Protein-Protein Interaction Domains and the Heterodimerization of Thyroid Hormone Receptor Variant
2 with Retinoid X Receptors
Yifei Wu,
Ying-Zi Yang and
Ronald J. Koenig
Division of Endocrinology and Metabolism University of Michigan
Medical Center Ann Arbor, Michigan 48109-0678
 |
ABSTRACT
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Heterodimerization between thyroid hormone
receptors (TRs) and retinoid X receptors (RXRs) is mediated by a weak
dimerization interface within the DNA- binding domains (DBDs) and a
strong interface within the C-terminal ligand- binding domains of the
receptors. Previous studies have shown that the conserved ninth heptad
in the TR ligand-binding domain appears to play a critical role in
heterodimerization with RXR. However, despite lacking the full ninth
heptad, TR variant
2 (TRv
2) can heterodimerize with RXR on
specific direct repeat response elements, but not on palindromic
elements or in solution. Two possibilities may account for TRv
2-RXR
heterodimerization on direct repeats. First, the DBD of TRv
2 may
play a critical role in heterodimerization with RXR. Second, a specific
sequence within the unique C terminus of TRv
2 may promote the
formation of TRv
2-RXR heterodimers. In this study, we used receptor
chimeras in which the DBD of RXR was replaced by either the TR DBD or
an unrelated DBD from the metalloregulatory transcription factor AMT1
to address the role of the DBD dimerization interface in TRv
2-RXR
heterodimerization. Gel mobility shift analyses showed that whereas
TR
1 formed heterodimers with these chimeras, TRv
2 failed to do
so. Deletion of the unique C terminus of TRv
2 had only a
marginal effect on heterodimerization with RXR. Mutations within the
DBD dimerization interface abolished heterodimerization of full-length
TRv
2 with RXR but only marginally affected heterodimerization of
full-length TR
1 with RXR. These data support the hypothesis that the
TR-RXR DBD dimerization interface plays a critical role in TRv
2-RXR
heterodimerization. Additional data show that the amino acid residues
that make direct TR-RXR contacts within the DBDs also may play a role
in receptor monomer binding to DNA, since mutations within these
residues severely impair this interaction.
 |
INTRODUCTION
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The thyroid hormone receptors (TRs), as well as the receptors for
retinoic acid and vitamin D, belong to the nuclear receptor superfamily
of ligand-dependent transcription factors that control diverse aspects
of development and cellular homeostasis (1). Transcriptional regulation
by TRs occurs via binding to thyroid hormone response elements (TREs)
in the promoter regions of target genes. TREs are usually composed of
two or more half-sites related to the sequence AGGTCA, arranged as
direct repeats with a 4-bp spacer (DR4) (2, 3), inverted repeats with
no spacer (4, 5), or everted repeats with a 6-bp spacer (4, 6).
Although TRs are capable of binding to TREs as monomers or homodimers,
TRs preferentially bind to most TREs as heterodimers with retinoid X
receptors (RXRs) (7, 8, 9).
Extensive structural and functional analyses have revealed that the
nuclear receptors contain functionally separable domains for DNA
binding and hormone binding. The DNA-binding domain (DBD) contains two
zinc fingers and is highly conserved among members of the superfamily
(1). The C-terminal region of the receptors is functionally complex,
performing a variety of activities including ligand binding, receptor
dimerization, and hormone-dependent transcriptional activation or
repression (1). It is known that heterodimerization between TR and RXR
is mediated by distinct dimerization interfaces within the DBDs and the
C-terminal ligand-binding domains (LBDs) of the receptors (10, 11). The
dimerization interface between the DBDs was elucidated through
crystallographic analysis (10). In general, the TR-RXR interaction
mediated through DBDs is believed to be relatively weak, since DBDs
alone cannot form heterodimers in solution. However, the DBD interface
specifies the requirement for the spacer of direct repeat response
elements (12, 13). In contrast to this weak DBD interface, the
dimerization domains within the C-terminal LBDs of TR and RXR have been
shown to be the major heterodimerization interface, since these domains
allow for the formation of dimers in solution before DNA targeting.
Although the dimerization interface appears to involve multiple regions
of the C-terminal LBD (11, 14, 15), a major role of the conserved ninth
heptad repeat [TR
1 amino acids 367374, part of helix 11 in the
crystal structure of the TR LBD (16)] in heterodimerization with RXR
has been documented by mutational analysis (11).
TR variant
2 (TRv
2) is an alternative splice product of the
TR
gene and is widely expressed (17). TRv
2 lacks a
full ninth heptad and possesses a unique 122-amino acid C-terminal
sequence in place of TR
1 amino acids 371410 (Fig. 1
). As TRv
2 is missing the C-terminal
40 amino acids of the TR
1 LBD, it is not surprising that TRv
2
does not bind T3 and is not a functional thyroid hormone
receptor. Although the physiological role of TRv
2 remains unknown,
its weak dominant negative activity in transfection systems has been
well demonstrated (18). Previous studies have shown that TRv
2 is
capable of forming heterodimers with RXR on specific DR response
elements in which a thymidine is present two nucleotides 5' to the
downstream hexameric half-site (TNAGGTCA) (19, 20, 21). Although many
natural TREs do not have a thymidine in this position, others do (such
as TREs in the spot 14 and human 5'-deiodinase type I genes). In
contrast, TRv
2-RXR heterodimers are not detected on inverted repeat
or everted repeat elements and are only marginally detectable in
solution. It is unclear why TRv
2 retains the ability to
heterodimerize with RXR on DRs. Two possibilities may account for this.
First, the DBD of TRv
2 may play a critical role in
heterodimerization with RXR. Second, a specific sequence within the
unique C terminus of TRv
2 may promote the formation of TRv
2-RXR
heterodimers.
In this report, we present evidence in support of the former
hypothesis. Moreover, we show that the amino acid residues within the
DBD that make direct TR-RXR contacts also may play an important role in
receptor monomer binding to DNA.
 |
RESULTS
|
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Mutations within the Dimer Interface of the TR or RXR DBD Severely
Impair the DNA Binding of TR or RXR Monomers
Previous studies have revealed that TRv
2 can form heterodimers
with RXR on a subset of DRs that contain a downstream octameric
TR-binding half-site (TNAGGTCA) (19, 20, 21). It also has been shown that
RXR in RXR-TR heterodimers occupies the 5'-half-site of a DR4 response
element (10, 12, 13). Based on the three-dimensional crystal structure
of the RXR-TR DBDs bound to a DR4, the amino acid residues that make
the dimer interface within the DBDs of TR and RXR have been defined
(10). Three arginine residues from the second zinc finger of RXR
interact with three amino acids from around the TR first zinc finger as
well as one amino acid in a more C-terminal region of the DBD known as
the T box. These residues were mutated to alanines singly or in
clusters (Fig. 2
) to assess whether the
DBD dimerization interface is required for the formation of TRv
2-RXR
heterodimers on the octamer-containing response element 8DR4 (Table 1
).
The wild-type and mutated DBDs were expressed either in rabbit
reticulocyte lysate or in Escherichia coli. In
vitro synthesized DBD proteins and a radiolabeled DNA probe (8DR4)
were used to perform electrophoretic mobility shift assays (EMSAs).
EMSAs revealed that the wild-type TR
DBD and RXR
DBD could bind
to 8DR4 as monomers (Fig. 3
, lanes 2 and
3) or as heterodimers (lane 7). Surprisingly, using reticulocyte
lysate-translated receptors, all mutant RXR
DBD proteins showed
virtually no binding to 8DR4 as monomers (Fig. 3
, lanes 46) or as
heterodimers with the wild-type TR
DBD (lanes 810). Similar
results were observed with bacterially expressed proteins (data not
shown). Furthermore, EMSAs revealed that the mutant TR
DBDs also
have greatly diminished DNA binding as monomers (Fig. 4
, lanes 24) or as heterodimers with
the wild-type RXR
DBD (lanes 79). The observed loss of monomer
binding to DNA complicates the use of full-length receptors bearing
these DBD mutations to study the role of the DBD interaction in
TRv
2-RXR heterodimerization, because loss of heterodimer-DNA
complexes could simply reflect defective TR
or RXR
monomer-DNA
binding. Furthermore, the loss of receptor monomer-DNA binding makes it
difficult to prove that these mutations do indeed disrupt DBD
heterodimerization on DNA.
At least two possibilities may account for the loss of the receptor
monomer binding to DNA. First, the mutations within the DBD may disrupt
the general conformation of the proteins. Second, the amino acid
residues that are proposed to make direct TR-RXR interactions may also
contribute to receptor monomer-DNA interactions. To begin to
distinguish between these two possibilities, we carried out a
proteolytic analysis by treating the wild-type and mutant DBDs of TR or
RXR with various proteases. We hypothesized that if the mutations
caused gross conformational changes in the DBDs, this would alter their
susceptibility to protease digestion. We were particularly interested
in three single DBD mutants (TR-D123A, RXR-R187A, and RXR-R177A), since
these proteins have mutations in residues that, by crystallography, are
not involved in DNA contacts, at least of the TR-RXR DBD
heterodimer.
The wild-type and mutant TR
and RXR
DBDs were translated in
reticulocyte lysate in the presence of both
[35S]methionine and [35S]cysteine. The
radioactively labeled DBDs were digested with increasing amounts of
trypsin, chymotrypsin, or elastase. The digestion products were
analyzed by Tricine-SDS-PAGE (22) and visualized by phosphorimager
analysis. Figure 5A
shows that the
trypsin digestion patterns of the wild-type and mutant TR
DBDs are
similar. Digestion with chymotrypsin or elastase also failed to reveal
differences between the wild-type and mutant proteins (data not shown).
Similar digestion patterns between the wild-type and mutant RXR
DBDs
also were obtained after incubation with chymotrypsin (Fig. 5B
),
trypsin, or elastase (data not shown). Thus, this technique failed to
provide evidence for gross conformational changes in the mutant DBDs.
This suggests that the mutated residues of the DBD dimer interfaces may
play a more direct role in protein-DNA interactions, at least for
receptor monomers.
The DBD Interaction Is Necessary for TRv
2-RXR Heterodimerization
on a Direct Repeat Response Element
Since mutations within the DBD dimer interface severely impaired
TR
or RXR
DBD monomer binding to DNA, full-length receptors with
these mutations were not initially used to study the role of the DBD
interaction in TRv
2-RXR heterodimerization. Instead, we constructed
receptor chimeras in which the RXR
DBD was replaced by either the
TR
DBD or an unrelated DBD from the yeast metalloregulatory
transcription factor AMT1 (23), referred to as RTR and RMR,
respectively (Fig. 6
). The AMT1 DBD was
used because AMT1 binds DNA as a monomer (23). Also, AMT1 is not a zinc
finger protein, and therefore it seemed unlikely that the AMT1 DBD
would heterodimerize with the TR
DBD. Similarly, since TR
could
not form homodimers on 8DR4 under our experimental conditions, it also
appeared unlikely that the TR
DBD in the chimeric RTR would
contribute to RTR-TRv
2 heterodimerization. Using RTR or RMR to
evaluate the role of the DBD in TR
-RXR
heterodimerization would
ensure that any observed failure of heterodimerization was not due to
impaired DNA binding.
The full-length receptor proteins were produced in reticulocyte lysate
in the presence of [3H]leucine, and the products were
analyzed by SDS-PAGE. EMSAs using these in vitro synthesized
full-length proteins showed that, as expected, TRv
2 and TR
1 are
both capable of heterodimerizing with RXR
(Fig. 7
, lanes 6 and 9) or RXR
-Sph (lanes 8
and 11). However, TRv
2 does not heterodimerize with RTR (lane 7),
although TR
1 does (lane 10). Somewhat unexpectedly, an RXR homodimer
band also was visible on this response element (e.g. lane
1).
To verify these data, gel supershift experiments were performed using a
specific anti-TRv
2 antibody (a generous gift from M. Lazar). Figure 8
shows that the TRv
2-RXR
and
TRv
2-RXR
-Sph heterodimer complexes were supershifted in the
presence of the TRv
2-specific antibody (lanes 7 and 11), but that no
supershifted complexes were observed when TRv
2 plus RTR was
incubated with the antibody (lane 9). This confirms that TRv
2 and
RXR
heterodimerize, but that TRv
2 and RTR do not.
As shown in Fig. 9
, TRv
2 also was
unable to heterodimerize with RMR on a DNA probe containing an upstream
AMT1 site followed by an optimal TR- binding half-site spaced by 4 bp
(lane 5), although RMR-TR
1 complexes did form on this probe (lane
6). Identical results were observed when the half-sites were spaced by
2 or 6 bp (data not shown).
In addition, EMSAs were performed using deletion mutants of TR
1 and
TRv
2 to assess the role of the unique C terminus of TRv
2 (amino
acids 371492) in heterodimerization with RXR. Deletion of TR
1
after amino acid 378, which leaves the full ninth heptad intact, had
minimal impact on heterodimerization with RXR (Fig. 10
, lane 12 vs. 4).
Similarly, deletion of TRv
2 after amino acid 378 had no significant
impact on heterodimerization (lane 10 vs. 6). Deletion after
amino acid 370, which leaves intact only those residues common to
TR
1 and TRv
2, also only marginally affected heterodimerization
with RXR (lane 14).
Finally, EMSAs were performed using full-length TR
1, TRv
2, or
RXR
bearing the alanine substitutions within the DBD dimerization
interfaces (see Fig. 2
). In the context of TR
1, these DBD mutations
only minimally impaired TR-RXR-DNA complex formation (Fig. 11A
, lanes 911 vs.
8), but the same mutations in the context of TRv
2 almost totally
disrupted heterodimer-DNA binding (Fig. 11B
, lanes 79 vs.
6). Similarly, mutations of the RXR DBD dimerization interface had no
effect on TR
1-RXR-DNA complex formation (Fig. 11A
, lanes 1213
vs. 8), but abolished TRv
2-RXR-DNA complex
formation (Fig. 11C
, lanes 67 vs. 5).

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Figure 11. Effects of DBD Mutations on TR-RXR-DNA
Interactions
A, Effect of DBD dimerization interface mutations on
heterodimerization of full-length TR 1 with RXR. EMSA was performed
using full-length TR 1 and RXR (with or without DBD mutations) and
a 32P-labeled 8DR4 oligonucleotide probe. The TR 1 and
RXR mutations are fully described in Fig. 2 . TR 1 proteins: wt,
wild-type; 123, D123A; 2, D2A; 3, 3A. RXR proteins: wt, wild-type; 177,
R177A; 2, R2A. M, TR monomer-DNA complexes; HTD, heterodimer-DNA
complexes. B, Effect of TRv 2 DBD dimerization interface mutations on
heterodimerization of full-length TRv 2 with RXR. EMSA was
performed using full-length TRv 2 and RXR (with
or without DBD mutations) and a 32P-labeled 8DR4
oligonucleotide probe. The TRv 2 mutations are fully described in
Fig. 2 . TRv 2 proteins: wt, wild-type; 123, D123A; 2, D2A; 3, 3A.
HMD, homodimer-DNA complexes; HTD, heterodimer-DNA complexes. C, Effect
of RXR DBD dimerization interface mutations on heterodimerization of
full-length TRv 2 with RXR. EMSA was performed using full-length
TRv 2 and DBD-mutated RXR and a 32P-labeled 8DR4
oligonucleotide probe. The RXR mutations are fully described in Fig. 2 . RXR proteins: wt, wild-type; 177, R177A; 187, R187A; 2, R2A. HTD,
heterodimer-DNA complexes. Additional lanes not relevant to this
experiment were deleted from the autoradiograph to produce this
figure.
|
|
Taken together, these findings strongly indicate that the DBD
interactions between TRv
2 and RXR
are essential for the formation
of RXR-TRv
2 heterodimers on 8DR4. The C-terminal unique sequence of
TRv
2 appears not to contribute to the dimerization interface. In
contrast to TRv
2, since a strong LBD interface (full ninth heptad)
is present in the TR
1, the TR
-RXR
DBD interaction is not
necessary for formation of TR
1-RXR
heterodimers on these DNA
probes.
 |
DISCUSSION
|
---|
It is believed that heterodimerization with RXRs is an important
mechanism by which TRs exert their biological activities. Mutational
and crystallographic analyses have revealed that distinct dimerization
interfaces within the DBD and the LBD of the receptors contribute to
the formation of TR-RXR heterodimers (10, 11). Mutational analyses have
shown that the conserved ninth heptad in the TR LBD is necessary for
heterodimerization with RXR (11). In contrast, the DBD interaction is
thought to be relatively weak and generally insufficient to support
heterodimerization with RXR in the absence of DNA (21).
It has been shown that TR
1-RXR heterodimer complexes can be formed
in solution, whereas TRv
2 is virtually incapable of heterodimerizing
with RXR in solution before DNA targeting (20, 21). This difference
reflects the incomplete ninth heptad of TRv
2 (20). Our data, as
shown in Figs. 7
, 9
, and 11
, indicate that TR
1 requires only the LBD
dimerization interface to form heterodimers with RXR
on DNA; the DBD
dimerization interface is dispensable. The fact that, despite lacking
the full ninth heptad, TRv
2 can still form heterodimers with RXR on
certain DRs raises the question as to which domain(s) within TRv
2
contribute to this heterodimerization. Two possibilities may be
considered. First, a novel strong dimerization interface may be located
in the unique 122-amino acid C-terminal sequence of TRv
2. Second,
the DBD interface of TRv
2 may promote heterodimerization with RXR.
In this study, we used receptor chimeras to examine the role of the DBD
interaction in the binding of TRv
2-RXR heterodimers to 8DR4. The
observations that TRv
2 failed to heterodimerize with the chimeras in
which the RXR DBD was swapped with the TR DBD (RTR) or the AMT1 DBD
(RMR), whereas TR
1 did heterodimerize, indicate that: 1) the DBD
interaction between TRv
2 and RXR is necessary for this
heterodimerization; and 2) a strong dimerization interface within the
unique C-terminal region of TRv
2 (similar to that of TR
1) is not
likely. To confirm these findings, a series of C-terminal truncation
mutants of TR
1 and TRv
2 were studied. Previous studies showed
that C-terminal deletion of TRv
2 to amino acid 387, which still
contains the truncated ninth heptad, does not impair heterodimerization
with RXR (21). However, further deletion to amino acid 347 abolished
TRv
2-RXR heterodimerization. These data do not exclude the
possibility that the unique TRv
2 sequence between amino acids
371387 may have a critical role in the TRv
2-RXR
heterodimerization, although, clearly, residues C terminal to 387 are
not required. Our studies demonstrate that deletion of the entire
unique C terminus of TRv
2 to create TR
-370 still does not prevent
heterodimerization with RXR. This indicates that the region between
amino acids 371 and 387 is not essential for the formation of
TRv
2-RXR heterodimers and thus substantiates the critical role of
the DBD interface in this process. Perhaps C-terminal deletion to
residue 347 abolishes TRv
2-RXR heterodimerization because this
removes the proximal half of the ninth heptad (see Fig. 1
)
In this model, because a strong LBD interaction between TRv
2 and RXR
is lacking, the binding of TRv
2 or RXR to the target DNA appears to
become an obligatory first step to promote the heterodimerization. The
DNA binding, in turn, allows two DBD interfaces to interact. It is
interesting to note that the unique C-terminal region of TRv
2 is
phosphorylated by casein kinase II, both when translated in
reticulocyte lysate and when expressed within cells (24). This
phosphorylation reduces the ability of TRv
2 to bind DNA as a
monomer. The optimized octameric TR binding half-site may be necessary
to compensate for this deficiency. Despite the low binding affinity of
TRv
2 monomers to DNA, the cooperative binding of TRv
2 and RXR to
the target DNA greatly promotes the protein-protein interaction and
thereby enhances the DNA binding of TRv
2-RXR heterodimers.
Furthermore, the inability of TRv
2 to heterodimerize with RXR on
inverted repeats or everted repeats (19, 20) presumably reflects the
inability of the DBD dimer interfaces to interact on these palindromic
elements.
It has been demonstrated that the DBD interface plays an important role
in discriminating differences in spacing between DR half-sites (12, 13), since the TR DBD can cooperatively and selectively bind to direct
repeat elements spaced by 4 bp as a heterodimer with the RXR DBD. Other
DBDs impose different spacing requirements. For example, the vitamin D
receptor DBD specifies binding to a DR3 element, and the retinoic acid
receptor DBD specifies binding to a DR5 (2, 3). Interestingly, we
observed that TR
1 could heterodimerize with RMR on half-sites spaced
by 2, 4, or 6 bp. This finding is consistent with the notion that the
precise spacing requirements for DR half-sites is intrinsic to the DBD
dimer interface, because the formation of TR
1-RMR heterodimers was
solely promoted through the strong LBD dimerization interface, not the
DBD interface.
An unexpected observation emerged when point mutations within the DBD
dimer interface of TR
and RXR
were studied. Our data showed that
the ability of DBD monomers to bind DNA was severely impaired or
abolished by these substitutions. Based on the crystal structure of the
RXR-TR heterodimer DBD bound to a DR4, these amino acids do not make
direct contact with the DNA (10). Although some of the mutated residues
are involved in water-mediated phosphate contact, others only
contribute to the protein-protein interaction in the crystal structure.
However, even the mutations that occurred within these latter residues
still resulted in the loss of receptor DBD monomer binding to DNA. A
trivial explanation for this finding would be global conformational
disruption of the receptors by these alanine substitutions. However,
proteolytic analysis showed that the digestion patterns of the
wild-type DBDs by various proteases are similar to those obtained for
the mutant DBDs, suggesting that the mutations may not disrupt the
global structure of the receptors DBDs. Furthermore, an alignment of
amino acid sequences among nuclear receptor superfamily proteins
revealed that the alanine substitutions should be tolerable, since
alanine and other small hydrophobic amino acids are used by several
naturally occurring members of the superfamily. For example, the amino
acid residue in the human glucocorticoid or androgen receptor
corresponding to mouse RXR
amino acid R177 is alanine (25, 26).
Therefore, these residues may not only participate in the intersubunit
interactions, but also may play a role in stabilizing the DNA-protein
complexes, at least for monomer-DNA interactions. Understanding
receptor monomer-DNA interactions is relevant since several nuclear
receptors can bind to DNA and activate transcription as monomers,
including SF-1 (27), NGFI-B (27), and TR
1 (28, 29). However, we
cannot exclude the possibility of conformational disruption caused by
these alanine substitutions, as proteolytic analysis may not be
sensitive enough to detect this. Efforts to study this
possibility by nondenaturing PAGE were uninformative (data not
shown). Further investigation will be required to better resolve this
issue.
 |
MATERIALS AND METHODS
|
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Expression of Proteins
Wild-type and mutated DBDs of mouse TR
(amino acids 40159)
and mouse RXR
(amino acids 133228) were expressed in
Escherichia coli as glutathione S-transferase
(GST) fusion proteins using vector pGEX-KG (a gift from K. Guan) as
described previously (30). The fusion proteins were purified by
glutathione-Sepharose beads (Sigma Chemical Co., St. Louis, MO), and
the receptor DBDs were cleaved from GST with thrombin. To produce the
receptor DBDs in reticulocyte lysate, PCR-amplified DNAs encoding the
wild-type and the mutated DBDs of TR
or RXR
were cloned into the
BamHI-SalI sites of pCITE-4a(+) (Pharmacia,
Piscataway, NJ). [3H]Leucine-labeled DBD proteins were
generated using TNT T7/T3-coupled transcription translation (Promega,
Madison, WI). For the generation of the full-length or truncated
receptors, TR
1 (31), rat TRv
2 (17), RXR
(9), and chimeric
cDNAs (see below), as well as point mutation or deletion mutant cDNAs,
were transcribed from pBluescript plasmids and then translated in
rabbit reticulocyte lysate (Promega) in the presence of
[3H]leucine. Trichloroacetic acid-precipitable protein
counts per minute were determined, and the products were analyzed by
SDS-PAGE.
Oligonucleotide-Directed Mutagenesis and Constructions of the
Chimeric Receptors and Deletion Mutants
The mutants with the alanine substitutions within the dimer
interface of the TR
or RXR
DBDs (Fig. 2
) were generated by PCR or
the Stratagene QuikChange mutagenesis kit (Stratagene, La Jolla, CA).
To construct the RTR and RMR chimeras, two SphI restriction
sites flanking the DBD of mouse RXR
were first created using the
Stratagene QuikChange kit. The resultant RXR
-Sph contained amino
acid substitutions at position 134 (S to C) and 234 (D to G), when
compared with wild-type RXR
. Next, PCR-generated DNAs encoding the
DBDs of TR
(amino acids 48150, with a residue substitution at
amino acid 149 from E to R) or AMT1 (amino acids 1115) (23) were
exchanged with the corresponding region of RXR
-Sph to create RTR or
RMR, respectively. The deletion mutants were constructed by insertion
of stop codons at the appropriate positions using the Promega Altered
Sites kit. To ensure the fidelity of the resulting constructs, all
predicted mutations and PCR-based constructs were verified by DNA
sequencing.
EMSAs
EMSAs were carried out essentially as previously described with
minor modifications (20). Briefly, 3 x 104 cpm of
32P-labeled DNA were incubated with 11.5 x
104 cpm of [3H]leucine-labeled, reticulocyte
lysate-translated full-length receptors or receptor DBDs; reticulocyte
lysate-translated empty vector was used as a control. (Where indicated,
315 ng of E. coli-derived receptor DBDs were used.)
Incubations were at room temperature for 40 min in a 35-µl reaction
mixture containing 20 mM HEPES, pH 7.8, 50 mM
KCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, 20%
glycerol, and 1.8 µg of
poly(deoxyinosinic-deoxycytidylic)acid. After incubation, the
samples were electrophoresed through 6% native polyacrylamide gels in
0.25x Tris-borate-EDTA buffer. The gels were dried and then
subjected to autoradiography. For antibody supershift studies, 1 µl
of antiserum was added to the EMSA reaction mixtures 30 min into the
above incubations and then incubated for another 15 min before gel
electrophoresis. The oligonucleotide probes used in the EMSA reactions
are listed in Table 1
.
Proteolytic Digestion Analysis
The limited proteolytic digestions were performed as described
elsewhere with minor modifications (32). One microliter of
35S-labeled (methionine plus cysteine) DBD proteins
(Promega TNT-coupled transcription translation system) was incubated
with different amounts of chymotrypsin in 20 µl of reaction buffer
(50 mM Tris, pH 7.8; 53 mM CaCl2)
at room temperature for 10 min. After incubation, the proteolytic
reactions were stopped by the addition of an equal volume of 2x SDS
sample buffer and heated for 5 min at 95 C. The samples were analyzed
on Tricine-SDS polyacrylamide gels (22). The gels were then incubated
in 25% isopropyl-7% acetic acid overnight and vacuum dried. The
radiolabeled proteolytic fragments were detected by PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) analysis. A similar
procedure was used for trypsin or elastase digestion except that the
digestion buffer for trypsin was 50 mM Tris, pH 7.5, and
for elastase the digestion buffer was 100 mM Tris, pH 8.0.
Preliminary experiments were performed with decreasing doses (in
0.5-fold decrements) of all these proteases until no digestion was
observed. These experiments did not reveal any bands in addition to the
ones seen in the data presented. Also, more prolonged electrophoresis
failed to resolve additional bands.
 |
ACKNOWLEDGMENTS
|
---|
We thank Mitchell Lazar for the TRv
2 antibody.
 |
FOOTNOTES
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Address requests for reprints to: Ronald J. Koenig, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Michigan Medical Center, 5560 MSRB-II, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0678. E-mail: RKoenig{at}umich.edu
This work was supported by NIH Grant DK-44155.
Received for publication March 5, 1998.
Revision received May 22, 1998.
Accepted for publication June 18, 1998.
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