Thyroid Hormone Resistance Syndrome Manifests as an Aberrant Interaction between Mutant T3 Receptors and Transcriptional Corepressors
Sunnie M. Yoh,
V. K. K. Chatterjee and
Martin L. Privalsky
Section of Microbiology (S.M.Y., M.L.P.) Division of Biological
Sciences University of California at Davis Davis, California
95616
Department of Medicine (V.K.K.C.) University of
Cambridge Level 5, Addenbrookes Hospital Cambridge, CB2 2QQ,
United Kingdom
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ABSTRACT
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Nuclear hormone receptors are hormone-regulated
transcription factors that play critical roles in chordate development
and homeostasis. Aberrant nuclear hormone receptors have been
implicated as causal agents in a number of endocrine and neoplastic
diseases. The syndrome of Resistance to Thyroid Hormone (RTH) is a
human genetic disease characterized by an impaired physiological
response to thyroid hormone. RTH is associated with diverse mutations
in the thyroid hormone receptor ß-gene. The resulting mutant
receptors function as dominant negatives, interfering with the actions
of normal thyroid hormone receptors coexpressed in the same cells. We
report here that RTH receptors interact aberrantly with a newly
recognized family of transcriptional corepressors variously denoted as
nuclear receptor corepressor (N-CoR), retinoid X receptor interacting
protein-13 (RIP-13), silencing mediator for retinoid and thyroid
hormone receptors (SMRT), and thyroid hormone receptor-associating
cofactor (TRAC). All RTH receptors tested exhibit an impaired ability
to dissociate from corepressors in the presence of thyroid hormone. Two
of the RTH mutations uncouple corepressor dissociation from hormone
binding; two additional RTH mutants exhibit an unusually strong
interaction with corepressor under all hormone conditions tested.
Finally, artificial mutants that abolish corepressor binding abrogate
the dominant negative activity of RTH mutants. We suggest that an
altered corepressor interaction is likely to play a critical role in
the dominant negative potency of RTH mutants and may contribute to the
variable phenotype in this disorder.
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INTRODUCTION
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Nuclear hormone receptors are hormone-regulated transcription
factors and include the thyroid hormone receptors (encoded by two
different loci, T3R
and T3Rß), steroid
receptors, and retinoid receptors (reviewed in Refs. 16). All nuclear
hormone receptors contain a variety of motifs involved in DNA binding,
hormone binding, receptor dimerization, and interactions with the
transcriptional machinery (Fig. 1
). On binding to
specific target DNA sequences, nuclear hormone receptors modulate the
expression of adjacent target genes (1, 2, 3, 4, 5, 6). The nature of the
transcriptional response (activation or repression) is dictated by cell
type, promoter context, and hormone status (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In most contexts
T3Rs are transcriptional repressors in the absence of
cognate hormone (T3) and activators in its presence (2, 7, 8, 11, 12, 13, 14, 15).

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Figure 1. Schematic Representation of the Human
T3Rß Protein and the Location of the RTH Mutations
The T3Rß is presented schematically from the N (n)
terminus to C (c) terminus, with a central zinc-finger domain (Zn)
indicated. The locations of the RTH mutations analyzed in this
manuscript are indicated below. The regions of the wt
receptor implicated in DNA recognition, hormone binding, receptor
dimerizaton, transcriptional (Tx) activation, and SMRT corepressor
binding are depicted above. X indicates the location of
the site-directed mutations introduced to disrupt SMRT association
(T3Rsad mutations; see text).
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Some aspects of this transcriptional regulation are probably
mediated by direct interactions between nuclear hormone receptors
and the general transcriptional machinery (5). However, a series of
specific ancillary proteins (denoted coactivators and corepressors)
also appear to play critical roles in transcriptional control. For
example, the transcriptional repression exhibited by T3Rs
in the absence of hormone appears to require an association of the
receptor with a family of corepressor proteins (variously denoted
TRACs, SMRT, RIP13, or N-CoR) (11, 12, 13, 14, 15, 16, 17, 18). T3R mutants unable
to associate with these corepressors are unable to repress (11, 12, 13, 14, 15).
Addition of T3 hormone converts T3Rs into
transcriptional activators, a process that correlates with dissociation
of the corepressors from the receptor and a corresponding recruitment
of a novel set of proteins believed to function as coactivators
(11, 12, 13, 14, 15, 19, 20, 21, 22, 23, 24, 25).
A variety of neoplastic and endocrine disorders are the consequence of
aberrations in nuclear hormone receptor function (26, 27, 28, 29, 30, 31). The syndrome
of Resistance to Thyroid Hormone (RTH) is an autosomal dominant human
endocrine disease (reviewed in Refs. 3234). Individuals with RTH
exhibit a failure to respond to elevated circulating thyroid hormone.
This disorder is associated with diverse mutations at the
T3Rß locus, resulting in the synthesis of abnormal
receptors that are impaired in hormone binding and in transcriptional
activation (35, 36, 37, 38, 39). As a result, the RTH-T3Rs appear to
function as dominant negatives, and interfere with the actions of the
normal T3Rs synthesized from the unaffected
T3R
- and ß-alleles (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50). More than 30 different
T3Rß mutations have been associated with the RTH
syndrome.
Nonetheless, the precise mechanism(s) by which RTH-T3Rs act
as dominant negatives remains uncertain. In addition, a given
RTH-T3R mutation can have very different phenotypic
consequences in different individuals or in different tissues in the
same individual, suggesting a potential involvement of other factors
modulating dominant negative function and the degree of resistance (32, 48, 51, 52, 53, 54). We report here evidence that the RTH syndrome is
associated with an aberrant interaction between the
RTH-T3Rs and the SMRT/TRAC corepressors and that this
corepressor interaction is important in the ability of
RTH-T3Rs to act as dominant negative inhibitors.
Furthermore, different RTH-T3Rs exhibit distinct
corepressor interactions. Conceivably, differential interactions with,
expression of, or genetic polymorphisms within the corepressors may
contribute to the highly variable presentation observed for RTH
disease.
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RESULTS
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RTH-T3Rß Mutants Exhibit Aberrant
Interactions with SMRT Corepressor
We wished to investigate whether corepressors play a role in the
dominant negative effect that is postulated to be the molecular basis
of RTH. We first tested the ability of wild type (wt)
T3Rß and a panel of RTH-T3Rß mutants to
associate with corepressor in vitro. We employed a
glutathione-S-transferase (GST) fusion of SMRT, and
35S-radiolabeled T3Rs obtained by in
vitro transcription and translation. All of our T3Rß
alleles (wt or mutant) produced three main forms of translation product
in vitro: a full-length molecule at approximately 55 kDa
(p55) and two truncated forms at approximately 48 and 33 kDa (p48, p33)
(Fig. 2
). The truncated T3R species are
likely to the products of translational initiation on internal ATG
codons and have been reported previously for both in vitro
and in vivo expression systems (26, 27, 55).

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Figure 2. Binding of wt and RTH Mutant Receptors to SMRT
Corepressor
Wild type and four different RTH mutant T3Rßs were tested
for the ability to bind to an immobilized GST-SMRT fusion protein or to
a nonrecombinant GST construct employed as a negative control, as
indicated above each figure. Radiolabeled
T3Rs was obtained by in vitro translation
and were incubated with the immobilized GST or GST-SMRT under the
hormone conditions indicated (expressed as nM
T3 in the incubation buffer). The GST or GST-TRAC agarose
was then extensively washed, and the radiolabeled receptor remaining
bound to the matrix was eluted and was analyzed by SDS-PAGE
and autoradiography. Input lanes display the in vitro
translation products used for the binding experiments; all receptors
were expressed at virtually identical levels and represent equivalent
specific activities.
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Wild-type T3Rß bound to the GST-SMRT construct in the
absence of hormone, but not to nonrecombinant GST employed as a
negative control (Fig. 2
). Addition of increasing amounts of
T3 hormone strongly inhibited binding of the
wtT3Rß to GST-SMRT, with a 50% inhibition observed at 56
nM hormone (Fig. 2
, and quantified in Fig. 3
and Table 1
). This interaction of wtT3Rß
with SMRT closely resembles that observed previously for
wtT3R
(11, 14, 15). Note that all three receptor
translation products (p55, p48, and p33) were bound by the GST-SMRT
construct and were released congruently by hormone, indicating that the
use of alternative initiation sites did not alter the interaction of
T3Rß with corepressor (Fig. 2
).

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Figure 3. Quantification of wt and RTH-T3Rß
Binding to SMRT
The electrophoretograms shown in Fig. 3 were quantified by
PhosphoImager (Molecular Dynamics, Sunnyvale, CA) analysis. The amount
of T3R bound to GST (squares) or to
GST-SMRT (circles) at a given hormone
concentration is expressed as a percentage of the total radiolabeled
T3R used in the binding reaction. Data for two different
RTH-mutants, G345S (open symbols) and P453A
(closed symbols), are shown in the third
panel. Note the change in scale of the ordinate for the
432G mutant.
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The RTH-T3Rß mutants also associated with GST-SMRT in the
absence of hormone, but in contrast to wtT3Rß, all the
RTH-T3Rßs tested exhibited aberrations in their ability
to dissociate on T3 treatment. The results for
representative mutants are presented in detail in Figs. 2
and 3
and are
summarized, along with data for additional mutants, in Table 1
. Nine of
the 11 RTH mutants analyzed here associated normally with SMRT in the
absence of hormone (i.e. at levels comparable to
wtT3R). Four RTH mutants in this group of nine (R320L,
M334R, R338W, and R438C) were eventually displaced from SMRT by
T3, but only at hormone levels significantly greater than
those required for the wt receptor/SMRT dissociation. For the remaining
five of these nine mutants (V264D, G344E, G345S, P453A, and P453H),
little or no receptor/SMRT dissociation (less than 50%) was observed
at up to 1 µM T3; notably the P453A and p453H
mutants fail to dissociate from SMRT even though these mutant receptors
are able to bind thyroid hormone (see Discussion).
The last two of the 11 RTH mutants analyzed (
430 M and
432G) were
not only impaired in their dissociation from SMRT in the presence of
T3, but also exhibited a dramatically enhanced SMRT
association at all hormone concentrations (Figs. 2
and 3
and Table 1
).
These mutants bound to GST-SMRT in the absence or presence of
T3 at levels some 8- to 10-fold greater than that seen with
wtT3Rß or the other RTH mutants (note the change in
ordinate in Fig. 3
); equal inputs of receptor were employed
in all experiments. All mutant and wt receptors were transcribed and
translated at equal efficiencies, utilizing identical conditions, and
therefore represent equal specific activities. Additionally, the
enhanced binding observed for mutants
430 M and
432G was
consistently seen in multiple experiments utilizing different
preparations of receptor and of GST-SMRT. Intriguingly, both of these
mutants represent single amino acid deletions mapping to the same
-helix of the receptor and present clinically in a similar fashion
(see Discussion).
We conclude that a common hallmark of the RTH receptors analyzed here
is an aberrant interaction with corepressor. The phenotypes of these
RTH mutants could be divided into three general categories: 1) normal
levels of SMRT association, but requiring higher than
normal levels of T3 for dissociation, 2) normal levels of
SMRT association, but with little or no T3-mediated
dissociation observed under the conditions employed, and 3) a
dramatically elevated level of SMRT association at all hormone
concentrations tested.
Altered Corepressor Association Correlates with the Dominant
Negative Properties of the RTH Mutants
We next tested the transcriptional properties of representative
RTH mutants from the categories defined above, using transient
transfections of CV-1 cells. We initially introduced the various
receptor mutants individually (Fig. 4A
). In the absence
of hormone, all five receptors tested functioned as repressors,
inhibiting reporter gene expression some 50% relative to that seen in
the absence of exogenous T3R. As expected, addition of 100
nM T3 converted the wtT3Rß from a
repressor into a strong transcriptional activator (Fig. 4A
). In
contrast, the R320L mutant exhibited an impaired ability to activate
reporter gene expression in the presence of 100 nM
T3, and the G345S,
432G, and P453A mutants functioned as
constitutive repressors unable to induce significant activation of the
reporter in response to T3 (Fig. 4A
).

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Figure 4. Transcriptional Properties of wt and
RTH-T3Rßs
A, Transcriptional properties of the receptors when analyzed
individually. CV-1 cells were transfected by lipofection with a DR4
luciferase reporter together with a pSG5 plasmid expressing either wt
or mutant T3Rß, as indicated (none indicates an empty
pSG5 vector). The cells were incubated in the absence (hatched
bars) or presence (filled bars) of 100
nM T3, and the cells were harvested. The
resulting luciferase activity was determined and normalized relative to
the activity of a pCH110-lac Z plasmid employed as an
internal control. Data represent the average and range of duplicate
experiments. B, Dominant negative activities of the different
RTH-T3Rß mutants when cointroduced with wt receptor. CV-1
cells were transfected by lipofection with the DR4 luciferase reporter,
together with different combinations of wt and RTH mutant receptors, as
indicated below the panel. Either a 1:1 or 5:1 ratio of
mutant to wt receptor were used, with the wt receptor at 100 ng per
plate in all cases. Empty pSG5 was employed to keep the total amount of
expression vector identical in all samples. The cells were incubated in
the absence (hatched bars) or presence (filled
bars) of 100 nM T3, and the relative
luciferase activity was determined as for panel A. Data represents the
average and range of duplicate experiments. C, Effect of hormone on
dominant negative properties of the R320L mutant. The same general
experimental design as in panel B was used, but employing no exogenous
receptor (open bars), wtT3Rß alone
(filled bars) or a 5:1 ratio of R320L to
wtT3Rß (horizontal striped bars) under a
range of T3 concentrations (indicated below
the figure). A calcium phosphate transfection method was used. Data
represent the average and range of duplicate experiments.
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We next examined the abilities of the RTH mutants to act as dominant
negatives when cointroduced together with the wt receptor (Fig. 4B
).
The G345S and
432G mutants were strong dominant negatives in this
cotransfection assay and significantly inhibited the functions of the
wt receptor in a dose-dependent manner. In contrast, the R320L mutant
acted only as a weak inhibitor of wtT3Rß at 100
nM T3. We retested the dominant negative
properties of R320L at a range of hormone concentrations (Fig. 4C
).
Consistent with the effects of hormone on the
SMRT/R320L-T3R association in vitro, the R320L
mutant functioned in cells as a strong dominant negative at low
T3 concentrations (i.e. at hormone levels that
fail to displace SMRT) but lost dominant negative activity at higher
T3 concentrations (presumably reflecting the release of
SMRT). We conclude that the RTH-T3Rs that exhibited a
strong constitutive association with SMRT in vitro
manifested the strongest dominant negative phenotypes, whereas the
mutant receptor that could be partially dissociated from SMRT by
T3 exhibited weaker, T3-sensitive dominant
negative properties.
Exogenously Introduced SMRT Derivatives Can Interfere with the
Dominant Negative Phenotype of the RTH-T3Rs
The N terminus of SMRT is required for transcriptional repression,
whereas the C terminus contains the domains necessary for
T3R association (Fig. 5A
and Refs. 1115).
As a consequence, ectopic expression of an N-terminally truncated SMRT
(
N-SMRT) interferes with the wtT3R-mediated repression
observed in the absence of hormone (Refs. 11 and 14 and Fig. 5D
),
presumably by displacing endogenous full-length SMRT from the receptor
(Fig. 5C
). In contrast, expression of
N-SMRT has little or no effect
on the transcriptional activation observed for wtT3Rß in
the presence of T3 (Fig. 5D
and Ref.14), conditions in
which endogenous SMRT is not bound to the wt receptor (Fig. 5A
). If the
dominant negative actions of the RTH-T3Rs reflect a
hormone-resistant association with SMRT (Fig. 5B
), cointroduction of a
N-SMRT deletion should counteract the dominant negative RTH
phenotype and partially restore the thyroid hormone response (Fig. 5C
).

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Figure 5. N-SMRT Expression Reverses the Dominant Negative
Actions of the RTH-T3Rßs
A, Model of the actions of SMRT corepressor on wtT3R.
SMRT is bound to receptor in the absence of T3 and mediates
transcriptional repression, but dissociates under physiological
T3 levels to permit transcriptional activation. The
N-terminal (n) SMRT domain required for transcriptional repression
(hatched) and the C-terminal (c) domain required for
receptor association are depicted. B, Model of the actions of SMRT on
RTH-T3Rs. SMRT is bound to receptor in the absence of
T3, but remains bound under physiological T3
conditions. As a result, the RTH-T3R behaves as a
constitutive repressor and interferes in a dominant fashion with the
actions of the wtT3R. C, Model of the actions of N-SMRT
derivatives on receptor-mediated repression. N-SMRT retains the
ability to bind receptor, but lacks the N-terminal domain required to
silence transcription. As a result, the ectopic expression of N-SMRT
displaces endogenous full-length SMRT from T3R and
interferes with receptor-mediated repression. D, N-SMRT interference
with the dominant negative phenotype of the 432G T3
mutant. CV-1 cells were transfected with the DR-4 luciferase reporter
and with various combinations of pSG5 vector expressing
wtT3Rß (0.5 µg per plate), the 432G mutant (2.5 µg
per plate), or N-SMRT (2.5 µg per plate), as indicated
below the panel. A calcium phosphate precipitation
procedure was employed. Empty pSG5 was introduced where necessary to
keep the total amount of expression vector the same for all samples.
The cells were incubated in the absence (hatched bars)
or presence (filled bars) of 100 nM
T3, and the relative luciferase activity was determined as
for Fig. 4 . Data represent the average and range of duplicate
experiments.
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We tested this hypothesis using the
432G mutant (which exhibited
both a very strong dominant negative phenotype in vivo and
an enhanced association with SMRT in vitro). As before,
introduction of the
432G mutant severely inhibited
T3-mediated gene activation by wtT3Rß (Fig. 5D
). However, these dominant negative properties of the
432G mutant
were strongly counteracted by the cointroduction of the
N-SMRT
derivative, resulting in significant restoration of reporter gene
activation by the wtTR3 (Fig. 5D
). These effects were
proportional to the amount of
N-SMRT expression vector introduced
and were not observed for an empty pSG5 vector (Fig. 5D
and data not
shown).
Artificial Mutants of RTH-T3Rs That Are
Impaired in SMRT Association Are Also Impaired in Dominant-Negative
Function
Although several distinct receptor domains interact with
corepressor, a conserved amino acid sequence in the receptor
ligand-binding domain is particularly important for corepressor
association (Fig. 1
and Refs. 1115). Receptors bearing mutations in
this conserved sequence are impaired for corepressor association
in vitro and for transcriptional repression in
vivo, but retain the ability to activate transcription (Refs. 11
and 14 and data not shown). To ask whether impairment of SMRT
association results in impairment of the RTH dominant negative
phenotype, we engineered analogous SMRT-association disruptive (sad)
mutations into four of our representative RTH mutant clones (R320L,
G345S,
432G, and P453A). When tested in vitro, these
RTH-T3Rsad mutants were all significantly
reduced in their ability to bind to GST-SMRT relative to the parental
receptors (Fig. 6A
). The residual SMRT binding displayed
by the T3Rsad proteins (Fig. 6A
) has been noted
before and is likely mediated by additional points of contact between
SMRT and receptor that are not altered in this mutagenesis scheme
(14).
In keeping with our hypothesis of an obligatory role for corepressor in
RTH dominant negative function, the RTH-T3Rsad
mutants were all severely compromised in their ability to interfere
with wtT3R function (compare Fig. 6B
to Fig. 4B
). Little or
no inhibition of T3-mediated reporter gene activation was
detected when T3Rsad versions of R320L, G345S,
432G, or p453A were introduced at 1:1 or 5:1 ratios relative to
wtT3R (Fig. 6B
). This contrasts with the strong inhibition
of wtT3Rß activation observed for the cointroduction of
the unmodified G345S,
432G, or P453A mutants (Fig. 6B
, last
lane, and Fig. 4B
). Note that repression of the reporter gene in
the absence of hormone, mediated by the unmodified
wtT3Rß, is still observed in Fig. 6B
, confirming that the
defects in transcriptional repression are specific for the receptors
bearing the sad mutations. The analogous sad
mutant form of the wtT3Rß fails to repress, but retains
the ability to activate, transcription when introduced individually
into CV-1 cells (data not shown), indicating that the sad
lesion does not abolish receptor expression, nuclear localization, or
DNA binding.
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DISCUSSION
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The RTH Syndrome Is a Complex Genetic Disorder Characterized by a
Diminished Physiological Response to Thyroid Hormone
More than 300 RTH kindreds have been identified, with most of the
known genetic lesions mapping to two domains (codons 310 to 349 and 429
to 461) within the T3Rß locus (32, 33, 34, 38). These domains
of the receptor play multiple roles in wtT3Rß function,
including hormone binding, receptor dimerization, and interactions with
the transcriptional machinery (1, 2, 3, 4, 5, 6); as a result an elucidation of the
precise molecular basis of the RTH phenotype has been complex.
Significantly, the vast majority of RTH mutants display a diminished or
complete loss of affinity for T3 hormone. Apparently as a
consequence, many RTH-T3Rs can repress gene transcription,
but are defective in gene activation in response to hormone (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).
Thus, it has been proposed that RTH-T3Rs function as
dominant negative inhibitors, interfering in trans with the
actions of the normal T3R
and -ß (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).
However, many features of RTH remain poorly understood, and its precise
molecular mechanism remains elusive. Various models by which
RTH-receptors could function as dominant negatives have been proposed,
including 1) the formation of inactive dimers between
RTH-T3Rs and wtT3Rs, 2) a competition between
RTH and wt receptors for essential cofactors, or 3) a competition
between RTH and wt receptors for DNA-binding sites. This complexity is
paralleled by the clinical phenotype of RTH syndrome, which varies in
its severity and characteristics from kindred to kindred. Thus, the
known biochemical properties of the RTH receptors do not invariably
predict the severity of the disease state, and individuals with the
same genetic lesion can display different symptoms (32, 33, 34, 48, 49, 51, 52). It has been suggested that additional, independently inherited
factors may be involved in RTH (32, 49, 53, 54).
We therefore asked whether the recently elucidated SMRT/N-CoR family of
corepressors might be involved in the pathogenesis of RTH. In this
manuscript, we present evidence that RTH-T3Rß mutants are
indeed altered in their interactions with SMRT and that these
alterations in corepressor association appear to play a role in the
dominant negative properties of the RTH receptors and may therefore
influence the clinical manifestations of the disease.
RTH Mutant Receptors Exhibit Defects in
T3-Mediated Dissociation from Corepressor
Wild type T3Rß binds to SMRT in the absence of
T3 but dissociates on binding hormone, a process paralleled
by a conversion of the receptor from a transcriptional repressor to an
activator (11, 12, 13, 14, 15). The RTH-T3Rs analyzed here share this
ability to associate with SMRT in the absence of T3 but are
notably impaired in the ability to dissociate from corepressor on
addition of hormone. Four of the RTH mutants tested required higher
than normal levels of hormone to dissociate from SMRT, whereas seven
others failed to dissociate significantly from SMRT at even 1
µM hormone. Given the strong transcriptional silencing
properties of SMRT (11, 14), the RTH-T3R/SMRT complexes
would be predicted to repress transcription under hormone conditions in
which wtT3Rß activates, a prediction consistent with the
dominant negative properties of the RTH mutants. Indeed, the RTH
receptors that were constitutively bound to SMRT exhibited strong,
hormone-refractory dominant negative properties in our transfection
studies. In contrast, an RTH-receptor (R320L) that exhibited impaired,
but detectable, release of corepressor at high hormone concentrations
displayed a similarly hormone-labile dominant negative phenotype.
Two RTH Mutations Appear to Uncouple SMRT Dissociation from Hormone
Binding
The failure of most of our RTH mutants to release from SMRT in the
presence of T3 could be attributed to the impaired
affinities of the mutant receptors for this hormone. For example, the
R338W mutant exhibits a 10-fold reduction in affinity for
T3 relative to wtT3Rß and requires some
6-fold more hormone for SMRT dissociation; similarly the G345S mutant
is virtually unable to bind hormone in vitro and fails to
significantly dissociate from SMRT at even 1 µM
T3. However, two striking exceptions were noted to this
general correlation between affinity for T3 and SMRT
release. The P453A and P453H mutants are only mildly impaired in
hormone binding in vitro (possessing even higher affinities
for T3 than the R338W mutant), yet fail to dissociate from
SMRT at any hormone concentration tested. Protease sensitivity assays
(56) confirm that the P453A and P453H mutant receptors are indeed
occupied by hormone at these T3 concentrations that fail to
displace SMRT (data not shown). Notably, the P453A and P453H mutants
represent different amino acid substitutions at the same codon, in a
region of the receptor that is proposed to undergo a conformational
change on binding hormone (57, 58). Codon 453 may therefore define a
receptor domain critical for corepressor release after hormone binding,
and lesions at this site may confer RTH by preventing SMRT dissociation
in a manner independent of hormone occupancy.
Consistent with these concepts, mutants at codon 453 possess dominant
negative properties that are relatively refractory to T3
(48). This region of the receptor has been suggested to operate as a
hinge, permitting a C-terminal tail of the receptor to rotate from an
exposed position in the absence of hormone to a new position nested
against the body of the receptor in the presence of hormone (57, 58).
Our preliminary evidence, employing protease sensitivity as a probe of
structure, supports the concept that this hinge function may be
impaired in the P453A and P453H mutants (B. Lin, S. Yoh, and M. L.
Privalsky, manuscript in preparation).
It should be noted that the hormone concentrations required for
inhibition of T3R/SMRT binding in our GST assay were higher
than the association constants (Ka) for T3
reported for the same receptors (Table 1
). This phenomenon was
consistently observed in multiple assays, and was also seen for
wtT3R
,-ß, and for retinoic acid receptors.
Intriguingly, analogous discrepancies exist between the Ka
values of these receptors and the significantly higher hormone
concentrations required for transcriptional activation in transient
transfection assays (e.g. Refs. 47 and 48). These
differences may simply be technical in origin, i.e. due to
sequestration of hormone by components of the transfection/binding
assays (47). On the other hand, this phenomenon may reflect a
physiologically relevant process. For example, SMRT association may
alter the affinity of T3Rs for hormone, analogous to the
effects reported for heterodimer formation with retinoid X receptor
(59, 60).
Two RTH Mutants Also Exhibit Dramatically Enhanced Levels of SMRT
Association
Two of the nine RTH mutants analyzed (
430 M and
432G) were
not only impaired in T3-mediated dissociation from SMRT,
but also exhibited a strongly elevated association with SMRT even in
the absence of hormone. Consistent with this enhanced SMRT binding, the
430 M and
432G mutants exert dominant negative phenotypes that
are among the strongest observed for any of RTH mutants tested; this is
particularly evident with certain promoters and under high hormone
concentrations (48). Notably, these two, independently derived
mutations represent in-frame, single-codon deletions that map to an
-helical domain in the T3Rß C terminus (based on the
crystallographic model of T3R
; Ref.57). This helix
appears to play important roles in the conformational changes
associated with hormone binding, in transcriptional activation, and in
dimer formation with other receptors, but has not been previously
identified as a direct contact site for corepressor. It appears that by
shortening or rotating this region by one amino acid residue, the
binding of corepressor can be greatly enhanced, either by directly
affecting an interaction surface or by a more indirect effect on the
global conformation of the receptor C terminus.
Corepressor Association Appears to be Required for the Dominant
Negative Actions of the RTH-T3Rs
Correlating with their dominant negative properties, all 11 RTH
mutants interacted with corepressor under hormone conditions in which
the wtT3R does not. Furthermore, mutations introduced into
RTH-T3Rs that disrupt SMRT association, or transfection of
N-SMRT derivatives that interfere with SMRT function, disrupted the
dominant negative phenotype. These results strongly implicate
corepressors in the ability of RTH-T3Rs to act as dominant
negative inhibitors of the T3 response. In these
properties, the RTH-T3Rß mutants closely parallel
v-Erb A, an oncogenic allele of T3R
that both
acts as a dominant negative allele and exhibits hormone-independent
corepressor association (11, 14). The ability of v-Erb A to
repress gene transcription and to function in oncogenesis closely
correlates with SMRT association (11, 14). However, it should be noted
that corepressors are a diverse family of protein factors, and the
dominant negative properties observed for v-Erb A and for
RTH-T3Rs may be mediated by SMRT itself, N-CoR, or some, as
yet unidentified, corepressor-like entity.
A Correlation May Exist between the SMRT Association Properties of
RTH-T3Rs in Vitro and the Clinical
Manifestations of RTH
Clinically, RTH has been broadly divided into a pituitary form
(PRTH) characterized by a predominant pituitary resistance but
preserved peripheral response to T3, vs. a
generalized form (GRTH) characterized by T3 resistance at
the level of both pituitary and peripheral tissues (reviewed in Refs.
3234). Despite these different clinical manifestations, receptors
isolated from PRTH and GRTH are often similar or identical to one
another in their biochemical properties. It is therefore interesting
that the RTH-T3Rs that display a highly elevated SMRT
association (
430 M and
432G) in the current study were both
isolated from patients presenting with PRTH. It is possible that this
constitutively elevated SMRT association may play a role peculiar to
the PRTH phenotype. However, the converse did not appear to be true:
not all PRTH-T3R mutants also displayed elevated SMRT
association in vitro (e.g. R338W).
Notably, none of the experiments described here were performed in
different tissues or in the genetic background of the original
patients. Therefore, our results do not address whether alterations in
corepressor, rather than in receptor, may also contribute to the RTH
syndrome. It is tempting to speculate that differences in corepressor
expression, perhaps coupled to genetic polymorphisms at the corepressor
loci, might account for the variable resistance of the RTH syndrome in
different individuals of the same kindred. Similarly, differences in
corepressor expression in different tissues could contribute to the
varying organ-specific effects of RTH. Clearly, however, RTH is a
complex clinical disease and is associated not only with a failure to
activate expression of certain genes in response to hormone, as tested
here, but also as a failure to suppress expression of others, most
notably that of pituitary TSH (32, 33, 34). Further work will be necessary
to determine the contributions of corepressors or related factors to
these different manifestations of this endocrine disorder.
 |
MATERIALS AND METHODS
|
---|
Molecular Clones
Wild type T3Rß was the generous gift of R. M.
Evans; the origins of the RTH mutant clones have been described
previously (7, 48). All T3Rß alleles were introduced as
BamHI to BamHI fragments into a pSG5 vector for
expression in vitro and for transient transfections. The
T3Rsad mutants (equivalent to an A223G, H224G,
T227A triple mutant, using the numbering system of Ref.12) were
created by a PCR protocol (61). Appropriate restriction fragments of
the resulting PCR product was used to replace the corresponding
sequences within the pSG5 clones of the RTH-T3Rß alleles.
Mutations were confirmed by restriction digestion/DNA sequence
analysis.
Assay of the SMRT/T3Rs Interaction
in Vitro
GST-SMRT (identical to the GST-TRAC-1 construct described
previously) was isolated from transformed Escherichia coli
and was immobilized by binding to glutathione agarose, as previously
described (14). 35S-Labeled receptors were synthesized by
in vitro transcription from pSG5 templates using T7 RNA
polymerase, coupled to in vitro translation using rabbit
reticulocyte lysates (TNT kit, Promega, Madison, WI). Each receptor was
subsequently mixed with approximately 2 µg immobilized GST-SMRT
(bound to 20 µl glutathione-agarose) in 200400 µl HEMG buffer
(14) containing 10 mg/ml BSA and a protease inhibitor cocktail
(Complete, Boehringer-Mannheim, Indianapolis, IN). T3
hormone (or an equivalent volume of ethanol carrier) was included in
the binding reactions where indicated. The binding reactions were
incubated for 6090 min at 4 C with gentle rocking, and the agarose
matrix was subsequently washed with 4 x 1 ml changes of HEMG
buffer. Bound proteins were eluted in 35 µl of 50 mM
Tris-Cl (pH 7.6) containing 10 mM glutathione, resolved by
SDS-PAGE, and visualized by autoradiography (14). Quantification of the
binding experiments were performed with a Molecular Biosystems Storm
phosphoimaging system (Sunnyvale, CA).
Transient Transfections
For calcium phosphate transfections, CV-1b cells (2 x
105 per 60-mm plate) were propagated overnight at 37 C in a
5% CO2 atmosphere in DMEM containing 10% heat-inactivated
FBS and penicillin-streptomycin (GIBCO/BRL, Gaithersburg, MD). The
cells were subsequently transferred to hormone-depleted medium and
incubated an additional 6 h. Calcium phosphate/DNA precipitates,
prepared by standard protocol (62), were then added, and the cells were
incubated for an additional 1618 h. Typically, each plate received 1
µg pCH110-lacZ (employed as an internal standard), 1 µg
of a DR4-luciferase reporter (63), various combinations of empty pSG5,
pSG5-T3Rß, or pSG5-
N-SMRT (as indicated), and
sufficient pUC18 or pUC19 to bring the total DNA to 10 µg. After an
overnight incubation with precipitate, the cells were washed and
transferred to fresh medium lacking or containing T3. The
cells were harvested 30 h later and lysed in 100 µl Reporter
Lysis Buffer (Promega) per plate. Luciferase activity was determined by
mixing 30 µl of extract with 100 µl Promega luciferase reagent in
an MGM luminometer (MGM Instruments, Cambridge, MA); ß-galactosidase
activity was determined by colorimetric assay (14, 64).
Lipofections were performed using 3 x 104 to 5
x 104 cells seeded per 1 cm-well in 24-well microtiter
plates. Lipofectin reagent (GIBCO/BRL) was diluted 10-fold into
serum-free medium, incubated 3045 min at room temperature, then mixed
with the DNA and incubated an additional 10 min. Typically, 300 ng
pCH110, 100 ng DR4-luciferase reporter, and 100 ng pSG5-T3R
construct were employed per well, with sufficient pUC19 DNA to bring
the total DNA concentration to 1 µg. The cells were overlayed with
the DNA and Lipofectin mixture, incubated 6 h at 37 C,
subsequently incubated 18 h with medium containing 20% FBS, and
then incubated 2448 h in medium containing 10% FBS with or without
hormone. Cells were lysed and assayed for luciferase and
ß-galactosidase activity in a similar manner as that described for
the calcium phosphate method.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. d. M. Vivanco Ruiz and R. M. Evans for generously
providing the luciferase reporter and wtT3Rß molecular
clones.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Martin L. Privalsky, Department of Microbiology, University of California at Davis, Davis, California 95616.
This work was supported by Public Health Services/NIH Grant R37
CA-53394.
Received for publication September 19, 1996.
Revision received January 27, 1996.
Accepted for publication January 27, 1996.
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