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
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ABSTRACT
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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
-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.
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INTRODUCTION
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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.
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RESULTS
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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 1
). 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 1
). Her basal heart rate was 110120 bpm and increased to 150156 bpm
on T3. Sex-hormone binding globulin levels changed
from 0.6 to 1.1 µg/dl (normal, 1.85.5 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 1
), 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.
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. 1A
). The
T3 binding properties of the L454S and additional mutant
receptors (L454A, E457A, F451X, G345R) are summarized in Table 2
. 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. 1B
). The T3-binding
affinity of the L454S mutant in transfected cells (27% wild-type) was
similar to that used in translated receptors (Table 2
).

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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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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. 2
). Homodimers of both wild-type (lanes
15) and L454S (lanes 610) 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.
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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. 3
).

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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 are shown in lane 1.
The locations of the TR-TR homodimers and RXR-TR heterodimers are
indicated by arrows.
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Using the positively regulated promoter TRE-tk-LUC, each of the TR
mutants exhibited strong transcriptional silencing in the absence of
T3 (Fig. 4A
). The AF-2 domain
mutants, L454S, L454A, and E457A, induced very little transcriptional
stimulation in the presence of T3 (Fig. 4B
). Complete
dose-response curves are shown in Fig. 4C
. 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.
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Using the negatively regulated promoter, TSH
-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. 5A
).
T3-induced repression was markedly impaired with each of
these mutants at 5 nM T3 (Fig. 5B
). At greater
doses of T3, transcriptional repression varied among the
mutants (Fig. 5C
). 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 -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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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 2
). The F451X and L454S mutants strongly
inhibited wild-type TRß1-regulated expression of TRE-tk-LUC (Fig. 6A
). 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
promoter, the
potency of inhibitory activity among mutants was similar to that seen
using the positively regulated promoter (Fig. 6B
). 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
-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 -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 -Luc. Cells were incubated
in the presence of 5 nM T3. Results are the
mean ± SEM from at least three transfections
performed in triplicate.
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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. 7
, 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 69) was decreased by
T3 in a dose-dependent manner. In contrast,
T3-dependent dissociation of NCoR from the L454S mutant
(lanes 25) 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 1012),
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.
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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 174461) 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. 8A
), and the degree of interaction was
reversed by the addition of increasing concentrations of
T3 (Fig. 8
, 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.
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Analogous experiments were performed to assess the interactions between
the CoAs and the TR mutants (Figs. 9
and 10
). In the absence of T3,
no interaction was seen between Gal4-SRC1 (Fig. 9A
) or Gal4-GRIP1 (Fig. 10A
) 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. 9
, B and C) or Gal4-GRIP1 (Fig. 10
, 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. 9C
and 10C
),
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.
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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. 11
. The dominant negative potency
of each mutant was calculated as fold inhibition (vector = 1) from
the data shown in Fig. 6A
(TRE-tk-LUC). NCoR interactions were
calculated from the data of Fig. 8B
as fold interaction (vector =
1). SRC1 and GRIP1 interactions in the presence of 5 nM
T3 were calculated from the data of Figs. 9B
and 10B
,
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. 11A
). In contrast, the correlation of dominant negative potency
with 1/SRC1 interaction (r = 0.051) (Fig. 11B
) or with 1/GRIP1
interaction (r = 0.093) (Fig. 11C
) 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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DISCUSSION
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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 (0.55
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. 12
) (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 (40 ).
The locations of various helices and TRß mutants are shown.
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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 (
430M and
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
|
---|
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. OMalley (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 15522453), 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
-Luc contains 846 bp of the 5'-flanking sequence and
44 bp of exon I from the human glycoprotein hormone
-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. OMalley 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 15709, 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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