TR Surfaces and Conformations Required to Bind Nuclear Receptor Corepressor
Adhirai Marimuthu1,
Weijun Feng,
Tetsuya Tagami,
Hoa Nguyen1,
J. Larry Jameson,
Robert J. Fletterick,
John D. Baxter2 and
Brian L. West1
Metabolic Research Unit (A.M., W.F., H.N., J.D.B., B.L.W.) and
Departments of Biochemistry and Biophysics and Cellular and Molecular
Pharmacology (R.J.F.), University of California San Francisco, San
Francisco, California 94143; Center for Endocrinology, Metabolism, and
Molecular Medicine (J.L.J.), Northwestern University Medical School,
Chicago, Illinois 60611; and Clinical Research Institute, Center for
Endocrine and Metabolic Diseases (T.T.), Kyoto National Hospital,
Kyoto 612-8555, Japan
Address all correspondence and requests for reprints to: Dr. Brian L. West, Molecular Biology Department, Plexxikon, Inc., 91 Bolivar Street, Berkeley, California 94710. E-mail: bwest{at}plexxikon.com
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ABSTRACT
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Residues of the TR that are critical for binding the nuclear
receptor corepressor (N-CoR) were identified by testing more than 100
separate mutations of the full-length human TRß that scan the surface
of its ligand binding domain. The primary inferred interaction surface
overlaps the surface described for binding of p160 coactivators, but
differs by extending to a novel site underneath which helix 12 rests in
the liganded TR, rather than including residues of helix 12.
Nonconservative mutations of this surface diminished binding similarly
to three isolated N-CoR receptor interaction domains (RIDs), but
conservative mutations affected binding variably, consistent with a
role for this surface in RID selectivity. The commonality of this
surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion
of helix 12 increased N-CoR binding by the TR modestly, and by the RXR
and ER to a much greater extent, indicating a competition between this
helix and the corepressor that regulates the extent of corepressor
binding by nuclear receptors. When helix 12 was deleted, N-CoR binding
by the ER was stimulated by tamoxifen, and binding by the TR was
stimulated by Triac, indicating that helix 12 is not the only feature
that regulates corepressor binding. Two additional
mutationsensitive surfaces were found alongside helix 1, near the
previously described CoR box, and above helix 11, nearby but separate
from residues that help link receptor in dimers. Based on effects of
selected mutations on T3 and coactivator binding, and on
results of combined mutations of the three sites on corepressor
binding, we propose that the second and third surfaces stabilize TR
unliganded conformation(s) required for efficient N-CoR binding. In
transfection assays mutations of all three surfaces impaired the
corepressor-mediated functions of unliganded TR repression or
activation. These detailed mapping results suggest approaches for
selective modulation of corepressor interaction that include the shape
of the molecular binding surface, the competitive occupancy by helix
12, pharmacological stimulation, and specific conformational
stabilization.
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INTRODUCTION
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NUCLEAR HORMONE RECEPTORS comprise a family
of related proteins that regulate transcription. Prototypical receptors
bind both DNA and hormone and can cause both positive and negative
changes in gene expression, depending upon the target gene promoter.
These receptors are single polypeptide chains containing an
amino-terminal domain, a centrally placed DNA-binding domain (DBD), and
a C-terminal ligand-binding domain (LBD) (1). Several
coregulatory proteins interact with nuclear receptors and contribute to
gene-regulatory effects (2). Coactivators are recruited to
the receptors in response to agonist binding and can participate in
gene activation or repression, depending on the response element
encountered. By contrast, corepressors mediate the functions of
unliganded receptors to repress the basal activity of promoters that
contain TR binding sites, but also to stimulate the basal activity of
certain promoters such as that for TSH (3).
Corepressors like nuclear receptor copressor (N-CoR) (4),
also called RIP13 (5), and silencing mediator of retinoid
and thyroid receptor (SMRT) (6), also called TRAC
(T3 receptor associating cofactor)
(7), were identified by their binding to the TR and RAR,
but binding of varying strength has been detected to a number of
different nuclear receptors including the RXR (5), VDR
(8), PPAR (9, 10), ER (11, 12),
PR (13, 14, 15), and the orphan receptors RevErb (16, 17), chicken ovalbumin upstream promoter transcription factor 1
(18), DAX-1 (19), steroidogenic factor-1
(20), and RVR (17). To understand
corepressor function it is important to determine how they interact
with receptors. Central to this is to define site(s) that bind these
proteins. Initially it was proposed that the corepressor binds to a CoR
box, which includes helix 1 of the TR LBD based on the observation that
deletion of this helix abolished N-CoR binding to the TR
(4). This notion received further support by the
observations that combined mutation of three conserved residues (A228,
H229, and T232 AHT) within the CoR box region and of a residue
preceding helix 1 (P214 in the hTRß) blocked binding of N-CoR and
SMRT (4, 6, 7). However, the AHT and P214R mutations are
of residues that are buried in the liganded TR structure
(21), and these mutations and deletion of helix 1 may
affect corepressor binding through conformational changes near to or
distant from the helix 1 region.
Alterations of nuclear receptors outside helix 1 have also been
reported to diminish binding of corepressors. In the TR these include
deletion of the helix 11 (22) and mutations located in
residues of helices 3, 4, and 5 that are surface exposed in the
ligand-bound structure (23, 24). Evidence for a role of
helices 3 and 5 has also been reported for the orphan receptors RevErb
and RVR (17) and helix 11 in RevErb
(16). Hormone binding stabilizes particular
conformations of helix 12 relative to helices 3 and 5
(21), and because mutations of helices 3 and 5 also
diminish binding to corepressors, it has been postulated that
conformational changes in helix 12 could also play a role in blocking
corepressor binding when the receptor binds ligand (23, 24). In further support of the notion that helix 12 can affect
corepressor were the observations with several nuclear receptors that
deletion (25) or mutation (10) of helix 12
increases corepressor binding. However, the boundaries of the
corepressor interaction surface have not been described, and the
precise structural role of helix 12 in destabilizing corepressor
binding has not been elucidated. Helix 12 has mostly been considered to
be passive in unliganded nuclear receptors and to serve its main role
in formation of the coactivator-binding surface. However, it is also
possible that this helix is important in the unliganded state.
Two receptor interaction domains (RIDs) have been described within both
SMRT and N-CoR (5, 16, 22, 26), and a third has been
described for N-CoR (27). All of these contain motifs of
IxxII that are important for binding (23, 24, 25, 27). The
separate RIDs exhibit specificity in their binding to different nuclear
receptors (18, 28), but the basis for this specificity is
unknown. The IxxII motifs are likely present within larger protein
structural domains that bind to the receptors. Differences between
these domains outside the consensus sequences could dictate specificity
for interacting with nuclear receptors, although there is no direct
evidence for this idea. It is also unclear to what extent the separate
RIDs recognize the same or different surfaces on TR or other nuclear
receptors.
In this study we applied scanning surface mutagenesis to identify TR
surfaces that interact with three fragments of N-CoR, each of which
contains one of the RIDs. We introduced mutations over the entire
surface of the TR LBD, using the x-ray structure of the hormone-bound
TR LBD (21) as a guide for placing mutations. Even though
corepressors bind unliganded TRs, we cautiously anticipated that use of
the liganded TR LBD structure would be suitable, because the available
evidence suggests that most of the overall fold of unliganded and
liganded structures would be similar. In comparative studies we used
x-ray structures of the unliganded human RXR (hRXR
) LBD
(29) and the human ER
(hER
) LBD bound to the
mixed-antagonist 4-hydroxytamoxifen (30), to place
mutations in these receptors. We used these mutated receptors in
glutathione-S-transferase (GST) pull-down assays to identify
the surface regions important for binding to N-CoR, and we used a
selection of the vectors encoding mutated TRs for mammalian cell
transfection to study the effect of the mutations on the TR unliganded
functions.
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RESULTS
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The TR LBD surface (Site 1) That Binds N-CoR Overlaps the
Coactivator-Binding Surface, but Extends Underneath Helix 12
To define the corepressor-binding site, more than 100 different
full-length hTRßs, each with single-point mutations distributed over
the TR LBD solvent-accessible surface, were assayed for in
vitro binding to GST-N-CoR fusion proteins. In most cases, a
charged Arg or Lys was chosen to replace each native TR residue, but
native Arg or Lys residues were mutated to Ala. All areas of the TR LBD
surface were sampled, and surface areas where mutational effects were
observed were characterized further by mutating neighboring residues
more densely. Binding of the [35S]-labeled TRs
were compared using three different N-CoR fragments linked to GST.
These fragments separately contained RIDs I and II described previously
(5), as well as a third receptor-binding N-CoR fragment
(RID III) described more recently (27).
The TR area in which mutations had the strongest effects on N-CoR
binding was on the surface formed by helices 3 and 5, overlapping the
surface previously shown to bind coactivators (31). We
refer to this as Site 1. Mutations T277R, I280K, V284R, K288A, I302R,
and C309K all showed decreased in vitro binding by N-CoR
(Fig. 1A
). By contrast, mutations of
residues of helix 12 that form part of the coactivator-binding surface
(L454R and E457K) did not decrease binding to N-CoR, indicating that,
unlike the case with coactivator, the N-CoR-binding surface does not
include the outside of helix 12. Further, a natural mutation of the TR
that deletes helix 12 [F451X (32)] increased TR binding
to N-CoR by about 2.5-fold for both the RIDs I and II (Fig. 1A
). Thus,
it appears either that N-CoR does not contact the residues of helix 12
or that such contact does not appreciably stabilize the
interaction.

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Figure 1. A TR LBD Surface (Site 1) That Overlaps the
Coactivator-Binding Surface, but Extends Underneath Helix 12,
Selectively Binds All Three RIDs of N-CoR
A, Binding of a selection of [35S]-labeled hTRß mutants
to mouse N-CoR protein fragments containing RIDs I (2,231/2,321,
solid), II (2,034/2,114, shaded), or III
(1,888/2,031, open) in GST pull-down assays. Binding is
expressed as the percentage of binding observed for the WT hTRß in
the same assay (typically 12% of the input for RIDs I and II and 30%
for RID III). Results are the average ± SD of
duplicates and are representative of at least two experiments. B, As
for panel A, except a selection of mostly conservative mutants was
examined. C, Space-filling models of TRß LBD showing the inferred
corepressor-binding surface. Residues that bind N-CoR normally after
mutation are shaded gray. Mutation-sensitive residues
are colored by residue type: hydrophobic, green; polar,
orange; basic, blue; and acidic,
red. Computer graphics prepared using MidasPlus (UCSF
Computer Graphics Laboratory) (53 ). Left
panel, View based on the TR LBD structure (21 ),
which contains the complete helix 12 (H12, line), but in which the
terminal two amino acids (E460 andD461) present on WT hTRß are
unstructured and therefore not visible. Right panel,
Model of the natural F451X mutant, in which 11 residues (451461) are
absent and therefore show the portion of the corepressor-binding
surface obscured by helix 12 in the ligand-bound state. The
corepressor-binding surface is comprised of a cluster of hydrophobic
residues (I280, V283, V284, I302, and C309) bordered by polar (T277 and
T281) and charged (K288 and K306) residues. The TR mutations tested and
found not to diminish binding to N-CoR include (with approximate
locations): Helix 1: E213R, D216A, E217R, E220R, K223A, T224R,
E227R, V230R, A231R; Helix 2: Q235R, S237R, K240A, Q241R, K242E; Strand
1: R243A; F245K; Loop: V256R; Helix 3: F269A, H271R, K274E, T281R,
D285A, K289A; Helix 4: M292K; Helix 5: E298R, E299A; Helix 6: R316H;
Strand 2/3: E324R; Strand 4: M334K; Helix 7: R338W; Helix 8: V348R,
M358A; Helix 9: D382R, P384R; Helix 10: A387R, V389K, E390R, E393R,
L400R, H413R; Helix 11: V414K; H416R, W418K, K420A, L422R, M423R,
K424A, T426R, D427A, M430R, A433R, C434R, S437R, L440R, V444R; Loop:
E449R, F451A; Helix 12: L454R, L456R, E457K. These views summarize
experiments of panels A and B and also experiments not shown.
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Two of the residues that form the inferred corepressor-binding surface,
I280 and C309, lie mostly underneath helix 12 and are solvent
inaccessible in the liganded TR-LBD structure, and a third, T277, is
partially obscured. Each of these residues would be predicted to be
fully solvent exposed when helix 12 is deleted, but because placement
of helix 12 in the unliganded TR is unknown, it is possible that
mutation of these residues might interfere with corepressor indirectly
by pushing helix 12 to an abnormal position. Therefore, as a control we
examined effects of the I280K and C309K mutations when helix 12 is
deleted and observed decreased corepressor binding with these double
mutations as well (Fig. 1A
, I280K/F451X and C309K/F451X). The finding
that residues obscured by helix 12 in the liganded TR contribute to the
binding surface provides support for the role hypothesized for helix 12
in destabilizing corepressor binding by demonstrating a mechanism:
helix 12 and corepressor compete for interactions with the same
residues.
The inferred corepressor-binding surface is partially obscured in the
x-ray structure model of the liganded TR LBD (Fig. 1C
, left
panel), and therefore it is more easily viewed when the 11
C-terminal residues are not displayed, so as to show a structure that
might occur in the natural deletion mutation, F451X (Fig. 1C
, right panel). This binding surface is relatively small and
is comprised of a cluster of hydrophobic residues I280, V283, V284,
I302, and C309) bordered by polar (T277 and T281) or charged (K288 and
K306) residues. The portion of this surface that overlaps with the
coactivator-binding surface includes residues V284, K288, I302, and
K306 (31).
Surface Structure of Site 1 Regulates RID Selectivity
Helices 3 and 5 are part of a region of
homology within the nuclear receptor family named "the signature
motif" (33). The fact that residues from these helices
appear to contact corepressor suggests that a large number of receptor
family members may bind corepressors. Binding of wild-type (WT) TR
occurs to all three N-CoR RIDs, but selectivity of other nuclear
receptors for separate corepressor RIDs has been reported (18, 28). The basis for such selectivity is unknown. We investigated
the effect of surface shape changes on selectivity by testing effects
of conservative mutations in which residues of helices 3 and 5 are
substituted with amino acids present in other nuclear receptors.
Although binding of the WT TR occurred to each of the three RIDs,
differential effects were found within this set of conservative
mutants, either specifically increasing (T281I, T281L, T281V, V284I,
and I302V) or decreasing (T281L, T281Q, T281V, V284I, and I302V)
binding to one of the individual RIDs (Fig. 1B
). Other mutants
affected binding to all N-CoR RIDs similarly, resulting in molecules
with decreased (I280M, V283M, V284A, I302A, I302M, C309A, and C309W) or
little changed (V283I and L305I) binding (Fig. 1B
). Because the tested
mutations are local and conservative, these results suggest that
variations in the shape of the interaction surface of different nuclear
receptors regulate their ability to bind corepressors and to define
different modes of binding.
Ligand Regulation of N-CoR Binding When Helix 12 Is Deleted
As demonstrated previously (25), we found that
deletion of the C-terminal helix of human RXR
(equivalent to helix
12 of the TR) results in a large increase in N-CoR binding (Fig. 2
, A and C). Strong binding occurred to
N-CoR RID I, but very little binding occurred to RID III and almost no
binding occurred to RID II (Fig. 2A
), results that are consistent with
previous reports (28). Mutations of RXR residues
equivalent to those forming the coactivator-binding surface of TR helix
3 have been shown to decrease corepressor binding (25),
and we extended this finding to show that mutation of a residue of the
RXR (W305), whose equivalent in the hormone-bound TR (C309) would be
buried by helix 12, markedly decreases N-CoR binding (Fig. 2C
). Thus it
appears that a surface of RXR similar to that of the TR is recognized
by N-CoR and that N-CoR binding to this surface is regulated by the
presence of helix 12 even in the absence of ligand.

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Figure 2. N-CoR Also Binds to Site 1 of the RXR and ER, and
N-CoR Binding Is Increased by Ligand When Helix 12 Is Deleted in the ER
and TR
Pull-down experiments examining the binding of labeled receptors to
mouse N-CoR protein fragment containing RIDs I (2,231/2,321,
solid) or II (2,034/2,114, shaded), or
III (1,888/2,031, open). Binding is expressed as the
percent of input labeled receptor. Results are the average ±
SD of duplicates and are representative of at least two
experiments. The control binding to GST is 0.3% (not shown). A,
Binding of [35S]-labeled hRXR deletion mutant spanning
residues 126447, containing the DBD and LBD, but lacking the N
terminus and H12 (labeled DBD-LBD H12). In all cases, duplicate
samples are performed to allow the average values to be quantified
after phosphorimaging. B, Binding of [35S]-labeled hER
deletion mutant spanning residues 250536, containing the DBD and LBD,
but lacking the N terminus and H12 (labeled DBD-LBD H12). TAMOX, -
or +, indicates the absence or addition of 10 µM
tamoxifen during the binding reaction. C, Binding of
[35S]-labeled hRXR deletion mutants spanning residues
126462, containing the DBD and LBD (labeled WT) or residues 126447,
which lack the C-terminal helix of the LBD (labeled H12). The W305K
mutation alters the hRXR residue equivalent to residue C309 in the
TR Site 1. D, Binding of [35S]-labeled hER deletion
mutants spanning residues 250595, containing the DBD and LBD (labeled
WT), or residues 250536, which lack the C-terminal helix of the LBD
(labeled H12). The I358R, K362A, and V376R mutations alter the
hER residues equivalent to residues V284, K288, and I302 in the TR
Site 1. TAMOX, - or +, indicates the absence or addition of 10
µM tamoxifen during the binding reaction. E, Binding of
[35S]-labeled hTRß deletion mutants spanning residues
202461, containing the LBD (labeled WT), or the 202450 mutant,
which lacks the helix 12 (labeled H12). TRIAC, - or +, indicates
the absence or addition of 50 µM Triac during the binding
reaction.
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Similar to the RXR, binding of the human ER
could be obtained using
N-CoR RID I but not RID II (Fig. 2
, B and D), but good binding occurred
with RID III (Fig. 2B
). Furthermore, binding of RID I to the ER was
enhanced by deletion of helix 12 (Fig. 2D
). However, binding of RID I
to the helix 12-deleted ER also required addition of the mixed
antagonist, tamoxifen (Fig. 2B
). This ligand stimulation was not
observed for binding to RID III (Fig. 2B
). Three mutations of the ER
(I358R, K362A, and V376R) showed diminished binding by the helix
12-deleted ER (Fig. 2B
), thus confirming that N-CoR binding likely
occurs to a surface similar to Site I of the TR. These three mutations
are located in the portion of Site 1 where corepressor and coactivator
binding overlap. A fourth mutation of ER (W382K), which would occur in
the part of Site 1 that is buried by the hormone-bound helix 12, also
abolished N-CoR binding (data not shown), but ER W382 likely is also
important for the binding of tamoxifen (30). The ligand
requirement for N-CoR RID I binding by the ER in the absence of helix
12 implies that mechanisms in addition to a conformation change in
helix 12 exist for regulation of corepressor binding. Presumably,
ligand binding causes conformation changes in parts of the nuclear
receptor in addition to helix 12.
We found that the TR can also be regulated by ligand-induced
conformational mechanisms. Although addition of Triac blocks binding of
N-CoR to the WT TR LBD that contains helix 12, it stimulates binding of
N-CoR to the helix 12-deleted TR LBD (Fig. 2E
). This effect required a
high concentration of Triac, consistent with the expected weak binding
affinity of Triac for the helix 12-deleted TR. We determined that the
F451X TR LBD binds with a dissociation constant
(Kd) of 100 nM in saturation
radioreceptor binding assays with
[125I]T3, and that this
binding is competed more effectively by T3
compared with reverse T3, and not competed at all
by tamoxifen (data not shown). Therefore, ligand-induced stimulation of
corepressor binding is likely a general property of nuclear receptors,
suggesting that pharmacological approaches may be more generally useful
than previously appreciated for regulating corepressor functions via
the stimulation of corepressor recruitment by nuclear receptors.
Mutations in Two Additional Surfaces of the TR LBD Influence N-CoR
Binding
To investigate the previously defined CoR box (4)
region, we mutated every surface residue on helix 1, and assayed these
for N-CoR binding. Confirming the previous report (4),
simultaneous mutation of three buried residues of helix 1 (A228, H229,
and T232, AHT) was defective in binding N-CoR RIDs I and II (Fig. 3A
). Most of the surface mutations of
helix 1 had no effect on N-CoR binding, although one surface mutation
located at the beginning of helix 1 (W219K, Fig. 3C
) had impaired
binding to RIDs I and II (Fig. 3A
), and RID III (data not shown). This
suggests there is little contribution to N-CoR binding by most of the
helix 1 surface but indicates the region near W219 may regulate
binding.

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Figure 3. A TR LBD Surface Forming a Cavity Along Helix 1
(Site 2) Contains a Cluster of Mutation-Sensitive Residues Affecting
N-CoR Binding
Binding of a selection of [35S]-labeled hTRß mutants to
mouse N-CoR protein fragments containing RIDs I (2,231/2,321,
solid) or II (2,034/2,114, shaded), or
III (1,888/2,031, open) in GST pull-down assays. Binding
is expressed as the percentage of binding observed for the WT hTRß in
the same assay. Results are the average ± SD of
duplicates and are representative of at least two experiments. A,
Results with mutations located on the surface of helix 1 and the loop
connecting helices 1 and 2 and the AHT triple mutation of the buried
helix 1 residues A228G, H229G, and T232G. B, Results with mutations
located on helices 6 (V319K) and 8 (S361R), the loop between helices 8
and 9 (N364R and D366R), and helices 9 (D367R) and 10 (Y406K, Y409K,
and R410A), which cluster to form surface Site 2. C, Space-filling
model of Site 2. The mutation-sensitive residues affecting N-CoR
interaction are colored by atom type (green for hydrophobic,
W219, V319, and Y406; blue for basic, R410; red
for acidic, D366 and D367; and orange for polar, S361 and
N364). Residues unresponsive to mutation are shown in gray
(see Fig. 1C ). The position of helix 1 (H1) is indicated by a
line. This view summarizes experiments of panels A and B and
experiments not shown. Site 2 is mixed nonpolar and polar in character.
Computer graphics prepared using MidasPlus (53 ).
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Examination of the region near residue W219 revealed eight additional
mutations, V319K, S361R, N364R, D366R, D367R, Y406K, Y409K, and R410A,
that impaired TR binding to N-CoR (Fig. 3B
), although not as strongly
as did some mutations of Site 1. Together with W219, these residues
define a concave surface running alongside helix 1 (Fig. 3C
), which we
refer to as "Site 2." Site 2 contains hydrophobic, polar, and
charged residues that derive from distant locations in the TR linear
sequence. Because mutation of the surface-exposed residues of helix 1
that lie over the buried AHT mutation do not affect N-CoR binding but
do define a border of Site 2, effects of the AHT mutation could be due
indirectly to effects on Site 2.
Amino acids on helix 11 have also been implicated in corepressor
binding, because deletions of the terminal portion of helix 11 affect
both N-CoR and SMRT binding (16, 22). Helices 10 and 11
participate in formation of the surface for TR-TR homodimerization and
TR-RXR heterodimerization, and mutation L422R has the strongest dimer
destabilizing effect (34). Most surface mutations of
helices 10 and 11, including L422R, and a buried mutation of helix 11
(L428R) did not diminish binding to N-CoR RIDs I and II (Fig. 4A
). Two mutations on the surface of
helix 11 (K424R and D427A) caused increases in binding to N-CoR RID II
(Fig. 4A
). Two mutations of helix 10 (Q396R and L401R) caused weak
(average 25%) decreases, and one mutation of helix 11 (R429A) caused
stronger (60%) decreases in N-CoR binding (Fig. 4A
). The Q396R, L401R,
and R429A mutations were also demonstrated to decrease the binding to
N-CoR RID III (data not shown). Whereas this surface influences N-CoR
binding, the structural pattern is discontinuous rather than clustered
(Fig. 4B
). Residue R429, the most sensitive to mutation, has its side
chain pointed above helix 11 toward residue Q396 of helix 10. We refer
to these two residues as "Site 3." Residue L401 does not contact
residues 396 or 429; its side chain points above helix 10 toward where
the DBD could be oriented in the full-length TR. In Fig. 4B
, residues
that give rise to decreased binding to all N-CoR RIDs are colored by
residue type. Residues K424 and D427, which increase binding to N-CoR
RID II, are colored gray; these two residues are directed
below helix 11, toward helix 8 (Fig. 4B
).

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Figure 4. A TR LBD Surface Above Helix 11 (Site 3) Contains
Residues Mutation Sensitive for N-CoR Binding
A, Binding of a selection of [35S]-labeled hTRß mutants
in helices 10 and 11 to mouse N-CoR protein fragments containing RIDs I
(2,231/2,321, solid) or II (2034/2114,
shaded) in GST pull-down assays. Binding is expressed as
the percentage of binding observed for the WT hTRß in the same assay.
Results are the average ± SD of duplicates and are
representative of at least two experiments. B, Space-filling model of
Site 3. The mutation-sensitive residues affecting N-CoR interaction are
colored by atom type (green for hydrophobic, L401;
blue for basic, R429; and orange for
polar, Q396). Residues unresponsive to mutation are shown in
gray (see Fig. 1C ). Computer graphics prepared using
MidasPlus (53 ). Two of the unresponsive mutations are
labeled: W418 is the residue examined as a control in Fig. 6 , and L422
is the residue known to block homo- and heterodimerization
(34 ). The basic residue K306 is part of surface Site 1.
The positions of helices 10 (H10) and 11 (H11) are indicated by
lines. This view summarizes experiments of Fig. 4A and
also experiments not shown.
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Loss of Corepressor Binding Correlates with Loss of Unliganded
Inhibition at Activating Thyroid Response Elements (TREs) and
Unliganded Activation at the Inhibitory TSH Promoter
The predominant function reported for N-CoR bound to unliganded TR
is repression of gene promoters that are activated by
T3. To test effects of the mutations on this
activity, we transfected CV-1 cells with vectors expressing each mutant
along with a synthetic gene containing two copies of the F2
(35) TRE linked to the thymidine kinase (TK) gene
promoter, with luciferase (LUC) coding sequences as the reporter. Cells
were incubated in culture medium deficient in thyroid hormones. As
compared with the level of expression using the vector without TR
coding sequences (CMX), the TR-WT decreased expression of this
reporter gene by 80%, and a selection of TR mutants quantitatively
varied in this effect (Fig. 5A
). When the
amount of unliganded repression is plotted against the amount of N-CoR
binding for each TR mutant, a significant correlation is obtained, with
r2 values of 0.52, 0.59, and 0.62 for RIDs I, II,
and III, respectively (Fig. 5C
). The mutations tested include those
from Sites I, II, and III, and therefore likely indicate that the
sensitivity to mutations for in vitro N-CoR binding have
functional significance in vivo. One mutation (L422R) had
strong effects in vivo without a correlated defect in the
in vitro binding assay. Because this mutation affects
receptor dimerization (34), this could suggest this
unliganded function requires in vivo formation of a TR dimer
or some other association at this site.

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Figure 5. The TR Mutants That Have Decreased N-CoR Binding
Exhibit Specific Correlated Defects in Unliganded TR Functions
A, Unliganded repression at an activating TRE. A reporter construct
consisting of two copies of the F2 element from the chicken lysozyme
gene (35 ) cloned upstream of the TK -109/+45 promoter,
linked to the LUC gene was examined after transfection into HeLa cells.
Compared with the empty CMX vector, cotransfection of a vector
expressing the WT hTR causes repression of LUC expression.
Cotransfection with vectors expressing mutant hTRß (as indicated)
resulted in variable loss of basal repression. Additional mutations
tested, but found to have no effect on unliganded repression include
E213R, D216A, E217R, E220R, T224R, Q235R, K240A, Q241R, F245K, L266K,
T281R, D285A, K289A, P291R, M292K, C294K, E295R, C298R, E299A, M334K,
Y389K, H413R, and Y414K. B, Unliganded activation at a negatively
regulated promoter. A reporter consisting of the TSH gene promoter
linked to LUC was transiently transfected into TSA-201 cells. Compared
with the empty CMX vector, cotransfection of a vector expressing the WT
hTR causes activation of LUC expression. Cotransfection with vectors
expressing mutant hTRß (as indicated) resulted in variable loss of
basal activation. C, Correlation between relative effects of mutations
on binding to N-CoR RIDs I, II, and III on relative unliganded
repression at the activating promoter (F2)2-TK109-LUC (from experiment
of panel A, but with the activation of WT set to 100 and each mutant
normalized accordingly). The correlation for each RID was significant,
with r2 values of 0.52, 0.59, and 0.62 for RIDs I, II, and
III, respectively. D, Correlation between relative effects of mutations
on binding to N-CoR RIDs I and II and on relative unliganded activation
at the repressing promoter TSH -LUC (from experiment of panel B, but
with the activation of WT set to 100 and each mutant normalized
accordingly). The correlation for each RID was significant, with
r2 values of 0.44, 0.66, and 0.56 for RIDs I, II, and III,
respectively.
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Corepressor-TR complexes are also thought to activate promoters whose
expression is inhibited by liganded TRs, such as the promoter for the
TSH gene (3). With the TSH promoter, overexpression of
N-CoR increases unliganded activation, and the P214R and the AHT
mutants, which block N-CoR binding, abolish unliganded activation
(3). We examined functional properties of TR mutants in
TSA-201 cells using the hTSH
promoter linked to LUC. The unliganded
TR increased LUC expression from this promoter by 2.5-fold, and
mutations that impair N-CoR binding also were deficient in this
unliganded activation (Fig. 5B
). There was a significant overall
correlation between defects in binding and this function
(r2 values of 0.44, 0.66, and 0.56 for RIDs I,
II, and III, respectively), although again, the L422R mutation was an
outlier (Fig. 5D
). There was also a significant correlation when the
relative TRE repression and relative TSH promoter activation results
for the various mutants were compared (r2 value
of 0.81, plot not shown), consistent with both functions requiring
interactions with corepressor. Thus defects in N-CoR binding have
functional importance in vivo, and regulation of the TSH
promoter by unliganded TR may require receptor dimers or some other
partner using this surface.
Specificity of Conformation Defects by Mutation of Sites 2 and
3
It has been suggested that the AHT triple mutation of buried
residues of helix 1 may decrease corepressor binding indirectly through
a change in conformation of the unliganded TR (36). The
AHT mutations are near the mutation-sensitive Site 2 alongside helix 1,
and therefore mechanism(s) for decreasing N-CoR binding may be similar
for all these mutations. Residues of Sites 2 and 3 are on the surface
of the hormone-bound TR LBD (21), making it less likely
that mutations at these sites cause strong global structural changes.
However, no unliganded TR structure has been determined, and the
disposition of the residues of Sites 2 and 3 could be different in the
unliganded conformation required for corepressor binding. Therefore it
is not obvious whether Sites 2 and 3 represent alternate direct binding
sites for N-CoR or whether the effects of mutations at these sites
involve structural changes that indirectly affect N-CoR binding to Site
1.
To test whether mutational defects in N-CoR binding might be mediated
by global structural changes, we examined abilities of the AHT triple
mutation and TR mutations from Site 1 (I280M and I302R), Site 2
(W219K), and Site 3 (R429A) to function in other capacities (Fig. 6
). Parts of Sites 1, 2, and 3 occur in
the TR LBD subdomain that does not bind hormone; therefore, as a
comparative control, mutation W418K of a partially buried residue of
helix 11 was created to intentionally destabilize this same subdomain.
The W418K mutation (displayed separately in Fig. 6
) did not diminish
binding to N-CoR (Fig. 4A
). All the mutants showed good binding of
[125I]T3 (Fig. 6A
),
although all exhibited a decreased affinity relative to WT TR.
Therefore, each mutation appears to have some global effect. The worst
[125I]T3 binder was the
AHT mutation with a Kd of 600 pM.

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Figure 6. Receptor Conformation Likely Does Regulate N-CoR
Binding, but the Effects Are Specific; Control Mutations Can Perturb
the Receptor and Not Affect N-CoR Binding
Six different TR mutants, including the I280M and I302R mutations of
Site 1, the AHT and W219K mutations of Site 2, and the R429A mutation
of Site 3, were compared in their effects to perturb receptor
functions. The W418K mutation of helix 11 (Fig. 4B ) is included as an
example of a mutation that does not affect N-CoR binding. A, Binding of
[125I]T3 to full-length TRs produced by
in vitro translation. Results are expressed as the
percentage of Ka observed for the TR WT. For each receptor
mutant, the Kd values (pM ± SD) are: WT
(35 ± 5), AHT (296 ± 30), W219K (71 ± 9), I280M
(240 ± 13), I302R (51 ± 6), R429A (138 ± 12), W418K
(137 ± 15). B, Binding of [35S]-labeled hTRß
mutants to GRIP1 (residues 5631,121). GST pull-down results are
expressed as the percentage of binding observed for the WT hTRß in
the same assay. Binding was performed in 30 µM
T3, and the TR WT binding was 77% of input. Results are
the average ± SD of duplicates and are representative
of at least two experiments. C, T3 activation after
transfection of TR WT and mutants vectors into HeLa cells. The reporter
consisted of two copies of the F2 element from the chicken lysozyme
gene (35 ) cloned upstream of the TK -109/+45 promoter,
linked to the LUC gene. Results are expressed as the percentage of fold
activation by 10 µM T3 of LUC expression for
the WT (47-fold). Empty CMX vector fold activation was 1.5-fold (data
not shown). Results are the average ± SD of
triplicates and are representative of two experiments.
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Effects of these selected mutations on binding to GR-interacting
protein 1 (GRIP1) were also assayed (Fig. 6B
), using excess hormone (50
µM). The I302R mutation, which participates in the p160
coactivator-binding surface (31), showed only background
binding. By contrast, the I280M and I280K (data not shown) mutations
that lie underneath helix 12 in the liganded TR conformation, and which
strongly block N-CoR binding to Site 1 (Fig. 1B
), showed almost no
defect in GRIP1 binding. These results are consistent with a model in
which hormone binding causes I280 to be covered by helix 12, so that
the corepressor binding Site 1 is blocked, and the coactivator binding
surface is created. Thus, mutation of the buried part of the
corepressor surface allows the best discrimination between corepressor
and the coactivator binding by the TR. Mutations from Sites 2 and 3 and
the control mutation all showed similar partial defects in GRIP1
binding, implying that these mutations have similar global defects in
recruitment of coactivator. A similar pattern was obtained for hormone
activation by these mutant receptors in transfection assays (Fig. 6C
);
the I302R mutant was inactive, but other mutants from Sites 2 and 3 and
the control W418K mutant, although capable of good activation, were
similarly slightly deficient. Thus, the mutations in Sites 2 and 3
exhibit indications of global structural changes, but no more so than
the control W418K mutation that had no effect on N-CoR binding.
Mutation of Site 2 Alters N-CoR Recognition by the TR LBD
Mutation of Site 1 diminished N-CoR binding to levels close to the
control GST protein, whereas the mutations of Sites 2 and 3 only
partially blocked binding. We asked whether binding that remains
in the W219K (Site 2) mutant could be blocked by secondary mutations of
Site 1 and found the secondary Site 1 mutations to be surprisingly
ineffective (N-CoR RID I; Fig. 7
).
Although low binding was observed for some of the single Site 1
mutants, these same mutants showed higher binding when present as
double mutants coupled with the W219K (Site 2) mutation. Thus,
mutation of residue W219 may cause a conformation change that affects
Site 1 recognition. A search was made for LBD residues outside of Site
1 that would more effectively block N-CoR binding in combination as
double mutants with W219K. The most effective of these secondary
mutations was R429A, which nearly completely blocked the remaining
binding (Fig. 7
and data not shown). When the Site 1 mutations were
combined with the R429A (Site 3) mutation, there was either no change
(V284R), a smaller increase (I280M), or increased (I302R) binding,
indicating that recognition of Site 1 by R429A is somewhat altered, but
mostly intact.

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Figure 7. Mutations in Sites 2 and 3 Alter the N-CoR
Recognition of Site 1
Binding of full-length [35S]-labeled hTRß mutants to a
mouse N-CoR protein fragment containing RID I (2,231/2,321) in GST
pull-down assays. The first bar represents binding by
the WT hTRß (typically 12% of the input), set to 100%. The
remaining bars represent binding by TRs with combined
mutations from Site 1 (I280M, V284R, and I302R), Site 2 (W219K), and
Site 3 (R429A), as indicated, with each expressed as the percentage of
binding observed for the WT hTRß. Results are the average ±
SD of duplicates and are representative of at least two
experiments. N-CoR binding is increased for all three Site 1 mutants
when combined with the Site 2 mutation, and N-CoR binding is increased
for the I302R mutant when combined with the Site 3 mutation.
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Representative mutants of Sites 1, 2, and 3 were also tested in the
context of the isolated TR LBD for effects of helix 12 deletion and
Triac addition (Fig. 8
). Deletion of
helix 12 caused a 5-fold increase in binding to N-CoR RID I, larger
than the 2.5-fold increase observed with full-length receptor, implying
that the N terminus and/or DBD affect binding. These effects may be
conformational because we could find no evidence of direct binding to
the N terminus or DBD using a mutation (A234X) that deletes the LBD
(data not shown). In the LBD the R429A (Site 3) mutation showed little
of the defect observed in the full-length TR, again implying that the N
terminus and/or DBD influence corepressor binding. In the LBD the W219K
(Site 2) mutation remained defective as for the full-length, and
deletion of helix 12 allowed little improvement, consistent with an
effect of Site 2 mutations to alter the recognition of Site 1. The
I302R (Site 1) mutation diminished binding of the LBD to background
levels, and deletion of helix 12 showed no improvement, consistent with
a requirement of residue I302 in contacting N-CoR RID I.

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Figure 8. Differential Effects on N-CoR Binding of Helix 12
Deletion and Ligand Addition in the TR-LBD Mutated at Sites 1, 2, and 3
Binding of [35S]-labeled hTRß LBD mutants to a mouse
N-CoR protein fragment containing RID I (2,231/2,321) in GST pull-down
assays. The first bar represents binding by the WT
hTRß LBD, expressed as percentage of labeled input. The
remaining bars indicate binding by TR LBDs, either WT or
mutated in Sites 1 (I302R), 2 (W219K), or 3 (R429A), and with deletions
of helix 12 (mutation to F451X) and treated with 50 µM
Triac, as indicated.
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Addition of Triac to the TRs with helix 12 deleted had different
effects for the three mutant types. The R429A mutant was fractionally
increased as for the WT TR, consistent with a lack of effect of this
mutant on binding of the LBD to RID I. The I302R mutant was little
changed by Triac addition, which would be predicted if residue I302 is
required for RID I binding. However, binding to the W219K mutant was
increased 6-fold, indicating that a portion of the decrease observed
with this mutation is due to a conformational effect that can be
corrected by ligand binding.
 |
DISCUSSION
|
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In this study we examined the mutational sensitivity of surface
residues of the TR LBD for impairment of in vitro binding of
full-length TR to each of the three RIDs of N-CoR. We mutated residues
representative of the entire LBD surface using the hormone-bound rat
TR
LBD (21) as a structural model. The results imply a
complex pattern of interaction between N-CoR and the TR, as reflected
by the fact that we found not one, but three sites of mutational
sensitivity (Sites 1, 2, and 3, Fig. 9
).
Site 1 overlaps partially the surface defined for coactivator binding
(31) but also extends underneath where helix 12 rests in
the ligand-bound conformation. Site 2 is separated from Site 1 by
helices 1 and 3 and runs along the side of helix 1; Site 3 is separated
from Site 1 by the turn between helices 9 and 10 and runs between
helices 10 and 11. Mutations in Site 1 nearly abolished binding, from
which we infer that Site 1 contacts corepressor directly. Mutations in
Sites 2 and 3 had partial effects, which could indicate that they form
weaker contacts to corepressor. Alternatively, Sites 2 and 3 could
function to define and maintain the presentation and stability of the
unliganded TR conformation required for corepressor binding.

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Figure 9. Model of the Nuclear Receptor LBD Indicating the
Surfaces and Conformation Changes That Appear to Regulate Corepressor
Binding
Ribbon diagram of the TR LBD. The residues of helices 3
and 5 comprising the inferred N-CoR interaction Site 1 are
blue at their Ca atoms. Two different conformations of
helix 12 are indicated; the conformation in the hormone-bound TR
structure (21 ) is cyan, whereas the
conformation modeled from the unliganded PPAR structure
(40 ) is green. In the hormone-bound
structure some of the residues of Site 1 are buried, whereas in the
unliganded model, Site 1 is fully exposed. The mutation-sensitive
surface residues of Site 2 occur behind helix 1 on the
right side of this view. The mutation-sensitive surface
residues of Site 3 occur above helix 11 on the
left side of this view.
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The current studies suggest that the mechanism of hormone-dependent
release of corepressor involves direct binding competition between
helix 12 and corepressor. This conclusion is derived from the
observation that residues buried by helix 12 in the
hormone-bound conformation form part of the corepressor-binding
surface of Site 1. In this model binding of the ligand induces a
conformation that favors occupancy by helix 12. This mechanism explains
previous studies that suggested a link between helix 12 conformation
and hormone-dependent corepressor release based upon the correlation of
increased protease attack of helix 12 in TRs with point mutations that
block the hormone-dependent release of corepressor (37),
the conversion of the TR into a constitutive repressor by deletion of
part of helix 12 (38), and the disruption of corepressor
binding by surfaceexposed residues near helix 12
(23, 24, 25).
The data demonstrate that helix 12 can regulate corepressor binding by
nuclear receptors in the unliganded state through its affinity for Site
1. This is based on the observation that deletion of helix 12 in the
unliganded TR increases corepressor binding by N-CoR RIDs I and II and,
in the RXR, has a profound effect on corepressor binding
(25), confirmed here. In some receptors, competition by
helix 12 might be strong enough to preclude the use of corepressors as
coregulators in the cell. However, it is conceivable these nuclear
receptors interact with corepressors in vivo if mechanisms
exist that reduce the competitive effect of helix 12, such as the
ability of helix 12 to be drawn away from one receptor by other
receptor molecules in a complex (39), or in
vivo proteolysis of helix 12.
Although binding of corepressor to Site 1 requires displacement of
helix 12 from its liganded conformation, the mapping of Site 1
indicates that the extent of required displacement is not large.
Complete extension of helix 12, as reported for unliganded RXR
(29), is probably not required. Sliding of helix 12 down
along helix 3, as reported for unliganded PPAR (40), would
provide sufficient exposure, as illustrated in Fig. 9
. Helix 12 could
also be uncoiled in the absence of ligand, because, even when
stabilized by ligand, the helix 12 of crystallized TR shows a B-factor
higher than the average for the entire LBD (21). It has
been predicted (24) that the IxxII motifs within
corepressors that interact with receptors (23, 25) extend
longer as amphipathic helices, compared with the LxxLL motifs of
coactivators. This prediction is consistent with our observation that
the corepressor-binding surface extends beyond that for
coactivator.
The residues of Site 1 are homologous between members of the nuclear
receptor superfamily, deriving from the signature motif sequences
between helices 3 and 5 (33). This observation suggests
that corepressors might dock to Site 1 of various nuclear receptors
more ubiquitously than previously thought. This is supported by our
observation that mutation of the residues of Site 1 in the ER and RXR,
including those under helix 12, also impaired corepressor binding.
Interestingly, previous mutation of this region in the orphan nuclear
receptor RevErb was found to impair corepressor binding
(17). This receptor lacks helix 12, and the role of helix
12 in restricting corepressor binding was not derived from this study.
Site 1 may be used generally, so long as it has a sufficient exposure
to, and affinity for, the corepressor.
Despite similarities between different nuclear receptors in Site 1,
receptors also show selectivity for the separate corepressor RIDs. Our
results indicate that the shape of the Site 1 surface is one
determinant of this selectivity. Whereas the helix 12 deleted
unliganded RXR and tamoxifen-liganded ER bound RID I, they did not bind
RID II, demonstrating that features other than helix 12 define
selectivity. By contrast, TR exhibited equal binding to N-CoR RIDs I
and II. We further observed that conservative changes in the residues
of TR Site 1, such as occur naturally between different nuclear
receptors, affected binding uniquely to N-CoR RIDs I, II, and III,
either decreasing or increasing the affinity of receptor binding. Thus,
the residues of Site 1 partially determine the selectivity. However,
none of the single point mutations of Site 1 conferred selectivity as
large as that observed for the RXR or ER, and no mutation reversed the
preference of TR to bind better to RID III than I or II, indicating
that either more than one mutation of Site 1 or contacts to or changes
outside of Site 1 are required to dictate the complete selectivity
observed when the natural receptors are compared. When previous reports
on receptor selectivity are compared with the current results, it
should be noted that differences often exist in the boundaries defined
for the LBD-interacting RIDs of N-CoR (27), and that
additional contacting surfaces of receptors and corepressors may yet be
elucidated in the future (41) that could influence binding
results in an assay-specific fashion.
Our study adds insights about the CoR box, which was originally
reported to be the TR site for interactions with corepressors
(4). The current results extend those of previous reports
(24, 42), which found that mutations of the surface
residues of helix 1 lying directly above the CoR box did not affect
corepressor binding, indicating that the CoR box, as postulated, does
not bind corepressor. We extended these studies by testing mutations at
every surface residue of helix 1 and found one mutation (W219K) that
diminished corepressor binding. Residue W219 on helix 1 forms part of
surface Site 2. Thus, the previous mutations supporting the existence
of the CoR box may have perturbed the receptor structure in a way that
affects Site 2.
The mechanisms for decreased corepressor binding due to mutations of
Sites 2 and 3 remain unclear. Some residues of Sites 2 and 3 are unique
to TR, but it is notable that residues D366 and R429 are relatively
conserved within some nuclear receptor family members
(33), consistent with roles in either supporting the TR
structure or providing protein-binding interfaces. The Site 2 and 3
mutants showed only small defects in both ligand binding and
coactivator binding that are comparable to defects of another TR mutant
(W418K) that is not defective in N-CoR binding. These results are
similar to previous reports that natural TR mutants found in patients
with the syndrome of Resistance to Thyroid Hormone
(43, 44, 45, 46, 47) generally show some ligand-binding defects, but
no loss of corepressor binding. Thus we conclude that if the effects of
mutations at Sites 2 or 3 on N-CoR binding are due to structural
perturbations, these perturbations are specific. More evidence
supporting a mechanism whereby Site 2 and 3 mutations are required to
specifically maintain the unliganded conformation essential for
presentation of Site 1 as the primary N-CoR interaction surface was
revealed by the observations that mutations of Sites 2 or 3 can alter
the mutational sensitivity of Site 1, and that under some conditions
ligand can reverse the effects of the Site 2 mutation.
Although conformation mechanisms likely contribute to the
corepressor-binding defects of Sites 2 and 3, a role for these surfaces
as direct contact sites should also be considered. Site 2 is separated
from Site 1 by one-third the circumference of the LBD (
30 Å),
whereas Site 3 is closer (
10 Å distance between residues R429 and
C309). The TR LBD has a molecular mass of 32 kDa, whereas the fragments
of N-CoR used to make the fusion proteins for GST pull-down experiments
are 9.1, 8.8, and 16.4 kDa for RIDs I, II, and III,
respectively. It seems unlikely the smaller N-CoR fragments
could contact Sites 1 and 2 simultaneously without making contacts to
the intervening residues of helices 1 and 3 simultaneously. By
contrast, it seems possible for the N-CoR fragments to contact Sites 1
and 3 simultaneously. Such a model would be consistent with our finding
that the N-CoR binding activity remaining in the partially defective
W219K (Site 2) mutant requires the integrity of residue R429A (Site 3)
more than it requires the residues of Site 1. The N-CoR binding
activity remaining in the R429A (Site 3) mutant is abolished by either
the W219K (Site 2) mutation or by some of the Site 1 mutations. Thus,
the Site 3 mutations could act through both conformational and direct
binding mechanisms.
Conceivably, Sites 2 and 3 could be interaction sites for other
molecules that modulate N-CoR binding through allosteric mechanisms.
The [35S]-labeled TRs used for the binding were
produced using reticulocyte lysates, and therefore numerous candidates
for putative allosteric regulators would have been present in our
pull-down assays. The effects of the Site 2 and 3 mutations primarily
appear not to be mediated through effects on the N terminus or DBD,
because deletion of these domains generally did not remove the effects
of the Site 2 or 3 mutations. However, the R429A (Site 3) mutation
failed to affect the binding to RID I in the context of the LBD, so
some role of the N terminus or DBD cannot be ruled out.
Effects of the mutations on the in vivo functions of the
full-length unliganded TR were assayed using both
T3-activated and
T3-repressed promoters. All mutations of Sites 1,
2, and 3 that diminished in vitro N-CoR binding also caused
correlated defects in unliganded repression and unliganded activation,
and these two functions were also highly correlated when compared
across the set of mutants. These results imply that the binding defects
measured in vitro are also responsible for defects in
corepressor binding and action measured in vivo.
Interestingly, one mutation that did not affect N-CoR binding in
vitro, L422R, strongly diminished the unliganded TR in
vivo functions. L422R strongly affects stability of both TR dimers
and heterodimers with RXR (34). N-CoR binding by DNA-bound
TRs has been reported to require homodimers (48). Thus it
is possible that the in vivo defects observed with L422R
indicate that formation of a TR dimer is essential for corepressor to
function at these promoters. As the TSH promoter contains no strong
binding site for the TR, the actions of such a putative dimer would
likely occur off the promoter DNA in this case.
N-CoR binding by the helix 12-deleted TR and ER was surprisingly
stimulated by ligand binding. N-CoR binding by the ER with helix 12
deleted was barely detectable unless tamoxifen was bound. In the case
of TR with helix 12 deleted, the agonist Triac stimulated N-CoR binding
by 30%. These observations provide evidence that influences on the
receptor extrinsic to helix 12 can regulate N-CoR binding. Tamoxifen
releases ER from its associated chaperone proteins that are present in
the reticulocyte lysates used to produce the radiolabeled receptors
(49). Thus, this release could free the receptor from
inhibitory influences of these proteins and thereby stimulate N-CoR
binding. However, the unliganded TR is not known to associate with
proteins other than corepressors. Thus, an alternative explanation is
that tamoxifen and Triac could stabilize the LBD in a manner that
favors N-CoR binding. The result that binding by the W219K/F451X mutant
to N-CoR RID I was stimulated 6-fold by Triac suggests the defect
caused by the W219K (Site 2) mutation can be appreciably corrected by
addition of ligand. Thus there could be a continuum of dynamic states
of the TR with different capacities of corepressor binding. In such a
model, specifically located mutations, such as those of Site 2, could
perturb the receptor conformation to disable binding, but other
influences, such as ligand binding or, conceivably, allosteric effects
with other domains of the TR or with TR dimerization partners, could
stabilize conformations to improve binding.
The current studies also suggest pharmacological approaches to modulate
selectively nuclear receptor functions through regulating corepressor
binding. Ligands that bind to the combined coactivator and corepressor
surface might impair or stimulate both corepressor and coactivator
action. Other ligands that bind to the corepressor-binding surfaces
might selectively impair or stimulate corepressor function. The fact
that mutations such as C309K and I280M have a much more profound affect
on corepressor than coactivator binding, despite their localization in
a region where they could impair packing of helix 12, implies that such
an approach might be successful. Ligands that bind in the
hormone-binding cavity might also be used for selective regulation. In
addition to the tamoxifen-stimulated binding of corepressors, it has
been reported that different retinoids vary in their capacity to
release corepressors (50). These results, coupled with our
finding that ligands can enhance corepressor binding by helix
12-deleted TRs, suggest that in some contexts ligands that range in
effect from corepressor recruitment to release for the same receptor
may be possible.
 |
MATERIALS AND METHODS
|
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Mutagenesis
The pCMX vector was used for expression of the full-length
hTRß and hRXR
, and the pSG5 vector was used for expression of the
full-length human ER
(hER
). Mutations within these nuclear
receptor-encoding sequences were created using the methods described in
the quick-change kit (Stratagene, La Jolla, CA). The same
mutagenesis methods were used in protocols to engineer the
Escherichia coli expression vectors encoding fragments of
N-CoR and TR. For the GST-N-CoR RID I, the sequences encoding amino
acids 2,2312,321 were cloned in frame to the C terminus of the 26-kDa
GST protein of the GEX-2T vector (Pharmacia Biotech,
Piscataway, NJ) between modified linker sequences encoding His-Met and
encoding Val-Ala-His-His-His-His-His-His. An NdeI site
encodes the His-Met and a SalI site encodes the Val-Ala. The
appropriate NdeI and SalI sites were introduced
into the mouse N-CoR cDNA sequence using sequential mutagenesis steps.
The same approach was used to make the GST-N-CoR RID II (encoding
residues 2,0342,114) and GST-N-CoR RID III (encoding residues
1,8882,031). Verification of the target sequences as well as the
flanking sequences was performed using both Sequenase kits
(Stratagene), and by automated DNA sequence (UCSF Cancer
Center Sequencing Facility).
GST Pull-Down Assay
The vectors encoding the GST-N-CoR-His fusion proteins of the
three N-CoR RIDs were used to transform the bacterial strain BL21,
using selection with ampicillin. One-liter cultures were grown using
2x LB medium to an OD600 of 0.6, and expression
was induced with room temperature shaking by addition of 0.5
mM isopropyl-ß-D-thiogalactopyranoside, for
3 h. Cells were harvested by centrifugation and after storage at
-80 C, extracted with 20 ml TST buffer [50 mM Tris, pH
7.5, 150 mM NaCl, 0.05% Tween-20, with 0.02%
monothioglycerol and 20 µM phenylmethylsulfonyl fluoride
(PMSF) added]. The lysate was centrifuged for 1 h at 17,000 rpm
in a JA20 rotor (Beckman Coulter, Inc., Palo Alto, CA),
and the lysates were mixed for 1 h at 4 C with 0.4 ml Talon Metal
affinity resin (CLONTECH Laboratories, Inc., Palo Alto,
CA) in the presence of 2 mM imidazole. Beads were
collected by low-speed centrifugation and washed twice batchwise with
10 ml buffer S (50 mM NaPO4, pH 8,
300 mM NaCl, and 10% glycerol, with 2 mM
imidazole added). Beads were then transferred to a 5 ml Econo column
and washed with 2 ml buffer S containing 2 mM imidazole.
The GST-N-CoR-His proteins were then eluted with 2 ml buffer S
containing 100 mM imidazole. Metal affinity-purified
GST-N-CoR His-tagged fragment (1.5 mg) was mixed for 1 h with 150
µl of glutathione Sepharose (Pharmacia Biotech), then
washed three times with 1 ml TST buffer with 1 mM
dithiothreitol added, and brought up to 150 µl of a 50% slurry,
typically containing 5 mg/ml protein.
For studies of binding to the GST-N-CoRs,
[35S]-Metlabeled nuclear receptors were
made using 1 µg of each receptor-encoding plasmid in 20 µl
reactions using TNT kits (Promega Corp., Madison, WI). For
each experiment, equivalent amounts (15 fmol) of labeled receptors were
diluted in 150 µl binding buffer (20 mM HEPES, pH 7.9,
150 mM KCl, 25 mM MgCl2,
10% glycerol, 0.1% Triton X-100, 0.1% NP-40, and 0.01%
dithiothreitol), of which duplicate 10-µl portions (20% of the
binding reaction inputs) were quantified by SDS-PAGE and
phosphorimaging (Molecular Dynamics, Inc., Sunnyvale, CA).
Binding buffer (100 µl) containing 4 µl of the glutathione
Sepharose-bound GST-N-CoR-His was added to duplicate 50-µl portions
of the diluted labeled receptors and mixed by mechanical inverting for
2 h at 4 C. The beads were washed three times with 1 ml binding
buffer with brief microcentrifugation in between. The washed beads were
boiled 3 min in SDS sample buffer and quantified by SDS-PAGE and
phosphorimaging.
Mammalian Cell Culture and Transfection
The unliganded functions of the TR were analyzed by comparing
the effects of a CMX vector encoding the WT and mutant receptors with
the effects observed for the empty parent CMX vector. For unliganded
repression studies, a LUC reporter plasmid was cotransfected that
contained two copies of the F2 TRE from the chicken lysozyme gene
(35) linked to the herpes simplex virus TK promoter
sequences extending from -109 to +48 of the transcription start site.
For these studies of the F2-TK109 promoter, CV-1 cells were harvested
from DMEM-H21 supplemented with 10% FBS and plated at 7,000
cells per well. The cells were washed 2 h later with 2 ml PBS, and
new medium was added containing serum stripped of thyroid hormone using
Dowex AG X-8 ion exchange resin (Bio-Rad Laboratories, Inc., Hercules, CA) (51). One and one-half hours
later, 0.5 ml suspension of DNA vectors precipitated with calcium
phosphate was added [1.7 µg LUC reporter, 80 ng CMX, or CMX-TR
expression vector, and 500 ng control ß-galactosidase vector]. After
15 h cells were washed twice with 2 ml DME-H21 and fresh DME-H21
containing stripped serum was added. After an additional 24 h,
cells were harvested in 200 ml lysis buffer and assayed for LUC (kit
from Promega Corp.) and ß-galactosidase (kit from
Tropix, Inc., Bedford, MA) activities.
For unliganded activation studies, a reporter consisting of the TSH
gene promoter linked to LUC (3) was transfected by the
calcium phosphate method (3) into TSA-201 cells and grown
in DMEM (Nikken Biomedical Laboratory, Kyoto, Japan) with 10%
Dowex resin-stripped FBS, penicillin (100 U/ml), and streptomycin (100
µg/ml). After exposure to the calcium phosphate-DNA precipitate for
8 h, DMEM with 10% Dowex resin-stripped FBS was added. Cells were
harvested after 40 h for measurements of LUC activity.
For T3 activation studies, a LUC reporter plasmid
was used containing two copies of a F2 thyroid response element linked
to the herpes simplex virus TK -109/+48 promoter. HeLa cells were
cultured and transfected as described previously
(31).
T3 Binding Assay
Affinity measurements for
125I-T3 binding to hTRß
mutant proteins produced by in vitro translation were
performed as described previously (52).
 |
FOOTNOTES
|
---|
This work was supported in part by a grant-in-aid for scientific
research (no. 11671103) from the Ministry of Education, Japan (to
T.T.), and by NIH Grants DK-53417, DK-51281, and DK-41842.
1 Current address for A.M., H.N., and B.L.W.: Plexxikon, Inc., 91
Bolivar Street, Berkeley, California 94710. 
2 J.D.B. has proprietary interests in and serves as a consultant and
Deputy Director to Karo Bio AB, which has commercial interests in this
area of research. 
Abbreviations: DBD, DNA-binding domain; GRIP,
GRinteracting protein; GST,
glutathione-S-transferase; h, human; LBD, ligand-binding
domain; LUC, luciferase; N-CoR, nuclear receptor corepressor; RID,
receptor interaction domain; SMRT, silencing mediator of retinoid and
thyroid receptor; TK, thymidine kinase; TRE, thyroid response element;
WT, wild type.
Received for publication February 26, 2001.
Accepted for publication October 3, 2001.
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