A Novel TRß Mutation (R383H) in Resistance to Thyroid Hormone Syndrome Predominantly Impairs Corepressor Release and Negative Transcriptional Regulation

R. J. Clifton-Bligh, F. de Zegher, R. L. Wagner, T. N. Collingwood, I. Francois, M. Van Helvoirt, R. J. Fletterick and V. K. K. Chatterjee

Department of Medicine (R.J.C.-B., T.N.C., V.K.K.C.) University of Cambridge, Level 5 Addenbrooke’s Hospital Hills Road, Cambridge, CB2 2QQ, United Kingdom
Department of Paediatrics (F.d.Z., I.F., M.V.H.) University of Leuven 3000 Leuven, Belgium
Graduate Group in Biophysics and Department of Biochemistry and Biophysics (R.L.W., R.J.F.) University of California at San Francisco San Francisco, California 94143-0448


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Resistance to thyroid hormone (RTH) is characterized by elevated serum thyroid hormones, failure to suppress pituitary TSH secretion, and variable T3 responsiveness in peripheral tissues. The disorder is associated with diverse mutations that cluster within three areas of the thyroid hormone ß (TRß) receptor. Here, we report a novel RTH mutation (R383H), which is located in a region not known to harbor naturally occurring mutations. Although the R383H mutant receptor activated positively regulated genes to an extent comparable to wild-type (WT), negative transcriptional regulation of human TSH{alpha} and TRH promoters was impaired in either TRß1 or TRß2 contexts, and WT receptor function was dominantly inhibited. T3-dependent changes in basal transcription with R383H were also impaired: on the TRH promoter, basal activation by unliganded R383H was not reversed by T3 to the same extent as WT; similarly transcriptional silencing by an unliganded Gal4-R383H fusion was not relieved at a T3 concentration that derepressed WT. In keeping with this, ligand-dependent corepressor release by R383H, either in a protein-protein interaction assay or as a DNA-bound heterodimer with retinoid X receptor on either positive or negative thyroid hormone response elements, was disproportionately impaired relative to its ligand-binding affinity, whereas its T3-dependent recruitment of coactivator was unimpaired. These properties were shared by another previously described RTH mutant (R429Q), and in the crystal structure of TR{alpha} the homologous residues interact in a polar invagination. Our data indicate a role for these residues in mediating negative transcriptional regulation and facilitating corepressor release and suggest that predominant impairment of these functions may be the minimal requirements for causation of RTH.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Resistance to thyroid hormone (RTH) is recognized when impaired thyroid hormone action within the hypothalamic-pituitary-thyroid axis produces characteristic biochemical features, i.e. elevated serum thyroid hormones together with nonsuppressed TSH levels (1). Clinical features of RTH depend on the degree of associated peripheral tissue resistance, with a spectrum of phenotypes ranging from generalized RTH, which may be relatively asymptomatic, to predominant pituitary resistance in which peripheral thyrotoxic features are present (1, 2). The effects of thyroid hormones (T4 and T3) are principally mediated by isoforms of the thyroid hormone receptor (TR{alpha}1, TRß1, TRß2) that bind to thyroid response elements (TREs) in target gene promoters and activate or repress gene expression (3, 4). After linkage of familial RTH to the TRß locus (5), a large number of TRß mutations have been described that are limited to the carboxy-terminal domain of the receptor, which mediates hormone binding, homodimerization, and heterodimerization with the retinoid X receptor (RXR) (6, 7). Functional studies have indicated that these mutations impair the transcriptional properties of TRß (8). Furthermore, since individuals heterozygous for deletion of the TRß gene do not have RTH (9), dominantly inherited RTH must result from the ability of the mutant TRß to inhibit its normal counterpart (10, 11). Although not fully understood, the dominant negative effect also requires that the mutant receptor is still able to bind DNA and to heterodimerize with RXR (8, 12). These properties correlate with the clustering of mutations within two ‘hot spots’ between amino acids ({alpha}{alpha}) 310–353 and between {alpha}{alpha} 429–461. The intervening ‘cold’ region between these clusters is devoid of natural mutations and encompasses a series of hydrophobic heptad repeats (13), which are implicated in both homo- and heterodimerization (6, 14). The ninth heptad is critical, as mutation of residues here abolishes heterodimerization and abrogates the dominant negative effect of RTH mutant receptors (8). In another study, systematic mutation of residues containing CpG dinucleotides within the remainder of the ‘cold’ region resulted in mutant receptors with only mild functional impairment and which exhibited weak or no dominant negative effect on positive thyroid hormone response elements (TREs) (15). These data suggested an explanation for the lack of natural mutations in this region, in that it appeared unlikely that they would cause the biochemical or clinical phenotype of RTH. A structural rationale for the clustering of mutations in RTH has recently been provided by the crystal structure of the rat thyroid hormone {alpha} receptor in which most RTH mutations are shown to approximate the hormone-binding cavity (16).

The molecular pathophysiology of RTH has been further clarified by the identification of nuclear cofactors that interact with distinct subdomains of thyroid receptors in a hormone-dependent manner and influence their contact with the basal transcriptional machinery (17) and/or participate in chromatin remodeling (18, 19, 20, 21) to promote or repress transcription. Several putative coactivators have been cloned, including receptor-interacting protein 140 (RIP-140), steroid receptor coactivator-1 (SRC-1), and CREB-binding protein (CBP) (22, 23, 24, 25, 26, 27, 28, 29), which interact with the highly conserved carboxy-terminal amphipathic {alpha}-helix in nuclear receptors in the presence of ligand, and a natural RTH mutation involving a critical residue in this helix impairs transactivation by disrupting this interaction (28). Two proteins, N-CoR (nuclear receptor corepressor) (30) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) (31, 32), have been identified which interact with unliganded thyroid and retinoic acid receptors to mediate repression of postively regulated genes, and many RTH mutants have recently been shown to interact aberrantly with corepressor. Moreover, this interaction is required for their dominant negative activity (33). Two groups have recently reported that corepressors may also mediate some aspects of negative transcriptional regulation by TR (34, 35), although their precise role remains to be elucidated.

Here, we describe the functional characteristics of a novel natural TRß mutation (R383H), predicted not to occur in RTH. This mutant receptor was predominantly impaired for negative transcriptional regulation and corepressor release. These properties are shared by another natural mutant (R429Q) (36) and, interestingly, the mutated residues interact in the crystal structure of TR{alpha}. We suggest that the functional abnormalities exhibited by these structurally colocalized mutants may represent the minimal derangements required to generate the RTH phenotype.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Novel Mutation in the TRß Gene in a Child with RTH
Although the proband was noted to have a low birth weight (2.65 kg), she presented at age 11 with headache, heat intolerance, and weight loss. Physical examination showed a mild goiter, hyperreflexia with clonus, and fine tremor; systolic blood pressure was elevated (138/70 mm Hg), and resting pulse rate was 92 beats/min. Her bone age was advanced (12 yr, 7 months) in the context of a normal growth pattern. Her serum free T4 and total T3 were elevated (Table 1Go), with a detectable TSH that responded normally to the administration of TRH (basal TSH, 1.4 mU/liter; 20 min after 200 µg TRH, 12.1 mU/liter; normal range, 0.15–4.6 mU/liter). Carbimazole treatment was commenced at age 12 yr, 9 months because of worsening headaches, insomnia, and hyperactivity with declining school performance. Although her symptoms resolved on this treatment, serum TSH concentrations rose and the goiter increased in size. Triiodothyroacetic acid (TRIAC), a thyromimetic with selective pituitary and hepatic action (37, 38, 39), had recently been shown to ameliorate symptoms in a number of RTH cases (40, 41). Consequently, thionamide therapy was withdrawn and TRIAC (0.7 mg twice daily) commenced. The patient has remained asymptomatic, with normal TSH levels and disappearance of goiter on this therapy. At age 15 yr, 7 months, while still taking TRIAC, a number of tissue markers of thyroid hormone action were measured and then repeated 2 weeks later after its discontinuation (Table 2Go). The withdrawal of TRIAC resulted in a rise in serum free (f)T3 levels, and, as expected, markers of pituitary and hepatic thyroid hormone action reflected RTH in these tissues (increased serum TSH, cholesterol, triglycerides; and reduced ferritin, angiotensin-converting enzyme). In contrast, there was a slight increase in BMR and peak 24-h heart rate when TRIAC treatment was interrupted. We interpret the combination of thyrotoxic symptoms and tissue marker measurements to indicate that RTH is predominant in the pituitary and liver in this patient.


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Table 1. Thyroid Function Tests in a Kindred with RTH

 

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Table 2. Tissue Markers of Thyroid Hormone Action in the Proband

 
Abnormal thyroid function tests were also noted in her father and grandfather (Table 1Go). Her father had been asymptomatic for many years although he had recently developed heat intolerance and palpitations, and scintigraphy revealed an enlarged thyroid gland with increased uptake in a nodule within the right lobe. The grandfather had never come to medical attention. PCR and direct sequencing of exon 10 of the TRß gene showed that all three individuals were heterozygous for a substitution at nucleotide 1433 (CGC to CAC), which corresponded to an arginine to histidine change at codon 383. Coding exons 4–9 of the TRß gene were also sequenced and no other abnormalities were detected. There was complete concordance between the nucleotide substitution and biochemical phenotype, such that five other family members with normal thyroid function did not harbor the receptor abnormality, which strongly suggested that the mutation was causally linked.

R383H Impairs Negative Transcriptional Regulation but Not Transactivation of Positively Regulated Genes
The properties of this unusual mutation were systematically examined to discover the extent of functional impairment. The T3 binding affinity of the R383H mutant receptor protein was 0.6 ± 0.1 x 1010 M-1 (mean ± SE) compared with 1.0 ± 0.01 x 1010 M-1 for the wild-type (WT) receptor; the ka mutant/kaWT ratio was 0.7 ± 0.1 (mean ± SE) which accords with a value determined previously (15).

The transcriptional properties of the R383H mutant were examined by transfection of either the mutant or WT receptor TRß1 isoform together with a reporter gene containing positively regulated TREs. The R383H mutant receptor transactivated everted repeat (F2TKLUC) or palindromic (PALTKLUC) TRE-containing reporter genes fully, comparable to WT recep-tor (Fig. 1Go, a and b) and achieved greater maximal activity than WT receptor on the direct repeat TRE (MALTKLUC) at higher T3 concentrations (Fig. 1cGo). Although the transcriptional response of mutant receptor appeared to be slightly delayed on all three response elements at low T3 concentrations (0.1 nM), this was not statistically different from WT. In keeping with its normal transcriptional properties on these TREs, when the R383H mutant and WT receptors were cotransfected in equal amounts, no dominant negative inhibition of WT receptor function was observed (Fig. 1Go, d–f).



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Figure 1. Positive Transcriptional Regulation by WT ({square}) and R383H ({circ}) Mutant Receptors

The data shown in this and subsequent figures are the mean (± SE) of at least three experiments each done in triplicate. a–c, Receptor function was assayed by transfection of JEG-3 cells with 50 ng TRß1 receptor expression vector, 500 ng (MALTKLUC, PALTKLUC) or 1 µg (F2TKLUC) reporter gene, and 100 ng BOS ß-gal internal control plasmid, followed by incubation with 0–1 µM T3 as indicated. Activation of F2 (a), PAL (b), and MAL (c) reporters by TRß receptor is expressed relative to the maximal induction by WT receptor. d–f, Dominant negative activity of the R383H mutant receptor was assayed in JEG-3 cells by cotransfection of 50 ng wild-type expression vector together with 50 ng WT (solid) or mutant (hatched) receptor, and reporter and reference plasmids as in panels a–c. Inhibition or stimulation of reporter gene activity was measured after incubating with low or high T3 concentrations as shown. Where less than 10% of the mean, error bars have been omitted for clarity.

 
The transcriptional activity of the R383H mutant was then examined with two negatively regulated target gene promoters, i.e. the human pituitary TSH{alpha} subunit and hypothalamic TRH genes. Ligand-dependent inhibition of TRHLUC by the R383H TRß1 mutant was significantly impaired compared with WT at low T3 concentrations, but maximal inhibition was achieved at higher T3 levels (Fig. 2aGo), and the mutant receptor showed a similarly impaired inhibitory profile with TSH{alpha}LUC (Fig. 2bGo). TRß2 is an amino-terminal splice variant of the TRß gene that is known to be highly expressed in the pituitary (42, 43) and hypothalamus (44) and may therefore be the critical receptor isoform involved in negative feedback effects of thyroid hormone at the hypothalamic-pituitary level. We therefore examined negative transcriptional regulation by the R383H mutant in a TRß2 context. Again, transcriptional inhibition of both TRH and TSH{alpha} reporter genes by the R383H mutant was significantly impaired compared with WT TRß2, analogous to that seen in a ß1-background (Fig. 2Go, c and d). Dominant negative activity was assayed by cotransfecting equal amounts of WT and mutant receptors with each negative TRE. The R383H mutant receptor was able to dominantly inhibit the function of its WT counterpart in either ß1- or ß2-contexts on the TRH promoter at low (0.1 nM) T3 concentrations, and the effect was relieved at 1 nM T3 (Fig. 2Go, e and g). With the TSH{alpha} promoter, the action of wild type TRß1 was inhibited by the R383H mutant in either TRß1 (Fig. 2fGo) or TRß2 (data not shown) backgrounds. However, WT TRß2 receptor function on this promoter could neither be inhibited by mutant R383Hß2 (Fig. 2hGo) nor by R383Hß1 receptors (data not shown).



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Figure 2. Negative Transcriptional Regulation by WT ({square}) and R383H ({circ}) Mutant Receptors

Inhibition of TRHLUC reporter activity was assayed by transfection of JEG-3 cells with 100 ng of either WT ({square}) or R383H mutant ({circ}) TRß1 (a) or TRß2 (c) expression vectors, 2 µg TRH LUC and 100 ng BOS ß-gal, and incubation with 0–1 nM T3. Inhibition of TSH{alpha}LUC reporter activity was assayed similarly using 50 ng WT ({square}) or R383H (O), TRß1 (b), or TRß2 (d) expression vector, 500 ng TSH{alpha}LUC, and 100 ng BOS ß-gal and incubation with 0–1 µM T3. Hormone-dependent inhibition is expressed relative to the activity in cells incubated without T3, and the background inhibition of TRHLUC and TSH{alpha}LUC reporter activity in control cells transfected with 100 ng (a and c) or 50 ng (b and d) RSV vector ({Delta}) is also shown. Dominant negative activity on TRHLUC of the R383H mutant ß1 (e) and ß2 (g) receptors was assayed using 100 ng WT expression vector, together with 100 ng wild type (solid) or mutant (hatched) receptor, and reporter and reference plasmids as in panel a. Dominant negative activity of the R383H mutant ß1 (f) and ß2 (h) receptors was assayed on TSH{alpha}LUC by cotransfection in JEG-3 cells with 50 ng WT expression vector, together with 50 ng WT (solid) or mutant (hatched) receptor, and reporter and reference plasmids as in panel b. Asterisks indicate a statistically significant difference (*, P <= 0.05; **, P < 0.01) between WT and R383H mutant receptor function. Where less than 10% of the mean, error bars have been omitted for clarity.

 
T3-Dependent Relief of Silencing or Reversal of Unliganded Activation Is Disproportionately Impaired with the R383H Mutant
TR is known to enhance the basal transcriptional activity of some negatively regulated promoters in the absence of ligand (34, 35, 45). In keeping with this, unliganded WT or R383H mutant ß1-receptors augmented the activity of the TRH gene promoter comparably (R383H = 3.22 ± 0.34 vs. WT = 3.03 ± 0.31-fold; mean ± SE) relative to levels seen in the absence of cotransfected receptor (Fig. 3aGo). For the R383H mutant receptor, this unliganded activation was not reversed by T3 comparably to WT, such that TRHLUC reporter activity remained elevated at T3 concentrations (0.05–0.1 nM) that returned the activity of this promoter by unliganded WT receptor to baseline (Fig. 3aGo). Impaired T3-dependent inhibition of TRHLUC reporter activity was also seen with the R383H mutant receptor in a TRß2 context (data not shown), and again the degree of unliganded activation did not differ between WT and mutant receptors (WT = 1.98 ± 0.21 vs. R383H = 1.89 ± 0.13-fold). In accordance with previous studies (36), we have not observed basal activation of the TSH{alpha} promoter by either unliganded TRß1 or TRß2 in JEG-3 cells.



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Figure 3. Basal Activation on a Negatively Regulated Promoter and Silencing on a Positively Regulated Reporter by Wild Type and Mutant TR

a, Transcriptional activity on TRHLUC for WT (solid bars) or R383H (hatched bars) ß1-receptor was assayed in the presence of 0–1 nM T3 as described above (Fig. 2aGo) and expressed as the activity relative to that seen in the absence of cotransfected receptor (using 100 ng RSV vector to control for the addition of DNA; stippled bar) and ligand. b, Silencing of basal transcription was assayed by cotransfecting 50 ng of either WT (solid bars) or R383H mutant (hatched bars) Gal4DBD-receptor ligand-binding domain fusions, together with 500 ng of UASTKLUC and 100 ng BOS ß-gal, into 1-BR cells in the presence of 0–10 nM T3. Repression is expressed relative to reporter activity of 1.0 with the Gal4DBD alone (stippled bar). Asterisks denote significant differences (*, P < 0.05, **, P < 0.01) between WT and R383H.

 
Two recent studies have suggested a possible correlation between ligand-independent activation by TR of negatively regulated promoters, and ligand-independent repression of positive TREs (34, 35). Accordingly, we assayed the silencing function of WT and R383H mutant receptors using a paradigm where repression is readily observed. The receptor ligand-binding domains were coupled to the heterologous DNA-binding domain of Gal4 and cotransfected with a reporter gene (UASTKLUC) containing Gal4-binding sites. In this system, unliganded WT and R383H mutant receptors inhibited basal transcription comparably, but T3-dependent derepression differed, such that the mutant receptor continued to repress transcription at a T3 concentration (0.5 nM) at which silencing by WT receptor was fully relieved (Fig. 3bGo). At higher T3 levels (10 nM) the R383H mutant activated reporter activity fully comparable to wild type.

The R383H Mutant Exhibits Impaired Ligand-Induced Corepressor Release but Recruits Coactivator Normally
The molecular events that occur after T3 occupancy of TR include release of corepressor together with recruitment of coactivators. Accordingly, to determine whether the abnormal T3-dependent changes in basal transcriptional activity observed above reflected aberrant TR-cofactor binding, we assayed the interaction between mutant receptor and corepressor or coactivator proteins in vitro. 35S-labeled R383H mutant receptor exhibited delayed ligand-dependent dissociation from glutathione-S-transferase (GST)-SMRT relative to WT receptor, and to a greater degree than predicted from its slightly decreased ligand-binding affinity (Fig. 4aGo). The EC50 for SMRT release was 7.5 nM T3 for WT receptor vs. 35 nM T3 for R383H. In contrast, T3-dependent recruitment of the R383H mutant receptor to a GST-SRC1 coactivator fusion was unimpaired (Fig. 4bGo). Ligand-dependent recruitment of other coactivators (CBP, RIP140) by the R383H mutant was also normal (data not shown).



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Figure 4. Protein Assays with Corepressor and Coactivator

Protein-protein interactions were assayed between bacterially expressed GST-SMRT (a) or GST-SRC1 (b) and in vitro synthesized 35S-labeled WT ({square}), mutant R383H ({circ}), or R429Q ({Delta}) TRß1. The amount of TR bound to the GST-SMRT protein in the presence of 0–1 µM T3 is represented by its ratio to the amount bound in the absence of ligand, and the amount of TR bound to GST-SRC1 is represented by its ratio to that maximally bound for each receptor in a given experiment. Each data point represents the mean ± SE of five (GST-SMRT) or three (GST-SRC1) experiments. The double asterisk indicates statistically significant differences (P < 0.01) for each of R383H and R429Q compared with WT, and a single asterisk indicates a significant difference (P < 0.05) between R383H and WT.

 
We subsequently examined the effect of DNA and RXR binding on aberrant corepressor release by the mutant receptor. In gel shift analyses, we used a negative TRE identified previously in the TRH gene promoter, which is independently capable of mediating negative regulation by TR (45). When bound to this response element as a heterodimer with RXR, unliganded WT TR recruited SMRT to generate a higher-order complex (Fig. 5AGo). This SMRT-TR/RXR complex was progressively attenuated in the presence of increasing concentrations of T3. With the R383H mutant, the addition of SMRT generated a greater amount of SMRT-mutant TR-RXR complex in the absence of ligand, and the corepressor then dissociated less readily with increasing T3 concentrations (Fig. 5AGo). In this representative experiment, the EC50 of corepressor release was 0.8 nM T3 for wild type vs. 2.3 nM T3 for R383H. Similarly, we observed impaired corepressor release when the R383H mutant receptor was bound to a direct repeat-positive TRE from the malic enzyme gene promoter (Fig. 5BGo), wherein the EC50 of corepressor release was 0.8 nM T3 for WT and 4.6 nM T3 for R383H.



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Figure 5. R383H and R429Q Impair Corepressor Release when Bound to DNA

Gel shift analyses were performed using the TRH negatively regulated promoter response element (TRH NTRE) (A) or a direct repeat positively regulated response element (ME PTRE) (B). Wild type (WT) or mutant receptors were preincubated with RXR, oligonucleotide probe, and 0–1 µM T3 before the addition of GST or GST-SMRT. The proportion of TR-RXR heterodimer supershifted by SMRT at each concentration was calculated by quantifying band intensities by electronic autoradiography. RT denotes receptor-RXR heterodimers, and SRT denotes heterodimers supershifted by SMRT.

 
R383H and R429Q Interact in the Crystal Structure of TR{alpha}, and Both Are Impaired for Corepressor Release and Negative Regulation
The crystal structure of rat TR{alpha} indicates that the residue arginine 329, which is homologous to arginine at codon 383 in TRß, forms a charge pair with a glutamic acid at codon 257, corresponding to codon 311 in TRß and that an arginine residue at codon 375, equivalent to codon 429 in TRß, also participates in this hydrophilic interaction (Fig. 6aGo). Interestingly, we and others have previously documented a natural arginine to glutamine TRß mutation at codon 429 (R429Q) in RTH (36, 46, 48). Despite a normal ligand-binding affinity (46), this mutant receptor exhibits significant functional impairment, particularly for negative transcriptional regulation (36, 46). Furthermore, homodimer formation by the R429Q mutant receptor has also been shown to be impaired, particularly with an everted repeat TRE configuration (8, 36). We have observed that the R383H mutant receptor also forms homodimers less readily than WT receptor on a palindromic TRE, although homodimer formation on an everted repeat TRE is normal (data not shown). In contrast, both R383H and R429Q mutants formed heterodimers with hRXR{alpha} readily on direct repeat (Fig. 5BGo), palindromic, and everted repeat response elements (data not shown).



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Figure 6. RTH Mutations Shown in the Crystal Structure of Rat TR{alpha}

A, A polar invagination is formed by the interaction between arginine at codon 329 (homologous to codon 383 in TRß), glutamic acid at codon 257 (codon 311 in TRß), and arginine at codon 375 (codon 429 in TRß). The water molecules that fill the invagination hydrogen bonds are shown as dotted lines between interacting residues. B, Mutations observed in patients with RTH mapped onto their homologous residues in the rat TR{alpha} ligand-binding domain. The R383H mutation is shown in red. The mutations shown are A234T, R243W/Q, T277A, A279V, R282S, M310T, M313T, S314Y/F, R316H, A317T, R320C/H/L, Y321C, D322H/N, G332R/E, E333Q, M334R, T337A, R338W/L, Q340H, G344E, G345R/S/V/D, G347E/W, V349M, I353T, R429Q, {Delta}430M, I431T, {Delta}432G, H435L/Q/Y, R438H/C, M442V, E445K, K443N/E, C446R, L450H, F451C/I/S, P453A/H/S/T, L454V, F459C, E460K (Refs. 2, 8, and 52–56 and our unpublished results), and those in residues between {alpha}{alpha} 234–282 are shown in green, mutations in residues between {alpha}{alpha} 310–353 are shown in orange, and mutations in residues between {alpha}{alpha} 429–461 are shown in blue.

 
In view of the structural proximity of the mutated R383 and R429 residues, and similarities in their DNA-binding and transcriptional properties, we also tested the interaction of the R429Q mutant with corepressor. As with R383H, ligand-dependent dissociation from GST-SMRT was disproportionately impaired for the R429Q mutant (Fig. 4aGo, R429Q EC50 28 nM T3), whereas its T3-dependent recruitment to GST-SRC1 was comparable to WT receptor (Fig. 4bGo). Corepressor release by this mutant was also markedly impaired when bound to negative or positive TREs [in representative experiments, EC50 for corepressor release by R429Q was 3 nM T3 on nTRE (Fig. 5AGo), and 5.2 nM T3 (Fig. 5BGo) on pTRE].


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have described a novel natural mutation (R383H) that is located outside the two main clusters of mutations in the TRß gene that give rise to RTH. The anomalous location of this mutation is particularly evident when compared with that of other RTH mutants that are mostly clustered around the ligand-binding pocket, when modeled on the crystal structure of TR{alpha} (Fig. 6BGo). The occurrence of this mutation in a kindred with RTH is significant as it indicates that at the very least, the negative feedback actions of thyroid hormones in the hypothalamic-pituitary-thyroid axis in vivo are impaired. The mild but consistent elevation of serum thyroid hormone concentrations in affected members of this kindred is congruent with the modest reduction in ligand-binding affinity of the R383H mutant receptor (70% that of wild type) together with the observation that the majority of RTH mutants demonstrate an inverse correlation between serum free T4 concentrations and their T3 affinity constants, defining the so-called group I RTH mutants (46), to which R383H may be added. The unexpected location of this mutation prompted a thorough study of the mutant receptor in which we have attempted to rationally explore its functional properties in a structural context.

The R383H mutant receptor was predominantly impaired for ligand-dependent inhibition of the pituitary TSH{alpha} and hypothalamic TRH gene promoters when studied in the context of TRß1 as well as TRß2—the particular receptor isoform that may mediate negative feedback effects in the pituitary and hypothalamus (42, 43, 44). Furthermore, this mutant receptor was capable of inhibiting WT receptor action on the TRH promoter in both TRß2 or TRß1 contexts or the TSH{alpha} promoter in a TRß1 context. It is interesting to note that the R383H mutant lacked dominant negative activity on the TSH{alpha} promoter in a TRß2 context. One explanation is that the presence of strong T3-dependent inhibition in the presence of empty Rous sarcoma virus (RSV) vector effectively masks any effect produced by cotransfection of the mutant R383H and WT receptors. Alternatively, recent studies indicate that transgenic mice, with expression of RTH mutant receptors targeted selectively to the pituitary, only exhibit marginally abnormal serum T4 levels (49). It is therefore possible that dominant negative inhibition by mutant receptors on hypothalamic target genes might contribute significantly to the abnormal biochemical phenotype of RTH. The R383H mutant activated positively regulated reporter genes comparably to wild type and exhibited negligible dominant negative activity with these TREs.

Recently, Yoh et al. (33) have shown that a number of RTH mutants interact aberrantly with corepressor. Some mutants bind SMRT more avidly than WT receptor when unliganded and T3-induced corepressor dissociation is impaired. For two mutations (P453A, P453H), involving a proline residue that precedes an amphipathic {alpha}-helix at the receptor carboxy terminus, ligand-dependent corepressor release was more markedly reduced than expected from their impaired ligand-binding affinities (33). Furthermore, when introduced into an RTH mutant background, artificial mutations that abolished corepressor interaction abrogated the dominant negative activity of RTH mutants. Corepressors have also been directly implicated in negative transcriptional regulation by WT TR. Tagami et al. (35) have observed that with negatively regulated promoters, cotransfected corepressor (SMRT or NCoR) augments basal promoter activation by unliganded TR. When tested in transfection assays, we found that the unliganded R383H mutant activated a negatively regulated promoter and repressed a positively regulated reporter gene comparably to WT receptor, although, in both contexts, T3-induced changes—either reversal of unliganded activation or relief of silencing—were impaired. We hypothesized that both these effects might reflect altered R383H-corepressor interaction, and indeed the mutant receptor dissociated less readily from SMRT than WT receptor (Fig. 4aGo). Furthermore, when tested as a heterodimer bound to TREs, the corepressor release by the R383H mutant was impaired on both negative and positive response elements. Accordingly, our data suggest that another receptor region distinct from the C-terminal amphipathic {alpha}-helix is involved in corepressor release, although the structural basis for this remains to be elucidated.

In a previous study, which tested the functional properties of artificial mutants generated by systematically mutating residues containing CpG dinucleotides within the ‘cold’ region of the TRß gene, the R383H mutant was predicted not to cause RTH because, when tested with positively regulated reporter genes, its transcriptional impairment was less than that observed with even comparatively mild RTH mutants (15), and another mutant (R429Q) was also predicted not to be involved in RTH on the same basis. However, both of these mutations have now been identified in association with RTH, and all affected individuals exhibited elevated serum thyroid hormones with nonsuppressed TSH levels (36, 46, 48), suggesting that they might manifest by impairing negative feedback regulation at hypothalamic and pituitary levels.

In the crystal structure of TR{alpha}, the residue homologous to Arg 383 interacts with another arginine, which corresponds to Arg 429 in TRß. Accordingly, it is intriguing that a naturally occurring RTH mutant involving this residue (R429Q) exhibits many functional similarities with R383H. Two groups have shown that the R429Q mutant exhibits greater functional impairment with negatively regulated than positively regulated promoters (36, 46), as we have documented with the R383H mutant. However, our studies do not exclude the possibility that, in different promoter or cell type contexts, these mutants can exhibit slightly impaired transactivation as has been observed (8, 15). We have also found that the R429Q mutant receptor is impaired for corepressor release in vitro and T3-dependent relief of silencing in vivo. Furthermore, with either mutant, these properties are not explicable on the basis of their T3 binding, as the R383H mutation results in a mild reduction in ligand-binding affinity, and the R429Q mutant binds ligand with wild-type affinity, and ligand-dependent coactivator recruitment by both mutants is normal. Our findings support the putative role for corepressors in negative regulation, in that impaired corepressor release seen with these mutants is associated with greater impairment of negative than positive transcriptional function. It remains equally possible that other, as yet unidentified, cofactors that bind TR in the same manner as corepressor mediate negative regulation in pituitary and hypothalamic contexts. The congruence of impaired corepressor release and ligand-dependent transcriptional inhibition in two natural TRß mutants, predicted not to occur in RTH, suggests that these properties might represent the minimal abnormalities necessary to cause this disorder.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical and Genetic Analyses
Serum free T4 and free T3 levels were measured with fluoroimmunometric assays using Delfia technology (Wallac, Milton Keynes, U.K.). Serum TSH concentrations were determined using a two-site assay (Wallac). BMR was measured over a 30-min period by ventilated hood indirect calorimetry. Pulse rate was monitored by Holter electrocardiography over 24-h periods. DNA was isolated from peripheral blood leukocytes from each family member using standard techniques. Exons 4–10 coding for the hormone-binding domain of the human TRß gene were amplified by PCR from the probands’ DNA using a forward primer tagged at the 5'-end with the universal M13 primer sequence, and automated dye primer (ABI/Perkin Elmer, Cheshire, U.K.) sequencing was then undertaken on an ABI 373 sequencer (ABI/Perkin Elmer, Foster City, CA). The presence or absence of the mutation in exon 10 was verified by at least two independent PCR and sequencing reactions for each family member. PCR primers and conditions have been described previously (48).

Ligand-Binding Assays
The R383H mutation was generated in vitro by site-directed mutagenesis of the WT TRß1 cDNA in M13 mp18 (8), subcloned into pGEM7Z, and verified by sequencing. WT and mutant receptor proteins were synthesized by in vitro transcription and translation in rabbit reticulocyte lysate (TNT, Promega, Southampton, U.K.). The T3 binding affinities of wild- type and mutant receptors were determined using a filter-binding assay (48), and Ka values are the mean (± SE) of at least four separate experiments performed in duplicate.

Transfection Assays
Transcriptional function of WT and mutant receptor was assayed by transient transfection with reporter genes as previously described (8). WT and mutant TRß1 cDNAs were cloned into a eukaryotic expression vector under the control of the RSV enhancer and promoter. The mutation was also introduced into the WT human TRß2 cDNA in the RSV vector, and all constructs were verified by sequencing. Reporter genes containing either a single copy (MALTKLUC, F2TKLUC) or two copies (PALTKLUC) of TRE upstream of the thymidine kinase (TK) promoter and luciferase gene have been described elsewhere (8). The TSH{alpha}LUC reporter gene contains the glycoprotein hormone {alpha}-subunit promoter (-846 to +44 bp) upstream of the luciferase gene (8). The TRHLUC construct contains the human TRH promoter from -900 to +55 bp upstream of the luciferase gene (34). JEG-3 cells were transfected by a 4- to 6-h exposure to calcium phosphate containing receptor expression vector(s), luciferase reporter gene, and internal control plasmid BOS-ßgal. Human fibroblast 1-BR cells were transfected by exposure to calcium phosphate containing Gal4-fusion receptor expression vector, UASTKLUC (50), and BOS-ßgal. In each case, after a 36-h incubation with L-T3 as indicated, cells were lysed and assayed for luciferase and ß-galactosidase activities. Significant differences (P < 0.05) were determined using Student’s t-test.

Protein-Protein Interaction Assays
Corepressor interaction assays were performed using a C-terminal construct of SMRT containing the two receptor interaction domains cloned in a GST-fusion bacterial expression vector (21). Full-length WT or mutant TRß1 receptors were synthesized in vitro in the presence of [35S]methionine, and equal counts of receptor were incubated at 4 C for 2 h with ~10 µg immobilized GST-SMRT in HEMG buffer (33) and L-T3 where indicated. After three washes with NETN buffer (20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0), reactions were analyzed by SDS-PAGE and Coomassie stained to verify that equal amounts of protein were recovered from the washing steps, followed by quantification on an Instant Imager (Packard, Meridan, CT) and subsequent autoradiography. Significant differences (P < 0.05) were determined using Student’s t-test. [pp]Coactivator interaction assays using a GST-SRC1 ({alpha}{alpha} 570–780) vector that contains the central three receptor interaction motifs (51) were performed as described above. Alternatively, the interaction of bacterially expressed wild-type or mutant receptor ligand-binding domains fused to GST was examined with in vitro translated RIP140 and CBP coactivators, using a similar method described previously (28). The vectors containing RIP140 and CBP have been described elsewhere (24, 27).

Gel Mobility Shift Assays with TR, RXR, and GST-SMRT
Electrophoretic mobility shift assays using a direct repeat TRE from the malic enzyme promoter were performed as previously described (8). A NTRE consisting of a single half-site has been described in the promoter of TRH gene (45), and the nucleotide sequence of the 32P-labeled oligonucleotide duplex used was: 5'- gcgacccctccccgcTGACCTcactcgagccgccgcctgg-3'. Supershift analyses were performed using GST-SMRT eluted from a Sepharose-G column with 20 mM glutathione in 50 mM Tris, pH 8.0. Equal amounts of in vitro translated receptor in a 1:1 ratio of in vitro translated hRXR{alpha} were incubated with 20 fmol 32P-labeled oligonucleotide probe and L-T3 where indicated for 30 min at room temperature before the addition of ~9 µg GST or GST-SMRT, such that the final reaction conditions were 8 mM glutathione, 20 mM Tris, pH 8.0, 16 mM HEPES, pH 7.8, 40 mM KCl, 8% glycerol, and 1 mM dithiothreitol. The incubation was continued for a further 30 min at room temperature. Reactions were analyzed by electrophoresis through a 5% acrylamide gel and quantitated using an electronic autoradiography Instant Imager (Packard, Meridan, CT).


    ACKNOWLEDGMENTS
 
We thank Professor R. Evans for providing the vectors containing SMRT and CBP, Dr. M. Parker for providing the vectors containing RIP-140 and GST-SRC1, and Drs. Wondisford and Hollenberg for providing the TRHLUC reporter gene.


    FOOTNOTES
 
Address requests for reprints to: Dr. V. Krishna Chatterjee, Department of Medicine, Level 5, Addenbrookes Hospital, Hills Road, Cambridge, United Kingdom CB2 2QQ.

This research was supported by the Wellcome Trust (V.K.K.C.) and Medical Research Council (U.K.). R.C-B. is a Commonwealth Foundation Research Scholar.

Received for publication February 21, 1997. Revision received January 30, 1998. Accepted for publication February 4, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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