Identification of Critical Residues for Heterodimerization within the Ligand-Binding Domain of Retinoid X Receptor
Soo-Kyung Lee,
Soon-Young Na,
Han-Jong Kim,
Jaemog Soh,
Hueng-Sik Choi and
Jae Woon Lee
College of Pharmacy (S.-K. L., H.-J.K., J.W.L.) Department of
Biology (S.-Y.N., J.S.) Hormone Research Center (J.S., H.-S.C.,
J.W.L) Chonnam National University Kwangju 500757, Korea
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ABSTRACT
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Nuclear receptors regulate transcription by
binding to specific DNA response elements as homodimers or heterodimers
with the retinoid X receptors (RXRs). The identity box (I-box), a
40-amino acid region within the ligand-binding domains of RXRs and
other nuclear receptors, was recently shown to determine identity in
the heterodimeric interactions. Here, we dissected this region in the
yeast two-hybrid system by analyzing a series of chimeric receptors
between human RXR
and rat hepatocyte nuclear factor 4 (HNF4), a
distinct member of the nuclear receptor superfamily that prefers
homodimerization. We found that the C-terminal 11-amino acid region of
the RXR I-box was sufficient to direct chimeric receptors based on the
HNF4 ligand-binding domain to heterodimerize with retinoic acid
receptors or thyroid hormone receptors. Furthermore, we identified the
hRXR
amino acids A416 and R421 of the 11-amino acid subregion as
most critical determinants of heterodimeric interactions;
i.e. mutant HNF4s incorporating only the hRXR
A416 or
R421 heterodimerized with retinoic acid receptor.
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INTRODUCTION
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The nuclear receptor superfamily is a group of
transcriptional regulatory proteins linked by conserved structure and
function (1). The superfamily includes receptors for a variety of small
hydrophobic ligands such as steroids, T3, and retinoids, as
well as a large number of related proteins that do not have known
ligands, referred to as orphan nuclear receptors (2). The receptor
proteins are direct regulators of transcription that function by
binding to specific DNA sequences named hormone response elements
(HREs) in promoters of target genes. Nearly all the superfamily members
bind as dimers to DNA elements. While some apparently bind only as
homodimers, thyroid hormone receptors (TRs), vitamin D receptor (VDR),
retinoic acid receptors (RARs), the peroxisome proliferator-activated
receptor, and several orphan nuclear receptors bind their specific
response elements with high affinity as heterodimers with the retinoid
X receptors (RXRs) (3, 4, 5, 6, 7, 8). Based on this high-affinity binding, such
heterodimers have been considered to be the functionally active forms
of these receptors in vivo. These heterodimers display
distinct HRE specificities to mediate the hormonal responsiveness of
target gene transcription, in that distinct HREs are comprised of
direct repeats (DRs) of a common half-site with variable spacing
between repeats playing a critical role in mediating specificity (2, 9). Accordingly, RARs activate preferentially through DRs spaced by two
or five nucleotides, whereas VDR and TR activate through DRs spaced by
three and four nucleotides, respectively. RXR-peroxisome
proliferator-activated receptor heterodimers as well as RXR homodimers
activate preferentially through DRs spaced by one nucleotide (referred
to as DR1). In addition to DRs, response elements composed of
palindromes as well as inverted palindromes referred to as everted
repeats (ER) have been shown to mediate transcriptional activation by
RXR-RAR and RXR-TR heterodimeric complexes (9). Such DNA-binding
flexibility stands in contrast to the steroid hormone receptors, which
bind exclusively as homodimers to inverted repeats (IR) spaced by three
nucleotides (10).
Some orphan nuclear receptors bind DNA as homodimers. In contrast with
the steroid receptor homodimers, orphan receptor homodimers can bind to
both palindromic and DR-response elements. In particular, the
hepatocyte nuclear factor 4 (HNF4) binds as a homodimer to DR1 and is a
strong constitutive transcriptional activator (11). In contrast with
HNF4, homodimers of the chicken ovalbumin upstream promoter
transcription factors (COUP-TFs) such as EAR2, EAR3, and ARP1 are
potent dominant repressors of both basal transcription and
transactivation by several receptors, including RXR, RAR, VDR, and TR
(12). Repression of these pathways by COUP-TF is believed to be
accomplished in part by direct competitive binding and by the presence
of a strong carboxy-terminal repressor domain (13). Interestingly,
COUP-TF has an ability to heterodimerize with RXR, thereby titrating
RXR into a transcriptionally inactive complex (14).
A dimerization interface has previously been identified within
the DNA binding domains (DBDs) of RXRs, RARs, VDR, and TRs that
selectively promotes DNA binding to cognate direct repeat HREs
(15, 16, 17, 18, 19, 20). An additional dimerization interface that mediates
cooperative binding to DNA, referred to as the I-box, has recently been
mapped to a 40-amino acid region within the carboxy-terminal ligand
binding domains (LBDs) of RAR, TR, COUP-TF, and RXR (21). In contrast
to the interface within the DBDs, this dimerization motif promotes
cooperative binding with similar efficiency to all three classes of
repeats, DR, IR, and ER. The two dimerization domains appear to work in
sequence and led to a two-step hypothesis for binding of heterodimers
to DNA (2, 21). Accordingly, the LBD dimerization interface initiates
the formation of solution heterodimers that, in turn, acquire the
capacity to bind to a number of differently organized repeats. However,
formation of a second dimer interface within the DBD restricts
receptors to bind to DRs.
The I-box sequences are fairly well conserved among a subset of
nuclear receptors including HNF4, a member of the homodimer subclass
(11). This led us to test whether the I-box region of the HNF4 plays a
similar role in the homodimeric interactions and, more importantly, to
determine whether differences between the I-box sequences contribute to
the preferences of HNF4 for homodimerization and RXR for
heterodimerization. To answer these questions without complications
associated with HRE binding and the dimerization interface within the
DBDs, we exploited the yeast two-hybrid system in which LBDs of nuclear
receptors are fused to either the DNA binding domain of the bacterial
repressor LexA or the B42 transcriptional activation domain, as
previously described (22, 23, 24). In this report, the HNF4 I-box was found
to be sufficient for the homodimeric interactions. In addition, an
11-amino acid subregion within the RXR I-box was found to be essential
for the heterodimeric interactions. From mutational analyses, the RXR
amino acids A416 and R421 of the 11-amino acid subregion were also
identified as particularly critical determinants of the heterodimeric
interactions.
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RESULTS
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The HNF4 I-Box Is Sufficient to Mediate the Homodimeric
Interactions
To characterize dimerization properties of nuclear
receptors, we exploited the yeast two-hybrid system that has been
previously described (22, 23, 24). In the host strain used in this system,
expression of ß-galactosidase (ß-Gal) is controlled by upstream
LexA DNA-binding sites (operators). Thus, this yeast strain depends on
transcriptional activation by a LexA protein for expression of ß-Gal.
A series of nuclear receptors were fused to the full-length LexA
repressor or the B42 transactivation domain. As previously described
(23, 24), LexA or its fusions to most nuclear receptors are
transcriptionally inactive in yeast. Protein-protein interactions
between nuclear receptors, however, can bring the B42 transactivation
domain to the LexA sites and activate the expression of the ß-Gal
construct. The LexA portion of such chimeras contains its own
dimerization domain and directs high-affinity binding to the LexA
operators as a dimer, regardless of the status of dimerization between
nuclear receptors. Therefore, an important merit of this system is the
fact that dimerization properties of nuclear receptors can be directly
assessed without complications associated with HRE binding. However, it
should be noted that the exact nature of these interactions, for
instance as to dimer vs. multimer, is not really known.
A dimerization interface referred to as the I-box has recently been
mapped to a 40-amino acid region within the carboxy-terminal LBDs of
RAR, TR, COUP, and RXR (21). The I-box sequences are moderately
conserved among a subset of nuclear receptors including the
homodimerizing HNF4, leading us to test whether the HNF4 I-box region
plays a similar role in the homodimeric interactions. To this end, we
constructed a series of chimeras between HNF4 and RXR. PCR was used to
construct B42/HNF, a chimeric protein consisting of the B42
transactivation domain fused to the entire hinge and LBDs (D, E and F,
amino acids 106455) of the rat HNF4 (Fig. 1A
). Chimeric receptors B42/H/X-298/389,
B42/H/X-338/429, B42/X-HHH, B42/H-XXX, and B42/X/H-428/339 were
similarly constructed (Fig. 1A
). Western blot analyses were executed to
confirm that expression levels for all the mutants constructed were
comparable (data not shown). LexA/HNF4 showed some constitutive
transcriptional activity in yeast, which was approximately 3.5-fold
higher than that of LexA alone (data not shown). As shown in Fig. 1B
, coexpression of B42/HNF led to approximately 5.5-fold enhancement of
this constitutive activity, faithfully reflecting the HNF4-HNF4
homodimeric interactions in this yeast system. Furthermore,
coexpression of B42/H/X-338/429 or B42/X-HHH led to approximately 4-
and 7-fold enhancements of the transcriptional activity of LexA/HNF4,
respectively. In contrast, the RXR I-box could not substitute the HNF4
I-box for the homodimeric interactions, as shown by inabilities of
B42/H/X-298/389, B42/H-XXX, and B42/X/H-428/339 to enhance the
transcriptional activity of LexA/HNF4. These results demonstrate that
the HNF4 I-box is indeed a sufficient and transferable interaction
interface for the homodimeric interactions.

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Figure 1. The I-box Directs Specificity in Dimerization
A, Schematic diagrams for chimeric nuclear receptors. Diagram shows
chimeras containing the indicated amino acids derived from RXR
(open) and HNF4 (stippled), where the
I-box domains are highlighted (dotted in RXR and
hatched in HNF4). Numbers refer to the amino acid
boundaries of the receptor fragments consisting of the hinge and ligand
binding D/E domains, the I-box domain, and the C-terminal F domain. B,
Dimerization probed by the yeast two-hybrid system. Host cells in which
ß-galactosidase expression is dependent on the presence of a
transcriptional activator with a LexA DBD were transformed with
plasmids expressing the indicated LexA or B42 chimeras and grown in
liquid culture containing galactose, since expression of the B42
chimeras is under the control of the galactose-inducible GAL1 promoter
(22). ß-Galactosidase readings were determined and corrected for
cell density and for time of development (A415 nm/A600
nm) x 1000/min. Fold-activations by each B42 chimera are
calculated by defining the reporter activity in the presence of B42/-
as 1. The result is the average of at least six different experiments,
and the SDs are less than 5%.
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As expected, B42/RXR was able to interact with LexA/RAR and LexA/TR
(Fig. 1B
), and similar results were observed with chimeric receptors
containing the RXR I-box sequences such as B42/H/X-298/389, B42/H-XXX,
and B42/X/H-428/339 (Fig. 1B
). In these interactions, LexA/RAR, which
includes a full-length RAR in contrast to LexA/TR and other LexA
chimeras consisting of only LBDs, shows higher ß-Gal activities than
LexA/TR, probably due to the AF1 activation domain in the RAR A/B
domains (25). Since LexA/TR and LexA/RAR show similar basal activities
in yeast, this difference results in higher fold-activations with
LexA/RAR than LexA/TR. For instance, coexpression of B42/RXR conferred
approximately 44-fold activation to LexA/RAR and approximately 11-fold
activation to LexA/TR (Fig. 1B
). It is noteworthy that incorporation of
only the RXR I-box sequences conferred to the resulting chimera the
ability to heterodimerize with RAR and TR (Fig. 1B
, compare
interactions with B42/HNF and B42/H-XXX). Thus, we conclude that the
RXR I-box was indeed a transferable interaction interface for
heterodimerization, as reported previously (21).
Localization of a Critical Region of the RXR I-Box for
Heterodimerization
As shown in Fig. 2
, there is a high
degree of sequence homology between the RXR I-box and the HNF4 I-box
sequences. Nevertheless, the HNF4 I-box directs homodimerization, while
the RXR I-box directs heterodimerization (21). Thus, we set out to
dissect the RXR I-box region to identify sequences critical for
heterodimerization by sequentially transferring part of the RXR I-box
sequences into corresponding regions of the HNF4, or vice
versa.

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Figure 2. Amino Acid Sequences of the I-Box Region
The I-box regions of USP (amino acids 429468), human RXR (amino
acids 389428), and rat HNF4 (amino acids 299338) are shown. Helix 9
(H9) and helix 10 (H10), which were recently shown to constitute an
interaction interface in the crystal structure of the RXR LBD (27), as
well as the three most C-terminal (7, 8, 9) of the nine heptad repeats
previously shown to be involved in dimerization (3032), are
indicated. Sequence homology determined with the NIH BLAST program is
indicated between sequences of two receptors, in which + indicates
conservative changes. Amino acids that are conserved between USP and
RXR but not in HNF4 are shaded. Three artificial blocks
designated to facilitate subsequent chimera constructions are
boxed.
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Examination of the I-box sequences reveals a high level of conservation
between RXR and its Drosophila counterpart ultraspiracle
(USP) (26), which heterodimerizes with many different members of the
superfamily. Therefore, amino acids of the I-box critical for
heterodimerization should be conserved between RXR and USP (Fig. 2
). To
facilitate analyses, we artificially divided the I-box region into
three separate subregions each containing two to four residues that are
conserved betwen RXR and USP but not in HNF4 (shaded in Fig. 2
). These subregions were sequentially exchanged between B42/HNF and
B42/X/H-428/339, resulting in a series of eight B42-fusion constructs
shown in Fig. 3A
. Western blot analyses
were executed to confirm that expression levels for all the mutants
constructed were comparable to each other (data not shown).

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Figure 3. An 11-Amino Acid Subregion of the RXR I-Box Directs
the Heterodimeric Interactions
A, Schematic diagrams for chimeric nuclear receptors. Diagram shows
chimeras containing the indicated amino acids derived from RXR
(dotted) and HNF4 (hatched). Three
smaller areas (stippled) around the three blocks
subjected to exchanges are identical between RXR and HNF4, as shown in
Fig. 2 . B, Dimerization properties of each chimera with LexA/HNF4,
LexA/TR, and LexA/RAR were probed by the yeast two-hybrid system, as
described in Fig. 1B . Fold-activations by each B42 chimera are
calculated by defining the reporter activity in the presence of B42/-
as 1. The result is the average of at least six different experiments,
and the SDs are less than 5%.
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As shown in Fig. 3B
, two things were evident from analyses of these
chimeric receptors in the yeast two-hybrid system (22, 23, 24). First, none
of the chimeric receptors was able to enhance the constitutive
transcriptional activities of the LexA/HNF4, demonstrating that
substitution of any of these HNF4 subregions with that of the RXR I-box
blocked homodimerization. This suggests that important residues for the
homodimeric interactions are present throughout the entire HNF4 I-box.
Second, the C-terminal 11-amino acid subregion of the RXR I-box was
sufficient to direct chimeric receptors to heterodimerize with RARs or
TRs; i.e. B42/H-HHX, B42/X-HHX, B42/H-XHX, and B42/X-XHX
efficiently interacted with LexA/RAR and LexA/TR (Fig. 3B
).
Identification of Critical Amino Acids Responsible for
Heterodimerization
As shown in Fig. 4
, the 11-amino
acid subregion of RXR is identical to that of HNF4 with the exception
of five residues (RXR A416, K417, R421, A424, and R426). Among these,
RXR K417, A424, and R426 (indicated by arrowheads in Fig. 4
)
are conserved changes from HNF4: i.e. HNF4 has glutamic
acid, threonine, and glutamine (HNF4 E327, T334, and Q336) at these
positions. However, RXR A416 and R421 (shaded in Fig. 4
)
that are conserved in USP are nonconserved changes from HNF4:
i.e. HNF4 has glycine and leucine (HNF4 G326 and L331) at
these positions. In contrast, COUP-TFs have glycine and arginine, and
RARs and TRs have proline and lysine at these positions (Fig. 4
).
Accordingly, these two residues were identified as primary candidates
for determining the heterodimerizing properties of the RXR I-box
and subjected to mutational analyses.

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Figure 4. Amino Acid Sequences of the 11-Amino Acid Subregion
of Various Receptors
Rat HNF4 (amino acids 326336), human RXR (amino acids 416426),
USP (amino acids 456466), human COUP-TF (amino acids 363373), human
RAR (amino acids 375385), and rat TRß (amino acids 419429) are
shown. Two amino acids of this subregion that are conserved between USP
and RXR but not in HNF4 are shaded. Three amino acids
that are conserved changes between RXR and HNF4 are indicated as
open arrows.
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As shown in Fig. 5A
, we introduced a
series of point-mutations into the 11-amino acid subregion in the
context of HNF4 resulting in three quadruple mutants (AR-AR, AR-KR, and
AR-KA), three triple mutants (AR-K, AR-A, and AR-R), two double mutants
(AR and PR), and three single mutants (A326, P326, and R331).
Interactions of these point-mutants with LexA/HNF4 were largely
unaffected among single, double, and triple mutants with an exception
of AR-K, as shown in Fig. 5B
. All the quadruple mutants were not able
to interact with LexA/HNF4 (Fig. 5B
). In contrast, quadruple mutants
AR-KA and AR-KR showed relatively strong interactions with LexA/RAR
(Fig. 5B
). Quaduple mutant AR-AR, all the triple mutants, double mutant
AR, and, most surprisingly, two single mutants (A326 and R331) were
also able to interact with LexA/RAR. These interactions were relatively
weak, but highly specific, as these mutants did not show any
interactions with LexA/GR either in the presence or absence of its
ligand, deoxycortisol (data not shown). Addition of the RAR ligands,
all-trans-retinoic acid or 9-cis-retinoic acid,
did not affect the interactions (data not shown). The inherently weaker
interactions of LexA/TR with the heterodimerizing chimeras, however,
were not evident with these point-mutants. In contrast, as expected
from their RAR-based mutations, P326 and PR that incorporate both P326
and R331 were not able to stimulate ß-galactosidase activities above
that of B42/HNF when coexpressed with LexA/RAR (Fig. 5B
). Western blot
analyses were executed to confirm that the expression levels for all
the mutants tested were indeed comparable (Fig. 5C
and data not
shown).

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Figure 5. Point Mutants of the 11-Amino Acid Subregion of the
HNF4
A, The HNF4 G326, E327, L331, T334, and Q336 subjected to point
mutagenesis are as indicated (conserved changes by open
arrows and nonconserved changes by closed
arrows, determined with the NIH BLAST program). All the point
mutants are identical to B42/HNF except the indicated amino acid
changes. Dashed positions are identical to HNF4
residues. B, Dimerization properties of point mutants with LexA/HNF and
LexA/RAR were probed by the yeast two-hybrid system, as described in
Fig. 1B . All the point mutants are identical to B42/HNF except the
indicated amino acid changes. Fold-activations by each B42 chimera are
calculated by defining the reporter activity in the presence of B42/-
as 1. The result is the average of at least six different experiments,
and the SDs are less than 5%. C, Western blot analysis of
the yeast strain EGY48 transformed with plasmids encoding indicated B42
chimeras was executed as described (23). All the point mutants are
identical to B42/HNF except the indicated amino acid changes.
Equivalent amounts (10 µg) of crude extracts for these samples as
well as null extracts were examined using an antibody directed against
Flu-tag, as described previously (22).
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To confirm the interactions observed in yeasts, we incubated a
glutathione S-transferase (GST) alone, GST-HNF, GST-RXR,
GST-H-HHX, GST-AR, GST-A326, or GST-R331 with TR-LBD labeled with
[35S]methionine by in vitro translation. All
the point-mutants were identical to GST-HNF except the indicated amino
acid changes. As shown in Fig. 6
, TR-LBD
bound specifically to GST-RXR and GST-H-HHX, but not to GST-HNF or GST
alone, independently confirming the yeast results. In addition, TR-LBD
was able to show relatively week but specific binding to GST-AR,
GST-A326, and GST-R331. In yeasts, these weak interactions were only
evident with LexA/RAR (Fig. 5
), probably due to the AF1 activation
domain in the A/B domains (25). As expected, luciferase labeled with
[35S]methionine interacted with none of the GST proteins.
Similar results were also obtained with RAR-LBD labeled with
[35S]methionine (data not shown). Overall, these results,
along with the yeast results, indicate that the RXR amino acids A416
and R421 are most critical to determine identity in the heterodimeric
interactions.

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Figure 6. Pull-Down Assays
Luciferase and TR-LBD labeled with [35S]methionine by
in vitro translation were incubated with glutathione
beads containing GST alone, GST fusions to HNF4-LBD (GST/HNF), RXR-LBD
(GST/RXR), H-HHX (GST/H-HHX), A326 (GST-A326), R331 (GST/R331), and AR
(GST/AR). All the point mutants are identical to HNF4-LBD except the
indicated amino acid changes. Beads were washed, and specifically bound
material was eluted with reduced glutathione and resolved by
SDS-PAGE.
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DISCUSSION
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A dimerization interface has been identified within the DNA
binding domains (DBDs) of RXRs, RARs, VDR, and TRs that selectively
promote DNA binding to cognate direct repeat HREs (15, 16, 17, 18, 19, 20). Another
dimerization interface referred to as the I-box has recently been
mapped to a transferable 40-amino acid region within the
carboxy-terminal LBDs of RAR, TR, COUP-TF, and RXR, which mediates
cooperative binding to DNA (21). In contrast to the interface within
the DBDs, this dimerization motif promotes cooperative binding with
similar efficiency to all three classes of repeats, DR, IR, and ER. In
the recently described crystal structure of the RXR LBD (27), a
rotationally symmetrical dimerization interface was formed mainly by
helix 10 (H10) and, to a lesser extent, helix 9 (H9) and the loop
between helix 7 (H7) and helix 8 (H8). The I-box nicely overlaps with
H9 and H10 (Fig. 2
), whose sequences are well conserved among a subset
of nuclear receptors including HNF4 (11) and COUP-TF (12) that prefer
to homodimerize. In addition, the crystal structures of RAR and TR LBDs
(28, 29) demonstrate that the I-box regions adopt a structure similar
to that of the RXR (27) in an overall common fold that can be
summarized as an
-helical antiparallel sandwich composed of 12
-helices (H1-H12). As expected from this common fold and the
conservation of the I-box sequences, the HNF4 I-box was expected to
play an important role in the HNF4-HNF4 interactions. As shown in Fig. 1
, the HNF4 I-box region is indeed sufficient for the homodimeric
interactions (see the results with B42/H/X-338/429 and B42/X-HHH in
Fig. 1
). However, residues critical for homodimerization are clearly
different from those for heterodimerization, since the HNF4-HNF4
interactions were not affected by any of the HNF4 point-mutations
replacing the two residues found to be critical for the heterodimeric
interactions (Fig. 5
). One potential explanation of the inability of
B42/H-HHX and similar mutants (shown in Fig. 3
) to interact with
LexA/HNF4 is that the overall structure of the chimeras is disrupted.
However, this is ruled out by the ability of the chimeras to interact
with LexA/RAR (Fig. 3
).
The results described here define an 11-amino acid subregion of the
I-box as a major determinant of dimerization specificity. This is
consistent with the fact that this subregion is contained within H10
(Fig. 2
), a major interface in the recently described crystal structure
of the RXR LBD (27) and overlaps with heptad 9 previously shown to be
critical for both homo- and heterodimerization of RXR (30, 31, 32). We also
found that a chimeric RAR construct containing the RXR 11-amino acid
subregion readily interacted with RAR, lending further support for the
importance of this subregion for the heterodimeric interactions (our
unpublished results). Within this subregion, RXR amino acids A416 and
R421 are particularly critical, consistent with the fact that these two
residues are conserved between RXR and USP, but not in the
homodimerizing HNF4. As shown in Fig. 4
, HNF4 has glycine and leucine,
while COUP-TF has glycine and arginine at these positions,
respectively. In contrast, RAR and TR, heterodimeric partners of RXR,
have proline and lysine at these positions. As expected, mutant HNF4s
incorporating only the RXR A416 or R421 heterodimerized with RAR.
However, these and other HNF4 point-mutants we constructed showed
distinct interactions with different receptors. R331, which resembles
COUP-TF at the two target positions, weakly interacted with LexA/RXR
(data not shown), consistent with a recent finding in which COUP-TF was
shown to have an ability to heterodimerize with RXR (14). In contrast,
PR, which resembles RAR and TR at these positions, did not interact
with LexA/RXR (data not shown). Similarly, AR with the RXR sequences at
these two positions interacted less efficiently with LexA/RAR than
single mutants A326 or R331 (Fig. 5B
). Overall, these results suggest
that the two residues (RXR A416 and R421) are clearly important for
identity in heterodimerization. Finally, it should be noted that
changes of these two residues alone were not sufficient to switch
specificity in the dimeric interactions (i.e. from
homodimerization to heterodimerization). The change of specificity in
dimerization was achieved with at least three mutations incorporated,
as shown by AR-K, which weakly interacts with LexA/RAR, but not with
LexA/HNF (Fig. 5
). Similarly, all the quadruple mutants changed
specificity in dimerization (Fig. 5B
; compare results with LexA/HNF4
and LexA/RAR). It is also noteworthy that the effects of substitutions
with the corresponding RXR sequences on the heterodimerizing potential
of the resulting HNF4 chimeras are largely accumulative:
i.e. A326, R331, AR, AR-K, AR-A, AR-R, and AR-AR show
relatively weak fold-activations with LexA/RAR (3- to 7-fold, as shown
in Fig. 5B
), while the quadruple mutants AR-KR and AR-KA, as well as
the quintuple mutant B42/H-HHX (Fig. 3B
), which incorporates all the
five RXR amino acids of the 11-amino acid subregion, showed much
stronger fold activations with LexA/RAR (23-, 21-, and 43-fold
activations, respectively). Accordingly, identity seems to be
determined in the context of the overall structures in relation to
neighboring residues, rather than solely by nature of the two residues.
In addition, it will be interesting to test effects of the identified
mutations in the context of full-length receptors.
In conclusion, we have identified an 11-amino acid subregion of the RXR
I-box as a critical domain for the heterodimeric interactions and
specifically identified two residues in this subregion as determinants
of heterodimerization. Recently, we introduced random mutations into
these two residues of RXR in an effort to make mutant RXR more
selective in the process of heterodimerization. Consistent with the
importance of these residues in heterodimerization, we were indeed able
to find a series of mutant RXRs with significantly altered specificity
(S.-K. Lee and J. W. Lee, unpublished). To understand in detail
how these two residues fit into the overall structure of the I-box in
each nuclear receptor and how they direct specificity in dimerization,
more mutational studies as well as more structural information will be
required.
 |
MATERIALS AND METHODS
|
---|
Hormones
Deoxycortisol, 9-cis-retinoic acid, and
all-trans retinoic acid were obtained from Sigma Chemical Co
(St. Louis, MO).
Yeast Cells, Plasmids, and Expressions
EGY48 cells
[MAT
leu2-his3-trp1-ura3-LEU2::pLexA-op6
LEU2(
UASLEU2)], the lexA-ß-galactosidase reporter construct, and
the LexA- and B42-parental vectors were as reported (22). LexA or B42
fusions to the full-length RAR and the LBDs of GR, RXR, HNF4, and TR
were as previously described (23, 24). Two subsequent PCR steps
according to a strategy that has been described (33) were employed for
all the following plasmid constructions, by using Vent Polymerase (New
England Biolabs, Beverly, MA) capable of proofreading to minimize
unwanted mutations. A forward vector primer (B42 primer; 5'-GCC TCC TAC
CCT TAT GAT-3') plus a reverse mutagenic primer (5'-CTT GAT CTT CCC GGG
GTC ACT CAG-3') and a forward mutagenic primer (5'-CTG AGT GAC CCC GGG
AAG ATC AAG-3') plus a reverse vector primer (ADH primer; 5'-GAC AAG
CCG ACA ACC-3') were used to amplify B42/HNF-wild type as a template
DNA at annealing temperature of 55 C. Both PCR products were mixed in
an equal molar ratio and amplified using the B42 and ADH vector
primers. Accordingly, B42/HNF includes a SmaI site just
upstream of the HNF4 I-box, in which codons for amino acids Pro 296
(CCA) and Gly 297 (GGC) were silently changed to CCC (Pro) and GGG
(Gly) to facilitate further subclonings. B42/H/X-298/389 encodes rat
HNF4 amino acids 106298 followed by human RXR
amino acids 389 to
462 to the C terminus. Similarly, B42/H/X-338/429 encodes HNF4 amino
acids 106338 followed by RXR amino acids 429462 to the C-terminus.
B42/X-HHH is identical to B42/RXR except that RXR amino acids 389428
were replaced by HNF4 amino acids 299338. B42/H-XXX is identical to
B42/HNF except that HNF4 amino acids 299338 were replaced by RXR
amino acids 389 to 428. B42/X/H-428/339 encodes RXR amino acids
198428 followed by HNF4 amino acids 339455 to the C terminus.
B42/H-XHH is identical to B42/HNF except that HNF4 amino acids 299- 308
were replaced by RXR amino acids 389398. Similarly, B42/H-HXH is
identical to B42/HNF except that HNF4 amino acids 312322 were
replaced by RXR amino acids 402412, while B42/H-HHX is identical to
B42/HNF except that HNF4 amino acids 326336 were replaced by RXR
amino acids 416426. B42/H-XHX is identical to B42/H-HHX except that
HNF4 amino acids 299308 were further replaced by RXR amino acids
389398. B42/X-XHH, B42/X-HXH, B42/X-HHX, and B42/X-XHX are identical
to B42/H-XHH, B42/H-HXH, B42/H-HHX, and B42/H-XHX, respectively, except
that HNF4 amino acids 106 and 299 were replaced by RXR amino acids
198389. A326 and P326 are identical to B42/HNF except that amino acid
Gly 326 was replaced by Ala and Pro, respectively. R331 is identical to
B42/HNF except that amino acid Leu 331 was replaced by Arg. AR and PR
are identical to R331 except that amino acid Gly 326 was further
replaced by Ala and Pro, respectively. AR-R, AR-A, and AR-K are
identical to AR except that amino acids Gln 336, Thr 334, and Glu 327
were replaced by Arg, Ala, and Lys, respectively. AR-AR is identical to
AR-A except that amino acid Gln 336 was replaced by Arg. Similarly,
AR-KR and AR-KA are identical to AR-K except that amino acid Gln 336
and Thr 334 were further replaced by Arg and Ala, respectively. All the
constructs described here were sequenced to prevent any unwanted PCR
mutations. In addition, expression levels of all the B42 chimeras were
determined using a monoclonal antibody directed against Flu-tagging
(gift of Dr. Kyung-Lim Lee) that resides just upstream of the B42
transactivation domain, as described (22). HNF4 sequences from B42/HNF,
B42/RXR, B42/H-HHX, A326, R331, and AR were transferred to pGEX4T1
(Pharmacia, Piscataway, NJ) to express GST fusions. Vector for in
vitro translation of the TR-LBD was as previously described
(24).
Yeast ß-Galactosidase Assays
The cotransformation and transactivation assays in yeast were
performed as described previously (23). Quantitative liquid
ß-galactosidase assays were performed with the following changes as
described (23). The yeast culture was initially diluted to an A600
nm of 0.05 and plated into 96-well culture dishes with the
various concentrations of hormone. The cultures were then incubated in
the dark at 30 C for 16 h. The A600 nm was determined,
and then cells were lysed and substrate was added and A415
nm was read after 1030 min. The normalized galactosidase
values were determined as follows: (A415 nm/A600
nm) x 1000/min developed. For each experiment, at least six
independently derived colonies expressing chimeric receptors were
tested.
Pull-Down Assays
GST fusion proteins were produced in Escherichia coli
and purified using glutathione-Sepharose affinity chromatography
essentially as described (24). GST proteins were bound to
glutathione-Sepharose 4B beads (Pharmacia) in binding buffer (50
mM KPO4, pH 6.0, 100 mM KCl, 10
mM MgCl2, 10% glycerol, 10 mg/ml E.
coli extract, and 0.1% Tween 20). Beads were washed once with
binding buffer and incubated for 60 min at 4 C in the same buffer with
equivalent amounts of various proteins labeled with
[35S]methionine by in vitro translation.
Nonbound proteins were removed by three washes with binding buffer
without E. coli extract, and specifically bound proteins
were eluted with 50 mM reduced glutathione in 0.5
M Tris, pH 8.0. Eluted proteins were resolved by PAGE and
visualized by fluorography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Kyung-Lim Lee for Flu-antibody and Drs. Wongi Seol
and David D. Moore for rat HNF4 cDNAs and critical reading of this
manuscript.
This research was supported by grants from KOSEF (960401-0801-3 and
Hormone Research Center) and Chonnam National University.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., College of Pharmacy, Chonnam National University, Kwangju, South Korea 500757.
Received for publication June 27, 1997.
Revision received November 24, 1997.
Accepted for publication November 26, 1997.
 |
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