From the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH and § Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
Received for publication, October 16, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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An Ligand-dependent gene expression mediated by nuclear
receptors involves the recruitment of transcriptional co-activators to the ligand binding domain
(LBD).1 The LBD is
structurally conserved among the NR family and consists of between 10 and 12 We have previously shown that a short The crystal structure of the LBD of PPAR In this study we have defined the minimal or core LXXLL
sequence capable of binding to NRs. By comparing the interactions of
LXXLL core motifs derived from three different co-activators with a panel of NR LBDs, we have identified elements of the
LXXLL core sequence that influence the affinity and
selectivity of co-activators for NRs.
Plasmid Constructions--
The two-hybrid vectors pBL1 and pASV3
(2) expressing the human ER Two-hybrid Interaction Assays--
The yeast reporter strain
used for all two-hybrid assays was W303-1B (HML Immunodetection of Proteins--
The expression of DBD fusion
proteins in yeast cell-free extracts was monitored by immunodetection
using a monoclonal antibody recognizing the human ER (a gift from P. Chambon, Strasbourg). The antibody recognizes the "F" region of the
LBD in the human ER and also the F region tag at the N termini
of the DBD fusion proteins (26). Equal amounts of protein were
separated by SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose for Western blotting. A peroxidase-linked goat
anti-mouse IgG was used to detect the primary antibody.
GST Pulldown and Peptide Inhibition Assays--
GST fusion
proteins were expressed in Escherichia coli DH5 Definition of a Minimal Functional LXXLL Sequence--
We have
previously shown that a 14-residue synthetic peptide corresponding to
the C-terminal LXXLL motif (motif 4) of SRC1e efficiently
competed the ligand-dependent binding of in
vitro translated SRC1 proteins to the LBD of ER LXXLL Motifs in SRC1 Display Differential Affinities for the LBDs
of Nuclear Receptors--
The human SRC1 protein is expressed in a
variety of cell lines as two major isoforms (SRC1a and SRC1e) which
differ at their C termini (16). Both isoforms contain the nuclear
receptor interaction domain (NID) consisting of three LXXLL
motifs (motifs 1-3) that are conserved in other p160 co-activators.
The SRC1a isoform contains a 4th motif at the extreme C terminus of the
protein that is responsible for the ability of this region of SRC1a to
interact with nuclear receptors (Figs. 1 and
2 and Refs. 3, 16, and 30).
Our previous results showed that upon addition of ligand, motifs 1-4
of SRC1 bind strongly to the LBD of ER High and Low Affinity LXXLL Motifs in RIP140--
We compared the
interaction of RIP140 motifs L1 to L9 with the LBDs of ER Low Affinity LXXLL Motifs Mediate the Interaction of CBP with
NRs--
Several groups have observed ligand-dependent
binding of N-terminal fragments of CBP to NRs (31-34). We compared the
ability of in vitro translated SRC1 or CBP full-length
proteins to bind to the LBD of ER
Weak interaction of the N terminus of CBP with RAR
To examine interactions of CBP with NRs in more detail, PCR products
spanning the regions 1-101, 345-381, and 1981-2165 (shown schematically in Fig. 4A) were
fused in frame with a heterologous DBD. These constructs were used in
yeast two-hybrid experiments to test for interactions with the LBDs of
ER
To confirm this weak interaction in vitro, the CBP-(1-101)
sequence was fused in frame with GST and expressed in E. coli for use in pulldown experiments. As shown in Fig.
4C, CBP-(1-101) bound to full-length ER
The second N-terminal fragment CBP-(345-381) also displayed weak
ligand-inducible interaction with the LBDs of ER
We next determined whether the CBP LXXLL core motifs
(designated CBP LXM-1-3) could bind the LBDs of steroid and retinoid receptors. We had previously demonstrated that CBP LXM-1 interacted weakly with the LBD of ER
The fold activation of reporter activities achieved with the
LXM-1 and LXM-3 core motifs (Fig. 5A) was slightly higher
than those obtained due to interaction of LBDs with DBD-CBP-(1-101) (Fig. 4B) and DBD-CBP-(1891-2165) (Fig. 4D). As
it has been shown that flanking sequences can influence the affinity of
LXXLL motifs for NRs (47, 50), this may indicate that core
sequences from CBP have a slightly increased affinity for steroid and
retinoid receptors compared with larger CBP fragments. Nonetheless, as shown in Fig. 5B, the reporter activity due to interaction
of LXXLL motifs derived from SRC1 with the LBD of the PR was
50-100-fold higher than that achieved with CBP motifs. This was not
due to lower expression of the CBP fusion proteins, as shown by Western blots (Fig. 4C). Taken together, our results indicate that
LXXLL motifs at the N and C termini of CBP and p300 account
for the low affinity binding of these proteins to steroid and retinoid receptors.
In this study we have defined the core LXXLL motif as
eight amino acids, requiring two residues N-terminal to the first
conserved leucine (+1) and at least one residue C-terminal to the 3rd
conserved leucine (+5) (Fig. 6).
Consistent with this, it has been previously observed that peptides of
13 and 14 amino acids in length encompassing motif 2 of GRIP1 strongly
compete the interaction of the GRIP1 NID with the TR-helical motif containing the sequence
LXXLL is required for the ligand-dependent
binding of transcriptional co-activators to nuclear receptors. By using
a peptide inhibition assay, we have defined the minimal "core"
LXXLL motif as an 8-amino acid sequence spanning positions
2 to +6 relative to the primary conserved leucine residue. In yeast
two-hybrid assays, core LXXLL motif sequences derived from
steroid receptor co-activator (SRC1), the 140-kDa receptor interacting
protein (RIP140), and CREB-binding protein (CBP) displayed differences
in selectivity and affinity for nuclear receptor ligand binding
domains. Although core LXXLL motifs from SRC1 and RIP140
mediated strong interactions with steroid and retinoid receptors, three
LXXLL motifs present in the global co-activator CBP were
found to have very weak affinity for these proteins. Core motifs with
high affinity for steroid and retinoid receptors were generally found
to contain a hydrophobic residue at position
1 relative to the first
conserved leucine and a nonhydrophobic residue at position +2. Our
results indicate that variant residues in LXXLL core motifs
influence the affinity and selectivity of co-activators for nuclear receptors.
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ABSTRACT
INTRODUCTION
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-helices folded in a globular domain (1). A central
hydrophobic pocket accommodates the cognate ligand, which upon binding
induces a conformational change in the LBD, exposing a co-activator
docking site on the LBD surface. Sequence conservation, mutational
analyses, and crystal structures have indicated that the co-activator
docking site is made up of residues from helices 3, 5, and 12, which
form a hydrophobic channel conserved among the NR family members. The
integrity of this surface is essential for co-activator binding and as
a consequence the transactivation function (AF-2) of the LBD (reviewed
in Ref. 2).
-helical sequence, the
LXXLL motif, is necessary and sufficient for mediating the
interaction of the co-activators RIP140, SRC1 and CBP/p300 with NRs
(3). LXXLL motifs have also been shown to mediate binding of
other co-activators including TIF1 (4), TIF2/GRIP1 (5, 6), p300/CBP-interacting protein/ACTR (7, 8), TRAP220/DRIP205 (9-11),
PPAR
co-activator-1 (12), and ASC-2/RAP250/NRC (13-15) to NRs. The
number and sequence of LXXLL motifs varies considerably among the co-activators and is likely to account for observed differences in binding of co-activators to selected NRs or classes of
NRs. The 160-kDa co-activators represented by SRC1, TIF2, and p/CIP
each have a nuclear receptor interaction domain (NID) containing three
LXXLL motifs. These sequences and the spacing between them are highly conserved, and we and others (3, 5, 7, 16, 17) have shown
that they mediate high affinity binding to NRs. In addition, the SRC1a
isoform has an additional LXXLL motif at its C terminus
(16). The 140-kDa protein RIP140 contains nine functional
LXXLL motifs that show variable affinity for the LBD of
ER
(3). We have also shown that CBP (and its paralog p300) contains
two conserved LXXLL motifs near the N terminus of the protein, which account for its weak interaction with ER and RAR (3).
Some nuclear receptor-binding proteins such as NR-binding SET
domain-containing protein contain variant motifs such as
FXXLL (18). In addition, a leucine-rich helix
(LXX(I/H)IXXX(I/L)) termed the co-repressor of
nuclear receptor box mediates the binding of the silencing mediator of
the co-repressors for retinoid and thyroid hormone receptors (SMRT) and
nuclear receptor co-repressor (NCOR) proteins to the unliganded forms
of NRs (19-21).
in complex with a portion
of the NID of SRC1 revealed that LXXLL motifs adopt an
-helical conformation that binds the AF-2 surface of the liganded LBD (22). A similar arrangement was observed in the crystal structures
of the liganded TR
and ER
LBDs complexed with a peptide corresponding to motif 2 of GRIP1 (6, 23). The three conserved leucines
were found to align on the face of the
-helix which packs against
the hydrophobic channel on the LBD surface. Two charged residues that
are highly conserved among LBDs (a glutamic acid in helix 12 and a
lysine residue in helix 3) and that are especially critical for AF-2
function appear to act as a charge clamp to hold the LXXLL
-helix in position (22). In all three structures, both AF-2 surfaces
in the LBD homodimer were occupied with one LXXLL
-helix.
The PPAR
/SRC1 NID structure indicates that, as a consequence of
having multiple LXXLL motifs, a single p160 co-activator
protein can contact both AF-2 surfaces in NR dimers. Consistent with
this, we and others (5, 16, 24, 25) have found a requirement for at
least two functional motifs to support high affinity binding of SRC1
and TIF2/GRIP1 NIDs or full-length proteins to steroid receptors.
Similarly, the co-activator TRAP220/DRIP205, which was isolated on the
basis of interaction with class II receptors TR and VDR, contains two
functional LXXLL motifs (NR-1 and NR-2), which have been
shown to make selective contacts with VDR and RXR in the VDR/RXR
heterodimer (10, 11). A strong interaction was identified between NR-2
and VDR, whereas a weaker interaction was observed between the NR-1
motif and RXR. In contrast, other putative co-activators appear to
contain a single functional LXXLL motif, e.g.
TIF1 (4) and ASC-2/RAP250/NRC (13-15). Thus, the number and sequence
of LXXLL core motifs, and possibly flanking sequences, will
influence the selectivity and affinity of co-activators for different NRs.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
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DNA binding domain (DBD) and the VP16
acidic activation domain (AAD), respectively, were gifts from R. Losson
and P. Chambon. The plasmid pCMV-
CBP was a gift from R. Goodman.
The plasmid pBSKS-mCBP-HA was a gift from A. Bannister and T. Kouzarides. DBD-LXXLL motif fusion proteins were generated
by ligation of phosphorylated, annealed oligonucleotide pairs into the
pBL1 vector. Fragments of RIP140 and CBP were generated by PCR using
elongase or Taq polymerase and cloned into pBL1 or a
modified GST-2TK vector containing cloning sites compatible with the
PCR constructs. The constructs AAD-RAR
(aa 200-462) and AAD-RXR
(aa 230-467) were generated by cloning PCR fragments into a modified
pASV3. All fusion constructs were fully sequenced. The constructs
pSG5-hSRC1e (16) GST-ER
LBD (previously termed GST-AF-2), AAD-ER
(aa 282-595), AAD-ER
Mut (ML-543/4-AA) (3), AAD-PR (aa 633-933),
and AAD-AR (aa 625-919) (27) have been described previously.
MAT
HMR
his3-11, 15 trp1-1 ade2-1
can1-100 leu2-3, 11 ura3) carrying the reporter plasmid
pRL
21-U3ERE, which contains a lacZ gene driven by
a modified URA3 promoter containing three estrogen response elements
(28). Transformants containing the desired plasmids were obtained by
selection for the appropriate plasmid markers and grown to late log
phase in 15 ml of selective medium (yeast nitrogen base containing 1%
glucose and appropriate supplements) in the presence or absence of up
to 1 µM of the appropriate ligand
(all-trans-retinoic acid; 9-cis-retinoic acid;
17-
-estradiol; R5020; mibolerone) or an equivalent volume of
vehicle. The preparation of cell-free extracts by the glass bead method
and the measurement of
-galactosidase activity in the extracts were
performed as described previously (28). Two-hybrid experiments were
performed three or more times, and reporter activities are expressed as nmol/min/µg protein.
using
isopropyl-
-D-thiogalactopyranoside induction as
described previously (3, 16). GST-Sepharose beads were loaded with GST
alone or GST fusion proteins prepared from bacterial cell-free extracts. 35S-Labeled mouse ER
and SRC1e proteins were
generated by coupled in vitro transcription/translation from
the plasmids pSP6-MOR (a gift from Roger White) and pSG5 hSRC1e (16)
and tested for interaction with GST proteins as described previously
(29). Binding was carried out overnight at 4 °C with gentle mixing
in NETN buffer (50 mM NaCl, 1 mM EDTA, 0.5%
Nonidet P-40, 20 mM Tris-HCl, pH 8.0) containing protease
inhibitors in the presence or absence of 1 µM estradiol
(E2) as required in a final volume of 1 ml. Peptides were dissolved in
sterile water at a concentration of 4 mg/ml and added to GST-binding
reactions at a final concentration of 50 µM, immediately
before the addition of ligand.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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in GST pulldown
experiments (3). This ability to compete the ER
/SRC1 interaction was
shown to be dependent on the integrity of the LXXLL motif,
as a peptide in which two conserved leucines (residues +4 and +5) were
replaced with two alanines failed to interfere with SRC1 binding to
ER
. In this study, we have used the peptide competition assay to
define the minimal LXXLL sequence required to bind the LBD
of ER
. A series of peptides spanning the high affinity binding motif
4 sequence were tested for their ability to compete the interaction of
SRC1e with GST-ER
LBD. As shown in Fig.
1, the ligand-dependent recruitment of SRC1e to GST-ER
LBD was strongly inhibited by the
peptides SLLQQLLTE and SLLQQLLT, respectively. However, omission of the
serine (LLQQLLTE) or threonine (SLLQQLL) residues at positions
2 and
+6 (relative to the first conserved leucine residue, designated +1)
resulted in a strong reduction in the ability of the resulting peptides
to compete SRC1e binding to ER
LBD. As expected, peptides in which
the LXXLL motif is disrupted or truncated showed no
competitive properties in these experiments. Similar results were
obtained using the SRC1a isoform (data not shown). These results are
consistent with our previous observation that as little as 8 amino
acids comprising the LXXLL motif is sufficient to bind to
the LBD of ER
in yeast two-hybrid assays (3) and define the core
NR-binding sequence as
2 to +6.
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Fig. 1.
Mapping the LXXLL core
motif. Glutathione-Sepharose-bound GST-ER LBD was incubated
with 35S-labeled in vitro translated SRC1e
protein in the presence or absence of 1 µM
17-
-estradiol (E2) as indicated. Binding was carried out
in the presence or absence of competitor peptides (final concentration
50 µM) as indicated. The sequences of the competitor
peptides are shown. The conserved leucine residues are
boxed, and the amino acids are numbered with
respect to the first conserved leucine residue.
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Fig. 2.
LXXLL motifs derived from
SRC1 display differential interactions with steroid and retinoid
receptor LBDs. Yeast two-hybrid experiment showing interaction of
LXXLL motifs from SRC1a with the LBDs of steroid and
retinoid receptors in yeast two-hybrid experiments. Control
(DBD) is the pBL1 vector containing the human estrogen
receptor DNA binding domain. Motifs 1-4 are bait constructs
in which the DBD is fused in frame with amino acids 632-642, 689-699,
748-758, and 1434-1441 of SRC1a, respectively. The prey vectors
consist of the acidic activation domain of VP16 (AAD) fused
in frame to the ligand binding domains (LBD) of estrogen
receptor (AAD-ER
), the androgen receptor
(AAD-AR), retinoic acid receptor
(AAD-RAR
)
and the retinoid X receptor (AAD-RXR
). Plasmids encoding
bait and prey proteins were co-transformed into the yeast strain L40
and cultured overnight in the presence or absence of 500 nM
of cognate ligand, E2, Mibolerone, all-trans-retinoic acid
(AT-RA), or 9-cis retinoic acid
(9C-RA). Reporter activity in cell-free extracts is
expressed as units of
-galactosidase activity. Shaded
columns indicate the reporter activity in the absence of ligand.
Black columns indicate the reporter activity in the presence
of cognate ligand.
with similar apparent
affinities, as determined by yeast two-hybrid experiments (3). In this
study, we compared the interactions of SRC1 LXXLL motifs
with the LBDs of hER
, hRAR
, mRXR
, hAR, and hPR receptors. As
shown in Fig. 2, motif 1 interacted strongly with the LBD of ER
as
expected but displayed a significantly reduced ability to bind to the
LBDs of RAR
and AR. In addition, the reporter activation due to
interaction of motif 1 with the LBDs of RXR
(Fig. 2) and PR (Fig. 5)
was consistently reduced in comparison to the other motifs. The motif 2 sequence displayed significant ligand-dependent interaction
with all LBDs tested, although a lower affinity for the AR LBD was
observed in comparison to other NR LBDs tested (Fig. 2). Motifs 3 and 4 exhibited strong interaction with the panel of LBDs tested in the
presence of ligand. However, motif 3 and to a lesser extent motif 4 displayed significant ligand-independent interaction with the LBD of
RXR
. The significance of the ligand-independent interactions with
RXR
LBD is unclear, as this was only observed in yeast two-hybrid
experiments. No significant ligand-independent interaction was observed
between GAL-RXR
and full-length SRC1, the intact NID or C terminus
of SRC1a in transiently transfected mammalian cells (51).
, RAR
,
and RXR
. As shown in Fig. 3, the
L1-L9 core motifs displayed differences in the strength of reporter activation due to binding to ER
LBD. In general, similar patterns of
interaction were observed for each core motif with the LBDs of ER
,
RAR
, and RXR
, with some exceptions as discussed below. By
comparison of the reporter activities induced in the presence of
ligand, we sought to determine how the sequences of individual core
motifs influenced the apparent strength of interactions with NR LBDs in
the two-hybrid system. Motifs showing strong NR binding in this assay
generally contained a hydrophobic residue at position
1, usually
leucine, valine, or tyrosine. However, the presence of a hydrophobic
residue at position +2 (as in motifs L3 and L5) appeared to reduce
affinity for NR LBDs. The motif L3 displayed weak interaction with the
LBDs of ER
and RXR
but negligible binding to the RAR
LBD. The
motif L5, on the other hand, displayed weak interaction with the LBDs
of ER
and RAR
but no detectable binding to RXR
. Similar
selectivity was observed using larger fragments of RIP140 spanning the
L3 or L5 motifs, in yeast two-hybrid experiments (data not shown).
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Fig. 3.
Differential interaction of
LXXLL motifs derived from RIP140 with the LBDs of
ER , RAR
, and
RXR
. Yeast two-hybrid experiments were
performed as in Fig. 2. DBD-L1 through DBD-L9 represent bait constructs
in which the DBD is fused in frame to RIP140 sequences, as indicated in
parentheses. Shaded and black columns
represent reporter activity in the absence and presence of 500 nM cognate ligand, respectively.
in GST pulldown experiments. SRC1e
exhibited strong binding to the LBD of ER
in the presence of
17-
-estradiol (Fig. 1). In contrast, full-length CBP showed only a
very weak ability to bind the ER LBD in similar experiments (51).
has been
localized to amino acids 1-101 (31, 32). In addition, a second region
comprising amino acids 356-495 has been reported to bind weakly to
RXR
, although this interaction was apparently independent of ligand
(32). Analysis of the sequence of CBP revealed it to contain three
potential LXXLL motifs (amino acids 68-78, 355-365, and
2067-2077), which are conserved in p300. Indeed, we have previously reported that the LXXLL motif located between amino acids 68 and 75 displays weak ligand-dependent binding to the LBD of
ER
in two-hybrid assays. However, the strength of binding of this
motif to ER
(as indicated by reporter activation) was between 10- and 50-fold lower than that achieved by motifs derived from SRC1 or RIP140, despite being expressed at comparable levels (3).
, RAR
, or RXR
(Fig. 4, B and D). The
CBP N-terminal 1-101 construct displayed potent transcriptional
activity in yeast, consistent with reports that the N terminus of CBP
functions as a transactivation domain in mammalian cells (35-37).
Reporter activation was slightly enhanced (~1.5-1.8-fold) upon
addition of ligand (Fig. 4B). To determine whether the
LXXLL motif mediates this weak ligand-induced interaction with NRs, the conserved leucine at residue 72 was mutated to alanine. This resulted in a loss of the weak ligand-induced response (Fig. 4B), supporting the hypothesis that this sequence is
required for ligand-induced NR binding. Western blots confirmed that
the wild type and mutant fusion proteins were expressed at similar levels (data not shown). The data shown in Fig. 4B were
pooled with data from replicated experiments to conduct an analysis of variance test using the SPSS statistics package. The increased reporter
activity due to interaction of DBD CBP-(1-101) with VP16-NRs in
the presence of ligand was found to be significant (F(1,
11) = 13.007, p < 0.01). In addition the
inability of the DBD CBP-(1-101) (L72A) to increase reporter
activity due to interaction with VP16-NRs in response to ligand was
also found to be significant (F(1, 11) = 37.309, p < 0.001).
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Fig. 4.
N- and C-terminal fragments of CBP interact
weakly with the LBDs of ER ,
RAR
, and RXR
.
A, schematic representation of CBP protein showing
approximate locations of sequences encoding LXMs 1-3, the CH1, CH2,
and CH3 domains, the bromodomain, and the histone acetyltransferase
(HAT) domain. Known binding sites for transcription factors
and other proteins are also shown. The black boxes represent
fragments of CBP used in yeast two-hybrid experiments, below.
B and D, yeast two-hybrid interaction of
DBD-CBP-(1-101), DBD-CBP-(1-101) (L72A), DBD-CBP-(342-381), and
DBD-CBP-(1891-2165) with AAD or AAD-LBDs, as indicated.
Shaded and black columns represent reporter
activity in the absence and presence of 500 nM cognate
ligand, respectively. C and E, GST pulldown
experiment showing interaction of 35S-labeled full-length
ER
with GST, GST-CBP-(1-101), GST-CBP-(1-101) (L72A),
GST-CBP-(342-381), and GST-CBP-(2058-2130) in the presence or absence
of 1 µM E2, as indicated.
in a
ligand-dependent manner. In contrast, the CBP-(1-101)
(L72A) mutant failed to bind to ER
LBD to a level above the
controls. However, it should be noted that the efficiency of binding in
the GST pulldown experiments was significantly lower than that achieved
with GST-NID of SRC1 (data not shown), indicating that ER
binds SRC1
with a higher affinity than to CBP.
, RAR
, and RXR
in yeast two-hybrid experiments (Fig. 4D). In contrast to
CBP-(1-101), no intrinsic reporter activation was observed with this
construct. The C-terminal fragment CBP-(1891-2165) displayed a weak
interaction with the LBDs of ER
and RXR
but not RAR
in these
experiments, enhancing the activity of the reporter 2-3-fold in the
presence of ligand (Fig. 4D). GST-pulldown experiments confirmed the weak ligand-dependent interaction of CBP
sequences 345-381 and 1891-2165 with full-length ER
(Fig.
4E). Note that the amount of ER
protein retained on the
beads was less than 10% of the input.
. As shown in Fig.
5A, CBP LXM-1 and CBP LXM-3
displayed ligand-induced binding to RAR
and PR. However, no
significant interaction with the LBD of the androgen receptor (AR) was
detected (Fig. 5A). Despite several attempts, we were unable
to achieve stable expression of the construct DBD-CBP-(356-366) as
determined by Western blots (data not shown). Thus, we were unable to
determine whether LXM-2 is sufficient to mediate binding to NRs in the
two-hybrid assay. However, the interaction of CBP-(345-381) fragment
with the LBDs of ER
, RAR
, and RXR
in this system (Fig. 4D) and in vitro (Fig. 4E) suggests
that LXM-2 also functions as a weak NR-binding motif.
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Fig. 5.
LXXLL motifs mediate weak
interaction of CBP with the LBDs of steroid and retinoid
receptors. A, yeast two-hybrid interaction of CBP
LXXLL motifs with AAD-RAR , AAD-ER
, AAD-RXR
, and
AAD-AR, performed as in Fig. 2. LXM-1 and LXM-3 are constructs in which
the DBD is fused in frame with amino acids 68-75 and 2067-2074 of
CBP, respectively. Shaded columns indicate the reporter
activity in the absence of ligand. Black columns indicate
the reporter activity in the presence of cognate ligand. B,
interaction of LXXLL motifs from SRC1 and CBP with the LBD
of the progesterone receptor (AAD-PR), in the presence or
absence of R5020 (500 nM) as indicated. C,
Western blot showing expression of the DBD-LXXLL constructs
in B. The antibody was a mouse monoclonal antibody derived
against the F-region epitope of the human ER
which is present at the
N terminus of the fusion proteins.
DISCUSSION
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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
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LBD, whereas
the hexapeptide LLRYLL did not (6). The crystal structure of the LBD of
TR
in complex with a synthetic peptide corresponding to motif 2 of
GRIP1 showed that the peptide adopts an amphipathic
-helix
conformation of nearly 3 turns from Lys-688 to Gln-695. Residues
outside this sequence were unstructured or in an extended coil
conformation. In contrast, the peptide in solution was found to be in a
random coil formation, thus
-helicity appears to be induced by the
interaction with the TR
LBD. The
-helical structure in motif 2 induced by binding the TR
LBD involves amino acids KILHRLLQ and
correlates exactly with the boundaries
2 to +6 that we have defined
as the minimum length of a LXXLL motif sufficient for
binding to AF-2. In addition, in a phage display screen for peptides
that bind to liganded ER
LBD, more than 30% of the isolated
LXXLL sequences commenced at position
2. The shortest
C-terminal boundary isolated in the same screen terminated at +7 (38).
Thus, we conclude that the minimal NR binding module is an 8-amino acid
-helix.
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Fig. 6.
Sequences of LXXLL motifs
showing differential affinities for steroid and retinoid
receptors. The sequences of the LXXLL motifs derived
from SRC1, RIP140, or CBP, and their respective amino acid numbers are
shown. The sequences are aligned with respect to the first conserved
leucine residue (numbered 1). The conserved leucine residues and the
amino acid at the 1 position are boxed.
A number of studies have indicated that co-activators show differential
but overlapping binding preferences for NRs. Although there are reports
of interactions of fragments of CBP with steroid and retinoid
receptors, we and others (51) have found these interactions to be far
weaker than the interaction of p160 proteins with steroid or retinoid
receptors (Fig. 1 and Fig. 5D). Fluorescence resonance
energy transfer experiments failed to show association of ER and CBP
in vivo, under conditions where the ER
/SRC1 and PPAR
/CBP association was readily observed (39). Similarly, others
(17, 40-42) have reported only very weak interactions between steroid,
thyroid, and retinoid receptors with CBP. In contrast interactions of
CBP and PPARs seem to be more robust, although still considerably
weaker than PPAR
/p160 interactions (39, 43-45).
We have demonstrated that LXXLL core motifs derived from
SRC1a, RIP140, and CBP show distinct preferences for steroid and retinoid receptors. By analysis of the core motif sequences, it is
apparent that positions 1 and +2 (and perhaps +3) have a strong impact on the ability of core motifs to bind to different NR LBDs. We
noted that the LXXLL motifs showing the strongest
interactions with the panel of LBDs selected tended to have a
hydrophobic residue at position
1 (Fig. 6). For example, motifs 2 and
3 of SRC1/TIF2/ACTR, and motif 4 of SRC1a, all of which display high
affinity for ER
and other NRs, contain a leucine or isoleucine
residue at the
1 position. In addition, in an affinity selection of
randomized peptides that bind the LBD of ER
, ~30% of
LXXLL sequences selected contained a hydrophobic residue at
1 (38). In another study using a "focused" peptide library, 12/17
sequences affinity selected with ER
LBD contained a leucine or
isoleucine residue at the
1 position (46). The SRC1 motif 1 has a
lysine residue at position
1. Although this does not appear to affect
its interaction with ER
in our experiments, motif 1 displayed
significantly reduced binding to RAR
, RXR
, AR, and PR (Fig. 2).
Consistent with this, it has been shown that a peptide based on motif 1 sequence was inferior to other SRC1 LXXLL motif peptides in
competition of ER
/SRC1 interaction in vitro (47).
CBP has been reported to interact weakly with RAR and RXR via the
N-terminal 400 amino acids (Fig. 5; see Refs. 31 and 32). However, it
has been shown that deletion of the first 400 residues of CBP did not
affect its ability to bind RAR (31, 41). This suggested that additional
binding site(s) distal to the N terminus are present in CBP or that CBP
interaction with NRs can be indirect. CBP contains three
LXXLL motifs, and we have demonstrated here that these
mediate weak ligand-dependent interactions of the N- and
C-terminal fragments of CBP with steroid and retinoid receptors. None
of the LXXLL sequences in CBP contain a hydrophobic residue at position 1. In a recent study of interaction of these sequences with the LBDs of PPAR
and RXR
, the CBP LXM-1 motif was shown to
bind to PPAR
but not RXR
(45). Furthermore, the same authors reported that the replacement of the glutamine at
1 with leucine significantly increased the ability of CBP LXM-1 to bind RXR
. This
is consistent with our finding that co-activators with high affinity
for steroid and retinoid receptors generally contain LXXLL
motifs with a hydrophobic residue at position
1. Thus, while we and
others (40, 48, 51) have shown CBP is required for the function of
steroid and retinoid receptors, we propose that at least some NRs
recruit CBP indirectly via the p160s.
Although 8 of the 9 nine LXXLL motifs present RIP140 have a
hydrophobic residue at position 1 (the exception being L4), not all
demonstrate high affinity binding to the panel of LBDs tested. For
example, we noted that motif L5 of RIP140 has very low affinity for
NRs, despite satisfying the requirement in having a leucine at position
1 (Fig. 3). This may be due to the presence of a leucine residue at
position +2, which is rare among the LXXLL motifs identified
to date. The presence of a bulky hydrophobic side chain at this
position may be detrimental to the formation of an amphipathic
-helix. However, there are examples of core motifs that tolerate a
hydrophobic residue at +2, notably motif 1 of SRC1 which shows reduced
affinity for NRs, and the functional LXXLL motif of human
ASC-2/NRC/RAP250, which has a valine at position +2 (13-15). Further
studies will be required to determine the repertoire of amino acids
tolerated within core motif sequences.
Structural studies have shown that lysine and glutamic acid residues in
the LBD that are almost universally conserved among the NR family may
act to clamp to the LXXLL -helix at the AF-2 surface (6,
22, 23). This involves contacts with residues outside the core motif.
In addition, it has been shown that CBP can acetylate a lysine residue
just upstream of motif 1 in ACTR and that this modification appears to
reduce the binding of ACTR to NRs (49). In addition, reports by several
groups (47, 50) indicate that sequences flanking the core motif affect
the affinity and selectivity of co-activators for NRs. Thus, sequence
flanking the core motif may also influence the interaction of
co-activators with NRs. Understanding the determinants of
LXXLL motif/LBD interactions will facilitate the design of
peptides and other antagonists capable of disrupting specific
NR/co-activator interactions.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Regine Losson, Pierre Chambon, Richard Goodman, and Tony Kouzarides for gifts of plasmids and antibodies and Graham Clark (ICRF) for automated sequencing. We also thank Eric Kalkhoven, Charlotte Bevan, and Frank Claessens for useful discussions and Paula Moran for advice on statistical analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wellcome Trust, the Imperial Cancer Research Fund, and a Marie Curie fellowship (to D. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-116-252-3474;
Fax: 44-116-252-3369; E-mail dh37@le.ac.uk.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M009404200
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ABBREVIATIONS |
---|
The abbreviations used are:
LBD, ligand binding
domain;
SRC1, steroid receptor co-activator;
RIP140, receptor
interacting protein;
CBP, CREB-binding protein;
NR, nuclear receptor;
AF, activation function;
TIF, transcriptional intermediary factor;
GRIP1, glucocorticoid receptor interacting protein;
ACTR, nuclear
receptor co-activator;
TRAP220, thyroid receptor-associated protein 220 kDa;
DRIP205, vitamin D receptor interacting protein 205 kDa;
ASC-2, activating signal co-integrator;
RAP250, nuclear receptor activating
protein 250 kDa;
NRC, nuclear receptor co-activator;
NID, nuclear
receptor interaction domain;
LXM, LXXLL motif;
ER, estrogen
receptor;
RAR, retinoic acid receptor;
RXR, retinoid X receptor;
TR, thyroid hormone receptor, AR, androgen receptor;
PR, progesterone
receptor;
PPAR, peroxisome proliferator activated receptor;
VDR, vitamin D receptor;
AAD, acidic activation domain;
DBD, DNA binding
domain;
PCR, polymerase chain reaction;
GST, glutathione
S-transferase;
E2, 17--estradiol;
aa, amino acids.
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