The Nuclear Receptor Corepressor (N-CoR) Contains Three Isoleucine Motifs (I/LXXII) That Serve as Receptor Interaction Domains (IDs)
Paul Webb,
Carol M. Anderson,
Cathleen Valentine,
Phuong Nguyen,
Adhirai Marimuthu,
Brian L. West,
John D. Baxter and
Peter J. Kushner
Metabolic Research Unit University of California School of
Medicine San Francisco California 94143-0540
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ABSTRACT
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Unliganded thyroid hormone receptors (TRs) repress
transcription through recruitment of corepressors, including nuclear
receptor corepressor (N-CoR). We find that N-CoR contains three
interaction domains (IDs) that bind to TR, rather than the previously
reported two. The hitherto unrecognized ID (ID3) serves as a fully
functional TR binding site, both in vivo and in
vitro, and may be the most important for TR binding. Each ID
motif contains a conserved hydrophobic core (I/LXXII) that resembles
the hydrophobic core of nuclear receptor boxes (LXXLL), which mediates
p160 coactivator binding to liganded nuclear receptors. Although the
integrity of the I/LXXII motif is required for ID function,
substitution of ID isoleucines with leucines did not allow ID peptides
to bind to li-ganded TR, and substitution of NR box leucines with
isoleucines did not allow NR box peptides to bind unliganded TR. This
indicates that the binding preferences of N-CoR for unliganded TR and
p160s for liganded TR are not dictated solely by the identity of
conserved hydrophobic residues within their TR binding motifs.
Examination of sequence conservation between IDs, and mutational
analysis of individual IDs, suggests that they are comprised of the
central hydrophobic core and distinct adjacent sequences that may make
unique contacts with the TR surface. Accordingly, a hybrid peptide that
contains distinct adjacent sequences from ID3 and ID1 shows enhanced
binding to TR.
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INTRODUCTION
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Nuclear receptors are a large family of conditional transcription
factors that include receptors for thyroid hormone
(T3), vitamins A (retinoids) and D, steroids,
various components of lipid metabolism, and a large number of orphan
receptors whose ligands, if any, have not been identified (1, 2, 3, 4). The
thyroid hormone receptors (TRs) bind to cognate DNA response elements
both in the absence and presence of ligand, most commonly as a
heterodimer with the retinoid X receptor (RXR) (5, 6, 7). Unliganded TRs
repress basal promoter activity, and addition of ligand both relieves
this repression and further enhances basal promoter activity.
Thyroid hormone-dependent enhancement of basal promoter activity
involves recruitment of two types of coactivator proteins. The TRs bind
to a closely related family of p160 coactivators, including GRIP1
(TIF2/NCoA-2), SRC-1 (NCoA-1), and ACTR (pCIP/Rac3/AIB1/TRAM-1)
(8, 9, 10, 11). The p160s, in turn, bind to other coactivators, including the
integrator molecule CBP/p300 and p/CAF, both of which possess histone
acetyltransferase activity. The TRs also bind TRAP220 (12), a component
of the TRAP (DRIP/ARC/SMCC) complex that potentiates TR-dependent
transcription from naked DNA templates in vitro, but also
regulates transcription from DNA templates that have been assembled
into chromatin in vitro (13, 14, 15, 16, 17). Thus, liganded TRs
activate transcription by recruiting large coactivator complexes, which
work by either modifying chromatin or via unspecified effects upon
general initiation factors. In contrast, unliganded TRs repress
transcription by recruiting corepressor proteins, which are released
upon hormone binding. The TRs bind to nuclear receptor corepressor
(N-CoR) (RIP-13) and to the closely related protein SMRT (TRAC-2)
(18, 19, 20, 21, 22, 23, 24). Both N-CoR and SMRT, in turn, bind a large complex that
contains mSin3a, SAP30, c-ski, and histone deace-tylases (HDACs)
(25, 26, 27) and also bind directly to class II HDACs (28, 29). Thus,
unliganded TRs repress transcription by recruiting large corepressor
complexes, which work, at least in part (30), by deacetylating
histones.
Recent studies have focused on the structural basis of coactivator and
corepressor recruitment. Like most nuclear receptors, TRs possess a
strong ligand- dependent transactivation function (AF-2) that is
located within the receptor ligand-binding domain (LBD) and serves as a
docking site for p160 coactivators (31, 32). A combination of x-ray
crystallography and site- directed mutagenesis has revealed that
the residues that comprise AF-2 form a small hydrophobic cleft upon the
surface of the T3-liganded TR-LBD (32, 33).
Mutational analysis of the p160s and crystallographic analysis of
nuclear receptor/p160 cocrystals revealed that AF-2 binds to short,
conserved
-helical motifs (termed NR boxes, consensus LXXLL)
(34, 35, 36, 37, 38, 39, 40, 41). Initial structure-function analysis of the TRs and retinoic
acid receptors suggested that key residues for corepressor binding were
located near the junction of the hinge and LBD (18). However, the
TR-LBD crystal structure revealed that these residues were not on the
surface of the molecule (33), suggesting that mutations of these
residues affect corepressor binding indirectly. Other evidence
indicated that coactivators and corepressors were in dynamic
equilibrium on the nuclear receptor (8, 11, 42) and that key residues
for transcriptional repression by v-erbA, an oncogenic viral homolog of
TR, and for transcriptional repression and corepressor binding by the
orphan receptors Rev-erbA
and RVR, actually resided within the AF-2
hydrophobic cleft (43, 44). This suggested that similar mechanisms
might underlie both coactivator and corepressor recruitment and
prompted us to search the N-CoR primary sequence for motifs that bear
similarities to nuclear receptor boxes. We find, in agreement with
recently published studies of others (45, 46, 47), that the N-CoR C
terminus contains a repeated receptor interaction domain (ID) that
contains the conserved hydrophobic core motif I/LXXII. More
surprisingly, we find that N-CoR actually contains three of these IDs,
rather than the previously reported two. We present several lines of
evidence that the hitherto unrecognized motif (ID3) is a fully
functional TR binding site and that the three IDs represent the
totality of TR binding activity. We also show that the IDs are
comprised of both a hydrophobic core and distinct adjacent sequences
and that a hybrid peptide containing distinct adjacent sequences from
ID3 and ID1 binds more tightly to TR.
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RESULTS
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Three Receptor IDs in N-CoR
We initially asked whether N-CoR contains sequence motifs that
resemble the p160 NR Box, consensus LXXLL. Previous studies of TR/N-CoR
interactions revealed two regions of TR binding (amino acids
2,2392,453 and 1,9442,239) in the N-CoR C terminus (18, 19, 22, 24, 48) (Fig. 1A
). The C-terminal binding
region contained a single short hydrophobic motif that resembled the NR
box (LEDII, amino acids 2,2762,281). The N-terminal binding region
contained two of these motifs (ICQII, amino acids 2,0732,077; IDVII,
amino acids 1,9491,953). We refer to these motifs as ID1, ID2, and
ID3 (from C-terminal to N-terminal). During the course of this study,
ID1 and ID2 were also identified by other groups (45, 46, 47) but ID3 was
not.

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Figure 1. Three Receptor IDs in the Carboxyl Terminus of
N-CoR
A, Schematic of the structural organization of N-CoR showing the
relative positions of the silencing domains (striped
boxes) and nuclear receptor IDs (black boxes).
The C-terminal portion of N-CoR is shown, below, on a larger scale. The
extent of previously defined TR binding regions are marked with
lines above, the positions of each ID motif are marked
with a black box, and the amino acid coordinates of each
ID motif are marked below. B, Sequences of the ID
motifs. The sequences of the ID motifs are presented and compared with
each other, and to those of SMRT. At the top, the p160
coactivator NR box consensus sequence is presented.
Underneath, the hydrophobic core motif of each ID is
also shown in a similar box. Homologies between N-CoR
ID1 and SMRT ID1 or N-CoR ID3, N-CoR ID2, and SMRT ID2 are
highlighted. Conserved residues are indicated with a
thick line, and conservative substitutions, or residues
displaced by a single position, are indicated with a thin
line.
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N-CoR ID1 and ID2 had counterparts at similar locations in the related
corepressor SMRT (46). Interestingly, both N-CoR IDs showed greater
homology to their SMRT counterparts than to each other (Fig. 1B
). The
strongest conservation between N-CoR and SMRT ID1 was C-terminal to the
core motif, whereas the strongest conservation between N-CoR and SMRT
ID2 was N terminal to the core motif. While N-CoR ID3 did not have an
obvious counterpart within SMRT, it did resemble N-CoR ID2. In
particular, ID3 and ID2 contained an IXXII core, a conserved Arg and
the sequence ITØA N-terminal to the cores, and a conserved Thr
immediately C terminal to the core. Because of these homologies, we
elected to examine all three IDs as candidate TR binding sites.
The ID Motifs Mediate TRß/N-CoR Interactions
To test whether each of the three IDs represented a functional TR
binding site, we synthesized short peptides that spanned the entire
region of conservation between N-CoR and SMRT ID1 or N-CoR ID3 and
N-CoR and SMRT ID2 (Fig. 2A
). We then
asked whether these short ID peptides would compete for TRß/N-CoR
interactions in vitro (Fig. 2B
). ID1 and ID3 showed
half-maximal competition at 0.31 µg of peptide. ID2 was weaker,
with half-maximal competition requiring 13 µg of peptide. Overall,
the efficiency of ID peptide competition for TRß/N-CoR interactions
was comparable to the efficiency of NR box peptide competition for
TRß/GRIP1 interactions (37). Moreover, several mutant peptides, some
of which conserved overall ID hydrophobicity, failed to compete for
TR/N-CoR interactions (data not shown and see Figs. 6
and 7
). Thus,
each of the three ID motifs could represent a functional TR binding
site.

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Figure 2. The ID Motifs Interact with TRß in
Vitro
A, Schematic of GST-N-CoR matrix and competitor peptides used in TRß
binding assays. B, Inhibition of TRß binding to GST-N-CoR C terminus
by synthetic peptides. An autoradiogram of SDS-PAGE gels showing
radiolabeled TRß input protein and TRß protein retained upon
bacterially expressed GST- or GST-N-CoR matrices, either in the absence
or the presence of T3 or in the presence of increasing
doses of competitor peptide. C, TR binds to N-CoR fragments that
overlap ID1, ID2, and ID3. The panel is an autoradiogram of an SDS-PAGE
gel showing radiolabeled TRß input protein and TRß protein bound to
N-CoR fragments that overlap ID3 (amino acids 1,9442,031), ID2 (amino
acids 2,0512,208), and ID1 (amino acids 2,2182,453). The
radiolabeled TR appears as a diffuse band in the ID1 lanes because the
TR migrates to approximately the same location as the GST-ID1 fusion
protein.
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Figure 6. Comparison of ID1 and NR Box Peptides
A, Schematic of GST-N-CoR and GST-GRIP1 fusion proteins and competitor
peptides. B, Peptide competitions for TRß binding to GST-N-CoR. The
left hand panel shows an autoradiogram of SDS-PAGE gels
showing radiolabeled TRß input protein and TRß protein retained
upon a bacterially expressed GST- or GST-N-CoR matrix, either in the
absence or the presence of T3 or in the presence of 30 µg
of each competitor peptide. The right hand panel shows a
similar experiment in which we examined the abilities of different
peptides to compete for the binding of TRß to bacterially expressed
GST-GRIP1.
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Figure 7. Sequence Composition of the IDs
A, Analysis of ID3 mutant peptides. A schematic of different competitor
peptides and GST-N-CoR is shown at the top.
Autoradiogram of a SDS-PAGE gel showing radiolabeled TRß protein
retained upon a GST-N-CoR matrix in the absence or presence of
T3 and 1 µg of competitor peptides. B, A similar analysis
of TR binding to GST-N-CoR (1,9442,453) in the presence of 30 µg
ID1 peptide or mutant derivative, with peptide sequences shown at
top. C, Concentration-dependent inhibition of
N-CoR/TRß interactions with an ID3/ID1 hybrid competitor peptide. A
schematic of the GST-N-CoR fusion protein and competitor peptide is
shown at the top. The arrows indicate the
extents of the ID3 and ID1 sequences. Below is an
autoradiogram of a SDS-PAGE gel, revealing the amount of radiolabeled
TRß retained on the GST-N-CoR matrix.
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We then asked whether N-CoR fragments that contained each of the
isolated IDs would bind to TR. Figure 2C
shows that, in agreement with
previous results (22, 24, 45, 46, 47), N-CoR fragments overlapping isolated
ID1 or ID2 bound the TR in the absence of ligand. A N-CoR fragment that
overlapped the ID3 IXXII motif (amino acids 1,9442,031) also showed
significant binding to TR. TR binding to ID3 was robust, even though
this N-CoR fragment lacks some of the conserved ID3 sequences that lie
to the N terminus of amino acid 1,944. Thus, the N-CoR C terminus
contains three distinct TR binding regions, each of which overlaps an
ID motif.
To determine whether the ID motifs themselves were required for
N-CoR/TRß interactions in vitro, we prepared a vector that
expresses the N-CoR C terminus (amino acids 1,6812,453) and
introduced mutations into the hydrophobic core of each ID. Figure 3
shows that the N-CoR C-terminal
fragment (WT) bound strongly to TRß in the absence of ligand, and
that binding was reduced by T3. Similar N-CoR
fragments that retained either ID3+ID2 or ID3+ID1 showed small
reductions in TRß binding in the absence of ligand, but an N-CoR
fragment that retained only ID2+ID1 showed a larger reduction in TRß
binding. Phosphoimaging of several experiments revealed that ID3+ID2
allowed more than 90% of the level of wild type N-CoR binding to
TRß, ID3+ID1 allowed up to 70%, and that ID2+ID1 allowed 4060%.
Thus, significant N-CoR binding is obtained with any two IDs, but the
combination of ID3+ID2 is preferred and the combination of ID2+ID1 is
weakest. By contrast, single IDs were insufficient to allow significant
binding to TRß. An N-CoR fragment that only contained ID3 did give
some weak binding to TRß (
5% of wild type), but fragments that
contained only ID2 or ID1, or no IDs failed to bind TRß. Taken
together, these results point toward several conclusions. First, the
N-CoR IDs are essential for TR binding. Second, because the N-CoR
triple mutant failed to bind to TRß, there is no additional TR
binding site within the N-CoR C terminus. Third, because each possible
ID pair allows significant TRß binding, yet isolated IDs do not, the
IDs must cooperate in TRß binding. Lastly, and most surprisingly,
because mutation of ID3 leads to the largest reduction in TRß
binding, and because isolated ID3, but not ID1 or ID2, is sufficient
for weak residual binding to the TRß-LBD, ID3 may be the strongest of
the three IDs for TRß binding in vitro.

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Figure 3. Mutations in ID Motifs Reduce N-CoR/TRß
Interactions
A schematic for the GST-TRß fusion protein and radiolabeled N-CoR
C-terminal fragment is shown at the top. Mutated IDs
contain double Ala substitutions within the C- terminal Iles of the
ID hydrophobic core (I/LXXII>I/LxxAA). The panels represent
autoradiograms of SDS-PAGE gels showing radiolabeled input wild-type
and mutant N-CoR proteins, and the amounts of each N-CoR protein
retained by GST-TRß, either in the absence or the presence of thyroid
hormone (T3). In each case, IDs indicated at the
left are those remaining after mutation.
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IDs Are Required For TRß/N-CoR Interactions in
Vivo
Next, we asked whether the ID motifs were needed for N-CoR/TRß
interactions in vivo. We first determined whether our N-CoR
C-terminal expression vector, which contains the IDs but lacks active
repression domains, would act as a dominant negative for
transcriptional repression. We transfected chicken embryo fibroblasts
(CEF) with a reporter containing a herpes simplex thymidine kinase
promoter and two GAL4 response elements (GAL-TK) and then determined
the level of transcription in the absence or presence of a yeast
Gal4-TRß fusion protein (Gal-TR) and N-CoR. Figure 4
shows that, as expected, Gal-TR
repressed transcription in CEF cells by 3-fold in the absence of
T3 (left panel) and enhanced
transcription by 15-fold in the presence of T3
(right panel). The N-CoR C-terminus (WT) reversed the
repression (left panel), but not the ligand-dependent
activation (right panel). Thus, consistent with previous
observations (24), the N-CoR C terminus interferes with the ability of
unliganded TRß-LBD to repress transcription.

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Figure 4. The IDs Are Needed for N-CoR Dominant Negative
Activity in Vivo
A schematic of the transfection components is shown at
top. The graphs below represent fold
repression by unliganded Gal-TRß (left panel, black
bars), or fold activation by liganded TRß (right
panel, gray bars), either in the absence or presence of
expression vectors for N-CoR C terminus or its mutated derivatives. To
determine fold repression or fold induction luciferase activities were
normalized to ß-galactosidase, and then compared with those obtained
in the absence of Gal-TRß, which is set at 1.
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We then asked whether the IDs were required for this dominant negative
activity. An N-CoR C-terminal fragment that retained ID3+ID2 retained
most of its dominant negative activity (left panel). By
contrast, fragments that retained either ID3+ID1 or ID2+ID1 were devoid
of dominant negative activity. Fragments that either retained single
IDs (ID3, ID2, and ID1), or no IDs, also lacked dominant negative
activity. None of the N-CoR expression vectors affected transcriptional
activation (right panel). Thus, the IDs are required for the
dominant negative activity of the N-CoR C terminus. Moreover, an N-CoR
fragment that retains ID3+ID2 showed significant dominant negative
activity indicating that in vivo, as in vitro,
TRß prefers ID3+ID2.
Next, we examined TRß binding to the IDs in two-hybrid assays in CEF
cells (Fig. 5A
). A N-CoR fragment that
contained all three IDs (ID3, -2, and -1) recruited unliganded
TRß-VP16, but not liganded TRß-VP16. By contrast, a similar
construction in which all three ID motifs were mutated (mID3, -2, and
-1) failed to recruit TRß-VP16, even when TRß-VP16 was
overexpressed (not shown). Thus, the three IDs constitute all of the TR
IDs within the N-CoR C terminus in vivo. In parallel, N-CoR
fragments that contained isolated ID3 or isolated ID1 both recruited
TRß-VP16, but their mutated equivalents did not. An N-CoR
fragment that contained ID2 only showed weak interactions with
TRß-VP16. However, the relative weakness of this interaction was
overcome when TRß-VP16 was overexpressed (Fig. 5B
). Thus, each ID
binds TRß-VP16 in vivo, but ID3 and ID1 are strongest.

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Figure 5. The IDs Are Needed for N-CoR/TRß Interactions in
Two-Hybrid Assays
A, A schematic of the transfection components is shown at the
top. The transfection was performed with 10 ng of
VP16-TRß fusion protein expression vector. The panel
below shows a comparison of the efficiency of either the
Gal-DBD (none) or different Gal-N-CoR fusions as baits. The regions of
N-CoR encoded by each fragment were as follows: IDs 3,2,1: amino acids
1,9252,308; ID3: amino acids 1,9251,994; ID2: amino acids
2,0492,091; ID1: amino acids 2,2392,308. Mutant N-CoR fragments
overlap the same regions but contain alanine substitutions within the
ID C-terminal isoleucines (I/LXXII>I/LXXAA). The panel shows
results from a single transfection experiment: each value is the
average of luciferase activities determined from three individual wells
and normalized to ß-galactosidase, the black bars from
cells maintained in the absence of ligand, the gray bars
in the presence of T3. B, Results of a similar transfection
which contained 1 µg of VP16-TRß expression vector. Here, ID2 is
seen to efficiently recruit TR.
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Further examination revealed other aspects of behavior of the Gal-N-CoR
fusion proteins. First, the N-CoR molecule that contained all three IDs
recruited TRß-VP16 more efficiently than the isolated IDs, implying,
once again, that the IDs cooperate in TRß binding. Second, TRß-VP16
showed strong T3-dependent release from all three
IDs, but its release from ID1 was incomplete (see Fig. 5A
, inset). This indicates that TRß/ID1 interactions possess
both ligand-dependent and ligand-independent components. Despite these
subtleties, our results suggest that all three IDs bind TRß in
vivo, and that the binding of each ID motif to TRß
recapitulates the pattern of N-CoR binding to TRß.
The IDs Are Composed of a Hydrophobic Core (I/LXXII) Along with
Distinct Adjacent Sequences
While we originally identified the IDs on the basis of their
resemblance to the coactivator NR box (LXXLL), it is clear that some
mechanism allows N-CoR to bind preferentially to unliganded TRs and
coactivators to bind preferentially to liganded TRs. We asked whether
the different types of hydrophobic residues within the N-CoR motifs
(Ile, I/LXXII) and the NR box motifs (Leu, LXXLL) were sufficient to
account for this preference. We synthesized a mutant N-CoR ID1 peptide
(ID1-LL), in which key Ile residues were substituted with Leu, so that
it resembled a p160 NR box, and a mutant GRIP1 NR box2 peptide in which
key Leu residues were substituted with Iles, so that it resembled an
N-CoR ID motif (Fig. 6A
).
Figure 6B
confirms the observations, shown above, that the N-CoR ID1
peptide competed for the binding of unliganded TRß to N-CoR
(left panel) and also shows that Box2, ID1-LL, and Box2-II
did not. In parallel (right panel), the ID1 peptide failed
to compete for the binding of T3-liganded TRß
to GRIP1, but Box2 competed efficiently. Both ID1-LL and Box2-II did
not. Thus, even Ile/Leu exchanges, which conserve the hydrophobicity of
the peptides, abolished their respective abilities to compete for
TRß/N-CoR and TRß/GRIP1 interactions. Moreover, the Ile/Leu
exchanges failed to allow the N-CoR ID peptide to compete for
TRß/GRIP1 interactions or the GRIP1 NR box peptide to compete for
TRß/N-CoR interactions. This indicates that the binding preferences
of N-CoR for unliganded TRß, and GRIP1 for liganded TRß, are not
dictated solely by the identity of the conserved hydrophobic residues
within their TRß binding motifs.
We next examined the sequence requirements for ID motif/TRß
interaction. In the case of ID3, Ala substitutions within the core
hydrophobic Ile residues (m1) abolished its ability to compete for
TRß/N-CoR interactions (Fig. 7A
).
Likewise, Ala substitutions at different locations within the conserved
ID3 N-terminal region either completely abolished (m2, m3, m5), or
partially reduced (m4), the ability of ID3 to compete for TRß/N-CoR
interactions. In the case of ID1, Ala substitutions within the Leu and
Ile residues of the ID1 core, and a Leu residue within the conserved
region of the ID1 C terminus, were sufficient to abolish competition,
whether they were placed within the same peptide (m1), or within
different peptides (m2m4) (Fig. 7B
). Substitution of the Glu residue
within the ID1 core, which is conserved between N-CoR and SMRT, also
abolished competition (m5), but substitution of Asp, which is not
conserved, did not (m6). Thus, residues both within and outside of the
hydrophobic core motifs are required for ID3 and ID1 peptides to
compete for TRß/N-CoR interactions.
Finally, because the area of best conservation between N-CoR ID3 and
N-CoR and SMRT ID2 lay N-terminal to the core motif and the area of
best conservation between N-CoR and SMRT ID1 lay C terminal to the core
motif, we prepared an ID3/ID1 hybrid peptide that contained both
regions (Fig. 7C
). Half-maximal competition was obtained with as little
as 1030 ng of hybrid peptide and complete competition was obtained
with as little as 300 ng to 1 µg of peptide. In parallel,
half-maximal competition for TRß/N-CoR interactions required 0.31
µg of the ID3 or ID1 peptides (see Fig. 2
). Thus, the hybrid peptide
competes for TRß binding to N-CoR more efficiently than the parental
peptides, suggesting that the binding of the IDs to TRß is suboptimal
and that the identity and position of the TR binding determinants
outside the core motif differs between the IDs.
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DISCUSSION
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N-CoR Contains Three ID Motifs, and a Hitherto Unrecognized ID
(ID3) Is Important For TR Binding
In this study we examined the structural requirements for the
interactions of the nuclear receptor corepressor, N-CoR, and the
unliganded TRß. We, along with others (18, 19, 22, 24, 45, 46, 47, 48),
noticed that two different regions of N-CoR that had been previously
shown to bind nuclear receptors contained the conserved core sequence
I/LXXII. However, unlike other groups, we found three of these ID
motifs, rather than two. Several lines of evidence indicated that all
three IDs bind to TRß. First, peptides corresponding to each ID motif
compete for TRß/N-CoR interactions in vitro. Second, TR
bound to short N-CoR fragments that overlapped each of the IDs. Third,
mutation of the individual ID motifs reduces, and mutations of any two
of the three ID motifs abolishes, N-CoR binding to TRß in
vitro. Fourth, each ID motif plays a role in the dominant negative
activity of an N-CoR C-terminal fragment in vivo. Finally,
all three IDs act as bait for a TRß-VP16 fusion protein in two-hybrid
assays. The previously unrecognized motif, ID3, shows TRß binding
activity that is good as, or more potent than, ID1 and ID2 in each of
these TRß binding assays. We therefore conclude that the C-terminal
region of N-CoR contains three separate receptor IDs and that the
previously unrecognized domain (ID3) is important for TRß
binding.
Our data also indicate that isolated ID3 and ID1 are stronger than
isolated ID2 and that any combination of two IDs is sufficient for
strong TR binding, although the particular combination of ID3 and ID2
is preferred both in vitro (Fig. 3
) and in vivo
(Fig. 4
). Nonetheless, there are some apparent discrepancies between
assays that need to be resolved. For example, any pair of IDs was
capable of binding TR in vitro, and isolated ID3 was
sufficient for residual interactions with the TR (Fig. 3
). However, the
specific combination of ID2 and ID3 was required for N-CoR dominant
negative activity in vivo, and other pairs of IDs or single
IDs were not functional (Fig. 4
). Furthermore, a GST-N-CoR fragment
that only contains ID2 binds strongly to TR (Fig. 2C
), even though
isolated ID2 appeared to bind relatively weakly to TR in other assays.
We suggest that these apparent discrepancies arise from differences in
the sensitivity and linear range of different assays. Thus,
glutathione-S-transferase (GST)-pull-down assays would
detect relatively weak TR/N-CoR interactions, whereas the dominant
negative interference assay would only detect strong TR/N-CoR
interactions. In accordance with the notion that it is important to
"tune" each assay for true quantitative comparisons, it was
possible to detect differences between the IDs in mammalian two-hybrid
assays in the presence of low levels of TR (Fig. 5A
), but these
differences disappeared when the TR was overexpressed (Fig. 5B
).
Our studies also raise the question of whether N-CoR might contain yet
more unrecognized IDs. We found that mutation of all three IDs
completely abolishes the ability of full length N-CoR to bind to the
TRß-LBD (not shown), suggesting that N-CoR lacks any unidentified
strong TRß-LBD binding site. Thus, we presently favor the idea that
N-CoR only contains three IDs. While we have not directly examined TR
interactions with SMRT in this study, sequence comparisons failed to
reveal an ID3 motif at a conserved position, or any other position, in
the SMRT primary sequence, nor any obvious homologies between other
surrounding ID3 residues and SMRT. We therefore suggest that SMRT only
contains two IDs. We stress that this conclusion is based only on
sequence comparisons and needs to be treated with caution. Strong
interactions between SMRT and TR can be observed even in the absence of
ID1 (46). While this binding may simply reflect the interactions of
SMRT ID2 with TR, it is also possible that SMRT contains both an ID2
and additional sequences that bind to TR, and that these sequences are
not recognizable from the sequence data alone.
The IDs Cooperate in TR Binding
Our studies indicate that the IDs cooperate in TR binding, both
in vitro and in vivo, just as the p160 NR boxes
cooperate in nuclear receptor binding (37). This cooperativity provides
obvious advantages for the sensitivity of hormone response: binding of
ligand to either receptor molecule within a homo- or heterodimer pair
would result in complete corepressor release. The reasons why N-CoR
would contain three distinct ID motifs, when two are sufficient for
high-affinity TRß interactions, are less clear. TRß shows some
preference for the pairing of ID3+ID2 (Figs. 3
, 4
, and 5
), even though
ID2 is relatively weak when examined in isolation (Figs. 3
and 5
).
Other nuclear receptors show a preference for ID1 (45, 46, 47). This
finding is reminiscent of the preference of different nuclear receptors
for different p160 NR boxes (34, 35, 36, 37, 38, 39). Thus, one possible explanation
for the presence of three IDs in the N-CoR C terminus is that they
might allow for higher order interactions between separate nuclear
receptors, e.g. a dimer and a monomer might be able to bind
simultaneously to a single N-CoR molecule.
The IDs Are Composed of a Hydrophobic Core and Distinct Adjacent
Sequences: Speculations on the Nature of the TR/N-CoR Interface
The same nuclear receptor hydrophobic cleft that mediates
interactions with p160 coactivators also mediates interactions with
corepressors (43, 44, 45, 46, 47, 49). Moreover, N-CoR and SMRT ID motifs contain
the core consensus sequence I/LXXI/VI, which resembles the NR box
consensus LXXLL. Despite these similarities, there must be some
mechanism that allows only unliganded TRß to bind N-CoR and only
liganded TRß to bind GRIP1. This preference cannot be accounted for
by the nature of hydrophobic residues within the ID motif and NR box
core motifs (Fig. 6
). N-CoR and GRIP1 must therefore recognize distinct
structural features of TRß that are regulated by ligand.
What are the mechanisms that N-CoR uses to recognize unliganded TR? Our
results, and the results of others (45, 46, 47) suggest that the IXXII
motif is important for recognition of the unliganded TR surface.
However, homologies between N-CoR ID3, N-CoR ID2, and SMRT ID2 and
N-CoR and SMRT ID1 extend beyond the conserved hydrophobic core (Fig. 1
), and we, and others (45, 46, 47), found that mutations within these
adjoining residues disrupted TR/N-CoR interactions. Thus, the IDs are
composed of both the hydrophobic core and adjacent sequences. We also
know that key residues for both coactivator and corepressor binding lie
within the nuclear receptor hydrophobic cleft (43, 44, 45, 46, 47, 49) and that
helix 12 forms a key part of the AF-2 surface (39, 40, 41), but is
dispensable or inhibitory for corepressor interactions (19, 45, 46, 47, 50, 51). Thus, it is likely that the choice between coactivator and
corepressor binding is regulated by ligand-dependent repositioning of
helix 12. Indeed, in the unliganded RXR-LBD crystal structure (52),
helix 12 extends away from the LBD rather than packing against the LBD
as in the T3-liganded TR (33). We therefore
propose that the conserved I/LXXII motif binds to a region of the cleft
that lies under helix 12 and is exposed by the repositioning of
helix 12 in the unliganded state and that TR/N-CoR interactions are
stabilized by interaction of adjoining sequences from the IDs with the
TRß surface.
Our results also give some indication that the structure of the N-CoR
ID motif may be different from the structure of the NR box. First, the
central hydrophobic residues of both motifs are important for TR
binding, but adjoining ID sequences play a more important role than
adjoining NR box sequences (39, 53). Second, while the NR boxes adopt a
two- turn
-helical structure (39, 40, 41), the IDs are longer and the
position of some of the TR binding residues is inconsistent with a
location on one face of an extended
-helix. Lastly, one of the
nonhydrophobic residues within the ID1 core motif (LDVII)
is required for ID1 peptide competitions (Fig. 7
). The nonhydrophobic
residues of the NR box core do not play a role in TR binding (39).
There may also be subtle differences in the way that the TR recognizes
distinct IDs. N-CoR ID1, but not ID2 or ID3, shows weak residual
hormone-independent binding to TR in two-hybrid assays. Moreover, the
region of best homology between N-CoR ID3, ID2, and SMRT ID2 lies
N-terminal to the core motif, and the area of best conservation between
N-CoR and SMRT ID1 lies C terminal to the core motif (Fig. 1
).
Recombining these two areas of conservation creates an artificial
hybrid peptide that competes very efficiently for TR/N-CoR interactions
(Fig. 7C
). Similar increases in TR binding efficiency have also been
obtained when distinct IDs are recombined and tested in two-hybrid
assays (45). Together, these results are consistent with the notion
that distinct adjoining sequences from different IDs make distinct
contacts with the TR surface. While we do not know what these contacts
are, one possibility is that the Leu-Met pair at the C terminus of ID1
(KALM) could bind to the upper part of the hydrophobic
cleft, with the LM in a similar position to the C-terminal leucines
(LXXLL) of the NR box (39). This interaction could also
have analogies to the way that estrogen receptor helix 12 folds into
the same region of the cleft in the presence of antiestrogens (40, 54)
and account for the weak ligand-independent component of TRß/ID1
interactions. It is likely that the full understanding of TRß/N-CoR
interactions will require resolution of TRß/N-CoR crystal structures.
We speculate that it may be possible to take advantage of synthetic
peptides, including the ID3/ID1 hybrid described here, for this x-ray
structural analysis.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The N-CoR C terminus expression vector was derived from a pBK
vector containing N-CoR sequences 1,6292,453 (18). The dual
mammalian/in vitro transcription-translation vector pSG5
(Stratagene, La Jolla, CA), was adapted with an
oligonucleotide containing SacII and XhoI cloning
sites. The N-CoR cDNA was isolated as a
SacII/XhoI fragment and ligated into SG5. N-CoR
C-terminal expression vectors containing mutated ID sequences were
prepared by standard PCR- based site-directed mutagenesis (Quickchange,
Stratagene). Oligonucleotides homologous to ID1, ID2, and
ID3, but containing sequences that code for Ala substitutions within
the Ile pair of each ID (L/IXXII>L/IXXAA), were used to generate the
mutations. Double and triple mutants were prepared by subsequent rounds
of mutagenesis. The vector encoding the GST-N-CoR ID3 fusion protein
(amino acids 1,9442,031) was prepared by introducing a
SalI site after residue 2031 into a GST-N-CoR fusion protein
encoding residues 1,9442,453 (18). This allowed the deletion of
residues 2,0322,453. The GST-N-CoR ID2 fragment (amino acids
2,0512,208) and ID1 fragments (amino acids 2,2182,453) were
isolated by PCR.
Gal-N-CoR expression vectors were derived from the pM expression vector
for the yeast Gal4 DNA-binding domain (CLONTECH Laboratories, Inc. Palo Alto, CA). N-CoR fragments were amplified by standard
PCR methods. All 5'- oligonucleotides contained an EcoRI
site for cloning. For Gal-N-CoR/1,9252,308 the 3'-oligonucleotide
contained a SalI site. The others were generated with a
3'-oligonucleotide that contained a HinDIII site. Similar
Gal-N-CoR expression vectors with mutated ID sequences were generated
using pSG5-N-CoR ID triple mutant as a template. GAL-RE-TK-luciferase
was prepared by cloning a double-stranded oligonucleotide containing
two copies of the GAL4 dimer binding site upstream of the fusion
reporter gene containing the Herpes simplex virus thymidine kinase
promoter (-109/+48) linked to the firefly luciferase gene.
The following plasmids have been previously described: GST-GRIP1
(5631,121) (37), GST-TRß (55), Gal-RE-e1b- luciferase (37),
cytomegalovirus (CMV)-TRß (32). The following were gifts: GST-N-CoR
fusions 1,9442,453 and 1,7442,453 from Dr. M. Lazar (University of
Pennsylvania School of Medicine, Philadelphia, PA), VP16-TRß from Dr.
R. Evans (University of California San Diego, San Diego, CA), Gal-TRß
from Dr. D. Moore (Baylor College of Medicine, Houston, TX),
actin-ß-galactosidase from Dr. M. Garabedian (New York University,
New York, NY).
Protein-Protein Interaction Assays
Labeled proteins, peptides, and GST fusion proteins were
prepared as previously described (37, 39). Peptide sequences were
as follows, ID1:- N-ASNLGLEDIIRKALMGSFDD-C; ID2:-
N-RTHRLITLADHICQIITQDFARNQ-C; ID3 N-RGKTTITAANFI-DVIITRQIASDK-C;
ID3/1 hybrid peptide:- N-RGKTTITAA NFI-
EDIIRKALMGSFDD-C.
Cell Culture and Transfections
Chicken embryo fibroblasts (CEF, UCSF Cell Culture Facility)
were grown in DMEM/F-12 Hams modified mix, without phenol red,
supplemented with 10% iron- supplemented newborn calf serum
(Sigma, St. Louis, MO) and pen-strep. CEF cells were
transfected, by electroporation (56), with 2 µg reporters, 1 µg of
Gal fusion protein expression vector or CMV vector control and, where
indicated, 5 µg of N-CoR or pSG5 control or 50 ng of TRß-VP16.
After electroporation, the cells were resuspended in medium containing
10% T3-depleted newborn calf serum. Luciferase
and ß-galactosidase activities were measured, using standard assays
(Promega Corp., Madison, WI; and Tropix, Bedford, MA) at
3648 h. Individual transfections (containing data from triplicate
wells) were repeated three to six times.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. R. Evans (University of California, San Diego) for
communicating unpublished results and for providing plasmids, Dr. M.
Lazar (University of Pennsylvania School of Medicine, Philadelphia,
PA), Dr. D. Moore (Baylor College of Medicine, Houston, TX) and Dr. M.
Garabedian (New York University, New York, NY) for providing plasmids,
Amber Boast for technical assistance, and Dr. F. Schaufele, Dr. R.
Fletterick, Dr. R. Price, and R. Wagner (University of California, San
Francisco) and Dr. B. Darimont (University of Oregon) for advice and
helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Peter Kushner, Metabolic Research Unit 1119 HSW, University of California San Francisco School of Medicine, San Francisco, California 94143-0540. E-mail:
kushner{at}itsa.ucsf.edu
Supported by NIH Grants DK-32129 and CA-30913 to P.J.K. Peter J.
Kushner is a shareholder and Director of KaroBio AB, a company with
commercial interests in this area of research. John D. Baxter has
proprietary interests in, and serves as a consultant to and deputy
director of, KaroBio AB.
Received for publication May 22, 2000.
Revision received August 28, 2000.
Accepted for publication August 30, 2000.
 |
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