A Novel Natural Mutation in the Thyroid Hormone Receptor Defines a Dual Functional Domain That Exchanges Nuclear Receptor Corepressors and Coactivators

Tetsuya Tagami, Wen-Xia Gu, Patricia T. Peairs, Brian L. West and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine (T.T., W.-X.G., J.L.J) Northwestern University Medical School Chicago, Illinois 60611
The Baton Rouge Clinic (P.T.P.) Baton Rouge, Louisiana 70806
Metabolic Research Unit (B.L.W.) University of California at San Francisco San Francisco, California 94143


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In a patient with severe resistance to thyroid hormone (RTH), we found a novel mutation (leucine to serine in codon 454, L454S) of the thyroid hormone receptor ß. This mutation is in the ligand-dependent transactivation domain that has been shown to interact with transcriptional coactivators (CoAs). The mutant protein binds T3, but its ability to activate transcription of a positively regulated gene (TRE-tk-Luc), and to repress a negatively regulated gene (TSH{alpha}-Luc), is markedly impaired. As anticipated from its location, the L454S mutant interacts weakly with CoAs, such as SRC1 and glucocorticoid receptor interacting protein 1 (GRIP1) in gel mobility shift assays and in mammalian two-hybrid assays, even in the presence of the maximal dose of T3. In contrast, in the absence of T3, the L454S mutant interacts much more strongly with nuclear receptor corepressor (NCoR) than does the wild-type receptor, and the T3-dependent release of NCoR is markedly impaired. By comparison, the NCoR interaction and T3-dependent dissociation of an adjacent AF-2 domain mutant (E457A) are normal. These findings reveal that the Leu 454 is involved directly, or indirectly, in the release of corepressors (CoRs) as well as in the recruitment of CoAs. The strong interaction with NCoR at a physiological concentration of T3 results in constitutive activation of the TSH genes as well as constitutive silencing of positively regulated genes. When the dominant negative effect was examined among various mutants, it correlated surprisingly well with the potency of NCoR binding but not with the degree of impairment in CoA binding. These findings suggest that the defective release of NCoRs, along with retained dimerization and DNA binding, are critical features for the inhibitory action of mutant thyroid hormone receptors. These studies also suggest that helix 12 of the thyroid hormone receptor acts as a dual functional domain. After the binding of T3, its conformation changes, causing the disruption of CoR binding and the recruitment of CoAs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Resistance to thyroid hormone (RTH) is a syndrome of reduced responsiveness of the target tissues to thyroid hormone (T3) (1, 2). RTH is an autosomal dominant disorder caused by mutations in the thyroid hormone receptor ß (TRß) gene. Although hormone resistance occurs to varying degrees in all tissues, the diagnosis is made primarily based on abnormalities in the TRH-TSH-T3 axis. Specifically, RTH is characterized by elevated levels of thyroid hormone without evidence of appropriate suppression of TSH. The degree of hypothalamic-pituitary resistance establishes the setpoint that defines the circulating hormone level that impacts all other tissues.

TRs function as ligand-regulated transcription factors that increase or decrease the expression of target genes (3, 4). In the unliganded state, TRs suppress the basal activity of promoters that contain positively regulated hormone response elements (5, 6, 7). Recently, two classes of nuclear corepressors (CoRs), termed nuclear receptor corepressor (NCoR) (8, 9) or retinoid X receptor (RXR)-interacting protein 13 (RIP13) (10) and silencing mediator for retinoid and thyroid hormone receptors (SMRT) (11) or T3 receptor-associating cofactor (TRAC) (12) were identified and have been shown to mediate ligand-independent repression. The addition of ligand reverses gene silencing and induces strong stimulation of these genes. Several transcriptional coactivators (CoAs) have also been identified and have been shown to mediate ligand-dependent activation. These include steroid receptor coactivator 1 (SRC1) (13), transcriptional intermediary factor 2 (TIF2) (14)/glucocorticoid receptor interacting protein 1 (GRIP1) (15), amplified in breast cancer 1 (AIB1) (16)/thyroid receptor activator molecule 1 (TRAM 1) (17)/receptor associated coactivator 3 (RAC3) (18)/p300/CBP cointegrator associated protein (p/CIP) (19)/nuclear receptor coactivator (ACTR) (20), cAMP response element binding protein (CREB) binding protein (CBP) (21)/p300 (22), among others. In contrast to positively regulated genes, negatively regulated genes are stimulated by unliganded receptor and are strongly repressed after the addition of T3 (23, 24, 25). Paradoxically, CoRs are involved in the basal activation of negatively regulated genes (25).

Studies of TRß mutations that cause RTH have provided valuable insights into the structure and function of the receptor (1, 2). Most RTH mutations reduce or eliminate T3 binding, thereby leading to defective T3-dependent transcriptional function. In rare instances, T3 binding is minimally altered, but the RTH mutants exhibit reduced transcriptional activation, probably because of altered interactions with CoAs (26, 27, 28, 29). Because the mutant receptors retain dimerization properties and the ability to bind to DNA, they are able to interfere with the function of normal TRs (dominant negative effect). In a previous study, we demonstrated that RTH mutants retain transcriptional silencing of positively regulated genes and basal activation of negatively regulated genes (30). The introduction of a TRß mutation (P214R) that disrupts interactions with CoRs eliminated these properties of the RTH mutants as well as their dominant negative activity, suggesting that CoRs play an important role in the inhibitory features of RTH mutants.

In this report, we describe a novel RTH mutation (leucine to serine in codon 454 in TRß) in a patient with severe RTH. Studies of this mutation reveal a dual functional domain that is required for T3-dependent release of CoRs and recruitment of CoAs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical Studies
The patient (MW) was the product of a precipitous vaginal delivery complicated by hypovolemia, apparently secondary to a tight nuchal cord. Her birth length was 20 inches and weight 6 lbs. Early infancy was notable for poor weight gain, dry skin, constipation, difficulty sleeping, and irritability. During the first year, she experienced four episodes of febrile seizures associated with recurrent otitis media. At age 13 months, she fractured her right tibia and fibula after a fall of 18 inches. At 15 months of age, she was evaluated because of developmental delay. She walked with assistance and spoke only a single word. Her weight was in the 5th percentile and height and head circumference were in the 50th percentile. Her blood pressure was 110/61 mm Hg, and the resting pulse rate was 141 beats per min (bpm). Skin was dry, the thyroid gland was palpable but not enlarged, and the remainder of her physical examination was unremarkable. Chromosome studies were normal and a magnetic resonance image of the head was normal. Thyroid function tests revealed T4 = 33 µg/dl, T3 resin uptake = 34%, TSH = 3.2 mIU/ml, and T4 binding globulin (TBG) = 5.4 mg/dl (normal = 2.1- 6.0 mg/dl) (Table 1Go). Retrieval of her neonatal T4 level, which had been misplaced in the laboratory, revealed a value of 24 µg/dl. There was no maternal or family history of thyroid disease. Methimazole therapy was begun but was discontinued 6 weeks later after a possible diagnosis of RTH was suggested. Several months later, the patient was admitted to the hospital to examine responses to T3, 25 µg orally three times per day. After 5 days of treatment, her thyroid function tests changed as decreased, but remained abnormally elevated (Table 1Go). Her basal heart rate was 110–120 bpm and increased to 150–156 bpm on T3. Sex-hormone binding globulin levels changed from 0.6 to 1.1 µg/dl (normal, 1.8–5.5 µg/dl) on T3. There was no change in resting energy expenditure. A TRH test on T3 showed the following TSH responses: 1.9 at 0 min, 53.2 at 30 min, and 28.2 at 60 min. T3 was discontinued and she was started on methylphenidate (Ritalin) for management of attention-deficit hyperactivity disorder. At age 7 yr, she developed several episodes of chest pain and shortness of breath, particularly after exertion. This was attributed to tachycardia (heart rate > 200 bpm). Methylphenidate was discontinued and ß-blockers were begun with some improvement. Methimazole (5 mg orally three times daily) was restarted because of persistent tachycardia and behavioral problems. Thyroid function tests showed a marked increase in TSH levels (Table 1Go), her behavioral problems and school performance improved, but a large goiter developed. Presently, the patient remains on a low dose of methimazole and ß-blockers.


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Table 1. Thyroid Function Studies in Patient with L454S TRß Mutation

 
Genetic Analyses
Direct sequencing of exon 10 of the TRß gene in the proband (MW) indicated that she was heterozygous for a T-to-C substitution of nucleotide 1361. The mutation results in the replacement of the leucine (TTG) with serine (TCG) in codon 454 (L454S). Both parents had normal TRß sequences, indicating that the mutation in MW was sporadic.

Analysis of T3 Binding
This mutation is located in the ligand-dependent AF-2 transactivation domain in the extreme carboxy terminus of TRß. Since most RTH mutants have reduced or a complete loss of binding to T3, the affinity of T3 binding was determined for the L454S mutation (Fig. 1AGo). The T3 binding properties of the L454S and additional mutant receptors (L454A, E457A, F451X, G345R) are summarized in Table 2Go. Using in vitro transcribed and translated proteins, the T3-binding affinities of L454S, L454A, and E457A were 22%, 59%, and 85% of wild-type receptor, respectively. Binding was below detection for the nonsense mutation, F451X (31), and for G345R (32). Because of unusual functional responses to T3 (see below), binding to L454S was also confirmed in intact cells (Fig. 1BGo). The T3-binding affinity of the L454S mutant in transfected cells (27% wild-type) was similar to that used in translated receptors (Table 2Go).



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Figure 1. Scatchard Analyses of T3 Binding to Wild-Type and the L454S Mutant TR

A, T3 binding to in vitro transcribed and translated receptors. B, T3 binding to TSA-201 cells transfected with 1 µg of TR expression vectors.

 

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Table 2. T3 Binding to Mutant Receptors

 
T3 has been shown to dissociate TR homodimers when bound to certain types of thyroid hormone response elements (TREs) (33, 34). The effect of T3 on homodimerization of the L454S was examined using the inverted palindrome-TRE (LAP-TRE) (Fig. 2Go). Homodimers of both wild-type (lanes 1–5) and L454S (lanes 6–10) were decreased by T3 in a dose-dependent manner. In contrast, F451X, which contains an 11-amino acid deletion at the carboxy terminus that deletes the AF-2 domain and eliminates T3 binding (31), was insensitive to T3 except at very high doses (103 nM). These results indicate that L454S binds T3 when bound to DNA, as well as in solution, and that T3 induces conformational changes to L454S analogous to those in the wild-type receptor.



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Figure 2. The Effects of T3 on Homodimerization of Wild-Type or AF-2 Mutants in Gel Mobility Shift Assays

DNA binding to in vitro translated TRs (wild-type, WT; L454S; F451X) was analyzed in the presence of increasing concentrations of T3. The DNA-binding site is 32P-labeled LAP-TRE. The amount of homodimer TR complex is decreased by T3.

 
Functional Properties of Mutant Receptors
The functional characteristics of mutant TRs were assessed based on their abilities to modulate expression of positively or negatively regulated reporter genes in TSA-201 cells. Expression of the wild-type or mutant receptors in transfected TSA-201 cells was analyzed using gel shift assays to detect expressed receptors bound to a DR4 response element (30). No binding was seen in mock-transfected cells, but similar amounts of binding were detected for wild-type TRß and the mutant receptors (F451X, L454S, L454A, E457A, and G345R) (Fig. 3Go).



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Figure 3. Expression and DNA-Binding Activity of TRs Expressed in TSA-201 Cells

TSA-201 cells were transfected with the indicated TR expression vectors. Lysates were bound to radiolabeled DR4-TRE and analyzed by electrophoretic gel mobility shift assays. The binding of control in vitro translated TR and RXR{alpha} are shown in lane 1. The locations of the TR-TR homodimers and RXR-TR heterodimers are indicated by arrows.

 
Using the positively regulated promoter TRE-tk-LUC, each of the TR mutants exhibited strong transcriptional silencing in the absence of T3 (Fig. 4AGo). The AF-2 domain mutants, L454S, L454A, and E457A, induced very little transcriptional stimulation in the presence of T3 (Fig. 4BGo). Complete dose-response curves are shown in Fig. 4CGo. Whether the slight T3-induced activation seen with these mutants reflects loss of CoR binding, recruitment of CoAs, or both, cannot be ascertained from these assays.



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Figure 4. Function of Wild-Type or AF-2 Mutant Receptors with Respect to a Positively Regulated Reporter Gene

TR expression plasmids (10 ng) for the indicated mutants were transfected into TSA-201 cells together with 100 ng of the positively regulated reporter gene, TRE-tk-Luc. A, Basal expression in the absence of T3. B, Expression in the presence of 5 nM T3. C, Expression in the presence of various concentrations of T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 
Using the negatively regulated promoter, TSH{alpha}-LUC, L454S, L454A, and the truncation mutant F451X exhibited strong basal activation in the absence of T3 compared with the wild-type TR or the E457A or G345R mutants (Fig. 5AGo). T3-induced repression was markedly impaired with each of these mutants at 5 nM T3 (Fig. 5BGo). At greater doses of T3, transcriptional repression varied among the mutants (Fig. 5CGo). Wild-type receptor showed half-maximal repression at 0.3 nM T3. The L454S and L454A mutants showed partial repression, but only at relatively high doses of T3 (half-maximal repression at 10 nM T3). The truncation mutant (F451X) was not repressed even at maximum doses of T3 (data not shown), consistent with its inability to bind hormone.



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Figure 5. Function of Wild-Type or AF-2 Mutant Receptors with Respect to a Negatively Regulated Reporter Gene

TR expression plasmids (100 ng) for the indicated mutants were transfected into TSA-201 cells together with 100 ng of the negatively regulated reporter gene, TSH{alpha}-Luc. A, Basal expression in the absence of T3. B, Expression in the presence of 5 nM T3. C, Expression in the presence of various concentrations of T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 
Dominant Negative Effect of Mutant Receptors on Positively and Negatively Regulated Promoters
Having established the functional properties of the individual receptor mutants, we next tested their dominant negative activities. Although the RTH mutants partially block the activity of the wild-type receptor at a 1:1 ratio (35) (data not shown), a 1:5 ratio of wild-type to mutant receptors was used to more clearly illustrate the dominant negative properties of the mutant receptors. The dominant negative activity was assessed at a dose of 5 nM T3, which is well above the saturating dose for both wild-type TR and the AF-2 domain mutants (Table 2Go). The F451X and L454S mutants strongly inhibited wild-type TRß1-regulated expression of TRE-tk-LUC (Fig. 6AGo). The G345R and L454A mutants moderately inhibited the activity of cotransfected wild-type receptor. By comparison, dominant negative activity of the E457A AF-2 domain mutant was minimal. Using the negatively regulated TSH{alpha} promoter, the potency of inhibitory activity among mutants was similar to that seen using the positively regulated promoter (Fig. 6BGo). The dominant negative effect was strongest using F451X, G345R, L454S, and L454A. It is notable that even though the E457A mutant induces little transcriptional activation of TRE-tk-Luc or repression of TSH{alpha}-Luc at this dose of T3, it is a relatively weak dominant negative inhibitor.



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Figure 6. Dominant Negative Activity of Mutant TRs

A, The dominant negative effects of mutant TRs on the positively regulated TRE-tk-Luc reporter gene. Wild-type (10 ng) and mutant TR (50 ng) expression plasmids were cotransfected into TSA-201 cells together with 100 ng of TRE-tk-Luc. B, The dominant negative effects of mutant TRs on the negatively regulated TSH{alpha}-Luc reporter gene. Wild-type (50 ng) and mutant TR (250 ng) expression plasmids were cotransfected into TSA-201 cells together with 100 ng of TSH{alpha}-Luc. Cells were incubated in the presence of 5 nM T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 
CoR and CoA Binding Properties of Mutant Receptors
Interactions of mutant receptors with NCoR or CoAs were examined using gel mobility shift assays and an interaction assay that is a variation of the two-hybrid assay performed in mammalian cells. For the gel mobility shift assays, the carboxyl-terminal half of the NCoR protein (NCoR-ID), which contains two nuclear receptor interaction domains (69), or GRIP1 (15) was used to supershift TR complexes bound to the LAP-TRE. As shown in Fig. 7Go, incubation of in vitro translated NCoR-ID with wild-type TR (lane 6) generated a slower migrating band corresponding to a complex between NCoR-ID and TR in the absence of T3. The NCoR interaction with wild-type TR (lanes 6–9) was decreased by T3 in a dose-dependent manner. In contrast, T3-dependent dissociation of NCoR from the L454S mutant (lanes 2–5) was impaired in comparison to the wild-type receptor. Incubation of in vitro translated GRIP1 with wild-type TR (lanes 14 and 15) generated a slower migrating band corresponding to a complex between GRIP1 and TR in the presence of T3. Using the L454S mutant, little or no interaction was observed (lanes 10–12), even in the presence of a maximal dose of T3.



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Figure 7. Interaction of NCoR or GRIP1 with Wild-Type or the L454S Mutant in Gel Mobility Shift Assays

NCoR-ID or GRIP1 binding to in vitro translated TRs (wild type, WT; L454S) was analyzed in the presence of increasing concentrations of T3. The DNA-binding site is 32P-labeled LAP-TRE. The positions of TR homodimer and the complexes supershifted by NCoR-ID and by GRIP1 are indicated. URL, Unprogrammed reticulocyte lysate.

 
A mammalian two-hybrid assay was used to more quantitatively assess interactions between mutant receptors and CoRs or CoAs. The NCoR-ID was fused to the DNA-binding domain (DBD) of the yeast transcription factor, Gal4. Similarly, the carboxyl-terminal interaction domains of the coactivators, SRC1 or GRIP1, were fused to Gal4. The ligand-binding domain (LBD) (residues 174–461) of wild-type or mutant TRßs was fused to the transcriptional activation domain of VP16 to allow detection of interactions between the Gal4 fusion proteins and the TRs. The reporter gene, UAS-tk-Luc, contains two Gal4-binding sites and was used to assay in vivo interactions between VP16-TR and Gal4-NCoR, -SRC1, or -GRIP1 in TSA-201 cells.

VP16-TRß exhibited T3 binding that was similar to native TRß (data not shown) and was used initially to test the interaction assay. The interaction between Gal4-NCoR and VP16-TRß was seen in the absence of T3 (Fig. 8AGo), and the degree of interaction was reversed by the addition of increasing concentrations of T3 (Fig. 8Go, B and C). T3-mediated dissociation of NCoR occurred with a half-maximal dose of about 0.3 nM. These results are consistent with the ability of T3 to dissociate NCoR from TR. The NCoR interaction with the VP16-L454S and VP16-L454A mutants was 2- to 3-fold greater than that seen with wild-type TR. Similar results were seen with the truncation mutant, F451X. The interaction of Gal4-NCoR and the E457A or the G345R mutants was, however, similar to wild-type TR. In the presence of 5 nM T3, NCoR was dissociated from the wild-type TR and the E457A mutants, but not from the L454S mutant. Dose-response experiments were performed to examine T3-mediated dissociation of NCoR in greater detail. Although T3 binding to the L454S mutant is about 25% of wild-type receptor, half-maximal dissociation of NCoR required greater than 10 nM T3, which is about 30-fold greater than that required by the wild-type receptor. It is notable, however, that dissociation of NCoR is complete at the highest dose of T3. Similar, but less pronounced, results were seen with the L454A mutant. T3 did not dissociate NCoR from the G345R or the F451X mutants, even at high doses (1 µM), consistent with their inability to bind T3 (data not shown). Using another CoR construct, Gal4-SMRT, essentially identical results were obtained. The SMRT interaction with the VP16-L454S mutant was 2.3-fold greater than that seen with wild-type TR in the absence of T3. T3-mediated dissociation of SMRT occurred with a half-maximal dose of about 0.2 nM with wild-type TR and 10 nM with the L454S mutant (data not shown).



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Figure 8. NCoR Interactions with Wild-Type and Mutant TRs in Mammalian Two-Hybrid Assays

The format of the mammalian two-hybrid experiment is shown at the top of the figure. The indicated VP16-TR expression plasmids (100 ng) were cotransfected into TSA-201 cells with 40 ng of Gal4-NCoR and 100 ng of the Gal4-responsive reporter gene UAS-tk-Luc. A, Basal expression in the absence of T3. B, Expression in the presence of 5 nM T3. C, Expression in the presence of various concentrations of T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 
Analogous experiments were performed to assess the interactions between the CoAs and the TR mutants (Figs. 9Go and 10Go). In the absence of T3, no interaction was seen between Gal4-SRC1 (Fig. 9AGo) or Gal4-GRIP1 (Fig. 10AGo) with any of the TR constructs. However, consistent with the ability of T3 to recruit CoAs to the TR, the interaction of Gal4-SRC1 (Fig. 9Go, B and C) or Gal4-GRIP1 (Fig. 10Go, B and C) and VP16-TRß was induced greater than 20-fold by T3. No interaction was observed between Gal4-DBD and VP16-TR in the absence or presence of T3 (data not shown). At a dose of 5 nM T3, there was little or no activation of any of the TR mutants except for L454A. Dose-response analyses revealed that wild-type TR recruitment of either SRC1 or GRIP1 was half-maximal at about 0.3 nM T3 (Figs. 9CGo and 10CGo), comparable to the dose required for NCoR dissociation. For the L454S or the L454A mutants, the recruitment of SRC1 or GRIP1 was partial, reaching only 25- to 30% of wild-type receptor, even at the highest doses of T3. Using Gal4-TRAM1 as a CoA, results almost identical to those using Gal4-SRC1 or Gal4-GRIP1 were seen (data not shown). Of note, the E457A mutant did not recruit any of these CoAs. Thus, the E457A mutation is permissive for T3-dependent dissociation of NCoR, but it lacks recruitment of CoAs.



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Figure 9. SRC1 Interactions with Wild-Type and Mutant TRs in Mammalian Two-Hybrid Assays

The format of the mammalian two-hybrid experiment is shown at the top of the figure. The indicated VP16-TR expression plasmids (100 ng) were cotransfected into TSA-201 cells with 40 ng of Gal4-SRC1 together with 100 ng of the Gal4- responsive reporter gene, UAS-tk-Luc. A, Basal expression in the absence of T3. B, Expression in the presence of 5 nM T3. C, Expression in the presence of various concentrations of T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 


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Figure 10. GRIP1 Interactions with Wild-Type and Mutant TRs in Mammalian Two-Hybrid Assays

The format of the mammalian two-hybrid experiment is shown at the top of the figure. The indicated VP16-TR expression plasmids (100 ng) were cotransfected into TSA-201 cells with 40 ng of Gal4-GRIP1 together with 100 ng of the Gal4-responsive reporter gene, UAS-tk-Luc. A, Basal expression in the absence of T3. B, Expression in the presence of 5 nM T3. C, Expression in the presence of various concentrations of T3. Results are the mean ± SEM from at least three transfections performed in triplicate.

 
Correlation of Dominant Negative Activity and Interaction of TR Mutants with CoRs and CoAs
The relationship between potencies of dominant negative effects and the interactions of TR mutants with NCoR or CoAs are depicted in Fig. 11Go. The dominant negative potency of each mutant was calculated as fold inhibition (vector = 1) from the data shown in Fig. 6AGo (TRE-tk-LUC). NCoR interactions were calculated from the data of Fig. 8BGo as fold interaction (vector = 1). SRC1 and GRIP1 interactions in the presence of 5 nM T3 were calculated from the data of Figs. 9BGo and 10BGo, respectively, as 1/fold interaction (vector = 1). When the dominant negative potency was plotted vs. TR interactions with NCoR, a very strong correlation was observed (r = 0.987) (Fig. 11AGo). In contrast, the correlation of dominant negative potency with 1/SRC1 interaction (r = 0.051) (Fig. 11BGo) or with 1/GRIP1 interaction (r = 0.093) (Fig. 11CGo) was very low. Studies using a variety of other TR mutants not included in this series confirm the correlation of TR binding to NCoR, but not to CoAs (data not shown). These results suggest that preserved and increased binding to NCoR, rather than the loss of binding to CoAs like SRC1 or GRIP1, may account for the severe RTH seen in this patient with the L454S RTH mutation.



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Figure 11. Correlation between Dominant Negative Activity and the in Vivo Interaction with NCoR, SRC1, or GRIP1

The dominant negative activity using TRE-tk-LUC is expressed as fold inhibition (data from Fig. 6AGo, vector = 1) by mutant TRs. It is plotted vs. (A) fold interaction (vector = 1) between VP16-mutant TR fusion proteins and Gal4-NCoR (data from Fig. 8Go), (B) 1/fold interaction (vector = 1) between VP16-mutant TR fusion proteins and Gal4-SRC1 (data from Fig. 9Go), or (C) Gal4-GRIP1 (data from Fig. 10Go).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we report a novel natural mutation (L454S) in the AF-2 domain of the TRß gene. The analysis of this mutant receptor, along with other RTH mutants, provides several lines of evidence that the dominant negative activity of mutant TRs correlates well with receptor binding to CoRs. The L454S mutant binds T3, but it does not interact with CoAs, such as SRC1 or GRIP1, even at maximal concentrations of T3. An interesting feature of this mutant is that it has a stronger interaction with NCoR than does the wild-type receptor in the absence of T3, and the release of NCoR by T3 is markedly impaired. This unusually potent interaction with NCoR, at a near-physiological dose of T3 (0.5–5 nM), likely contributes to the potent dominant negative activity of the receptor mutant in vivo.

Based on the x-ray crystal structure of the TR and other nuclear receptors, the AF-2 domain in helix 12 of the LBD has been proposed to undergo induced conformational changes after binding to the ligand (Fig. 12Go) (38, 39, 40). The major change is that the AF-2 domain in helix 12 comes into close contact with helices 3, 5, and 6, which create a scaffold that is relatively unaltered by the binding of ligand. The apposition of helix 12 with these other regions of the receptor creates a small hydrophobic cleft that has recently been shown to be a binding site for transcriptional CoAs (41). This cleft is bordered by helix 3 (I280, V284, K288), helix 5 (I302, L305, K306), helix 6 (C309), and residues L454 and E457, and V458 in helix 12. It has been postulated that this region of the receptor interacts with the conserved hydrophobic LXXLL motif that is found in several different CoAs (19, 41, 42, 43). It is notable that Leu 454, the site of the RTH mutation described here, is a key residue for CoA contact (41). In our study and in others, CoAs such as SRC1 and GRIP1 do not interact well with the helix 12 AF-2 transactivation mutants, L454 or E457 (28, 29, 41, 44). The L454V mutant, which was also identified in a patient with severe RTH, does not bind to SRC1 or RIP140 using in vitro pull-down assays (28).



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Figure 12. Three-Dimensional Model of the TR LBD

The structure of the LBD of TRß is shown as a ribbon diagram based upon the x-ray crystal structure of TR{alpha} (40 ). The locations of various helices and TRß mutants are shown.

 
Although TR domains important for CoA interactions have been partially defined, there is less information about regions involved in CoR binding. Mutations within the hinge region, or CoR box, disrupt CoR binding (8, 11). However, these effects are likely to be indirect since several of these residues appear to be buried in the TR protein (40). In addition, mutations within the so-called ninth heptad repeat of the TR that disrupt receptor dimerization with RXR also alter interactions with CoRs (45), suggesting that more distal regions of the receptor may also be involved in CoR binding. Our studies with the AF-2 domain mutants indicate that this region also plays a critical role in CoR binding. Although the AF-2 domain mutants, L454S/L454A and E457A, still bind T3 and fail to interact with CoAs, their interactions with NCoR were very different. The NCoR interaction with either L454S or L454A was increased. In contrast, the binding of E457A to NCoR was similar to that of wild-type receptor in the absence of T3. In addition to the enhanced binding to NCoR in the absence of T3, the L454S mutant exhibits impaired NCoR dissociation by T3, whereas E457A dissociates from T3 in a manner similar to the wild-type receptor. These alterations in NCoR dissociation are not easily accounted for by differences in T3 binding affinity. Although T3 binding to the L454S and the L454A mutants is lower than that of the E457A mutant, they remain strongly bound to NCoR even at T3 doses 10- to 20-fold greater than those required to dissociate NCoR from the E457A mutant or the wild-type TR receptor. Because of the apparent alteration in T3-mediated dissociation of NCoR from the L454S mutant in the mammalian two-hybrid assays, we confirmed T3 binding in intact cells, and it was similar to that seen using in vitro translated receptors. The difference in NCoR interactions between these two mutants is also not accounted for entirely by the locations of the mutations. Another mutant, E457R, also exhibited strong interactions with NCoR (data not shown), suggesting that the type of amino acid substitution, as well as its location in the AF-2 domain, may influence interactions with NCoR. Taken together, these findings suggest that in the absence of T3, the AF-2 domain, directly or indirectly, represents an important site for NCoR binding. These results are consistent with recent studies of TR interactions with CoRs in vitro (46, 51). In these studies, mutations at P453 uncoupled CoR dissociation from T3 binding. Our findings suggest that helix 12 acts as a dual functional domain. After the binding of T3, its conformation changes, causing the disruption of CoR binding and the recruitment of CoAs.

The studies of CoR interactions with RTH mutants have provided some unanticipated insights into the pathophysiology of this disorder. Previously, the dominant negative effects of mutant receptors in RTH were thought to result primarily from the loss of T3 binding and/or a loss of transcriptional activation (2, 26, 47). The preservation of DNA binding and receptor dimerization suggested that the inhibitory effects of the mutant receptors might be accomplished by the binding of transcriptionally inactive receptors to target genes (48, 49). However, more recent studies suggest a previously unappreciated role for CoRs in the inhibitory activity of mutant receptors (30, 50, 51). For example, the introduction of mutations that disrupt CoR binding into the background of RTH mutants essentially eliminates the dominant negative effects of these receptors (30, 51). Yoh et al. (51) also reported that two RTH mutants that contained in-frame, single-codon deletions ({Delta}430M and {Delta}432G) in helix 11 exhibited increased association with glutathione-S-transferase (GST)-SMRT in vitro. They did not bind T3 and possessed strong dominant negative activity. These deletions likely have similar effects as the F451X deletion of the AF-2 domain. These findings raise the possibility that the retention of CoR binding is important, if not necessary, for the dominant negative activity of RTH mutants. The current studies showing that several potent RTH mutants (e.g. F451X; L454S) have increased binding to CoRs are consistent with this role of CoRs.

The relationship of different mutations to the severity, or the nature, of the clinical phenotype of RTH has been difficult to establish (2). In part, the absence of a clear-cut genotype-phenotype correlation appears to reflect the influence of genetic background or nongenetic factors in the clinical features of the disorder (52, 53). Despite these difficulties, mutations in helix 12, particularly mutations that truncate the receptor (31), appear to cause a particularly severe form of hormone resistance. The patient reported here, with the L454S mutation, has an unusual presentation of RTH. In addition to typical features such as goiter and attention-deficit hyperactivity disorder, she also experienced repeated hospitalizations for infectious diseases and had difficulty maintaining her weight (54). These problems were accompanied by unusually high T4 (33 µg/dl) levels compared with other patients with RTH (1). When given T3 (25 µg three times daily for 5 days), T4 and TSH levels decreased, consistent with some degree of suppression of the hypothalamic-pituitary axis. However, there was still a marked TSH response to TRH, indicating strong central RTH, even after the administration of exogenous hormone. Evidence of peripheral resistance was also seen with minimal response of the sex hormone-binding globulin and resting energy expenditure. However, in addition to an already-high resting heart rate, the patient developed increased tachycardia on T3, suggesting a relative sensitivity of the cardiac system, at least from the perspective of heart rate. This finding was in keeping with her later presentation of exercise-induced tachycardia and dyspnea. These features emphasize the need to individualize treatment approaches in patients with RTH. Although this patient appears to have benefited from ß-blockers and methimazole-induced reduction of thyroid hormone levels, it remains unclear whether her improvements in interpersonal relations and school performance will persist, and the long-term effect of lowered thyroid hormone levels on other aspects of her growth and physiology are not known. The clinical manifestations in this patient are remarkably similar to another patient with a mutation in the same amino acid, but to a different residue (L454V) (28).

In a previous study, we found that some carboxy-terminal mutations exhibited greater dominant negative activity (e.g. P453S, R438H) and were generally associated with more severe RTH than mutations in the central region of the LBD that eliminated T3 binding (e.g. G345R, G345S) (35). These results seemed paradoxical at the time because it seemed possible that the helix 12 mutants (which bind T3 with reduced affinity) might acquire partial function as T3 levels increase in RTH. However, in light of the current studies, we postulate that the strength of CoR interactions may explain differences in the potency of various RTH mutants. The extent to which the strength and reversibility of NCoR binding to RTH mutants accounts for variability in the phenotype of RTH now warrants further study. It has been shown that the levels of SMRT and NCoR mRNAs vary in different tissues (55). Therefore, it is possible that the variable resistance that occurs in different tissues may reflect the levels or activity of CoRs.

The strong relationship between CoR interaction and dominant negative activity raises a question regarding the role of decreased CoA binding. Loss of CoA binding can occur either because mutant receptors fail to bind T3 (and therefore cannot dissociate CoRs or recruit CoAs) or because specific mutations interfere with CoA binding to the TR. Our studies suggest that the loss of CoA binding, while common among RTH mutants, may not play a central role in hormone resistance. For example, two types of TR mutants are deficient in CoA binding, but exhibit minimal dominant negative activity. In one case, the E457A helix 12 mutant fails to bind CoAs, but has weak dominant negative activity. In another example, helix 3 transactivation mutants K288I and K288A (56) interact weakly with CoAs, but also fail to bind to NCoR (data not shown). These mutants exhibit weak dominant negative activity (data not shown) and have not been found in naturally occurring cases of RTH. As additional RTH mutants are identified, it will be useful to establish their relative abilities to bind to CoRs and CoAs and to assess their dominant negative properties.

Current models for thyroid hormone action for positively regulated genes suggest that the silencing activity of unliganded TR is mediated by the interaction with CoRs and associated histone deacetylases (57, 58), whereas transactivation is induced in the presence of T3 in part by the recruitment of CoAs and associated histone acetylases (8, 11, 57, 58). For promoters that are negatively regulated by T3, basal activation in the absence of hormone is dependent on the interaction of CoRs with TR, and T3-dependent repression is induced by the release of CoRs plus the association of CoAs in the presence of T3 (25). The strong interaction with NCoR of the L454S mutant may therefore retain constitutive repression of positively regulated genes as well as constitutive activation of negatively regulated genes such as TSH and TRH.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic Studies
After obtaining informed consent, genomic DNA was extracted from leukocytes obtained from the patient and her parents. The coding exons of the human (h)TRß gene were amplified using PCR and were sequenced in the sense and antisense direction using a 373 DNA Sequencer (Applied Biosystems, Perkin-Elmer Corp., Foster City, CA) (59).

Plasmid Constructions
The mutant hTRß1 cDNAs were prepared by oligonucleotide-directed mutagenesis and verified by DNA sequencing as described previously (60). The numbering of the amino acid residues of TRß is based on a consensus nomenclature (61). Mutant and wild-type receptor cDNAs were subcloned into pCMX (62) for in vitro transcription/translation and for transient expression in transfected cells. The VP16 constructs for wild-type and mutant TRs contain the LBD of the receptor downstream of the VP16 activation domain in-frame in pCMX. The NCoR construct was provided by M. G. Rosenfeld (University of California, San Diego, CA) (8). SRC-1 cDNA was provided by B. W. O’Malley (Baylor College of Medicine, Houston, TX) (13) and was transferred into pCMX. The pCMX-NCoR interaction domain (ID) (internal ATG at amino acid 1579) expression vector was created by deleting the NotI-BstI fragment from pCMX-NCoR. The Gal4-NCoR (residues 1552–2453), Gal4-SRC1 (residues 213-1061), and Gal4-GRIP1 (residues 480-1462) contain the indicated TR interaction domains of these proteins fused downstream of the Gal4 DBD in-frame in pSG424 (64).

The plasmid TRE-tk-Luc contains two copies of a palindromic TRE upstream of the thymidine kinase promoter (tk109) in the pA3 luciferase vector (48). TSH{alpha}-Luc contains 846 bp of the 5'-flanking sequence and 44 bp of exon I from the human glycoprotein hormone {alpha}-subunit gene in pA3-Luc (60). The Gal4 reporter plasmid, UAS-tk-Luc, contains two copies of the Gal4 recognition sequence (UAS) upstream of tk109 in pA3-Luc.

T3 Binding Studies
Mutant or wild-type TR was transcribed and translated using the TNT-coupled reticulolysate system (Promega, Madison, WI). T3-binding affinity was determined using a filter-binding assay (65). Whole cell lysates from TSA-201 cells transfected with 1 µg of TR expression plasmids were prepared by three cycles of freeze-thaw lysis in 20 mM Tris-HCl, pH 7.5, 0.5 M KCl, 2 mM dithiothreitol (DTT), 20% glycerol, and 1 mM phenylmethylsulfonylfluoride. Cell extracts were prepared by centrifugation at 10,000 x g for 30 min at 4 C, and supernatants were stored at -20 C. The T3-binding affinity using cell extracts was determined as described previously (66).

DNA Binding Studies
Reticulocyte lysates expressing TR (2.5 µl), in the presence or absence of NCoR-ID (4 µl) or GRIP1 (4 µl) with various amounts of T3, were preincubated at room temperature in a 24-µl reaction with a binding buffer consisting of 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, and 50 µg/ml poly(dI-dC) for 15 min. 32P-labeled LAP-TREs (sense strand, agtcTGACCTgacgtcAGGTCActcga) were added, and the mixture was incubated for an additional 20 min. The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel containing 2.5% glycerol in 0.5x TBE (45 mM Tris borate, 1 mM EDTA).

Whole cell lysates from transfected TSA-201 cells were prepared by three cycles of freeze-thaw lysis in 20 mM Tris-HCl, pH 7.5, 0.5 M KCl, 2 mM DTT, 20% glycerol, and 1 mM phenylmethylsulfonylfluoride. Cell extracts were prepared by centrifugation at 10,000 x g for 30 min at 4 C, and supernatants were stored at -20 C. Cell extracts (5 µg) were preincubated in 24 µl of a modified binding buffer (20 mM HEPES, pH 7.8, 100 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, and 100 µg/ml poly dI-dC) at 4 C for 15 min. The sequence of the DR4-TRE oligonucleotide is: 5'-agcttcAGGTCActtcAGGTCAc-3' (sense strand).

Transient Expression Assays
TSA-201 cells, a clone of human embryonic kidney 293 cells (67), were grown in Optimem (BRL-GIBCO, Grand Island, NY) with 4% Dowex resin-stripped FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) and were transfected by the calcium phosphate method (48). The total amount of expression plasmid DNA was kept constant in the different experimental groups by adding corresponding amounts of the same plasmids without receptor. After exposure to the calcium phosphate-DNA precipitate for 8 h, Optimem with 1% Dowex resin-stripped FBS was added, with various concentrations of T3. Cells were harvested after 40 h for measurements of luciferase activity (68).


    ACKNOWLEDGMENTS
 
We are grateful to R. M. Evans, M. G. Rosenfeld, and B. W. O’Malley for providing plasmids and to A. Sakurai and L. G. DeGroot for TRß sequence information.


    FOOTNOTES
 
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry Building 15–709, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu

This work was supported by NIH Grant DK-42144 (to J.L.J).

Received for publication June 1, 1998. Revision received August 3, 1998. Accepted for publication August 25, 1998.


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