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 Addenbrookes 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
|
---|
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
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
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
|
---|
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
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
(
) 310353 and between 
429461. 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
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
-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
. We suggest that
the functional abnormalities exhibited by these structurally
colocalized mutants may represent the minimal derangements required to
generate the RTH phenotype.
 |
RESULTS
|
---|
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 1
), 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,
0.154.6 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 2
). 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.
Abnormal thyroid function tests were also noted in her father and
grandfather (Table 1
). 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 49 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. 1
, a and b) and
achieved greater maximal activity than WT receptor on the direct repeat
TRE (MALTKLUC) at higher T3 concentrations (Fig. 1c
).
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. 1
, df).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 1. Positive Transcriptional Regulation by WT ( ) and
R383H ( ) Mutant Receptors
The data shown in this and subsequent figures are the mean (±
SE) of at least three experiments each done in triplicate.
ac, 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 01 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. df, 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 ac. 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
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. 2a
), and the
mutant receptor showed a similarly impaired inhibitory profile with
TSH
LUC (Fig. 2b
). 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
reporter genes
by the R383H mutant was significantly impaired compared with WT TRß2,
analogous to that seen in a ß1-background (Fig. 2
, 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. 2
, e and g). With the TSH
promoter, the
action of wild type TRß1 was inhibited by the R383H mutant in either
TRß1 (Fig. 2f
) or TRß2 (data not shown) backgrounds. However, WT
TRß2 receptor function on this promoter could neither be inhibited by
mutant R383Hß2 (Fig. 2h
) nor by R383Hß1 receptors (data not
shown).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 2. Negative Transcriptional Regulation by WT ( ) and
R383H ( ) Mutant Receptors
Inhibition of TRHLUC reporter activity was assayed by transfection of
JEG-3 cells with 100 ng of either WT ( ) or R383H mutant ( ) TRß1
(a) or TRß2 (c) expression vectors, 2 µg TRH LUC and 100 ng BOS
ß-gal, and incubation with 01 nM T3.
Inhibition of TSH LUC reporter activity was assayed similarly using
50 ng WT ( ) or R383H (O), TRß1 (b), or TRß2 (d) expression
vector, 500 ng TSH LUC, and 100 ng BOS ß-gal and incubation with
01 µM T3. Hormone-dependent inhibition is
expressed relative to the activity in cells incubated without
T3, and the background inhibition of TRHLUC and TSH LUC
reporter activity in control cells transfected with 100 ng (a and c) or
50 ng (b and d) RSV vector ( ) 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 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. 3a
). 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 (0.050.1
nM) that returned the activity of this promoter by
unliganded WT receptor to baseline (Fig. 3a
). 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
promoter by either
unliganded TRß1 or TRß2 in JEG-3 cells.

View larger version (20K):
[in this window]
[in a new window]
|
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 01 nM T3 as
described above (Fig. 2a ) 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 010 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. 3b
). 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. 4a
). 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. 4b
). Ligand-dependent
recruitment of other coactivators (CBP, RIP140) by the R383H mutant was
also normal (data not shown).
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. 5A
). 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. 5A
). 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. 5B
), wherein the EC50 of corepressor release was 0.8
nM T3 for WT and 4.6 nM
T3 for R383H.

View larger version (73K):
[in this window]
[in a new window]
|
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 01
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
, and
Both Are Impaired for Corepressor Release and Negative Regulation
The crystal structure of rat TR
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. 6a
). 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
readily on direct repeat (Fig. 5B
), palindromic, and everted repeat
response elements (data not shown).

View larger version (51K):
[in this window]
[in a new window]
|
Figure 6. RTH Mutations Shown in the Crystal Structure of
Rat TR
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 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, 430M, I431T, 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 5256 and our
unpublished results), and those in residues between  234282 are shown in
green, mutations in residues between  310353 are
shown in orange, and mutations in residues between
 429461 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. 4a
, R429Q
EC50 28 nM T3), whereas its
T3-dependent recruitment to GST-SRC1 was comparable to WT
receptor (Fig. 4b
). 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. 5A
), and 5.2
nM T3 (Fig. 5B
) on pTRE].
 |
DISCUSSION
|
---|
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
(Fig. 6B
). 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
and hypothalamic
TRH gene promoters when studied in the context of TRß1 as well as
TRß2the 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
promoter in a TRß1 context. It is interesting to note that the
R383H mutant lacked dominant negative activity on the TSH
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
-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 changeseither reversal of unliganded
activation or relief of silencingwere 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. 4a
). 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
-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
, 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
|
---|
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 410 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
LUC
reporter gene contains the glycoprotein hormone
-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
Students 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 Students t-test.
[pp]Coactivator interaction assays using a GST-SRC1 (
570780)
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
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.
 |
REFERENCES
|
---|
-
Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of
resistance to thyroid hormone. Endocr Rev 14:348399[Medline]
-
Refetoff S, Weiss RE, Usala SJ, Hayashi Y 1994 The syndromes
of resistance to thyroid hormone: update 1994. In: Braverman LE,
Refetoff S, Negro-Vilar A (eds) Endocrine Reviews Monographs 3:
Clinical and Molecular Aspects of Diseases of the Thyroid. The
Endocrine Society Press, Bethesda, MD, pp 336343
-
Evans RM 1988 The steroid and thyroid receptor superfamily.
Science 240:889895[Medline]
-
Chin WW 1991 Nuclear thyroid hormone receptors. In: Parker MG
(ed) Nuclear Hormone Receptors. Academic Press, London, pp 79102
-
Usala SJ, Bale AE, Gesundheit N, Weinberger C, Lash RW,
Wondisford FE, McBride OW, Weintraub BD 1988 Tight linkage between the
syndrome of generalised thyroid resistance and the human c-erbAß
gene. Mol Endocrinol 2:12171220[Abstract]
-
Forman BM, Yang C-R, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel
in vivo interactions between the thyroid hormone and
retinoic acid receptors. Mol Endocrinol 3:16101626[Abstract]
-
Glass CK 1994 Differential recognition of target genes by
nuclear receptor monomers, dimers, and heterodimers. Endocr Rev 15:391407[Medline]
-
Collingwood TN, Adams M, Tone Y, Chatterjee VKK 1994 Spectrum
of transcriptional, dimerization, and dominant negative properties of
twenty different mutant thyroid hormone ß-receptors in thyroid
hormone resistance syndrome. Mol Endocrinol 8:12621277[Abstract]
-
Takeda K, Sakurai A, DeGroot LJ, Refetoff S 1992 Recessive
inheritance of thyroid hormone resistance caused by complete deletion
of the protein-coding region of the thyroid hormone receptor-ß gene.
J Clin Endocrinol Metab 74:4955[Abstract]
-
Chatterjee VKK, Nagaya T, Madison LD, Datta S, Rentoumis
A, Jameson JL 1991 Thyroid hormone resistance syndrome: inhibition of
normal receptor function by mutant thyroid hormone receptors.
J Clin Invest 87:19771984[Medline]
-
Sakurai A, Miyamoto T, Refetoff S, DeGroot LJ 1990 Dominant
negative transcriptional regulation by a mutant thyroid hormone
receptor-ß in a family with generalized resistance to thyroid
hormone. Mol Endocrinol 4:19881994[Abstract]
-
Nagaya T, Jameson JL 1993 Thyroid hormone receptor
dimerization is required for dominant negative inhibition by mutations
that cause thyroid hormone resistance. J Biol Chem 268:1576615771[Abstract/Free Full Text]
-
Forman BM, Samuels HH 1990 Interactions among a subfamily of
nuclear hormone receptors: the regulatory zipper model. Mol Endocrinol 4:12931301[Abstract]
-
Perlmann T, Umesono K, Rangarajan PN, Forman BM, Evans RM 1996 Two distinct dimerization interfaces differentially modulate target
gene specificity of nuclear hormone receptors. Mol Endocrinol 10:958966[Abstract]
-
Hayashi Y, Sunthornthepvarakul T, Refetoff S 1994 Mutations of
CpG dinucleotides located in the triiodothyronine (T3)-binding domain
of the thyroid hormone receptor (TR) ß gene that appears to be devoid
of natural mutations may not be detected because they are unlikely to
produce the clinical phenotype of resistance to thyroid hormone. J
Clin Invest 94:607615[Medline]
-
Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD,
Fletterick RJ 1995 A structural role for hormone in the thyroid hormone
receptor. Nature 378:690697[CrossRef][Medline]
-
Nakajima T, Uchida C, Andreson SF, Parvin JD, Montminy M 1997 Analysis of a cAMP-responsive activator reveals a two-component
mechanism for transcriptional induction via signal-dependent factors.
Genes Dev 11:738747[Abstract]
-
Ogryzko VV, Shiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953960[Medline]
-
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a
histone acetyltransferase. Nature 384:641643[CrossRef][Medline]
-
Heinzel T, Lavinsky RM, Mullen T-M, Söderström M,
Laherty CD, Torchia J, Yang W-M, Brard G, Ngo SD, Davie JR, Seto E,
Eisenman RN, Rose DW, Glass CK, Rosenfeld MG 1997 A complex containing
N-CoR, mSin3 and histone deacetylase mediates transcriptional
repression. Nature 387:4348[CrossRef][Medline]
-
Nagy L, Kao H-Y, Chakravarti D, Lin RJ, Hassig CA, Ayer DE,
Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a
complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373380[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Cavaillès V, Dauvois S, LHorset F, Lopez G, Hoare S,
Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates
transcriptional activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
Oñate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and characterization of a coactivator for the steroid
hormone receptor superfamily. Science 270:13541357[Abstract]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG,
Juguilon H, Montminy M, Evans RM 1996 Role of CBP/p300 in nuclear
receptor signalling. Nature 383:99103[CrossRef][Medline]
-
Collingwood TN, Rajanayagam O, Adams M, Wagner R,
Cavaillès V, Kalkhoven E, Matthews C, Nystrom E, Stenlof K,
Lindstedt G, Tisell L, Fletterick RJ, Parker MG, Chatterjee VKK 1997 A
natural transactivation mutation in the thyroid hormone ß receptor:
impaired interaction with putative transcriptional mediators. Proc Natl
Acad Sci USA 94:248253[Abstract/Free Full Text]
-
Chrivia JC, Kwok RPS, Lamb N, Hagiwara M, Montminy MR, Goodman
RH 1993 Phosphorylated CREB binds specifically to the nuclear protein
CBP. Nature 365:855859[CrossRef][Medline]
-
Hörlein AJ, Näär AM, Heinzel T, Torchia J,
Gloss B, Kurokawa R, Ryan A, Kamei Y, Söderström M, Glass
CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid
hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Sande S, Privalsky ML 1996 Identification of TRACs
(T3 receptor-associating cofactors), a family of cofactors
that associate with, and modulate the activity of, nuclear hormone
receptors. Mol Endocrinol 10:813825[Abstract]
-
Yoh SM, Chatterjee VKK, Privalsky ML 1997 Thyroid hormone
resistance syndrome manifests as an aberrant interaction between mutant
T3 receptors and transcriptional corepressors. Mol Endocrinol 11:470480[Abstract/Free Full Text]
-
Hollenberg AN, Monden T, Madura JP, Lee K, Wondisford FE 1996 Function of nuclear co-repressor protein on thyroid hormone
response elements is regulated by the receptor A/B domain. J Biol
Chem 271:2851628520[Abstract/Free Full Text]
-
Tagami T, Madison LD, Nagaya T, Jameson JL 1997 Nuclear
receptor corepressors activate rather than suppress basal
transcription of genes that are nega-tively regulated by thyroid
hormone. Mol Cell Biol 17:26422648[Abstract]
-
Flynn TR, Hollenberg AN, Cohen O, Menke JB, Usala SJ, Tollin
S, Hegarty MK, Wondisford FE 1994 A novel C-terminal domain in the
thyroid hormone receptor selectively mediates thyroid hormone
inhibition. J Biol Chem 269:3271332716[Abstract/Free Full Text]
-
Sherman SI, Ladenson PW 1992 Organ-specific effects of
tiratricol: a thyroid hormone analog with hepatic, not pituitary,
superagonist effects. J Clin Endocrinol Metab 75:901905[Abstract]
-
Bracco D, Morin O, Schutz Y, Liang H, Jequier E, Burger AG 1993 Comparison of the metabolic and endocrine effects of
3,5,3'-triiodothyroacetic acid and thyroxine. J Clin Endocrinol
Metab 77:221228[Abstract]
-
Sherman SI, Ringel MD, Smith MJ, Kopelen HA, Zoghbi WA,
Ladenson PW 1997 Augmented hepatic and skeletal thyromimetic effects of
tiratricol in comparison with levothyroxine. J Clin Endocrinol
Metab 82:21532158[Abstract/Free Full Text]
-
Radetti G, Persani L, Molinaro G, Mannavola D, Cortelazzi
D, Chatterjee VKK, Beck-Peccoz P 1997 Clinical and hormonal outcome
after two years of triiodothyroacetic acid treatment in a child with
thyroid hormone resistance. Thyroid 5:775778
-
Takeda T, Suzuki s, Liu R-T, DeGroot LJ 1995 Triiodothyroacetic acid has unique potential for therapy of resistance
to thyroid hormone. J Clin Endocrinol Metab 80:20332040[Abstract]
-
Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen
PR, Moore DD, Chin WW 1989 Identification of a thyroid hormone receptor
that is pituitary-specific. Science 244:7678[Medline]
-
Ercan-Fang S, Schwartz HL, Oppenheimer JH 1996 Isoform-specific 3,5,3'-triodothyronine receptor binding
capacity and messenger ribonucleic acid content in rat
adenohypophysis: effect of thyroidal state and comparison with
extrapituitary tissues. Endocrinology 137:32283233[Abstract]
-
Lazar M 1993 Thyroid hormone receptors: multiple forms,
multiple possibilities. Endocr Rev 14:184193[Medline]
-
Hollenberg AN, Monden T, Flynn TR, Boers ME, Cohen O,
Wondisford FE 1995 The human thyrotropin-releasing-hormone gene is
regulated by thyroid-hormone through 2 distinct classes of negative
thyroid-hormone response elements. Mol Endocrinol 9:540550[Abstract]
-
Hayashi Y, Weiss RE, Sarne DH, Yen PM, Sunthornthepvarakul T,
Marcocci C, Chin WW, Refetoff S 1995 Do clinical manifestations of
resistance to thyroid hormone correlate with the functional alteration
of the corresponding mutant thyroid hormone-ß receptors? J Clin
Endocrinol Metab 80:32463256[Abstract]
-
Hollenberg AN, Monden T, Wondisford FE 1995 Ligand-independent
and -dependent functions of thyroid hormone receptor isoforms depend
upon their distinct amino termini. J Biol Chem 270:1427414280[Abstract/Free Full Text]
-
Adams M, Matthews C, Collingwood TN, Tone Y, Beck-Peccoz P,
Chatterjee VKK 1994 Genetic analysis of 29 kindreds with generalized
and pituitary resistance to thyroid hormone: identification of thirteen
novel mutations in the thyroid hormone receptor ß gene. J Clin
Invest 94:506515[Medline]
-
Abel ED, Kaulbach HC, Boers M-E, Wondisford FE, Transgenic
expression of mutant thyroid hormone receptor isoforms reveal
differential effects on pituitary thyroid hormone action in
vivo. Program of the 79th Annual Meeting of The Endocrine Society,
Minneapolis, MN, 1997, p 294 (Abstract)
-
Tone Y, Collingwood TN, Adams M, Chatterjee VKK 1994 Functional analysis of a transactivation domain in the
thyroid hormone ß receptor. J Biol Chem 269:3115731161[Abstract/Free Full Text]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Beck-Peccoz P, Chatterjee VKK, Chin WW, DeGroot LJ, Jameson
JL, Nakamura H, Refetoff S, Usala SJ, Weintraub BD 1994 Nomenclature of thyroid hormone receptor ß-gene mutations in
resistance to thyroid hormone: consensus statement from the first
workshop on thyroid hormone resistance, July 1011, 1993, Cambridge,
United Kingdom. J Clin Endocrinol Metab 78:990993[Medline]
-
Tsukaguchi H, Yoshimasa Y, Fujimoto K, Ishii H, Yamamoto T,
Yoshimasa T, Yagura T, Takamatsu J 1995 Three novel mutations of
thyroid hormone receptor ß gene in unrelated patients with resistance
to thyroid hormone: two mutations of the same codon (H435L and H435Q)
produce separate subtypes of resistance. J Clin Endocrinol Metab 80:36133616[Abstract]
-
Onigata K, Yagi H, Sakurai A, Nagashima T, Nomura Y, Nagashima
K, Hashizume K, Morikawa A 1995 A novel point mutation (R243Q) in exon
7 of the c-erbAß thyroid hormone receptor gene in a family with
resistance to thyroid hormone. Thyroid 5:355358[Medline]
-
Pohlenz J, Schönberger W, Wemme H, Winterpacht A, Wirth
S, Zabel B 1996 New point mutation (R243W) in the hormone binding
domain of the c-erbA ß1 gene in a family with generalised resistance
to thyroid hormone. Hum Mutation 7:7981[CrossRef][Medline]
-
Kijima H, Kubo M, Ishizuka T, Kakinuma M, Koike T 1996 A novel
missense mutation in the thyroid hormone receptor ß gene in a kindred
with resistance to thyroid hormone. Hum Genet 97:407408[CrossRef][Medline]