Download Spectrum of transcriptional, dimerization, and dominant negative

Spectrum of Transcriptional,
Dimerization,
and Dominant Negative
Properties of Twenty Different
Mutant Thyroid Hormone
,&Receptors in Thyroid Hormone
Resistance Syndrome
T. N. Collingwood*,
M. Adams*,
Y. Tone, and V. K. K. Chatterjee
Department of Medicine
University of Cambridge
Level 5 Addenbrooke’s
Hospital
Cambridge, CB2 2QQ, United Kingdom
Resistance to thyroid hormone
(RTH) is usually dominantly inherited and characterized
by elevated thyroid hormone levels, impaired feedback inhibition of
pituitary TSH production,
and variable hormonal responsiveness
in peripheral
tissues. We have identified 20 different mutations in the thyroid hormone
B-receptor
(TRB) gene in RTH and assayed mutant
receptor properties
using the TSHa subunit gene
promoter
or promoters
containing
three different
types of positive thyroid response
element (TRE).
Dominant negative inhibition of wild type TRB action
by mutant receptors
was also tested. The mutant
receptors
exhibited
differing transcriptional
inhibitory properties and dominant negative potential with
the TSHa promoter
that correlated
with their impaired hormone
binding,
whereas
transactivation
and dominant negative effects with promoters
containing positive TREs varied depending
on their configuration. Heterodimeric
mutant receptor-retinoid
X
receptor (RXR) interactions,
either in cultured cells
or as TRE-bound
complexes
in gel retardation
assays, were uniformly preserved,
whereas
homodimerit receptor interactions
could not be detected in
viva, and in vitro homodimer
formation on TREs was
variably reduced or absent for some mutant proteins. We correlate these findings with the distribution of receptor mutations that cluster in two areas
within the hormone binding domain outside putative
dimerization
regions and show that artificial mutations that impaired
heterodimerization
abrogated
dominant
negative activity. Therefore,
we suggest
that the dominant negative effect of mutant recep
tors in the pituitary-thyroid
axis generates
the characteristic biochemical
abnormality
of RTH and that
variable resistance
in other tissues may be due to
response
element-dependent
differences
in their
088s8809/94/1282-1277$03,00/O
Molecular Endocrinology
Copyright 0 1994 by The Endocrine
dominant negative potential.
ogy 8: 1282-1277,1994)
(Molecular
Endocrinol-
INTRODUCTION
Thyroid hormones (T4 and T3) have a number of actions
such as the regulation of growth and metabolic processes, augmentation
of myocardial contractility,
and
control of brain development. The synthesis of T4 and
T3 in the thyroid is controlled by TSH from the pituitary.
In turn, these hormones inhibit TSHa and P-subunit
gene transcription as part of a classic negative feedback
loop (1). The biochemical
hallmark of resistance to
thyroid hormone (RTH) is abnormally elevated serum
thyroid hormone levels together with a failure to suppress pituitary TSH secretion. In addition, affected individuals exhibit a variable degree of resistance to
hormone action in peripheral tissues. Some patients
are asymptomatic,
with few clinical signs other than a
goiter, leading to a diagnosis of generalized resistance
(GRTH), whereas peripheral thyrotoxic features in other
cases suggest
more selective pituitary
resistance
(PRTH) to the effects of thyroid hormone (2). The actions of thyroid hormone are known to be mediated by
a nuclear receptor protein which binds to regulatory
DNA sequences [thyroid response elements (TREs)] in
target gene promoters and activates or inhibits their
expression in a hormone-dependent
manner (3). After
the demonstration
of tight linkage between the thyroid
hormone receptor p (TRP) gene locus and generalized
resistance (4) several groups have identified a number
of different receptor mutations in families with this
disorder (2). Recently, receptor mutations have also
been documented
in cases of selective pituitary resistance (5, 6) suggesting that it is not a separate entity
but part of the variable phenotypic spectrum of a single
dominantly inherited genetic disorder.
Society
1262
Dominant
Negative
Mutant
Receptors
in Thyroid
Hormone
Resistance
Genetic analyses of a large number of individuals
with RTH indicate that they are heterozygous for TRP
mutations that localize to the hormone binding domain
of the receptor (7, 8). Previous studies have examined
the functional properties of a small number of mutant
receptors and established that their ability to bind ligand
and modulate target gene expression is impaired (9,
10). In addition, when coexpressed, the mutant proteins
are capable of inhibiting the action of their wild type
counterparts in a dominant negative manner (9, 11).
Evidence to support the importance of dominant negative activity of mutant receptors in the pathogenesis
of the disorder is provided by the observation that
individuals who are heterozygous for a complete deletion of a single TRP allele are completely normal (12)
whereas one individual who was homozygous for a
dominant negative receptor mutation exhibited severe
resistance (13).
In this study we have characterized the properties of
20 different receptor mutants that we have identified in
European kindreds with RTH. As a correlate of the
defective feedback in the pituitary-thyroid axis, we have
assayed the ability of mutant receptors to repress transcription and exert dominant negative effects on the
human TSHa: subunit gene promoter. The variable resistance in other tissues is likely to be mediated by the
action of thyroid hormone on a number of other target
genes and to depend on factors such as the nature of
the response element, promoter context, and cell type.
Analyses of the promoter regions of T,-inducible genes
indicate that they contain TREs that consist of two or
more hexameric nucleotide motifs, of consensus sequence A/GGGTCA, arranged as a direct repeat (14)
an everted repeat (15) or a palindromic sequence (16).
We have examined the influence of response element
configuration on mutant receptor function and dominant
negative activity using these three types of TRE linked
to a common heterologous promoter in the same functionally receptor-deficient cell line. Finally, we have correlated these properties with the ability of wild type and
mutant receptor proteins to interact either as homodimers, or as heterodimers with the RXR. In vivo interactions of receptors in cultured cells were assayed by
coexpression of hybrid proteins containing the DNA
bindingdomain of the yeast transcription factor GAL4
linked to wild type or mutant TR@together with the
activating domain of VP16 linked to RXR. In addition,
as wild type receptor has also been shown to interact
with TREs either as a homodimer (17, 18) or as a
heterodimer with RXR (19-21) we have examined
these mutant receptor-DNA interactions.
We find that the mutant receptors exert variable
dominant negative effects both with the TSHa gene
promoter as well as with positive TRE containing promoters. Our analyses also indicate that the reduced
ligand binding and transcriptionalproperties of mutant
receptors, together with preservation of their ability to
bind DNA and form heterodimerswith RXR, are essential for dominant negative activity. We suggest that
these observationsprovide an explanation for the clus-
1263
tered distributionof mutationswithin the hormonebinding domain of the receptor as well as the biochemical
and variable clinical phenotype of this disorder.
RESULTS
Transcriptional Activity of Mutant Receptors Is
Response Element-Dependent
In every case of RTH, the diagnosis was made by
finding abnormallyelevated serumthyroid hormonelevels together with nonsuppressedpituitary TSH secretion (Table 1). Full detailsof the clinicaland biochemical
features in these kindreds will be publishedseparately
(M. Adams, C. Matthews, T. N. Collingwood,V. K. K.
Chatterjee, manuscript in preparation).We have documented 20 different TRP receptor mutations in unrelated families,including17 missensemutations,two inframe deletions (A430M, A432G), and a single sevennucleotide insertion mutation (frameshift). Based on
their TBbindingaffinities(Table l), the mutant receptors
can be grouped into three categories. The majority
showed a mild to moderate impairment in TS binding
Table 1. Clinical
Receotors
Mutation
Wild type
R320H
P453S
R438C
R438H
R429Q
P453T
P453A
R320L
R338W
P453H
M334R
Y321C
1431T
V264D
R316H
G345S
G344E
A430M
A432G
Frameb
Normal
range
Data
and T3 Binding
FT4*
pmol liter-’
29
35
25
36
31
44
25
34
71
45
72
49
25
29
27
See Ref. 62
64
53
39
69
9-20
TSH’
mU liter-’
2.0
1.5
5.6
2.7
7.8
2.0
1.9
4.2
4.1
9.4
3.2
2.1
1.4
2.5
0.7
4.1
1.0
3.6
2.2
Affinity
of Mutant
Phenotype
GRTH
GRTH
GRTH
GRTH
GRTH
GRTH
PRTH
GRTH
PRTH
GRTH
PRTH
PRTH
PRTH
GRTH
GRTH
GRTH
GRTH
PRTH
PRTH
GRTH
2.2 (0.2)
0.83 (0.12)
0.79 (0.19)
0.67 (0.15)
0.51 (0.20)
0.46 (0.11)
0.44 (0.08)
0.38 (0.03)
0.21 (0.06)
0.21 (0.06)
0.15 (0.01)
0.11 (0.03)
0.04 (0.02)
0.02 (0.01)
co.02
co.02
co.02
BND
BND
BND
BND
0.4-4.0
Codon nomenclature
is based upon a predicted
protein sequence containing
461 residues
(63). BND, Binding not detected.
’ Thyroid function tests and clinical phenotype
are in the index
case from each kindred.
b An insertion of ACTCTTC
after nucleotide
1638 leading to a
shift in reading frame and addition of residues to the extreme
carboxyterminus.
MOL ENDO. 1994
1264
[association constant (KJ values ranging from 0.83 to
0.02 x 10” M-‘1; three (V264D, R316H, G345S) retained very low TB binding capabilities with affinities that
could not be quantified (K, < 0.02 x 10” M-‘), and four
mutants (G344E, A430M, A432G, frameshift) exhibited
no detectable TJ binding.
In the first instance we assayed the ability of wild
type and mutant receptors to inhibit transcription of the
human TSHa-gene promoter, and the results are shown
in Fig. 1A and B. The wild type receptor inhibited
TSHaLUC activity in a T3 dose-dependent
manner with
maximal inhibition occurring at 1 nM TJ. In keeping with
their impaired ligand affinities, the majority of mutant
receptors exhibited a reduction in sensitivity to hormone
such that higher TS concentrations
were required to
exert a comparable inhibitory effect. With mutant receptors that retained TS binding, the rank order of their
inhibitory potencies (Fig 1, A and B: WT > R338W =
R320H = Y321C > R316H = R320L > M334R >
V264D > G345S; WT > P453S = R438C = R438H =
P453T = P453A > R429Q = l431T = P453H) was
broadly comparable to their relative K, values (Table 1:
WT > R320H > R338W = R320L = M334R > Y321C
> R316H = V264D = G345S; WT > P453S = R438C
> R438H = P453T = P453A = R429Q > P453H >
1431 T). The four non-hormone-binding
mutants (G344E,
A430M, A432G, frameshift) showed almost flat T3dependent inhibition profiles that did not differ significantly from inhibition in mock transfected cells.
We next performed
similar studies with reporter
genes, which are all activated by wild type receptor in
a ligand-dependent
manner. MAL-TKLUC
contains a
natural direct repeat TRE from the malic enzyme gene
(14) and is maximally stimulated by wild type receptor
at 5 nM TJ (Fig. 1, C and D). As with negative transcriptional regulation, most mutants showed a right-shift in
activation profiles and achieved the same maximal induction as wild type receptor at high TJ concentrations.
However, several mutants were unable to attain full
activation, despite the presence of supramaximal
TJ
concentrations.
Interestingly, this effect was observed
with several different mutations
at a single codon
(P453A, P453H, P453S P453T). In addition, two mutations at codon 438 (R438C R438H), each with similar
K. values, showed divergent activation profiles; R438C
stimulated MAL-TKLUC activity to a greater extent than
wild type receptor whereas activation by R438H was
markedly impaired. Mutant Y321 C, which binds T3 with
a greatly reduced affinity, also exhibited a higher maximum activation level than the wild type receptor. The
four non-T3-binding
mutants (G344E, A430M, A432G,
frameshift) did not activate gene expression through
the malic enzyme TRE. These results were compared
with activation profiles obtained using a palindromic
response element (PAL), which was derived by optimizing the TRE in the rat GH gene (22). When tested with
PAL-TKLUC,
the mutant receptors again showed a
range of Tadependent
activation profiles (Fig. 1, E and
F). As with MAL-TKLUC,
mutant Y321C was supraactive compared to wild type receptor, and the four non-
Vol8 No. 9
hormone-binding
mutants did not activate even in the
presence of 1 FM TJ. However, some TRE-dependent
differences were apparent. In contrast to their profiles
with the malic enzyme TRE, mutants 1431T, R438H,
P453A, P453H, P453S and P453T all stimulated PALTKLUC to the same extent as wild type receptor.
Conversely, mutants R338W, G345S and R429Q were
all less active on the palindromic vs. the malic enzyme
TRE. These response
element-specific
differences
were investigated further using a third type of TRE. F2TKLUC contains the F2 response element from the
chicken lysozyme gene, which consists of two hexamer
motifs arranged as an ever-ted repeat separated by six
base pairs (15). As the maximum activation of this
reporter gene was less than that achieved with other
TRE configurations
(maximal -15-fold
induction with
wild type receptor at 5 nM T3), it was not possible to
generate TJ dose-dependent
profiles for each mutant.
The activity of wild type and mutant receptors at intermediate (1 nM) and supramaximal(1
PM) T3 concentrations is shown in Fig. 2. Once again, at 1 nM TB
transactivation
by each mutant receptor was diminished, reflecting their reduced hormone responsiveness. With the exception of the non-Ts-binding
mutants
(G344E, A430M, A4326, frameshift), this impairment
was reversible with supramaximal
levels of T3 (1 PM).
However,
a number of mutants (V264D,
R338W,
G345S R429Q 1431T, P453H) again showed an inability to activate fully, despite the presence of sufficient
T3 to overcome their reduced affinity for ligand.
Our results indicate that the reduction in TJ binding
affinities of mutant receptors leads to a right shift in
hormone-dependent
repression or activation profiles on
the response elements tested. With respect to negative
regulation of the TSHa gene promoter, it appears that
impaired transcriptional
activity is mainly due to a reduction in T,-binding
affinity. However, on positively
regulated TREs additional factors are involved such
that a number of mutants exhibit impaired transactivation properties, depending on the configuration
of the
response element.
Variable Dominant
Receptors
Negative
Inhibition
by Mutant
Using the same reporter
genes, we investigated
whether mutant receptors also varied in their ability to
inhibit wild type receptor action in a dominant negative
manner. In these studies, wild type and mutant receptors were coexpressed,
and reporter gene activities
were assayed at two hormone concentrations.
A submaximal concentration
of TS (0.3 nM for TSHa LUC, 1
nM for F2-TKLUC, MAL-TKLUC,
and PAL-TKLUC), at
which impaired mutant receptor function would be expected, was used to compare their relative dominant
negative potencies, and a supramaximal T3 level (10 nM
for TSHCILUC, 1 PM for FBTKLUC, MAL-TKLUC,
and
PAL-TKLUC)
was used to assess the reversibility of
these effects. Although mutant to wild type receptor
ratios of 5:l were used with some reporter genes to
I’\1
0. I
I hl
I
10
I?
I’\1
I (
I WJ
-‘I hl.l
C
1ooU
In\11
Fig. 1. T,-Dependent
Activity of Wild Type and Mutant Receptors
on the TSt& Promoter,
Mallc Enzyme.
and Palindromic
TREs
The data shown in this and subsequent
figures are the mean of at least three separate
experiments,
each done In duplicate
In
on the TSHcc promoter.
JEG-3
each case the SEM was less than 1 200 Panels a and b. Actlvlty of wild type and mutant receptors
cells were transfected
with 2 pg TSHtrLlJC
100 ng receptor
expresslon
vector. and 200 ng BOS-3gal
and incubated
with various
concentrations
of TB (O-l PM) Hormonedependent
InhIbItion of luclferase
actlvlty by wild type and mutant receptors
IS expressed
relative
to the activity
In cells Incubated
without
T, after normalization
for ;r-galactosldase
actlvlty
Typically
35”., InhIbItion
of
TSHaLUC
acbvtty was observed
In control cells transfected
with RSVCAT
at 1 PM T3 Panels c and d. Actlvlty
of wild type and
mutant receptors
on the direct repeat mailc enzyme
TRE JEG-3 cells were transfected
with 2 pg MAL-TKLUC.
100 ng receptor
expresslon
vector
and 200 ng of BOS-$gal
Luciferase
activity was determlned
after lncubatlon
with O-l PM T3 and normalized
for
$-galactosdase
activity
In each experiment
the maximum
corrected
luciferase
acbvlty with wild type receptor
was taken as 100%
and activation
by each mutant receptor
calculated
relative to this Wild type receptor
Induced this reporter
gene approximately
1 OO-fold in the presence
of 5 nM T9 Panels e and 1. Actlvlty of wild type and mutant receptors
on a pallndromlc
TRE Cells were
transfected
with 2 pg PAL-TKLUC.
100 ng receptor
expression
vector,
and 200 ng BOS3gal
and incubated
with various
concentrations
of T3 PAL-TKLUC
was induced approximately
80.fold by wild type receptor
with 10 nM T:, Hormone-dependent
activation
was calculated
as for panels c and d
MOL
1266
ENDO.
1994
Vol8
F2-TKLUC
is
g
&
2
40
30
20
10
140,
0
20
n
Fig. 2. Activity
of Wild Type and Mutant
Receptors
on the
Ever-ted Repeat F2 TRE
JEG3 cells were transfected
with 4 pg FBTKLUC,
100 ng
receptor
expression
vector, and 200 ng BOS-Pgal.
Corrected
luciferase
activity induced by wild type TRPl in the presence
of 1 PM T3 was taken as lOO%, and activation
by mutant
receptors
at 1 nM (hatched
bars) and 1 FM TS (solid bars) was
calculated
relative to this. Wild type receptor
induced
F2TKLUC activity by approximately
15fold
in response
to 1 PM
T3 stimulation.
demonstrate optimum dominant negative activity, these
inhibitory effects were also apparent with equal ratios
of receptors.
At 0.3 nM TJ, each mutant exerted a dominant negative effect on expression of TSHLULUC, although the
magnitude of this effect varied. At 10 nM TJ the majority
of mutants ceased to exert an inhibitory effect (Fig. 3A).
However the dominant negative effects of P453H and
the non T,-binding mutants (G344E, A430M, A432G,
frameshift) were still apparent. Subsequent experiments indicated that P453H loses dominant negative
activity at between 10 and 100 nM TB (see also Ref. 9)
whereas the non-T3-binding mutants still inhibit wild
type receptor activity at 1 @I TS (our unpublished data).
Detailed TJ dose-response studies with three mutants
(R316H, R438C, and P453H) also showed that the
concentration of hormone required to abrogate dominant negative activity correlated with the ligand binding
affinity of each mutant (data not shown).
At low (1 nM) hormone concentrations, the mutant
receptors also exhibited a variable spectrum of dominant negative activity when investigated using the three
positively regulated reporter genes (Fig. 3, 8, C and D).
At supramaximal TB levels, these inhibitory effects were
reversible for most mutants but with some notable
exceptions. The non-Ts-binding mutants (G344E,
A430M, A4326, frameshift) showed potent dominant
negative activity on all elements with little reversal at 1
PM TJ; in addition, the effects of codon 453 mutants
(P453A, P453H, P453S, P453T) were also poorly reversible, particularly on MAL-TKLUC and FBTKLUC,
No. 9
consistent with their impaired activation profiles on
these TREs. However, dominant negative activity did
not necessarily correlate with the impairment of activation potential, as exemplified by the weak dominant
negative activities of l43lT and R338W mutants on
PAL-TKLUC (Fig. 3C). Finally, mutant R429Q illustrated
properties that varied with response element configuration, being a strong inhibitor on FBTKLUC but considerably weaker on the other two response elements.
These results suggest that the dominant negative
effects of mutant receptors with TSHL~LUC appear to
correlate with the degree of impairment of hormonedependent repression of this promoter. However, with
positively regulated TREs, the dominant negative effects are variable, and factors other than the reduction
in ligand-dependent transactivation are involved.
Wild Type and Mutant Receptors Interact
hRXRa When Coexpressed
in Viva
with
A seriesof nine heptad repeatsof hydrophobic residues
mediatingdimerization has been delineatedwithin the
hormonebindingdomainof the thyroid hormonereceptor (23), raisingthe possibilitythat the mutationsin RTH
also modulate this function. Although TR can interact
with responseelementseither as a homodimeror as a
heterodimer with RXR, whether such protein-protein
interactionscan occur in cellshas not beendetermined.
Accordingly, an assay used previously to demonstrate
interactions between the retinoic acid receptor (RAR)
and RXR (24) was used to detect TR and RXR interactions in viva in cultured cells. Expression vectors
(GAL4-TRs), encoding the DNA-binding domain of the
yeast transcription factor GAL4 (GAL4-DBD) fused to
the hormone binding domains of wild type or mutant
TR@(residues 174-461) or RXR (residues198-467),
were cotransfected with a secondvector containingthe
activating domain of VP16 fused to either hRXRa
(VP16-RXRa) or TRP (VP16-TR). Interaction between
these fusion proteins results in VP18mediated transcriptional activation of a reporter gene (UAS-TKLUC)
which contains GAL4 binding sites. In addition, we also
studied the dimerization propertiesof two artificial mutants, L42lR and L428R, in which the hydrophobic
leucine residuesat each end of the ninth heptad have
been changedto arginine.UAS-TKLUCactivity was not
stimulated upon cotransfection with wild type GALC
TR, VP1~-RXRLU,or VP16-TR expressionvectors individually (data not shown), or with a combination of
VP16-RXRa and GALCDBD (Fig. 4). However, cotransfection of VP16-RXRa with wild type GALCTR resulted
in a marked induction of luciferase activity (Fig. 4), as
did cotransfection of VPlG-TR and GAL4-RXRLU
expression vectors (data not shown), indicating a heterodimeric interaction between the carboxy-terminal
domainsof TR and RXRa. Similarly, cotransfection of
vectors encoding mutant GAL4-TRs with VP16-RXRa
also increased reporter gene activity, indicating that
each mutant retained the ability to heterodimerizewith
Dominant
Negative
Mutant
Receptors
*IL a
in Thyroid
Hormone
Resistance
TSH 0. LUC
1267
m-lr
11
MAL-TKLUC
l:i?-TKLUC
PAL-TKLUC
Fig. 3. Dominant
Negative
Inhibition of Wild Type Receptor
Activity by Mutant Receptors
Is T3-Dependent
In this and subsequent
experiments
no significant
differences
in luciferase
activity were obtained
after transfection
of either 600
ng wild type receptor
expression
vector or 100 ng wild type expression
vector
plus 500 ng RSVCAT.
a, JEG3
cells were
transfected
with 2 pg TSHaLUC,
200 ng BOS-@gal,
and 100 ng wild type plus 500 ng of either wild type or mutant
receptor
expression
vectors.
Inhibition
of normalized
luciferase
activity at 0.3 nM TB (upper panel) or 10 nM TB (rower panel) is expressed
relative to values in the absence of hormone.
b, Cells were transfected
with 2 pg MAL-TKLUC,
200 ng BOS-@gal,
100 ng wild type
plus 500 ng wild type or mutant receptor
expression
vectors.
Normalized
luciferase
activity after incubation
in 1 nM T3 (upper
panel) or 1 PM TB (lower pane/) is expressed
relative to values in cells transfected
with wild type receptor
with 1 PM TB. c, Cells
were transfected
as in panel b except 2 pg PAL-TKLUC
were used. Dominant
negative
effects of mutant receptors
at 1 nrv (upper
pane/) or 1 PM T3 (lower pane/) were calculated
as in panel b. d, Dominant
negative
effects of mutant receptors
on FP-TKLUC.
Cells were transfected
with 4 pg F2-TKLUC,
200 ng BOS-Bgal,
100 ng wild type plus 100 ng wild type or mutant
receptor
expression
vectors.
The upper and lower pane/s show the effects of mutant receptors
on induction
by wild type receptor
at 1 nM
or 1 PM TS, respectively.
Results were calculated
as in panel b.
RX!% in ceils. However, both of the heptad mutants,
L421R and L428R, were inactive in this assay reflecting
a lossof dimerizationcapacity. Importantly, cotransfection of wild type VPlG-TR with either wild type or
mutant GAL4-TR expression vectors did not result in
any detectable increase in UASTK-LUC reporter gene
activity (data not shown), suggesting an absence of
homodimericinteractionsin vivo.
Homodimerization
of Receptor
Response Element-Dependent
Previous
studies
have
established
Mutants
Is
that
the thyroid
hor-
mone receptor can interact with TREs as a homodimer
(25). On the basisthat variable dimerizationof mutant
receptors could influencetheir transcriptionaland dominant negative properties, we studied the interaction of
these proteins with TREs using gel mobility shift assays. In accordance with previous observations (25)
we observed homodimerformation most readily with
the everted repeat (F2) and palindromicconfigurations
of responseelement but were unable to demonstrate
significant homodimer formation on the direct repeat
TRE from the malicenzyme gene.
On the F2 element most mutants showed binding
comparableto wild type receptor but a few (R338W,
R438C P453A) exhibited moderately reduced homo-
MOL
1268
ENDO.
1994
Vol8
No. 9
Fig. 4. In Viva Interaction
between
RXRa and Wild Type and Mutant TRs
Cells were transfected
with 5 fig UAS-TKLUC,
200 ng VPlG-RXRa
expression
vector,
1 rg GAL4-TR
(wild type or mutant)
expression
vectors,
and 300 ng BOS-pgal.
Reporter
gene activation
by wild type or mutant TR fusion proteins and VP1 6-RXRa
is
expressed
relative to normalized
activity
in cells cotransfected
with VPlG-RXRa
and GAL4 DNA-binding
domain (GALCDBD)
vectors. Each point is the mean (+SEM) of three experiments,
done in triplicate.
dimer formation. For two mutants (R316H, R429Q),
homodimer formation was severely diminished or absent, and three others (M344R, A430M, 1431T) showed
enhanced homodimer binding (Fig. 5A). A similar study
using the palindromic response element generated a
markedly different profile of homodimer
formation by
mutant receptors (Fig. 58). In this case dimerization of
R316H and R429Q was preserved, whereas R338W
and P453H showed impaired binding. Interestingly,
homodimer formation by A432G appeared to be significantly enhanced on PAL, contrary to F2, while M334R
and l431T exhibited increased dimerization
on both
response elements. Lastly, dimer formation by R438C
and P453A was moderately weakened on PAL, as with
the F2 element. These results suggested that the mutant receptors differed in their ability to form homodimers and that these differences varied with the type of
TRE.
Mutant Receptors Retain the Ability to Form
Heterodimers
with RXRa and Show Differential
Homodimer Dissociation
in Response to Ligand
Previous studies have established
that TR interacts
with response elements not only as a homodimer but
also as heterodimers with receptor-associated
auxiliary
proteins (TRAPS) (26, 27) and several groups have
shown that the retinoid X receptor meets the criteria
for this activity (20, 28). We therefore investigated the
heterodimeric
interaction of mutant receptors with human RXRa, the predominant retinoid X receptor isoform
present in the cell line (JEG-3) used in our transfection
assays (29). We tested the ability of mutant receptors
to form homodimers
and heterodimers
on F2 and palindromic response elements and examined the influence of T3 on these processes, since it was known
that ligand decreases homodimer but not heterodimer
formation by wild type TR (18, 25).
On the F2 response element (Fig. 6, A and B) the
variable homodimer
profile of mutant receptors was
attenuated to differing extents by TS, in keeping with
their altered ligand binding affinities. In contrast, the
interaction of each mutant with RXRL~ was uniformly
preserved and was not affected by TJ. Using the palindromic TRE (Fig. 6, C and D), the formation of TRRXRa complexes was favored over homodimeric interactions and again preserved for all mutant receptors.
In contrast to F2, we were unable to demonstrate any
effect of ligand on homodimer formation with this element, in accordance
with the observations
of other
groups (25). Once again, heterodimer formation on this
element was not affected by the presence of thyroid
hormone. Lastly, mutant receptor interactions with the
malic enzyme TRE were evaluated (Fig. 7, A and B).
We were able to document only heterodimeric
receptor
interactions with this configuration of TRE, and this was
not influenced by the mutations or the presence of TJ.
Overall, these studies show that for each RTH mutant
the formation of TR-RXRa heterodimers
is preserved
on each of three different configurations
of TRE. In
contrast, the stability of a TRP mutant homodimer is
dependent upon its affinity for ligand and the type of
Dominant Negative Mutant Receptors in Thyroid Hormone Resistance
FZ
-
nnnnnn
1269
_
B
Pal
--
Fig. 5. Gel Mobility Shift Assays Showing Differential Homodimer Formation by Receptor Mutants on TRE-FP and TRE-PAL in the
Absence of Ligand
Equal amounts (-35 fmol) of in vitro translated mutant receptors were incubated in the presence of the specified labeled DNA
oligonucleotide. RL, Nonprogrammed reticulocyte lysate; Wt, wild type receptor. All other lanes contain receptor mutants as
indicated. A, Complexes formed with TRE-FP (40 fmol probe). B, Complexes formed with TRE-PAL (20 fmol probe). So/id arrows
show specific receptor complexes. Open arrows show nonspecific complexes. T/r denotes TRj3 homodimer.
responseelement to which it is bound, in addition to
the integrity of its dimerizationregions.
Loss of Dominant Negative Activity Correlates with
Loss of Heterodimerization Function
In order to test the hypothesis that dimerization is
importantfor dominantnegative activity, we studiedthe
propertiesof two artificialmutant receptors, L421R and
L428R which were shown to markedly reduce heterodimeric interactions between TR and RXR in vivo. Ligandbindingstudiesshowedthat mutant L421R bound
T3 weakly with a K, c 2 x 1OSM-‘, whereasthe L428R
mutant exhibited negligibleT3 binding. Consistentwith
these properties, the L421R mutant activated or repressedreporter genes fully at supramaximalT3 concentrations, whereasthe L428R mutant was transcriptionally inactive (our unpublisheddata). These mutants
also exhibited divergent dimerization potential that was
responseelement dependent. On the F2 TRE, both
homo- and heterodimerization of mutant L421R was
markedly impaired in the absence of ligand (Figs. 5A
and 66) whereas the L428R mutant formed homodimers despite an inability to heterodimerize with RXR
(Fig. 6B). A similar pattern was observed with the
palindromicTRE, with both mutations retaining homodimer formation yet showing weak (L421R) or absent
(L428R) heterodimerization(Figs. 5B and 6D). On the
malicenzyme TRE, the ability of both mutants to form
heterodimerswas also impaired(Fig. 7B). Interestingly,
the addition of T3 altered the dimerizationpropertiesof
L421R, restoring its ability to form heterodimerson F2
(Fig. 6B) and to a lesser extent on the malic enzyme
element, but did not modulate the DNA-binding of
L428R. While these studies were in progress, similar
properties were described for analogousmutations introduced into the retinoic acid and chick a-thyroid hormone receptors (30).
To determine the relative importance of home- vs.
heterodimerizaton in dominant negative activity, we
carried out further studies with two “double mutant”
receptors containing a resistance mutation together
with either the artificial L421R or L428R dimerization
mutations. The frameshift mutant was used in these
studies,owingto its negligibletranscriptionalresponses
to TJ, ability to form both homo- and heterodimers,and
its strong dominant negative potency. We examined
the DNA bindingpropertiesof each individualmutation,
and those of the double mutants on the F2 response
element(Fig. 8), and assesseddominantnegative activity on F2-TKLUC and TSH~YLUC(Fig. 9A and B). The
frameshift mutation showed strong dominant negative
effects with both reporter genes, whereas neither
L421R nor L428R mutants inhibitedwild type receptor
function significantly. The properties of individual mutations in gel shift assays were also as shown previously, except that the effects of ligandin reducing wild
MOL ENDC. 1994
1270
Vol8 No. 9
RXR
A
RXR
1
RLTWT
wI.m1.,1
w-r
V2S4D
n.
R316H
R32OH
,,.I,.
R320L
FZ _
B I R42W
T3:I.
nnnnnn
M334R
R33W
I+, I-
G344E
03465
I _ + I I .
+,
f
-
RXR
A43OM
+I,-
14311
A432G
+I I-*II-.II-+II-v1I-
R438C
F2
C
Y3ilC
_
R438H
-
“““““n
RXR
RLI;;:WY
T3:I.l-Fl1--*11-+11-R)-
I
P453A
P463H
I-.
P453S
I-
P4632
Prime
L42?R
441..
V2640
R316H
I
l 1
4
RXR
WT
L428R
+a,.
I
R32OH
R320L
Y32lC
M334R
R338W
G344E
+a,.
+,I+I,+a,*II.
0345s
PaI --
D ,
RI.
T5:rnb.
RXR
RWQ
AA3OM MIT
+a,.
+II.+II.*II,,.
M32G
R438C
Pal
R43SH
P453A
P483H
P463S
+,,+,I+1,.,.
P&32
+,I.
Frame
L421R
L428R
+,I+,I+,
I
_
Rg. 6. Gel mobility shii assay showing differential effects of ligand on the stabllity of mutant receptor homodimers with TRE-FP
and TRE-PAL and preservation of TRB/RXRa heterodimers. RL, Nonprogrammed reticulocyte lysate; Wt, wild type receptor. All
other lanes contain receptor mutants as indicated. Equal amounts (35 fmol) of in vitro translated mutants were incubated with the
spscified labeled DNA oligonucleotide and in vitro translated RXRcr (-6 fmol in panels A and B; -3 fmol in panels C and D) in the
absence (-) or presence (+) of 10 nM T3. A and B, Complexes formed on TRE-F2 (40 fmol probe). C and D, Complexes formed on
TRE-PAL (20 fmol probe). So/id arrows show specific receptor complexes. Open arrows show nonspecific complexes. T/T denotes
TR@ homodlmer. R/T denotes TRB/RXRa hetercdimers.
Dominant Negative Mutant Receptors in Thyroid Hormone Resistance
A
RXR
I
RL RL
T3:l.
.,I.
B
,
RL
W
+,1.,.
R4ZBQ
TJ:rnI.
VZ64D
A430M
em,.
MIT
+,a.
R316H
R320H
R33OL
V32,C
M334R
R33SW
63441
G345S
+,,.
+,I.
+,,.
+,,.
+I,+,,+,,.
+,
1271
I
RXR
I
A433G
R43SC
R43SH
P4S3A
P4S3H
P4S3S P4S3T
Frame
L431R
L42SR
+I, _ +a,,I#.
‘II.
+I,.
+a,.
*,I.
+,I.
.,I_
,I,.
+I
Fig. 7. Gel Mobility Shift Assay Showing Preservation of Mutant TRP/RXRCY Heterodimers on the Malic Enzyme TRE (TRE-ME)
RL, Nonprogrammed reticulocyte lysate; Wt, wild type receptor. All other lanes contain mutant receptors as indicated. Equal
amounts of in vitro translated mutants (35 fmol) were incubated with TRE-ME (40 fmol) with 6 fmol of in v/fro translated RXRa in
the absence (-) or presence (+) of 10 nrv T3. Solid arrows show specific receptor complexes. Open arrows show nonspecific
complexes. R/T denotes TRfi/RXRa heterodimers.
RXR
I
13:
RL
mt.
WT
L421R
+,I.
F2 -
L428R
+,I.
Frame
+,, _ *,I
L421R
L428R
Frlme
_
+,I
Frime
_ +l
’
nnnnnn -
Fig. 8. Gel Mobility Shift Assay Showing Modulation of Dimerization in a Dominant Negative Mutant Receptor
Equal amounts (-35 fmol) of in vitro translated TRB mutants
were incubated with -6 fmol in vitro translated RXRCYand
TRE-F2 (40 fmol) in the absence (-) or presence (+) of 1 @I
T3. The lanes labeled L421 R+Frame and L428R+Frame contain double mutants. So/id arrows show specific receptor
complexes. Open arrows show nonspecific complexes. T/r
denotes TR@ homodimers. R/T denotes TRB/RXRa heterodimers.
type homodimerformation and augmentingheterodimer
formation were more marked at this TOconcentration
(1 PM). However, L421R/frameshift showed markedly
impaired homo- and heterodimerization that was not
T3-reversible,and L428R/frameshift showed a marked
reduction in heterodimerformation with preservation of
homodimerizationthat was not modulatedby ligand. In
transfection studies both frameshift/L421R and frameshift/L428R exhibited negligibledominant negative activity (Fig. 9, A and 6).
These results suggest that, in order to exert a dominant negative effect, a mutant receptor should exhibit
impairedtranscriptionalproperties yet retain the ability
to form heterodimerswith RXR. Thus, L421R has no
dominant negative activity because it both forms heterodimersand is transcriptionallyactive when liganded.
Although L428R is transcriptionally inactive, it is not a
dominantnegative inhibitor,presumablydue to severely
attenuated heterodimerizationwith RXRa. Both these
mutations abrogate the dominant negative effect of
frameshift; although L421R/frameshift remains transcriptionallyinactive it is unableto form either homo-or
heterodimers. The L428R/frameshift mutant is even
more informative as homodimerformation is preserved,
indicating that loss of dominant negative activity is
associated with selective impairmentof heterodimerization.
MOL
1272
ENDO.
1994
Vol8
140
=
FZ-TKLUC
60,
b
TSHa
No. 9
LUC
1
Fig. 9. Dominant
Negative
Effects
of
Dimerization
a, JEG3
cells were transfer&d
with
mutant expression
vectors and incubated
TSHaLUC,
200 ng BOS-pgal,
and 100
with 0.3 nM T3. Results were calculated
the
Frameshift
Mutant
Regulatory
Abolished
by Additional
Receptor
Mutations
That
Prevent
4 pg F2-TK-LUC,
200 ng BOS+gal,
and 100 ng wild type plus 100 ng of wild type or
with 1 PM TS. Results were calculated
as for Fig. 3d. b, Cells were transfected
with 2 pg
ng wild type plus 500 ng wild type or mutant receptor
expression
vectors
and incubated
as for Fig. 3a.
DISCUSSION
Transcriptional
TRB Receptors
Are
Properties
of Mutant
In this paper we describe the properties of 20 different
mutant thyroid hormone p-receptors
identified in our
series of unrelated European kindreds with RTH. Eight
of these receptor mutations have been documented
previously in this disorder (2) but a further 12 are novel
mutations not previously described. In a comprehensive
survey of mutant receptor function, we have compared
the ligand binding and dimerization
properties of these
proteins with their variable transcriptional
and dominant
negative potential on both positively and negatively
regulated reporter genes.
Our studies have shown that, consistent with their
location in the hormone binding domain, the ability of
these mutant receptors
to bind ligand is variably
affected. Consequently,
with the exception
of nonhormone-binding
mutants, their ability to inhibit transcription of the TSHa promoter was impaired at low
levels of Tg but generally reversible at higher ligand
concentrations.
In contrast, qualitatively
different responses were obtained with positively regulated target
genes, where transactivation
was not always linked to
hormone binding. Furthermore,
a single mutant could
exhibit markedly divergent profiles that varied with response element configuration,
suggesting that these
differences were unlikely to be due to variations in
expression of mutant proteins. In general, four types of
response profile were seen: 1) right-shifted responses
with attainment of a maximum activation equivalent to
wild type receptor but requiring higher TB levels; 2)
negligible response to ligand with non-T3 binding mutants; 3) mutants exhibiting higher levels of activation
relative to wild type receptor; and 4) mutant receptors
that were unable to transactivate
to wild type levels
despite the presence of saturating concentrations
of
TB. It is interesting to note that several different mutations of the proline residue at codon 453 exhibited this
last type of profile on the malic enzyme and F2, but not
the palindromic, TREs. This residue precedes a putative
amphipathic a-helical sequence at the extreme carboxy
terminus of the receptor which is highly conserved
among many members of the nuclear receptor family
and is deleted in the transcriptionally
inactive oncogene
verbA (31). Indeed, mutational analyses of a homologous sequence motif in c-erbAcy and the retinoic acid
receptor (32) as well as in the estrogen and glucocorticoid receptors (33) suggest it is involved in transactivation. We propose that the various codon 453 mutants
may represent natural examples of receptors whose
ability to transactivate
is selectively impaired on some
response elements (F2, MAL) but not on others (PAL).
Whether these effects are due to response elementspecific differences in mutant receptor interaction with
basal transcription
factors such as TFIIB, which interacts with both TRa and TRP (34, 35) or other adapter
proteins, remains to be determined.
The ability of mutant receptors to exert dominant
negative inhibitory effects also differed depending on
the nature and configuration
of response elements.
Thus, with the TSHU promoter the dominant negative
potential of mutant receptors correlated with their impaired ligand binding and transcriptional
properties such
that, at near saturating levels of hormone, dominant
negative effects were abrogated for mutants with detectable hormone binding. In contrast, dominant negative effects on positive TREs were variable and correlated poorly with transactivation,
being unexpectedly
weak (e.g. R338W on PAL) or strong (e.g. R429Q on
Dominant
Negative
Mutant
Receptors
in Thyroid
Hormone
Resistance
F2) in some cases. In addition, we observed that the
dominant negative activity of a number of different
codon 453 mutants was poorly reversible on the malic
enzyme, F2, and palindromic response elements, which
agrees with a previous study of a single mutant at this
position (36).
Dimerization
Function
of Mutant
TRB Receptors
In X-linked androgen insensitivity syndrome, mutations
have been described throughout the androgen receptor
protein (37) and in autosomal
recessive vitamin D
resistance they are largely confined to the DNA binding
and hinge domains (38). In contrast, with one exception,
the RTH mutations we have described in this study
localize to two regions of the ligand binding domain
(Fig. lo), and an analysis of other published RTH mutations indicates that they cluster within these areas
also (2). Amino acid sequence alignments of the carboxy-terminal domains of nuclear receptors have delineated a series of nine conserved heptad repeats of
hydrophobic residues potentially capable of mediating
protein-protein
interactions
(23, 39). Previous mutational analyses of the estrogen receptor (40) as well as
deletion mutants of TRB, have indicated that the integrity of the carboxy-terminal
ninth heptad is important
for dimerization (41). In this context, it is significant that
one cluster of mutations (codons 316-345)
overlaps
the first heptad, whereas the second (codons 429-461)
is carboxy-terminal
to the ninth heptad (Fig. 10). In
addition, the mutations lie outside a conserved sequence motif (residues 286-305)
that is involved in
receptor interactions with TRAPS as well as heterodimer formation with RXRP (29). Accordingly, our analyses of the dimerization properties of mutant receptors
provides a further insight into the structural determinants for these interactions.
By coexpressing
hybrid GALQTRP
receptors and
VP18tagged
RXR, we have demonstrated
an interaction between the carboxy-terminal
domain of TRP and
1273
RXR in viva in cultured cells, as has previously been
shown for RXR and RAR (24). We have also shown
that each resistance mutant retains this property, although the strength of interaction varies slightly. However, homodimeric TR solution interactions in vivo were
not detected, in accordance with previous observations
using either RAR or RXR (24). In subsequent experiments, which examined TR-TR and TR-RXR interactions in the presence of DNA, we have shown that
homodimer formation by the mutant receptors is variably altered relative to wild type receptor and, furthermore, that the ability of individual mutants to form
homodimers differed depending on the configuration of
TRE. In contrast, heterodimer formation between mutant receptors and RXRa was fully preserved on all
response elements tested.
These results provide indirect evidence to support
the importance
of the heptad repeats in mediating
dimerization, although they suggest that the first heptad
repeat is either not required for this interaction or that
its functional integrity is not compromised
by these
mutations. The ability of some mutant receptors (e.g.
R316H, R429Q) to form heterodimers
but not homodimers, as well as differences in homodimer formation
with different TRE configurations,
suggests that the
two interactions may be subject to different structural
constraints. Evidence in favor of this notion is provided
by the recent observation that the isolated DNA binding
domains of TR and RXR are capable of heterodimer
formation on direct repeat TREs, whereas the intact
receptors are required for interactions on an inverted
palindromic
response element (42). It is intriguing to
note that, apart from the V264D mutation described in
this study and another mutation at codon 234 (43) no
RTH mutations have been described in the hinge region,
although a transcription
activation function has been
mapped to the equivalent area in TRal (44). This region
also includes the A box sequence motif (Fig. lo), which
facilitates DNA binding and heterodimeric
interactions
of TR (45) as well as sequences necessary for nuclear
FS
316
264
*
174
H
*A*A**
Hl+--il-f-l-i
I+-1
*i
domain
box
193
429
*******
*
0
186
345
286
461
lW-%l
305
421
428
Fig. 10. A schematic
representation
of the hormone
binding domain of human TRP (amino acids 174-461).
The location of the
mutations
described
in this study are shown by an asterisk
together
with their codon boundaries.
The two single amino acid
deletion mutants are denoted by triangles and the arrowhead
shows the position of the insertion mutation that leads to a frameshift
(FS) and a predicted
protein of 465 amino acids. Regions that have been shown to be important
for receptor
dimerization
are
indicated
as follows: The A box extends
from residues
186-l 93 (45); the TRAP domain has been shown to be necessary
for
interaction
with auxiliary
proteins (64); a series of heptad repeats (23) are also shown,
and hydrophobic
leucine residues in the
ninth heptad, which were mutated
in this study, are highlighted.
MOL
1274
ENDO.
1994
Vol8
localization (46). It is tempting to speculate that the lack
of observed mutations within this region may be due to
the need to preserve the integrity of these latter functions in order for mutant receptors to exert a dominant
negative effect.
Variables
Influencing
Dominant
Negative
No. 9
tected in our gel retardation
assays. Possible differences in mutant receptor interactions with the various
RXR isoforms have yet to be determined.
In addition,
the role of other proteins such as TRAPS or COUP-TF
(53) which interact with TR, also remains to be elucidated.
Potential
Pathogenesis
We have shown that reduced ligand binding with consequent impaired hormone-dependent
transcriptional
regulation, accompanied
by the preservation of DNA
binding and dimerization functions, are universal properties of these mutant TRs. These features are consistent with two models in which either nonfunctional
homodimeric
or heterodimeric
mutant receptor complexes compete with their wild type counterparts
to
exert a dominant negative effect. However, we do not
favor occlusion of TREs by mutant homodimer
complexes as a model for dominant negative activity for
several reasons. First, we find that with all response
elements, both wild type and mutant receptors exhibited a preference for heterodimeric
interactions
with
RXR, and we suggest that this may reflect preferential
solution interactions
between TR and RXR in cells.
Second, although we have demonstrated
impaired dissociation of mutant receptor homodimer complexes in
the presence of TJ, in keeping with a previous study
with a single RTH mutant (47) we also find that some
mutants (R316H, R429Q on F2; R338W on PAL) have
virtually lost their ability to form homodimers, yet retain
their capacity for dominant negative activity on these
response elements. Finally, we have demonstrated
that
addition of an artificial mutation in the ninth heptad
abrogates the dominant negative activity of the frameshift mutant by attenuating
heterodimerization
while
homodimer formation is preserved, confirming a recent
similar observation made with two other mutant receptors (48). In this context, we note that two other studies
have also correlated the dominant negative activities of
unliganded
RAR (30) or TR (49) with their ability to
interact with RXR. In addition, it has been shown that
dominant negative inhibition is not altered by coexpression of RXR, favoring competition
between wild type
and mutant TR-RXR heterodimers
for DNA binding,
rather than the sequestration
of limiting amounts of
RXR by mutant receptors (50).
In our studies we have not examined receptor interactions with regulatory elements in the TSHa promoter.
Although earlier work (51) had delineated a single negative TRE, several other putative TREs have since been
documented (52) and the relative importance of these
in mediating negative regulation remains unclear. However, by analogy with the F2 data, we infer that heterodimer formation may also be critical for dominant
negative activity on the TSHa promoter. Our studies
have not addressed all the variables that may contribute
to dominant negative activity. We cannot exclude the
possibility of small differences in the ability of mutant
receptors to interact with RXR, as suggested by the
solution interaction experiments,
which were not de-
of Phenotype
Our studies indicate that RTH is associated with diverse
receptor mutations
resulting in functionally impaired
proteins that inhibit the action of wild type receptor on
the TSHa subunit gene promoter. We propose that this
dominant negative effect leads to a failure of negative
feedback in the pituitary-thyroid
axis, resulting in raised
serum thyroid hormone levels with nonsuppressed
TSH
secretion, which is the key biochemical feature of this
disorder. However, the dominant negative potency of
mutant receptors in vitro does not correlate closely with
thyroid hormone levels in vivo. This discrepancy may
be explained in part by our recent observation that the
biological activity of TSH is enhanced in RTH (54) and
is a further variable which modulates thyroid hormone
production. Attempts to correlate the functional properties of mutant receptors with other clinical features of
RTH are hampered
by a lack of precise indices of
thyroid hormone action on peripheral
target genes.
Nevertheless, it is interesting to note that, in our own
series (manuscript
in preparation)
as well as in two
other cases (5,6), the R338W receptor mutation, which
exerts significantly weaker dominant negative effects
on all configurations
of positive TRE compared to the
TSHa promoter, was associated with peripheral thyrotoxic symptoms despite resistance in the pituitarythyroid axis. Therefore we suggest that the variable
dominant negative potential of mutant receptors on
different response element configurations
may provide
a basis for the variable effects of elevated thyroid
hormone levels on target genes in peripheral tissues.
MATERIALS
AND METHODS
Expression Vectors and Reporter
Constructs
Receptor mutations were generated by site-directed mutagenesis of TRPl and verified by sequencing as described previously (51). The full length wild type and mutant human TRfll
cDNAs
were
expressed
using
a vector
containing
the Rous
sarcoma virus (RSV) enhancer and promoter (51). RSVCAT is
a vector containing
the same viral sequences
driving expression of the chloramphenicol
acetyltransferase
gene. Fusion
proteins
were generated
after the introduction
of artificial
EcoRl sites within TRfll and RXRa cDNAs. The GALCTRPl
or GAL4-RXRCZ
expression
vectors
were created by insertion
of EcoRl fragments
encompassing
the hormone
binding domains of TR>l(residues
174-461j
or RXRa (residues
?98467) in frame into pSG424
(55). The same EcoRl fragment
of
TR/31 was cloned into AASV (56) in order to express
VP16TRfll
VP1 6-RXRa
contains
the full length
hRXRa
cDNA
introduced
into the EcoRI site of AASV. TSHaLUC
contains
the 5’-flanking
region of the human TSHa-subunit
gene, from
-846
to +44 base pairs, coupled
to the luciferase
reporter
Dominant
Negative
Mutant
Receptors
in Thyroid
Hormone
Resistance
gene of P&Luc (57). The reporter
plasmids
MAL-TKLUC,
PALTKLUC, and FP-TKLUC
each contain a different
TRE configuration upstream
of the viral TK promoter
and luciferase
(LUC)
gene and were constructed
in a similar manner.
MAL-TKLUC
contains the TRE from the malic enzyme
gene (14) cloned into
the BarnHI site of pBLCAT2
(58). A cassette
containina
this
TRE and the thymidine
kinase
(TK) promoter
region
of
pBLCAT2
was then amplified using polymerase
chain reaction
and cloned into the Hindlll site of PA3Luc. The plasmid
F2TKLUC was constructed
by transferrinq
the chicken lysozvme
silencer element TRE (FP).and
TK promoter
from F2-TKCAT
(15) into the HindIll site of PAaLuc. PAL-TKLUC
contains
two
copies of a palindromic
response
element
(22) plus the TK
promoter
of pBLCAT2
inserted into the HindIll site of P&Luc.
The GAL4 reporter
plasmid, UAS-TKLUC,
contains the 17 mer
UAS sequences
and TK promoter
of 17 MX2-tk-CAT
(59)
polymerase
chain reaction-amplified
and cloned into the HindIll
site of p&LUC.
The internal control plasmid BOS-flgal
contains
the EF-1 a promoter
region of pEF-BOS
(60) driving expression
of the IacZ gene.
Cell Culture
and Transfection
Assays
JEG3
cells were grown
in Optimem
(GIBCO-BRL,
Paisley,
Scotland) supplemented
with 2% (vol/vol)
fetal calf serum and
1% (vol/vol)
Penicillin, Streptomycin,
Fungizone
(GIBCO-BRL,
Paisley,
Scotland).
Eighteen
hours before
transfection
the
medium was replaced
with Optimem
containing
2% fetal calf
serum
depleted
of thyroid
hormones
by treatment
with
DOWEX
l-X8 resin (61). Duplicate
plates of cells were transfected by a 4-h exposure
to calcium phosphate.
Cells were
exposed
to 2 pg (MAL-TKLUC,
TSHorLUC,
PAL-TKLUC)
or 4
fig (F2-TKLUC)
of reporter
plasmid, together
with 100-600
ng
wild type or mutant
receptor
expression
vector and 200 ng
BOS-Bgal.
For solution
heterodimerization
assays cells were
cultured
in medium
containing
1% Penicillin,
Streptomycin,
Fungizone
and transfected
with 5 pg UAS-TKLUC,
1 fig mutant
or wild type GALCTRp,
and 200 ng VP1 6-RXRa fusion protein
expression
vectors,
in addition to 300 ng BOS-@GAL.
After a
38-h incubation,
with T3 as appropriate,
cells were lysed in
buffer containing
25 mM glycine-glycine,
15 mM MgSO.,, 4 mM
NaEGTA,
1 mM dithiothreitol,
and 1% (vol/vol)
Triton X-100,
pH 7.8. Luciferase
activity was measured
using an Autolumat
LB 953 luminometer
(Berthold,
Stevenage,
UK). p-Galactosidase activity was also determined
and used to normalize
luciferase values for transfection
efficiency.
In Vitro Translation
of Mutants
and T3 Binding
Assays
For the measurement
of ligand binding affinity, mutant receptors were synthesized
in rabbit reticulocyte
lysate from cDNA
templates
in pGEM7Z
using the Promega
TNT coupled transcription and translation
system (Promega,
Southampton,
UK).
The T3 binding affinity of each in vifro translated
mutant was
measured
using a modification
of a filter binding assay described previously
(62). Data are the mean of at least two
separate
determinations,
each performed
in duplicate.
For gel
retardation
assays, mutant proteins were synthesized
using a
noncoupled
transcription
and translation
system,
according
to the manufacturer’s
recommended
protocol
(Promega).
Capped
mRNAs transcribed
from the above templates
were
used to program
translation
of [35S]methionine-labeled
protein
in rabbit
reticulocyte
lysate.
After
translation,
0.2 mg/ml
RNaseA was added followed by 5 min incubation
at 30 C, and
aliquots of each translation
product were analyzed
by sddium
dodecyl sulfate-polyacrylamide
gel electrophoresis.
The level
of incorporated
[%S]methionine
was measured
using a trichloroacetic
acid precipitation
method
according
to the recommended
protocol
(Promega).
An estimate
of total methionine
incorporation
was calculated
on the basis that the rabbit
reticulocyte
lysate contained
approximately
5 WM methionine
(see Promega
technical
manual).
These
data were subsequently used to calculate the total amount of receptor
synthe-
1275
sized, taking into account
residues
for some mutants.
efficiency
for each mutant
intensity of the major band
(Molecular
Dynamics,
Seal,
equal amounts
of ?S-labeled
shift assays.
Gel Mobility
Shift
the addition or loss of methionine
In addition,
relative translation
was quantified
by measuring
the
(-52 kDa) using a phosphorimager
UK). This enabled the addition of
mutant receptor
in gel mobility
Assays
The nucleotide
sequences
of the 32P-labeled
oligonucleotide
duplexes
used in gel mobility
shift assays (with flanking
sequences shown in lower case) were:
(F2)5’-aaggggatccl’TATTGACCCCAGCTGAGGTCAAGTTAC
Gagatcttcct-3’;
(PAL)5’-aaggggatccTAAGATTCAGGTCATGACCTGAGGAG
ATagatcttcct-3’;
(ME)5’-aaggggatccAGGACGTTGGGGTTAGGGGAGGACAG
TGGACagatcttcct-3’.
Equal amounts of each 35S-labeled in vitro translated
mutant
protein (2-4 ~1) were preincubated
at room temperature
for 20
min in buffer (20 mM HEPES, 50 mrv KCI, 10% glycerol, 2 mM
dithiothreitol,
pH 7.8) in the presence
of 1 pg poly (dl-dC). In
vitro translated
hRXRa
and L-T3 were also present
where
specified. The total amount of reticulocyte
lysate in each assay
was standardized
by the addition of nonprogrammed
translation mixture.
32P-lab&d
oligonucleotides
were added followed
by a further
15-min incubation
at room temperature
in a total
volume of 20 ~1. The protein-DNA
complexes
were then analyzed using polyacrylamide
gel electrophores
in 0.5X TBE (44
mM Tris, 44 mM boric acid, 1 mM EDTA). The same amount of
each mutant receptor was used in all experiments.
The assays
were replicated
using mutant proteins from at least two independent translation
reactions.
Acknowledgments
We thank R. Renkawitz
and R. Evans for providing
us with
F2TKCAT
and RXRa plasmids,
respectively.
We are indebted
to the many physicians
who referred cases of thyroid hormone
resistance
to us, without whom these studies would not have
been possible.
Received
February
2, 1994. Revision
received
April 27,
1994. Accepted
May 12,1994.
Address
requests
for reprints
to: V. Krishna
Chatterjee,
Department
of Medicine,
University
of Cambridge,
Level 5,
Addenbrooke’s
Hospital,
Hills Road, Cambridge
CB2 2QQ,
United Kingdom.
This work was supported
by the Wellcome
Trust and the
Medical Research
Council (U.K).
‘T. N. Collingwood
and M. Adams contributed
equally to
this work and should both be considered
first authors.
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