate reporter expression even in the absence of hormone, was further stimulated by addition of T3, with maximum reporter expression obtained at 33 nM hormone and above (Fig. 1A
). Notably, reporter activation by TRß2 was significantly stronger than by TRß1 over a wide range of subsaturating hormone concentrations, with the TRß1 and TRß2 activation curves converging only at the highest hormone concentrations (Fig. 1A
). Stated reciprocally, TRß2 required significantly less T3 to produce the same level of reporter activation as did TRß1. Immunoblotting confirmed that the TRß1 and ß2 isoforms were expressed at comparable levels in our cell transfections (Fig. 1B
); furthermore, the enhanced activation properties of TRß2 relative to TRß1 were observed over a wide range of receptor plasmid inputs (data not shown). All transfections were normalized to an internal lac Z reporter control to exclude possible variations in transfection efficiency or recovery.
The Differences in Hormone Responsiveness of the TRß1 and ß2 Isoforms Were Not Due to Differences in T3 Binding, Assayed as Hormone-Induced Protease Resistance
The enhanced responsiveness of TRß2 to T3 in reporter gene assays might reflect a higher affinity of TRß2 for hormone. Binding of hormone by nuclear receptors results in a compaction of the protein chain and produces a protease-resistant polypeptide core (9). This hormone-mediated gain of protease resistance, determined over a range of hormone concentrations, is a useful measure of hormone binding avidity (66, 67, 68, 69), and we applied this methodology to the wild-type TRß1 and TRß2 isoforms (Fig. 2
, top panel). The unliganded TRs were highly susceptible to protease degradation in the absence of T3, with little or no intact receptor of either isoform remaining after protease incubation. Introduction of increasing amounts of T3 into the assay was paralleled by the formation of a protease-resistant polypeptide of the size expected for the compacted hormone binding domain. Notably, very similar patterns of protease resistance were observed for both wild-type TRß1 and wild-type TRß2, with no statistically significant difference noted between the two (Fig. 2
, top panel). These results agree with published studies, using radiolabeled ligand, that similarly conclude TRß1 and TRß2 possess near identical affinities for T3 (26, 69). Our results indicate that the different responsiveness of TRß1 and ß2 to hormone in reporter gene assays is not due to isoform-specific differences in hormone affinity.

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Fig. 2. Comparable Hormone Binding by TRß1 and TRß2 Using a Protease-Protection Assay
35S-radiolabeled TRß1 and TRß2, synthesized by in vitro transcription/translation, were preincubated with each of the T3 concentrations indicated. The proteins were then exposed to elastase for 10 min at 25 C, the incubations were terminated, and the amount of protease-resistant core polypeptide was determined by SDS-PAGE and PhosphorImager analysis, as described in Materials and Methods. Maximum protection was defined as 100%. The wild-type, R338L mutant, R429Q mutant, and P453S mutant receptors were analyzed as indicated. Mutations associated with PRTH are italicized. The average and SE of two or more experiments are presented.
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TRß2 Displays an Enhanced Binding of Transcriptional Coactivators Relative to TRß1 at Subsaturating Hormone Concentrations
We next examined whether the TRß1 and TRß2 isoforms displayed distinct interactions with corepressors or coactivators that might account for their different transcriptional regulatory properties in vivo. We used a pull-down assay employing a glutathione-S-transferase (GST)-SMRT fusion (containing the S2 interaction domain) and 35S-radiolabeled, full-length TRß1 or TRß2. Both TRß1 and TRß2 bound to this corepressor construct in the absence of hormone and released at comparable T3 concentrations (Fig. 3A
). Similar results were observed using a GST-nuclear receptor corepressor (N-CoR) construct, although, in agreement with prior studies, TRß2 displayed a somewhat stronger interaction with this corepressor than did TRß1 in the absence of hormone (Ref. 63 and data not shown). Little or no binding of TRs was observed to a nonrecombinant GST control (data not shown). These experiments suggest that the enhanced transcriptional response of TRß2 is not due to a preferential release of corepressor in response to T3.

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Fig. 3. Corepressor and Coactivator Binding and Release by TRß1 and TRß2
A, Binding of native TRß1 or TRß2 to a GST-SMRT construct. 35S-radiolabeled TRß1 or TRß2, synthesized by in vitro transcription/translation, were incubated with glutathione-agarose beads bearing an immobilized GST-SMRT fusion (representing the S2 receptor interaction domain of the corepressor) under the different T3 concentrations indicated. After a 2-h incubation, the GST-SMRT construct (and any radiola beled receptors bound to it) were washed, and the radiolabeled receptors were eluted with soluble glutathionine and were quantified by SDS-PAGE and PhosphorImager analysis. The average and SE of two or more experiments are presented; the amount of receptor introduced into each incubation reaction (i.e. input) is defined as 100%. B, Binding of native TRß1 or TRß2 to a GST-SRC1(LXXLL) construct. A similar protocol was employed as in panel A, except a GST-construct containing the internal receptor interaction domains of the SRC1a coactivator (containing three LXXLL motifs) was used instead of the GST-corepressor construct. Binding to a nonrecombinant GST was less than 2% of input. C, Binding of full-length SRC1a to GST-TRß1 or GST-TRß2 fusion constructs. The same general protocol as in panel B was employed, except in a reciprocal fashion. A full-length 35S-radiolabeled SRC1a protein, synthesized by in vitro transcription/translation, was incubated with immobilized GST fusions of either TRß1 or TRß2. The overall procedure and analysis were as in panel B. An SRC1a construct in which the LXXLL motifs had been mutated to LXXAA was also employed as a negative control. The average and SE of two or more experiments are presented for each panel.
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We next examined the interaction between the different TRß isoforms and the p160 coactivators, using a GST fusion containing the LXXLL receptor-interaction domains of the glucocorticoid receptor-interacting protein (GRIP) 1 coactivator (Fig. 3B
). Wild-type TRß2 displayed an elevated ability to bind to the GST-GRIP1 construct in the absence of hormone compared with wild-type TRß1 (Fig. 3B
, compare black vs. unfilled bars). Addition of T3 increased binding by both TRß1 and TRß2 to the GST-GRIP1 construct. Significantly, TRß2 exhibited a greater interaction with the GRIP1 construct at any given T3 concentration than did TRß1 (Fig. 3B
). Similar results were observed with a GST-fusion of steroid receptor coactivator (SRC) 1a, a second member of the p160 coactivator family (data not shown). Analogous results were obtained in a reciprocal experiment using GST-TR constructs and full-length, 35S-radiolabeled SRC1a coactivator; GST-TRß2 exhibited a hormone-independent interaction with SRC1a not seen with GST-TRß1 and displayed an enhanced interaction with SRC1a over a range of T3 concentrations compared with GST-TRß1 (Fig. 3C
; compare TRß2, squares, to TRß1, triangles). SRC1a binding by either TR isoform was abolished if the LXXLL receptor interaction motifs in the coactivator were mutated to LXXAA (Fig. 3C
; compare the wt SRC1a, closed symbols, to the LXXAA SRC1a mutant, open symbols). Our results indicate that the enhanced transcriptional activation properties of the TRß2 isoform in vivo parallels an enhanced ability of this isoform to bind to p160 coactivators in vitro.
Mutations Associated with PRTH Syndrome, But Not with Generalized Resistance, Selectively Impair the Enhanced Hormone Response of the TRß2 Isoform
We repeated our transfection experiments using a panel of mutant TRs isolated from individuals with either generalized (G345S and P453S) or pituitary-specific (R338L, R338W, and R429Q) RTH syndrome (70, 71). We introduced each mutation into either the TRß1 or TRß2 isoform background and determined the ability of the encoded receptors to repress or to activate the DR4-TK-luciferase reporter in CV-1 cells (Fig. 4
). All the RTH-receptor mutants examined retained the ability to active target gene expression at high T3 levels, but (consistent with published reports) were impaired for hormone binding and required from 2 to more than 100 times more T3 than the corresponding wild-type isoform to achieve half-maximal activation of the DR-4-TK-luciferase reporter (Fig. 4
). Notably, the G345S and P453S receptor mutants shifted the response curve of both the TRß1 and ß2 isoforms to higher hormone concentrations yet retained the preferential hormone responsiveness of the TRß2 isoform observed for the wild-type receptors (Fig. 4
). That is, these GRTH mutants impaired the hormone response of both the ß2 and ß1 isoforms, while preserving or exaggerating the ability of the TRß2 isoform to activate transcription in the absence of hormone and to respond at lower hormone concentrations than the corresponding TRß1 mutant (Fig. 4
).

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Fig. 4. Transcriptional Regulation in Response to Differing T3 Concentrations by TRß1 or TRß2 Forms of RTH Mutant Receptors
Transcriptional regulation by each RTH mutant, expressed as a TRß1 or TRß2 isoform, was assayed in CV-1 cells using the same DR-4-tk-luciferase reporter and procedures as in Fig. 1 . Mutations associated with PRTH are italicized. The average and SE of two or more experiments are presented for each panel.
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In contrast, although the R338L, R338W, and R429Q mutations impaired the activity of both the ß1 and ß2 isoforms, the impact appeared to be disproportionate on the TRß2 isoform, with these PRTH mutants reducing or abolishing the preferential response characteristic of the wild-type TRß2 isoform (Fig. 4
). The R338L and R429Q TR mutants manifested the most dramatic effect in this regard and displayed near identical hormone responsiveness whether expressed as the ß1 or the ß2 isoform (Fig. 4
). Although the R338W mutation also selectively suppressed the transcriptional activation properties of the ß2 isoform at very low hormone concentrations, this selective impairment was lost at higher T3 concentrations, with the ß2 isoform becoming more active than the corresponding ß1 isoform at the highest hormone concentrations (Fig. 4
).
We also experimentally created and tested an artificial TRß mutation, E457D, known to interfere with hormone-dependent coactivator recruitment (6, 9). The E457D mutation mimics many of the properties of the naturally occurring RTH-TR mutants and can function as a dominant-negative inhibitor of wild-type TRs. As anticipated, the E457D TRß1 receptor repressed the DR4-TK-luciferase reporter in the absence of hormone but was dramatically impaired in activation of the DR4-TK-luciferase reporter, even at quite high T3 concentrations (Fig. 4
). Notably, the same E457D mutation in the TRß2 context retained the hormone-independent transcriptional activation seen for the wild-type TRß2 isoform and displayed detectably stronger reporter activation in the presence of T3 than did the same mutation in the TRß1 background. These results suggest that disruption of activation function-2 significantly impairs transcriptional activation but retains elements of the preferential TRß2 > TRß1 response observed for the wild-type receptors (Fig. 4
).
The Mutant TRs Associated with Pituitary RTH Syndrome Display Isoform-Selective Defects in Coactivator Binding in Vitro
We wished to determine the basis for the disproportionate impairment of TRß2 transcription regulation in the R338L, R338W, and R429Q mutants. A reduced affinity for hormone is a frequent molecular defect underlying both forms of disease (39, 70), so we repeated the hormone-mediated protease resistance assay with representative PRTH (R338L and R429Q) and GRTH (P453S) mutants (Fig. 2
, bottom three panels). Consistent with these prior studies, both the PRTH and GRTH mutants were impaired for T3 binding, requiring a greater T3 concentration to confer protease resistance than did the corresponding wild-type receptors (Fig. 2
; note the change of scale of the ordinate from the top panel). Notably, however, the RTH-TR mutants displayed comparable defects in T3 binding whether expressed as the ß1 or as the ß2 isoform, regardless of PRTH or GRTH origin (Fig. 2
). We conclude that isoform-specific changes in hormone affinity are unlikely to account for the disproportionate effect of the PRTH mutants on the T3 response of TRß2.
We also tested the corepressor interaction properties of the various mutant receptors (Fig. 5
). All receptors, wild-type, GRTH mutant, and PRTH mutant, bound strongly to our GST-N-CoR construct in the absence of hormone (Fig. 5
). Consistent with their impaired hormone binding properties, the RTH-TR mutants required higher T3 concentrations to release from corepressor than did wild-type TR (e.g. note the incomplete release of the G345S mutant from corepressor even at 1 µM T3), but no pattern was observed in corepressor binding or release consistent with the selective loss of TRß2 transcriptional activation observed for the PRTH mutants (Fig. 5
and data not shown).

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Fig. 5. Comparison of Corepressor Binding by Wild-Type and RTH Mutant Forms of TRß1 or TRß2
35S-radiolabeled TRß1 or TRß2, wild-type or mutant, were synthesized by in vitro transcription/translation and were incubated with glutathione-agarose beads bearing an immobilized GST-N-CoR (N1+N2+N3) construct. Two hormone concentrations (0 and 1000 nM) were used. The washing, elution, and quantification were as in Fig. 3A . Mutations as-sociated with PRTH are italicized. The average and SE of two or more experiments are presented.
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We next compared the ability of representative GRTH and PRTH mutants to bind to our GST-GRIP1 coactivator constructs. Notably, the P453S mutation, when expressed as the TRß2 form, retained an enhanced coactivator binding at limiting T3 relative to the same mutation expressed as the TRß1 isoform (Fig. 6A
). In contrast, the R338L mutant, although retaining the wild-type TRß2 ability to bind GRIP1 in the absence of hormone, lost the preferential ß2 response to hormone, with both the TRß2 and TRß1 isoforms of this mutant displaying very similar coactivator binding under limiting T3 concentrations (Fig. 6A
). Similar results were obtained using an analogous GST-SRC1a fusion (data not shown).

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Fig. 6. Comparison of Coactivator Binding by Wild-Type and RTH Mutant Forms of TRß1 or TRß2
A, LXXLL-domain coactivator binding by wild-type, R338L mutant, or P453S mutant TRs was determined. The same general protocol as in Fig. 3B was employed, using a GST-coactivator fusion containing the three internal LXXLL motifs of SRC1 and incubating this construct with 35S-radiolabeled native receptors. The average and SE of two or more experiments are presented. B, Q-rich coactivator domain binding by wild-type or RTH mutant TRs was determined. The same general protocol as in Fig. 3B was employed, but using a GST-GRIP1 fusion representing the Q-rich domain of the GRIP1 coactivator and incubating this construct together with 35S-radiolabeled native receptors. Mutations associated with PRTH are italicized.
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The interaction between p160 coactivators and TRs is primarily mediated by docking of the LXXLL coactivator motifs, tested above, with a cleft comprised of helices 3, 5/6, and 12 of the receptor hormone binding domain (11, 49, 65). The N terminus of the TRß2 isoform, but not of ß1, can also interact with a glutamine-rich region of SRC1 and GRIP1; this secondary interaction contributes to the ability of the ß2 isoform to activate transcription in the total absence of hormone ligand (19, 62, 64). Significantly, the GST-GRIP1 and GST-SRC1a constructs employed here contain LXXLL domains but lack this Q-rich region; mutating the LXXLL motifs in these constructs to LXXAA abolished all interaction with both the TRß1 and TRß2 isoforms (e.g. Fig. 3C
). Conversely, all the TRß2 mutants bound to the isolated Q-rich coactivator domain with the same affinity as wild-type TRß2, whereas none of the TRß1 receptors bound to the Q-rich region above background levels (Fig. 6B
). These results suggest that the enhanced coactivator binding (and transcriptional activity) of TRß2 vs. ß1 at subsaturating hormone concentrations reflects an enhanced affinity for the LXXLL coactivator motifs themselves, and this enhanced LXXLL interaction is selectively lost in the PRTH mutants. In contrast, interaction of the TRß2 N-terminal domain with the Q-rich coactivator domain may stabilize coactivator binding by TRß2 in low or no T3 but is not the sole basis of the phenomenon studied here.
The Enhanced T3 Response of Wild-Type TRß2 Extends to a Negative Response Element and Is Selectively Impaired in the PRTH TRß2 Mutants
More than half of T3-responsive genes are negatively regulated by T3 (72), including the TSH genes that play a central role in the negative feedback regulation of T3/T4 synthesis. We therefore tested the effects of TR isoform and the different RTH-TR mutations on the ability of TRs to negatively regulate the TSH
promoter. Consistent with a prior study (73), cointroduction of wild-type TRß1 activated a TSH
promoter-luciferase reporter in the absence of T3, whereas T3 reversed this phenomenon and repressed the TSH
-luciferase reporter to below basal levels (Fig. 7A
). Interestingly, the unliganded wild-type TRß2 isoform failed to activate the TSH
-luciferase receptor, and TRß2 repressed TSH
-luciferase expression more effectively than did TRß1 over a range of subsaturating T3 levels (Fig. 7A
). In essence, the negative-acting TSH
promoter appeared to act reciprocally to the positive-acting DR4-TK promoter. These data suggest that the TRß2 isoform is capable of regulating target gene expression, either up or down, at T3 levels below those required for comparable regulation by the TRß1 isoform.

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Fig. 7. Comparison of Negative Transcriptional Regulation by Wild-Type and RTH Mutant Forms of TRß1 or TRß2
A TSH promoter-luciferase reporter was introduced into CV-1 cells together with a pSG5 vector expressing wild-type or RTH-mutant forms of TRß1 or TRß2, as indicated and as described in Materials and Methods. After 24 h, T3 hormone (or ethanol carrier alone) was added to the concentrations indicated, the cells were incubated an additional 24 h and were harvested as noted in Materials and Methods. Luciferase levels were calculated and normalized to the levels of expression of a ß-galactosidase reporter cointroduced as an internal control. The average and SE of two or more experiments are presented. A, Wild type. B, P453S mutant. C, R338L mutant. D, R429Q mutant. Mutations associated with PRTH are italicized.
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We also examined the effects of the RTH mutations on negative regulation. Although the precise T3 response of these mutants on the negative-acting TSH
-luciferase reporter differed in its details from that observed on the positive-acting DR4-TK-luciferase reporter, a common theme emerged. The P453S mutant retained the distinction between ß1 and ß2 isoforms observed for the wild-type receptors, with the TRß2 mutant isoform displaying stronger repression at subsaturating T3 concentration than the TRß1 mutant isoform (Fig. 7B
). In contrast, the R338L, R338W, and R429Q mutants lost all or much of the differential T3 response seen for the wild-type receptor (Figs. 7
, C and D, and data not shown); little or no difference was observed in the ability of these PRTH mutants to repress the TSH
promoter whether expressed as either the TRß1 or the TRß2 isoform. These results suggest that, when expressed as TRß2 in the pituitary and hypothalamus, these PRTH mutants may have lost the ability to respond to and suppress rising T3/T4 levels in advance of the ability of the peripheral TRß1 receptors to respond to these elevated hormone levels.
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DISCUSSION
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The Wild-Type TRß2 Isoform Displays an Enhanced Response to T3 Compared with TRß1
An important feature of TR expression is the generation of receptor diversity through use of multiple genetic loci and alternative mRNA splicing (10, 26). The TR
1, TRß1, and TRß2 isoforms serve as the key mediators of the physiological actions of thyroid hormone (2, 10, 26, 27). Notably, these three isoforms display distinctive developmental and tissue-specific expression patterns (2, 10, 26, 27). TR
1 can be detected early in embryogenesis, is widely expressed in fetal and adult tissues, and plays a key role in the control of heart rate and of linear growth. TRß1 expression occurs later in embryogenesis, coinciding with the appearance of circulating T3/T4, and can found in a wide variety of adult tissues; knockouts of TRß1 are associated with hearing loss and retinal malformation. TRß2 displays the most narrow expression pattern of all the TR isoforms and is restricted primarily to the pituitary and hypothalamus; TRß2 function is important for the negative feedback regulation of thyroid hormone synthesis, with TRß2 knockouts resulting in elevated TSH, TRH, and circulating T3/T4 levels (17, 18, 19, 20, 21, 22, 23, 25).
We wished to better understand the molecular properties of the TRß2 isoform that might account for its unique contributions to regulation of the thyroid/pituitary/hypothalamus axis. We report here that TRß2 displays a significantly stronger ability to activate positive response reporters in response to T3 than does TRß1, and this is observable over a wide range of subsaturating T3 concentrations. As a result, the activation curve of TRß2 parallels that of TRß1 but is shifted to lower hormone concentrations, such that at any given subsaturating T3 concentration TRß2 mimics the activity seen for TRß1 at an approximately 5-fold higher level of hormone. In essence, TRß2 is presensitized to hormone agonist, such that target genes in cells expressing the TRß2 isoform will respond to a rise in T3 levels significantly before cells expressing the TRß1 isoform. This enhanced ability of TRß2 to activate target gene expression in response to T3 is paralleled by an enhanced ability of TRß2 to recruit p160 coactivators in vitro in response to hormone.
The enhanced response of TRß2 to T3 on a positive response element was also observed, but reciprocally, on the negative thyroid hormone response element (TRE) found in the TSH
promoter. TSH plays an important role in the negative feedback regulation of T3/T4 synthesis (3). Under limiting T3 conditions, the TRß2 isoform repressed the TSH
reporter more efficiently than did the TRß1 isoform, resulting in a shift of the repression curve of TRß2 to the left of that of TRß1. Negative response element function is not well understood; it has been suggested that the actions of corepressors and coactivators on negative response element may be somehow reversed to those on positive response elements (e.g. Refs. 13, 14, 15, 16). Our results are generally consistent with this hypothesis. Other investigators have also reported an enhanced ability of TRß2 to repress the TSH
promoter relative to TRß1 (21), although this was observed as an elevated activation of the negative TRE in the absence of hormone, rather than an increased repression in the presence of T3. Differences in the details by which these experiments were performed may account for these similar, but imperfectly congruent results.
We do not yet fully understand the molecular basis for the enhanced sensitivity of the TRß2 isoform to limiting T3 concentrations. TRß1 and TRß2 differ in sequence only within their N-terminal A/B domains. The TRß2 A/B domain has been shown to make contacts with a variety of coactivators and corepressors (14, 19, 62, 63, 64). One such contact occurs between the TRß2 A/B domain and a glutamine-rich region of the p160 coactivators (62, 64). However, as shown here, the TRß2 isoform displays an enhanced interaction with the LXXLL motifs of the p160 coactivators even in the absence of this glutamine-rich coactivator domain. We suggest that the enhanced interaction of the TRß2 isoform with the p160 coactivators under subsaturating T3 conditions is mediated jointly through the hormone-independent interaction of the TRß2 A/B domain with the glutamine-rich region of the p160s, and through an elevated interaction of the hormone binding domain of the TRß2 isoform with the LXXLL motifs in the coactivator. Consistent with this suggestion, a multiplicity of inter- and intramolecular interactions have been reported between coactivators, nuclear receptor A/B domains, and nuclear receptor hormone binding domains, and these interactions can enhance, or suppress, transcriptional activity of different receptors.
Mutations Associated with GRTH Impair both TRß1 and ß2 Function Comparably, Whereas the PRTH Mutations, R338L, R338W, and R429Q, More Severely Impair TRß2 Function
RTH syndrome is associated with mutations in the TRß locus that interfere with corepressor release and/or prevent coactivator acquisition (16, 44, 45, 46, 47, 48, 49, 50, 51, 52). These mutant TRs, when coexpressed, interfere with the ability of wild-type receptors to activate-positive response elements and to repress negative response elements. RTH syndrome is manifested clinically as strongly elevated levels of TSH, TRH, and circulating T3/T4, indicative of a disruption in negative feedback regulation of the pituitary/hypothalamus/thyroid axis (5, 27, 37, 38, 39, 40, 41, 42, 43, 74). In GRTH, both the negative feedback suppression of T3/T4 synthesis, and the ability of the peripheral tissue to respond to this elevated hormone, are similarly impaired (5, 27, 37, 38, 39, 40, 41, 42, 43, 74). Circulating levels of T3/T4 are therefore elevated, but without corresponding thyrotoxicity; GRTH, in fact, can present with characteristics of hypothyroidism, including impaired growth, hearing defects, and depressed mentation. In PRTH, the impaired negative feedback response of the pituitary/hypothalamus/thyroid axis again leads to elevated circulating T3/T4 but without a proportional impairment in peripheral T3 sensitivity; as a result, patients with PRTH often display symptoms of peripheral hyperthyroidism and thyrotoxicity (53, 54, 55).
We compared the properties of representative TR mutations, isolated from either PRTH or GRTH index cases, when expressed as either TRß1 or TRß2. An intriguing distinction became apparent. Two TRß mutations associated with GRTH, G345S, and P453S, required higher than normal T3 concentrations to activate positive response reporters and to repress negative response reporters in transfected cells. Nonetheless, both the G345S and P453S mutants retained a preferential response to T3 when expressed as the ß2 isoform relative to the corresponding ß1 isoform. Therefore, these GRTH syndrome mutants are impaired for T3 binding, but preserve, or further exaggerate, the preferential ß2 > ß1 T3 response seen for the wild-type receptors. The three PRTH mutants, R338L, R338W, and R429Q, were also impaired in T3 binding and in reporter gene regulation. In contrast to the GRTH mutants, however, these PRTH mutants disproportionately affected the response of the TRß2 isoform to T3. As a result, these PRTH mutants displayed more nearly congruent T3 responses whether expressed as the ß2 or ß1 isoform than did the wild-type or GRTH mutants.
The G345S and P453S mutations map to the hormone binding pocket and to the pivot between helix 11 and 12, respectively, and presumably disrupt the T3-driven repositioning of helix 12 required for corepressor release and for coactivator binding (6, 9). Apparently this hormone-dependent activation function-2 of TRß can be impaired without disrupting the preferential response of the TRß2 isoform to T3 relative to ß1; this hypothesis is supported by our observation that an artificial mutation, E457D, that disrupts the coactivator binding surface on helix 12 displays some of the same characteristics as those of the naturally occurring R345S and P453S mutants.
The structural basis for the R338L, R338W, and R429Q phenotype is more difficult to ascertain. These PRTH mutations lie outside of the domains directly implicated in hormone binding or in the corepressor/coactivator exchange (6, 9). Instead, these three mutations disrupt salt-bridge interactions that may contribute to the global conformation of the wild-type receptor (6, 9, 75, 76, 77). The R338L, R338W, and R429Q mutations may therefore destabilize aspects of the overall receptor conformation that are more important for TRß2 function than for TRß1 function. Consistent with this hypothesis, mutation of the aspartic acid in these acid/base salt bridge pairs generates a TRß2-selective impairment similar to that seen for the arginine mutations reported here (Wan, W., and M. L. Privalsky, unpublished observations). We suggest that the salt bridges in the wild-type TR help stabilize a receptor conformation necessary to bind coactivator under limiting T3 concentrations, and that this stabilized receptor conformation functions together with the unique A/B domain of the ß2 isoform so as to enhance the transcriptional response under subsaturating hormone conditions. The TRß2 A/B domain may participate by interacting directly with the receptor hormone binding domain, as proposed for the androgen receptors, or indirectly by helping to recruit coactivators through a multiplicity of intermolecular A/B and hormone-binding domain contacts (e.g. Refs. 78 and 79). In this model, elimination of either the salt-bridge interactions (i.e. in the PRTH mutants) or the ß2 N terminus (i.e. in the ß1 isoform) would disrupt this synergy and disrupt the preferential transcriptional activity of the TRß2 isoform.
Implications for Physiology and for Understanding Human RTH Syndrome
The enhanced ability of TRß2 over TRß1 to respond to rising T3 levels is intriguing given the role of TRß2 as a sensor in the negative feedback regulation of thyroid hormone synthesis. The enhanced response of TRß2 would be expected to result in the pituitary and hypothalamus being more sensitive to T3 than are peripheral tissues that express TRß1. We suggest that, as a result, the pituitary/hypothalamus/thyroid axis is able to respond to, and thereby suppress, rising T3 levels before the periphery responds to the increase in T3. In this fashion, fluctuations in circulating T4/T3 may be damped and their effect on peripheral gene expression smoothed so as to minimize large, unregulated swings in circulating hormone that would otherwise lead to peripheral thyrotoxicity.
Supporting this model, three TR mutants that selectively impair the elevated TRß2 response to T3 (R338L, R338W, and R429Q) were initially identified in individuals presenting with PRTH (70, 71). We suggest that in PRTH the pituitary/hypothalamus axis has lost part or all of its preferential ability to sense rising T3 levels, resulting in incompletely suppressed TSH and TRH levels and an increase in circulating thyroid hormone; in the same individuals, the mutant TRß1, less affected than TRß2, would retain the ability to respond to this increase in T3 and would produce a peripheral thyrotoxicity. Conversely, mutations that impair the T3 response of both TRß1 and TRß2 proportionally or that exaggerate the ß2 response, such as G345S and P453S, were identified from GRTH patients. We propose that these latter mutants disrupt the TRß2 negative-feedback regulation of the pituitary/hypothalamus/thyroid axis, elevating TSH, TRH, and circulating T3 levels; however, these GRTH mutants also impair (proportionally or greater still) the TRß1 peripheral response to these hormones, thereby preventing thyrotoxicity and even mimicking some aspects of hypothyroidism. Additionally, the TRß1 mutants can have dominant-negative activity on TR
in peripheral tissues. Certain PRTH mutants have been reported to function efficiently as dominant negatives only when expressed in the TRß2 form (36); this observation may reflect, in part, the TRß2-selective impairment caused by these mutations at limiting T3 concentrations, as noted here, or may represent an distinct phenomenon possibly related to receptor homo- and heterodimerization (36).
It should be emphasized that RTH syndrome is complex and multideterminant, with PRTH and GRTH representing extremes within a more continuous spectrum of possible disease phenotypes (32, 42, 56, 57, 58, 59, 60, 61). The genetic background and physiological state of the individual patient can influence the clinical presentation of this disorder, and a given mutation can manifest as either PRTH or GRTH within different individuals of a single kindred (32, 42, 56, 57, 58, 59, 60, 61). It may be significant in this regard that the T3 response curves of the ß1 and ß2 isoforms of several of our PRTH mutants, such as R338W, are not fully parallel, but instead diverge at either low or high hormone concentrations. As a result, the TRß2 and TRß1 isoforms of these mutants may behave more alike, or more different, depending on the T3 concentration, and might manifest as a more central, or a more generalized, resistance depending on thyroid hormone levels. Undoubtedly, additional genetic and physiological events also contribute to determining the overall clinical presentation of a given patient.
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MATERIALS AND METHODS
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Molecular Clones
The pSG-5 wt-TRß1, pSG5 RTH-TRß1, the pTK-DR4-luciferase, the pCH110-lac Z, the GEX-KG-SRC1 (codons 11721441), and the pGEX-KG-GRIP (codons 11211462) plasmids were previously described (51, 63, 64). The corresponding TRß2 alleles were introduced into the pSG5 vector as EcoRI to EcoRI fragments using standard recombinant DNA methodologies. Appropriate restriction fragments representing the full-length wild-type or mutant TRß1 and TRß2 isoforms were also introduced into a pGEX vector to allow expression of glutathione-S-transferase (GST) fusion proteins in bacteria (80). The pGEX-KG-N-CoR (codons 18172453) construct was created in a similar manner. The GRIP1 (LXXLL to LXXAA) mutant was created by a QuikChange oligonucleotide-mediated mutagenesis procedure, employing the protocol recommended by the manufacturer (Stratagene, La Jolla, CA).
Transient Transfection Assays
CV-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum in a 5% CO2 atmosphere at 37 C. For reporter gene assays, the cells were rinsed with PBS lacking magnesium and calcium (PBS), placed in DMEM supplemented with 10% hormone-depleted fetal bovine serum, and then transfected using an Effectene protocol as recommended by the manufacturer (QIAGEN, Valencia, CA). Approximately 5 x 105 cells were transfected with 10 ng of the appropriate pSG5-TRß1 or TRß2 plasmid, 100 ng of the DR4-TK-luciferase reporter, and 60 ng of a pCH110-lac Z plasmid (used as a internal normalization control); pUC18 plasmid was employed in all cases to bring the total transfected DNA per sample to 200 ng. The culture medium was replaced 24 h later with fresh medium containing either T3 or an equivalent amount of ethanol carrier. The cells were harvested 24 h later, and the luciferase and ß-galactosidase activities were determined as previously described (51, 64, 81). Assays using a negative response TSH-
promoter-luciferase construct (80) were performed in a similar fashion, except 1 mM 8-bromo-cAMP was included in the media to enhance the basal levels of expression of the reporter.
Protein-Protein Interaction Assays
GST-fusion proteins were synthesized in Escherichia coli strain BL-21 containing the corresponding pGEX vector, the bacteria were lysed, and the GST fusion proteins were bound to a glutathione-agarose matrix. The pSG5- or pCR3.1-based TR or coactivator plasmids were transcribed and translated into 35S-radiolabeled proteins in vitro by using a T7 RNA polymerase-coupled TnT Quick kit (Promega Corp., Madison WI). Each radiolabeled protein (typically 25 µl of TnT reaction product per assay) was subsequently incubated at 4 C with the immobilized GST fusion protein of interest (
50 ng of GST fusion protein immobilized to 10 µl of agarose matrix per reaction) in a total volume of 120 µl of HEMG buffer [4 mM HEPES (pH 7.8), 0.2 mM EDTA, 5 mM MgCL2, 10% glycerol, 100 mM KCl, 0.1% Nonidet P-40, and 1.5 mM dithiothreitol] containing 10 mg/ml of BSA and 1x Complete Proteinase Inhibitor (Roche Molecular Biochemicals, Indianapolis, IN). The binding reactions were performed in 96-well multiscreen filter plates (Millipore, Bedford, MA) placed on a rotating platform to ensure constant mixing. After a 3-h incubation at 4 C, the filter wells were washed four times with 200 µl of ice-cold HEMG buffer each, and any radiolabeled proteins remaining bound to the immobilized GST fusion proteins were subsequently eluted with 50 µl of 10 mM glutathione in 50 mM Tris-HCl (pH 7.8). The eluted proteins were resolved by SDS-PAGE and were visualized and quantified using a PhosphorImager/STORM system (Molecular Dynamics, Sunnyvale, CA).
Protease Resistance Assay
35S-radiolabeled wild-type TRß1 and TRß2 proteins were synthesized in vitro using the coupled TnT system (Promega), For each time point, 1 µl of the TnT reaction products was diluted to 16 µl in ice-cold 50 mM Tris-HCl (pH 7.5) containing the indicated concentration of T3, or an equivalent amount of ethanol carrier. After 10 min on ice, 4 µl of elastase (type IV, from Sigma) was added to each sample, the tubes were transferred to 25 C, and were incubated for 10 min (68). The reactions were terminated by addition of 20 µl of 2x SDS-PAGE sample buffer and rapid freezing on dry ice. The samples were subsequently quickly thawed and denatured by heating to 95 C for 10 min, were resolved by SDS 15%-PAGE, and the proteolytic degradation products were visualized and quantified by PhosphorImager analysis.
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FOOTNOTES
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This work was supported by the Public Health Service Grant DK53528 from the National Institute of Diabetes, Digestive, and Kidney Diseases.
First Published Online March 31, 2005
Abbreviations: DR-4, Direct repeat-4; GRIP, glucocorticoid receptor-interacting protein; GRTH, generalized resistance; GST, glutathione-S-transferase; N-CoR, nuclear receptor corepressor; PRTH, pituitary resistance; RTH, resistance to thyroid hormone; SRC, steroid receptor coactivator; TK, thymidine kinase; TR, thyroid hormone receptor; TRE, thyroid hormone response element.
Received for publication January 7, 2005.
Accepted for publication March 23, 2005.
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