(Received for publication, July 30, 1996, and in revised form, December 9, 1996)
From the Laboratory of Molecular Biology, Human thyroid hormone nuclear receptor isoforms
(TR Thyroid hormone receptors (TRs)1 are
the products of two genes, TR Comparison of the sequences between the human TR At present, the molecular basis of isoform-specific gene regulation is
not understood. It was suggested that the different homodimerization
potentials of the two isoforms may underlie the functional differences.
TR Dulbecco's modified essential
medium was purchased from BioWhittaker (Walkersville, MD). Fetal bovine
serum and lipofectamine transfection reagents were from Life
Technologies, Inc. [ The T7-expression plasmids of the six chimeric receptors (see
Fig. 2) were derived from the T7 expression plasmids of w-TR
The mammalian expression plasmids of the TR The probe,
F2, was 32P-labeled similarly as described (20). Briefly,
two complementary oligonucleotides containing the F2 sequences as shown
in Sequence 1 below,
Laboratory of Biochemical Pharmacology, NIDDKD,
National Institutes of Health, Bethesda, Maryland 20892 and
§ Department of Biochemistry, Chang-Guang Medical College,
Kwei-San, Tao-yuan, Taiwan
1 and TR
1) express differentially in a tissue-specific and
development-dependent manner. It is unclear whether these
two isoforms have differential functions. We analyzed their interaction
with a thyroid hormone response element with half-site binding motifs
arranged in an everted repeat separated by six nucleotides (F2).
Despite extensive sequence homologies, the two isoforms bound to F2
with different affinities and ratios of homodimer/monomer. Using
F2-containing reporter gene, we found that the transcriptional activity
of TR
1 was ~6-fold higher than that of TR
1. The lower activity
of TR
1 was not due to differences in expression of the two isoforms
because similar nuclear localization patterns were observed. To
understand the structural determinants responsible for these
differences, we constructed chimeric receptors in which hinge regions
(domain D), hormone binding domains (domain E), and domains (D + E)
were sequentially interchanged and their activities were compared. Chimeric TRs containing the domains D, E or (D + E) of TR
1 showed increased propensities to form homodimers and mediated higher transactivation activities than TR
1. Thus, differential
transactivation activities of TR isoforms are mediated by interplay of
their domains and could serve as an important regulatory mechanism to
achieve diversity and specificity of pleiotropic T3
effect.
and TR
, located on chromosomes 17 and 3, respectively. Alternate splicings of their primary transcripts
produce isoforms of the protein (
1,
2,
1, and
2), which
regulate the transcription of their target genes by binding to specific
DNA sequences, known as thyroid hormone response elements (TREs). These
contain repeats of a half-site binding motif with the sequence AGGTCA.
Naturally occurring TREs can include these sequences as adjacent
palindromic repeats, as direct repeats separated by 4 nucleotides, and
as everted repeats separated by 6 nucleotides (F2) (1, 2). The
sequences of TRs have been divided into four separate domains, A/B, C,
D, and E. Domain C contains two zinc fingers and is involved in binding
of the receptors to TREs. Domains D and E are structurally linked, in
so far as part of domain D is required for the biological function of
domain E, which is to bind thyroid hormones (3). Domains D and E are
also involved in binding to co-repressors and dimerization,
respectively (4). The crystal structures of TRE-bound domains C of
TR
1 and the retinoid X receptor (5) and of domains D/E complexed
with a thyroid hormone agonist (6) have recently been solved. These
structures give important information on interaction within domains but
reveal nothing about the modes and roles of the interaction between
domains in intact receptors, which may have important biological
significance.
1 (w-TR
1) and
human TR
1 (w-TR
1) indicates that except domain A/B, there is
extensive sequence homology between the two isoforms, specifically 88%
in domain C, 71% in domain D, and 86% in domain E. Despite this high
sequence homology, biochemical evidence suggests that they could have
isoform-specific roles in mediating the action of thyroid hormones.
TR
and
genes are expressed at different stages during embryonic
development (7, 8) and during amphibian metamorphosis (9). Moreover,
these two isoforms are expressed differentially in different tissues
(8, 10). More direct evidence to support the isoform-specific
functional role of the TR
1 and TR
1 was provided by using gene
transfer experiments. Strait et al. (11) showed that the
gene encoding PCP-2 is regulated by TR
1 but not by TR
1. The
3,3
,5-triiodo-L-thyronine
(T3)-dependent negative regulation of thyroid
tropin releasing hormone promoter was shown to be mediated by TR
1
but not by TR
1 (12). Recently, using stably transfected neuronal
cell line, Lebel et al. (13) showed that only cells that
overexpress TR
1, but not TR
1, can respond to T3 to
exhibit morphological and functional characteristics indicative of
neural differentiation.
1 is known to bind to F2 and the TRE site on cardiac
-myosin
heavy chain mainly as a homodimer, whereas TR
1 forms homodimer
poorly (14, 15). These differences, however, are not eliminated by
removal of A/B domains from the molecules (16) and consequently must
arise from the remainder of the receptors. However, it is not clear
that they are a consequence only of differences in sequence. They may
also be caused by changes in interactions between domains in the intact
receptors. Because of their marked effects on the properties of the
receptors as transcription factors, we have investigated their origins
by construction of a series of six chimeric receptors, in which
domains A/B/C, D, and E from the two isoforms are joined in all
possible combinations. We have measured their affinities for
T3 under identical conditions and their binding to an F2
TRE. We also determined the T3-dependent transcriptional activity of the wild type and chimeric receptors. We
found that the domains C, D, and E are functionally linked, and the
differential transcriptional activity of the two isoforms is mediated
by interplay of their domains.
Chemicals and Materials
-32P]dCTP was obtained from
Amersham Life Sciences, Inc. [14C]Chloramphenicol and
[3
-125I]T3
([125I]T3) were purchased from DuPont NEN.
TNT-coupled reticulocyte lysate in vitro translation kits
were from Promega (Madison, WI).
1 and TR
1
1 (pLC13) (17) and w-TR
1 (pCJ3) (18). For cloning purpose, two
restriction enzyme sites, NsiI and BamHI, were
introduced into the boundaries between domains C and D and domains D
and E of w-TR
1 (nucleotide positions 412-414 (AAG/Lys to AAA/Lys) and 616-621 (GGCAGC/Gly-Ser to GGATCC/Gly-Ser)) to yield a new T7
expression plasmid of w-TR
1, pCH
. Only one restriction site, BamHI, was required to introduce into the boundary between
domains D and E of w-TR
1 (nucleotide positions 991-996
(GGCAGC/Gly-Ser to GGATCC/Gly-Ser)) because in pCJ3 (w-TR
1) the
NsiI already existed which yielded a new T7 expression
plasmid of w-TR
1, pCH
. The introduction of these restriction
sites was carried out by in vitro mutagenesis kit according
to the manufacturer's instructions (Bio-Rad). The introduction of
these two new restriction sites into TR
1 and the BamHI
site into TR
1 did not change the amino acid sequences of TR isoform
proteins. The six chimeric receptors were constructed by exchanging the
domains between w-TR
1 and w-TR
1 using NsiI,
BamHI, and the 3
EcoRI site immediately
downstream of the termination codons of w-TR
1 (nucleotide position
1306 for TR
1 and 1672 for TR
1 (pCJ3)) to yield T7 expression
plasmids pCH1, pCH2, pCH3, pCH4, pCH5, and pCH6 for
,
,
,
,
, and
, respectively
(see Fig. 2). The coding sequences for the six chimeric receptors were
verified by restriction map analyses and direct DNA sequencing.
Fig. 2.
Schematic representation of w-TR1,
w-TR
1, and their chimeric receptors. I, the domain
structure of the two isoforms and the extent of sequence homology.
II, the chimeric receptors are designated by
three-letter symbols, the first letter represents A/B and C domains; the second
letter represents D domain, and the third
letter represents E domain. The amino acid positions at
the boundaries of domains C, D, and E
are shown.
[View Larger Version of this Image (47K GIF file)]
1 and TR
1 chimeric
receptors were derived from the corresponding w-TR
1 and w-TR
1 expression plasmids, pCLC61 and pCLC51 (19), respectively. The expression of w-TR
1 and w-TR
1 is driven by cytomegalovirus
promoter. To prepare the mammalian expression plasmids of chimeric
receptor of TR
1, pCLC51 was restricted by NotI followed
by filling in with Klenow in the presence of deoxynucleotides. The
coding sequence of the w-TR
1 in pCLC51 was then released by treating
the linearized and blunt-ended pCLC51 with HindIII, thereby
providing the vector for ligation to the proper chimeric TR coding
fragments. The wild type and chimeric TR
1 coding fragments were
derived from the above T7 expression plasmids (pCH
, pCH1, pCH2, and
pCH3) by treating the plasmids with EcoRI. After filling in,
the fragments were released by treating with NdeI. An
adaptor (HindIII/NdeI) was used in the final
ligation of TR coding fragments to the vectors to yield plasmids
pCDMCH
, pCDMCH1, pCDMCH2, and pCDMCH3 for W-TR
1,
,
and
, respectively. The mammalian expression
plasmids of w-TR
1 and its chimeric receptors were prepared similarly
except that the vector was derived from pCLC61. The resulting mammalian plasmids were pCDMCH
, pCDMCH4, pCDMCH5, and pCDMCH6 for w-TR
1,
,
, and
, respectively.
were annealed and the recess 3
-end filled with DNA polymerase
(Klenow fragment) in the presence of [
-32P]dCTP. The
labeled oligonucleotides were separated on a 12% polyacrylamide gel
and purified by electroelution.
For EMSA, unlabeled TRs synthesized by in vitro
transcription/translation were used. The synthesized receptor proteins
were quantified by measuring the intensity of the
35S-labeled protein bands after SDS-polyacrylamide gel
electrophoresis using PhosphorImager (Molecular Dynamics, CA). The
35S-labeled protein was synthesized concurrently by using
amino acid mixture minus methionine but with
[35S]methionine (4 µl; 1190 Ci/mmol). Based on the
quantitation of the labeled receptors, the amounts of the unlabeled
receptors were calculated. For the determination of the binding
constants of TRs to F2, equal amounts of the in vitro
translated unlabeled receptors were incubated with increasing
concentrations of the labeled probes (0.2-120 fmol) in the binding
buffer (25 mM Hepes, pH 7.5, 5 mM
MgCl2, 4 mM EDTA, 10 mM
dithiothreitol, 0.11 M NaCl, and 0.4 µg of
single-stranded DNA). In some experiments, RXR prepared as described
by Meier et al. (20) was added. After incubation for 30 min
at 25 °C, the reaction mixture was loaded onto a 5% polyacrylamide
gel and electrophoresed at 4 °C for 2-3 h at a constant voltage of
250 V. The gel was dried and autoradiographed. The intensities of
retarded bands and free probes were quantified by PhosphorImager. The
binding data were analyzed based on the equations and considerations as
described below.
Binding of glucocorticoid nuclear receptors to response elements with adjacent identical half-sites has been successfully analyzed using a simple two-site cooperative model, which ignored dimerization of free receptors in solution (21). Since dimerization of unbound TR's has never been detected, we interpret our results in a similar way (Equation 1). Receptor (R) can bind to either TRE half-site (D) to give monomeric complexes (DR and RD) or to both yielding a dimeric complex (RDR).
![]() |
(Eq. 1) |
We assume that a receptor molecule can bind to either half-site on an empty TRE (D) with a binding constant K1 and on a monomeric complex (DR or RD) with a binding constant K2. If K2 = s·K2, then s is the cooperativity parameter. Positive cooperativity implies s > 1, i.e. stronger binding of the second TR than the first to the TRE. The concentration of monomeric complexes
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
![]() |
(Eq. 9) |
![]() |
(Eq. 10) |
For each combination of recombinant TR and TRE, values of K1 and K2 were estimated by fitting the measured concentrations [monomer] and [dimer] simultaneously to Equations 9 and 10 as functions of [D]0 and r, with the constraints K1 > 0, K2 > 0. Analyses were performed using the PC-MLAB program (Civilized Software, Bethesda, MD). It must be pointed out that this procedure violates one basic assumption of least squares curve fitting, i.e. that experimental uncertainties in plotted data parallel the y axis (22). Here we have uncertainties along both axes. Together with the problems of the gel retardation method, which requires separation of reactants and products, perturbing the system from equilibrium, as discussed previously (16), could result in some uncertainty in K1 and K2. Consequently, the values given in Table I may be only approximate.
|
The binding was carried out by incubating the in vitro translated TR proteins with 0.4 nM [125I]T3 in the presence or absence of increasing concentrations of unlabeled T3 (0.1 to 10 nM) in 0.25 ml of buffer B (50 mM Tris·HCl, pH 8.0, 0.2 M NaCl, 0.01% Lubrol, and 20% glycerol) for 90 min at 25 °C. TR-bound [125I]T3 was separated from the unbound radioligand in a Sephadex G-25 (fine) column (5.5 × 1 cm), as described (3).
The binding data were analyzed by using Equation 11 based on direct competition between [125I]T3 and the unlabeled T3 for a single site on the receptor. The concentration of radioactive complex is given by Equation 11:
![]() |
(Eq. 11) |
CV1 cells (4 × 105 cells/60-mm dish) were plated 24 h before
transfection in Dulbecco's modified essential medium containing 10%
fetal bovine serum. Cells were transfected with appropriate expression
plasmids (0.2 µg) for w-TR1 (pCDMCH
), w-TR
1 (pCDMCH
), or
the chimeric receptors (pCDMCH1, pCDMCH2, pCDMCH3, pCDMCH4, pCDMCH5,
and pCDMCH6), TRE-containing TK-CAT reporter plasmid (0.2 µg), and
pCH110 (0.2 µg; an expression plasmid for
-galactosidase) by using
the lipofectamine transfection method according to the manufacturer's
procedure (Life Technologies, Inc.). pBluescript SK II (+) Strategene,
La Jolla, CA) was used to bring the total DNA transfected to 3 µg.
After 6 h, the medium was replaced by fresh Dulbecco's modified
essential medium containing 10% thyroid hormone-depleted serum.
Fifteen hours before cells were harvested, T3 (100 nM) was added to the appropriate dishes. After an
additional 18 h, cells were lysed and chloramphenicol
acetyltransferase (CAT) activity was determined as described previously
(23, 24). CAT activity was normalized by using equal amounts of lysate
proteins.
Cultured CV-1 cells were transfected as described above. Two days later, cells were processed for immunofluorescence studies as described previously (25). Briefly, cells were fixed in 3.7% formaldehyde in phosphate-buffered saline for 5 min at 25 °C. After washing, cells were incubated with monoclonal antibody C4 (10 µg/ml; 26) in phosphate-buffered saline containing 0.1% saponin and 4 mg/ml normal goat globulin for 30 min at 25 °C. After being washed with phosphate-buffered saline, cells were incubated with affinity-purified goat anti-mouse immunoglobulin conjugated with rhodamine (25 µg/ml) for 30 min at 25 °C. Cells were viewed and photographed using microscope equipped with rhodamine epifluorescence optics.
Western BlottingCell lysates (25 µg) from transient transfection experiments as described above were loaded onto a 10% SDS gel. After electrophoresis, proteins were transferred onto a nitrocellulose membrane (PH79 membrane; Schleicher & Schuell). The membrane was gently shaken in 5% non-fat milk in TBS (25 mM Tris, pH 7.4, 150 mM NaCl) for 20 h and was subsequently washed three times with TBS. The membrane was incubated with monoclonal antibody C4 (1 µg/ml) for 1 h. After washing, the membrane was incubated with affinity-purified rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase (1:1,000 dilution). TR protein bands were visualized by chemiluminescence using ECL kit (Amersham Life Sciences, Inc.).
Previously it has been shown that TR1 binds to F2 mainly as
a homodimer, whereas TR
1 binds to F2 both as a homodimer and as a
monomer (14, 15). However, there was no quantitative comparison in the
differential binding of F2 to the two isoforms. We therefore compared
the binding affinities of F2 to the two isoforms. Fig. 1
shows the binding of TR
1 and TR
1 to F2 in a concentration-dependent manner. Consistent with previous
observations (14, 16), TR
1 bound to F2 predominantly as a homodimer.
Interestingly, when F2 concentration was higher than 15 fmol, weak
binding of TR
1 to F2 as a monomer was clearly detected (lanes
13-16). However, TR
1 bound to F2 differently from TR
1. As
shown in Fig. 1, at all corresponding F2 concentrations, TR
1 bound
both as a homodimer and as a monomer. It clearly had a higher
propensity to form monomer than TR
1 (lanes 1 versus 9;
lane 2 versus 10; lane 3 versus 11; lane 4 versus 12; lane 5 versus 13; lane 6 versus
14; lane 7 versus 15; and lane 8 versus
16).
The binding data shown in Fig. 1 were analyzed, and the
Ka values of homodimeric (K2)
and monomeric (K1) binding for TR1 were found
to be 400 and 0.1 × 106 M
1,
respectively, indicating an increase of 4000-fold in the binding affinity when TR
1 was bound to F2 as a homodimer (s = 4000;
Table I). Thus, binding of the first monomer of TR
1
to F2 facilitated the binding of the second monomer. We designated
"s" as the ratio of
K2/K1 to measure the
extent of positive cooperativity in the binding of TR to TREs. The
Ka values of homodimeric and monomeric binding of
TR
1 to F2 were 300 and 3 × 106
M
1, respectively, which gave a substantially
lower cooperativity (s = 100; see Table I) than that for
TR
1.
To identify the molecular basis of the differential
interaction of TR1 and TR
1 with F2, we interchanged the domains
between the two isoforms and evaluated the effects of domain swapping on the F2 and T3 binding activity. An examination of the
sequences between the two isoforms indicates that there is no
sequence homology in the A/B domain, whereas there is an 88, 71, and
86% homology in sequence in domains C, D and E, respectively (Fig.
2I). We have previously shown that the
removal of domain A/B has no effect on the interaction of TR
1 with
TREs (16). Therefore, we grouped domain A/B together with domain C as a
unit and constructed the chimeric receptors by swapping domains A/B/C,
D, and E (Fig. 2II). The sequences encoding the chimeric
receptors in the constructs were confirmed by restriction map analyses
and DNA sequencing.
To assess the T3 binding activity, we prepared the
receptors by in vitro transcription/translation and carried
out competitive T3 binding assays. The displacement curves
for w-TR1 and its chimeric receptors are shown in Fig.
3A and for w-TR
1 and its chimeric
receptors are shown in Fig. 3B. Binding data were analyzed, and the Kd values are shown in Table
II. The Kd values for the binding of
w-TR
1 and w-TR
1 to T3 were 0.36 ± 0.06 and
0.10 ± 0.037 nM, respectively, indicating that
w-TR
1 bound to T3 with an approximately 3-fold higher
affinity than that of w-TR
1. The 3-fold difference is very
significant as indicated by the t test (p < 0.01). The difference in the binding affinity was not due to the
different protein expression level by the in vitro
transcription/translation. As shown in Fig. 3C, lane 2 shows the two translation products of w-TR
1 initiated from the ATGs (Met-5
and -32) with the molecular weights of ~55,000 and ~52,000 (26, 27)
that have the combined intensity similar to that of w-TR
1 shown in
lane 6 (Fig. 3C). Similar binding experiments were carried out for the chimeric receptors, and as shown in Fig. 3, A and B, no significant differences were
observed in the binding curves within the same subtype. The
Kd values for the chimeric receptors are virtually
identical to those of the wild type receptors (Table II), indicating
that the domain swapping between the two isoforms had no effect on the
T3 binding activity.
|
In contrast to the T3 binding activity, domain swapping had
a dramatic effect on the interaction of chimeric receptors with F2.
Lanes 2-5 of Fig. 4 compare the binding of
w-TR1 (
; see Fig. 2) and its chimeric receptors to F2 by
EMSA. Replacement of domains D or E of TR
1 by that of TR
1 had no
significant effect on the binding of
or
receptor to
F2 as a homodimer, but an increase in the formation of monomer was seen
(lanes 3 versus 2; lanes 4 versus 2). However,
when both domains D and E were swapped, a dramatic increase in the
monomer formation was detected. The extent of monomer formation was
similar to that seen for w-TR
1 (
, lane 6 of Fig.
4A versus lane 5). We further measured an F2
concentration-dependent binding to each chimeric receptor
(
,
, and
), similar to the experiments shown
in Fig. 1, and determined their affinity constants. The binding data
were analyzed, and the Ka values are shown in Table
I. Swapping of domain D or E of TR
1 by that of TR
1 led to a 3- and 4-fold increase in the binding affinity of
or
to F2 as a monomer, respectively (K1 = 0.3 and
0.4 × 106 M
1, respectively,
versus 0.1 × 106
M
1 for w-TR
1), but with little change in
the binding affinity of these two chimeric receptors as a dimer
(K2 = 500 × 106
M
1). On the other hand, when both domains D
and E were swapped, a dramatic 20-fold increase in monomer binding
affinity (K1 = 2 × 106
M
1) was detected. Thus, inclusion of domain D
or E of TR
1 facilitates the binding of TR as a monomer.
Lanes 7-9 of Fig. 4 show that replacement of either domain
D or E alone or both domains D and E of TR1 by the corresponding regions of TR
1 resulted in a similar reduction in the monomer formation (lane 6 versus lanes 7-9). The ratios of monomer
to homodimer were clearly reduced in
,
, and
. A more detailed analysis was carried out by determining the
affinities in the binding of F2 to the chimeric TR
s. Their
K1, K2, and s values are
shown in Table I which indicate that there were only small changes in
the values of positive cooperativity in
,
, and
as compared with
(s = 50-100).
RXRs have been shown to heterodimerize with TRs and modulate the
activity of TRs (1, 2). We therefore also examined the effect of domain
swapping on the heterodimerization activity of the chimeric receptors.
Similar to w-TR1 and w-TR
1, all chimeric TRs were capable of
forming dimers with RXR
on F2. No significant differences in the
extent of formation of heterodimers were detected among the chimeric
TRs (data not shown).
To assess the role of the domains in
the transactivation activity of TRs, we constructed the mammalian
expression vectors in which the expression of the wild type and
chimeric TRs was driven by the cytomegalovirus promoter. We
co-transfected the TR expression plasmids with F2-containing reporter
into CV1 cells. Fig. 5 shows that w-TR1 (
)
had a ~6-fold higher T3-dependent transactivation activity than w-TR
1 (
; bars 2 versus
6 of Fig. 5). The lower transactivation of w-TR
1 was not due to
the lower expression of w-TR
1 proteins in CV1 cells. Using high
titer monoclonal antibody C4 (26), we had concurrently carried out immunocytochemical localization of TRs in CV1 cells and Western blotting for quantitation of the expressed TRs. w-TRs and their chimeric TRs were similarly expressed in the nuclei (data not shown).
Thus, the lower transactivation activity was not due to the inability
of w-TR
1 to be translocated into the nuclei. Furthermore, the
Western blots show that w-TR
1 (
), surprisingly, was
expressed ~2-fold higher than w-TR
1 (lanes 6 versus lane
2 of Fig. 5), indicating that the lower transactivation activity
was not due to the lower protein expression level of w-TR
1.
To identify which domain in the TRs mediated the differential
transactivation activity between these two isoforms, we further examined the transactivation activity of the chimeric TRs. On F2,
swapping of domain D of w-TR1 by that of w-TR
1 (bars 3 versus 2 of Fig. 5) reduced the transactivation activity of
by 35%. As shown bars 4 and 5, the
T3-dependent transactivation was reduced by
~60% when domain E alone or domains D + E of w-TR
1 was replaced by that of w-TR
1. Swapping of domains E and D + E in w-TR
1 by the corresponding regions of TR
1 led to a 1.8- (bar 8)
and 2.5-fold (bar 9 of Fig. 5) increase in the
transactivation in
and
, respectively. The
differences in the transactivation activity of the chimeric TRs were
neither due to the differences in the ability of the chimeric TRs to be
translocated into the nuclei because similar nuclear localization
patterns were seen (data not shown) nor due to the TR expression levels
because the transactivation activities shown in Fig. 5 were normalized
against the amounts of proteins detected in Fig. 6.
Taken together, these results indicate that domains D and E of TR
1
had a propensity to mediate a higher transactivation activity, and
those of TR
1 mediated a lower transactivation activity.
To evaluate whether the differential transactivation activity of the
two TR isoforms is mediated by TR/RXR heterodimer pathway, we
co-transfected RXR expression plasmid with F2-CAT reporter and
w-TR
1 or w-TR
1 expression plasmids into CV1 cells. Consistent with the previous findings (24), the
T3-dependent transactivation activity of
w-TR
1 mediated by F2 was repressed ~60% by RXR
. A similar
extent of repression was also seen for the
T3-dependent transactivation activity of
w-TR
1 by RXR
(data not shown). Therefore, the higher
transactivation activity of w-TR
1 was not due to the TR/RXR
heterodimer pathway.
In an important study, Rastinejad et al. (5) recently
determined the crystal structure of a heterodimeric complex of two proteins, made up from domains (C + D) derived from the RXR and TR1, respectively, bound to a direct repeat TRE. The DNA in the complex is undistorted, with regular B-DNA geometry. Many amino acid
side chains are involved in the TR
1-DNA interactions. The specificity of binding is determined by side chains from the 11-residue long C domain "recognition helix," which starts at the third metal coordinating cysteine of the first zinc finger. These make direct contacts with the base pairs and backbone phosphates in the major groove of the half-site binding motif. Domain D contains a long
-helix, which makes extensive interactions with the minor groove between the half-site and upstream spacer sequence. The dimerization interface between RXR and TR
1 lies across the minor groove of the
spacer, involving mainly residues from the first zinc finger in TR
1
and the second zinc finger in RXR. Of the many side chains identified
as making DNA contacts, only one, K193R, is changed in TR
1.
Mutagenesis experiments indicate that at least one other conserved
region of TRs, Leu-367-Leu-374, located in domain E, is involved in
dimerization of intact receptors. The structure of the separate ligand
binding domains D + E of rat TR
1 has recently been determined (6).
The isolated protein is monomeric and gives no indication as to how
this sequence, which forms "an extensive hydrophobic patch,"
participates in dimerization. The analogous sequence in human RXR-
does form a dimer interface in crystals of its isolated ligand binding
domain (29). It has been suggested that this dimerization sequence from
domain E has no selective pressure on response element recognition but
only serves to stabilize these homodimer complexes (30), being active
in all dimerization interfaces. Biochemical data, on the polarity of
binding and the specificity for particular spacings in DNA
response elements shown by heterodimers formed by various
members of the steroid/thyroid hormone receptor family, were readily
explained using the crystal structure solved by Rastinejad
et al. (5). This indicates the generality of the binding
mode which they detected and predicts that homodimers formed by TRs on
the three different types of TREs will have distinct dimerization
interfaces. Homodimers formed on F2 elements will be symmetrical, with
a dimerization interface including the first zinc fingers of domain C
and the D domain
-helices of both proteins. The situation is further
complicated by the spatial arrangements of the binding sites. For DR4,
the centers of the two binding motifs are on the same face of the DNA,
one turn of the DNA helix apart (5). We can predict that for F2 they
will fall a little further apart, on opposite faces of the DNA. Under
most conditions, binding of TR
1 to an F2 response element occurs as
a dimer complex. Since the two half-sites involved are basically
identical, the observed low levels of a 1:1 DNA/protein monomer complex
indicates high positive cooperativity between the two half-sites
(i.e. K2
K1). There
are two extreme ways in which positive cooperativity between two
intrinsically identical half-sites can be achieved (31). Relative to
the empty TRE we can detect (i) stabilization of the dimer complex by a
large positive free energy of interaction between both sites occupied
by proteins, with no interaction between occupied and unoccupied sites,
or (ii) destabilization of the monomer complex by a large negative free
energy of interaction between occupied and unoccupied sites, with no
interaction between two occupied sites. In general, less extreme
situations, where both occupied-occupied and occupied-unoccupied interactions occur, must also be considered. The binding of TR
1, TR
1, and their chimeric receptors to F2 shows positive cooperativity (K2
K1). Table I
clearly shows that the enhanced positive cooperativity of binding shown
by TR
1 over TR
1 (i.e. its greater tendency to bind as
a dimer) results mainly from the second of these causes. TR
1 monomer
complex is much less stable than the TR
1 form, and this lower
stability is relieved by formation of the dimer complex. As noted
above, the sequences involved in DNA binding are essentially identical
in TR
1 and TR
1. Consequently, the instability of the TR
1
monomer complex must result from the overall structure of the receptor
molecule and its effect on the binding interfaces. The data obtained
with the chimeric receptors show that all proteins in which the DNA
binding domain C is of the
form show monomeric binding to F2, like
TR
1 (Table I, Fig. 4). However, exchange of only this domain to the
form (
) is not sufficient to significantly
enhance binding cooperativity. In the case of F2, some destabilization
of the monomer complex is detected when both domains C-D or C-E are
exchanged, with a full effect with all three domains derived from
.
In monomer complexes, these all fall on the occupied-unoccupied site
interface, where the destabilization must occur. The physical origin of
this destabilization interaction may only be revealed by determination
of the structure of suitable complexes.
The hormone binding site of TRs is located in domain E. In earlier
work, we showed that domain E, isolated from h-TR1 with part of
domain D, can still bind T3 but with reduced affinity (3).
Addition of domains C and D restored the molecule's affinity to that
of the intact receptor, indicating regulatory interactions between
these domains. The results shown in Fig. 3 indicate that the chimeric
receptors derived from TR
1's domain C all have higher T3 binding affinity than the chimeric receptors derived
from domain C of TR
1, at least in the absence of DNA (see Table II).
Thus this aspect of the behavior of a chimeric receptor is determined exclusively by the origin of its domain C, the DNA binding domain, reinforcing the importance of interdomain interactions.
The results on the effect of domain swapping on the transcriptional
activity of the TR isoforms revealed that despite a higher T3 binding affinity, TR1 and its chimeric receptors had
lower transcriptional activity (Fig. 5). This was unexpected,
suggesting that the mode of DNA binding to TRs overrides the advantage
gained from higher T3 affinity. Inspection of the data
further indicates that the extent of transcriptional activation by a
receptor correlates better with the source of its domains than with its
affinity for DNA. It is clear that domains D and E of TR
1 tend to
impart higher transcriptional activity than those of TR
1. This in
turn correlates well with the propensity of the chimeric receptors to
form homodimers (see Fig. 4). The fact that w-TR
1 and its chimeric
receptors formed heterodimers with the RXR as well as w-TR
1 and its
chimeric receptors suggests that the higher transcriptional activity of TRs which contain the domains D and E of TR
1 probably was not mediated by the heterodimer pathway. This notion is further supported by the findings that the transfected RXR repressed the
T3-dependent transactivation activity of the
two isoforms with similar extent. Therefore, this higher
transcriptional activity of TR
1 lies most likely in the interactions
of domains D and E with domain C in homodimers. Genetic experiments
have shown that the hormone-dependent transactivation
activity depends on a short amphipathic
-helix at the extreme
carboxyl terminus of domain E, which undergoes a large conformational
change on hormone binding (6, 26, 30). The sequence is conserved in
both isoforms but is probably located in different sequence contexts in
relation to the DNA binding domain or in the context of the entire
molecule. Thus, it may function with differing efficacies in the two
different environments. Recently, several co-repressors and one
co-activator for several members of the receptor superfamily including
TRs have been reported (4, 32-35). Their function has been proposed to
act as bridging factors between the TRs and the basal transcriptional machinery (4). It is possible that the resultant tertiary structure of
domains D and E of TR
1 is less favorable to bind to a co-repressor. It is also possible that the structure of domains D and E of TR
1 is
such that the carboxyl-terminal
-helix is more easily accessible to
a co-activator. Thus, domains D and E of TR
1 may have a higher efficacy in transmitting the effects of conformational change of the
carboxyl-terminal
-helix upon binding the hormone to regulate the
interaction of the domain C with the target genes. These possibilities can only be distinguished when the x-ray crystallographic structures of
ligand-bound intact TR
1 and TR
1 are solved and compared. Our
present studies indicate that domains C, D, and E are functionally linked and the interplay of these domains underlines the differential transcriptional activity of the two isoforms.