From the Women's Health Research Institute, Wyeth
Research, Collegeville, Pennsylvania 19426 and the ¶ Department of
Molecular and Cellular Biology, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, October 29, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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Nuclear receptor (NR)-mediated transcription is
driven by dynamic multiprotein coactivator complexes, the composition
of which is thought to determine the biological activity of NRs at
specific promoters. The extent to which NRs discriminate between a
spectrum of potential binding partners is intuitively a function of the inherent affinities of these individual interactions. Using real time
interaction analysis with BIAcore, we evaluated the affinities and
kinetics of the interactions of full-length members of the SRC/p160
coactivator family with estrogen receptor Nuclear receptors (NRs)1
mediate the transcriptional response to a variety of lipophilic
ligands and metabolic derivatives (1). Recent years have seen the
identification and characterization of many accessory factors, or
coactivators, that mediate the functions of activated NRs at their
cognate promoters. One subclass of coactivators, the SRC/p160
coactivator family, is largely distinct from others, such as the
p300/CREB-binding protein cointegrators (2, 3) and thyroid
receptor-interacting protein/vitamin D receptor-interacting protein/Mediator (4-7). Common to most coactivator subclasses, however, are conserved LXXLL motifs (NR boxes/NR interaction
domains), which mediate in part the binding of the coactivators to
liganded NRs (8, 9). Mechanistic data indicate multiple modes of action
for coactivators, including bridging of receptors with basal
transcription factors, and enzymatic modification of histones and other
transcription factors (for recent reviews, see Refs. 10-12).
Whereas coactivators often exhibit similar properties in in
vitro assays, increasing evidence suggests that they are
differentially utilized in vivo in cell type- and
promoter-selective fashions. Moreover, their ubiquitous expression
suggests that a spectrum of distinct potential interacting partners
exists for a given receptor in a specific cell type. The formation of
NR-coactivator complexes is principally a function of (i) the
intracellular concentrations of the NR and coactivators and (ii) the
inherent affinities of specific NR-coactivator interactions. The
development of models within which the discrimination by NRs between
this array of coregulators can be understood therefore requires the
measurement not only of their expression levels in various tissues but
also of the affinity of individual receptor-coactivator interactions.
An area of increasing interest is the molecular basis of the complex
tissue-specific pharmacology of natural NR ligands and their synthetic
derivatives, the selective receptor modulators. Several studies support
the notion that this tissue specificity may be attributed in part to
selective coactivator recruitment. In the case of the ER, selective ER
modulators induce distinct conformational changes (13, 14) and patterns
of SRC/p160 recruitment (13) by ER Strategies for characterizing receptor-coactivator interactions have
relied in large part upon oligopeptide fragments corresponding to
minimal coactivator NR interaction domains (16, 17). We have previously
shown that NRs interact with isolated NR boxes with low (micromolar)
affinity and that mutation of individual SRC/p160 NR boxes does not
abrogate ligand-dependent ER-SRC/p160 interactions (15).
These data suggest that distinct NR interaction domains can substitute
for each other and that regions remote from these motifs also
contribute energetically to supplement complex formation (9, 15, 18,
19). Moreover, they indicate that analysis of the interactions of
full-length molecules might place measurements of receptor-coactivator
affinities in a more informative context.
Mindful of these caveats, we have designed an assay system in which the
interaction affinity and kinetics of full-length NRs and coactivators
can be evaluated. Intact SRC-1, TIF2/SRC-2, and RAC3/SRC-3 were
immobilized on the surface of a BIAcore sensor chip. Coactivator
interactions with purified ER Equipment and Reagents--
The BIAcore 2000 system, sensor
chips CM 5 (certified), Tween 20, and amine coupling kit were obtained
from BIAcore Inc. (Piscataway, NJ). Purified recombinant human ER Plasmids--
The construction of the SRC-1 (20), TIF2/SRC-2,
and RAC3/SRC-3 (21) Xenopus expression vectors has been
described previously.
Synthesis of Full-length Coactivators--
N-terminal FLAG
fusions of SRC-1, TIF2/SRC-2, and RAC3/SRC-3 were expressed in
Xenopus oocytes by injection of in vitro
synthesized mRNA encoding FLAG-tagged SRC/p160s into 1000 stage VI
Xenopus oocytes (50 ng of mRNA/oocyte). Oocytes were
lysed as previously described (20).
Interaction Analysis--
The buffer used for all experiments
was 50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.2 mM EDTA, 0.005% Tween 20, 1 mM dithiothreitol. FLAG protein immobilization on the BIAcore sensor chip surface was
carried out as described previously (15, 22). Binding was measured in
arbitrary response units (RU). A signal of 1000 RU corresponds to a
surface concentration change of ~1 ng/mm2 (15). Anti-FLAG
antibody was immobilized using an amine coupling kit, typically
resulting in the binding of 8000-10,000 RU of antibody. Crude
Xenopus lysates containing FLAG-tagged coactivators were then injected at a flow rate of 5 µl/min over a surface coated with
immobilized anti-FLAG antibody. Unprogrammed oocyte lysate was used in
each run to establish background binding to the FLAG antibody-coated
sensor chip and generate a baseline for each individual assay. To
determine whether mass transport limitations might affect receptor-coactivator interactions, binding experiments were performed at the following rates: 5, 10, 30, and 50 µl/min. At flow rates of 10 µl/min and above, the values of kon and
koff determined by analysis of interaction data
using BIAevaluation software were independent of flow rates. All
subsequent experiments except coactivator immobilization were performed
with a constant buffer flow of 30 µl/min at 25 °C. Samples of
receptor were injected across the surface using a sample loop. After
the injection plug had passed the surface, complexes were washed with
buffer for an additional 1000 s. Following each injection, the
surface was regenerated with one 30-µl injection of 0.05% SDS. To
remove immobilized FLAG coactivator, a 30-µl injection of 10 mM glycine, pH 2.0, was used. Data were collected at 1 Hz
and analyzed using the BIAevaluation program 3.1 (BIAcore, Inc.) on a
PC. This program uses a global fitting analysis method for the
determination of rate and affinity constants for each interaction.
Kinetic rate errors were determined during data analysis in which
immobilized coactivator was subjected to multiple interaction cycles
with ER Full-length SRC/p160s Can Be Isolated from Xenopus
Oocyte Extracts--
Western blotting analysis of the
Xenopus oocyte extracts expressing SRC-1, TIF2/SRC-2, and
RAC3/SRC-3 is presented in Fig. 1A. We have recently shown
that FLAG-tagged SRC-1 expressed in Xenopus oocytes enhanced
progesterone receptor transactivation in an in vitro
transcription assay (20), indicating that SRC/p160s expressed in this
system retain functionality. To evaluate the affinity and kinetics of
the interactions between the full-length SRC/p160 family members and
ER
Crude extracts prepared from SRC/p160 mRNA- programmed
Xenopus oocytes were directly injected at a flow rate of 5 µl/min over flow cells coated with immobilized anti-FLAG antibody.
The binding of SRC-1 (flow cell 1; Fig. 1B), TIF2/SRC-2
(flow cell 2; Fig. 1C), and RAC3/SRC-3 (flow cell 3; Fig.
1D) to immobilized anti-FLAG antibody in a typical
experiment is shown in Fig. 1. Each extract injection was followed by
several washes with 10 µl of 0.05% SDS to strip SRC/p160s of any
interacting proteins and to remove other proteins that bind
nonspecifically to the surface. After washing, 1126 RU of SRC-1 was
immobilized in flow cell 1 (Fig. 1B), 830 RU of TIF2/SRC-2
was immobilized in the flow cell 2 (Fig. 1C), and 971 RU of
RAC3/SRC-3 was immobilized in flow cell 3 (Fig. 1D). Flow
cell 4 was exposed to unprogrammed Xenopus oocyte extract and used as a control (data not shown). Binding of ER Intact ER The ER-SRC/p160 Interaction Fits a Two-step Interaction
Model--
We next titrated immobilized SRC/p160s with ER
We then evaluated several possible interaction models for the
ER-SRC/p160 binding. Fig. 4 shows the
theoretical progress of receptor-coactivator binding following a
Langmuir interaction model (Fig. 4A) and a two-state
interaction model (Fig. 4A) upon which have been overlaid
the experimental data obtained for ER Selective Recruitment of Full-length SRC/p160 Family
Members by ER Current models of receptor-coactivator interactions have been
based in large part upon interpretations of observations of molecular
fragments made in a wide range of assays and experimental conditions.
Whereas such studies have gone a long way toward shaping our
understanding of mechanistic aspects of coactivator function, they are
of more limited use in designing integrated models of the biology of
these factors. For this reason, we sought to develop an approach in
which kinetic and affinity measurements of NR-coactivator interactions
could be made in the context of full-length molecules in a routine,
controlled assay.
In this study, we evaluated the affinities and kinetics of the
interaction of full-length SRC/p160 family members with ER The affinities of full-length SRC/p160 interactions with ERs are
appreciably higher in our assay than those of the NR interaction domains of the same coactivators (15). At present, the exact reason for
the observed difference is unclear, but it probably involves additional
contacts made in the context of the complex between full-length
coactivator and receptor. It has been consistently demonstrated that
molecular determinants distinct from the LXXLL motifs are
important influences on the affinity of NR-coactivator interactions (9,
18, 19). Moreover, the involvement of discrete domains in the C
terminus of NRs in recruitment of coactivators has been noted in a
number of crystallographic studies (25). Taken together with these
studies, our results reiterate that models based upon observations of
individual NR box-containing peptides, by failing to account for steric
aspects of interactions between full-length molecules, afford a less
than complete perspective on fundamental aspects of coactivator biology.
This assertion is borne out by our studies of the effect of the ligand
on the interaction of ERs with full-length SRC/p160s. In our previous
study using coactivator NR interaction domains, 17 Our data indicate that interactions between full-length ERs and intact
SRC/p160s are bipartite, involving an initial, rapid association of a
transitional intermediate and a slower terminal phase. The bipartite
interaction can be interpreted in at least two ways. First, it may
reflect the dynamics sketched by Nolte and co-workers (25), who
postulated an initial docking between a glutamate/lysine NR charge
clamp and ionic residues N- and C-terminal of the SRC-1/NCoA-1 NR box.
This may be followed by an induced fit interaction between the leucine
side chains of the NR box and the hydrophobic cavity in AF-2.
Conceivably, the fast-slow interaction kinetics we have observed might
be generated by an initial rapid compatibility between charged
residues, followed by intricate conformational changes accompanying the
apposition of two extended hydrophobic surfaces.
An alternative model arising from the bipartite reaction kinetics
described here requires consideration of the stoichiometry of the
interaction between NRs and SRC/p160s. A model proposed for the
interaction of SRC-1 with a heterodimer of the receptors for
all-trans-retinoic acid and 9-cis-retinoic acid
suggests that binding of tandem NR boxes occurs in a cooperative manner
(26). Specifically, binding of the second NR box to the
9-cis-retinoic acid moiety is pursuant to a conformational
change arising from the interaction of all-trans-retinoic
acid receptor with ligand and recruitment of the first NR box. Whether
such a scenario can be accommodated in the context of the interaction
of a single SRC/p160 molecule with an ER Evidence accumulated from our own and other laboratories has indicated
that coactivators are organized in vivo into large multiprotein complexes (4, 6, 29). In this respect, it could be
justifiably argued that our analysis of individually purified proteins
might not be directly comparable with the complexities of
transcriptional complexes in living cells. Evidence suggests, however,
that contacts between NRs and other transcription factors in these
complexes are discrete, typically occurring through only one or two
adaptor subunits (30, 31). Given that quantitative analysis of the
dynamics of such complexes is technically challenging, we reasoned that
a logical first step would be to evaluate the interactions between key
components of these complexes and proceed to interpret this information
in the context of the larger complex.
Several studies have demonstrated the capacity of post-translational
modifications of NRs and/or their coactivators to influence cyclicity
of molecular interactions at transcriptionally active promoters (32,
33). For example, the acetylation of an activator of thyroid
receptor/SRC-3 by p300/CREB-binding protein neutralizes the positive
charges of two lysine residues adjacent to the core LXXLL
motif and disrupts the association of activator of thyroid receptor/SRC-3 with promoter- bound ERs. In addition, coactivators have
been shown to be substrates for several kinases (34, 35), and the
notion is emerging that phosphorylation might influence coactivator
affinities for NRs and other coregulators. We anticipate that future
studies will determine whether the significance with which
post-translation modifications have been invested is sustainable in the
context of full-length receptor-coactivator interactions on DNA. In
conclusion, we suggest that the discriminatory relationships we have
identified may contribute to the combinatorial diversity by which
NR-mediated signaling pathways are so clearly characterized.
(ER
) and ER
bound
to a variety of ligands. We substantiate that 17
-estradiol enhances
the affinity of ER-SRC/p160 interactions, whereas 4(OH)-tamoxifen, raloxifene, and ICI-182,780 inhibit these interactions. We show that a
well defined, ER isoform-specific hierarchy governs the association of
liganded ERs with full length SRC/p160 family members. Moreover, our
data indicate that the interaction affinities of the full-length
SRC/p160s with ERs are significantly higher then those of
the NR interaction domains of the same coactivators, indicating
that portions of coactivator molecules distinct from NR interaction
domains might participate in receptor-coactivator complex formation.
Finally, the interaction kinetics of SRC/p160s with ERs are consistent
with a bipartite model, involving initial rapid formation of an
unstable intermediate complex, and a subsequent slower reaction leading
to its stabilization. We interpret our results as evidence that
hierarchical coactivator interaction affinities are an important source
of diversity in NR-mediated signaling and that the complexity of
receptor-coactivator cross-talk might be best understood in the context
of full-length molecules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
. Moreover, we have
described a synthetic ligand (WAY164397) that binds to both ER isoforms
but selectively enhances the interaction of ER
with SRC-1 and SRC-3
while exhibiting little effect on ER
interaction with these proteins
(15). In the same study, genistein was shown to enhance the interaction of ER (
/
) with SRC-1 and SRC-3 but demonstrated a minimal effect on ER (
/
) interaction with vitamin D receptor-interacting protein 205 and CREB-binding protein (15).
and ER
were studied over a range of
ER concentrations in the absence and presence of the ER ligands
17
-estradiol, 4(OH)-tamoxifen, raloxifene, and ICI-182,780 using
real time interaction analysis. Our data indicate that SRC/p160
proteins interact with liganded ERs with affinities higher than those
of individual NR interaction domains. They show that ER
and ER
have robust 17
-estradiol-induced affinity preferences for particular
SRC/p160s and that formation of the ER-SRC/p160 complex involves a
transitional intermediate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
were obtained from PanVera Corp. (Madison, WI).
17
-Estradiol and 4(OH)-tamoxifen and FLAG reagents were obtained
from Sigma. Raloxifene and ICI-182,780 were synthesized at Wyeth
(Radnor, PA).
or -
at different receptor concentrations. Five or six
binding cycles at different receptor concentrations were used to obtain
the affinity and kinetic rates. Each titration experiment was repeated
two or three times for each receptor-coactivator combination.
Independent experiments were separately analyzed. The differences in
affinity and kinetic rates did not exceed 10%. Refractive index
differences for the ERs at different protein concentrations were
adjusted using Sigma Plot 5.0 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
, we utilized real time interaction analysis with
BIAcore. In this technology, one of the interacting molecules is
immobilized onto a solid phase, the chip surface, while the interacting
partner is injected into the flow cell. When a soluble macromolecule
binds to the immobilized species, it leads to an increase in the
macromolecule concentration at the sensor surface, generating a change
in the refractive index, which is measured by BIAcore. Binding,
measured in RU, is recorded in real time, and obtained data can be used
to evaluate the kinetics and affinity of macromolecule interaction.
View larger version (58K):
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Fig. 1.
Full-length SRC/p160 family members can be
directly extracted from oocyte extracts. A, Western
blotting analysis of the Xenopus oocyte extracts containing
SRC-1, TIF2/SRC-2, and RAC3/SRC-3 with anti-FLAG antibody
showing unprogrammed lysate (lane 1), SRC-1
(lane 2), TIF2/SRC-2 (lane
3), and RAC3/SRC-3 (lane 4). The
arrow indicates specifically reacting FLAG-tagged protein.
The lower band represents a cross-reacting
species. B-D, typical SRC/p160 immobilization experiment.
Anti-FLAG antibody (8000-10,000 RU) was immobilized in all four flow
cells as previously described (36). B, immobilization of
SRC-1 in flow cell 1. SRC-1-programmed oocyte extract was injected at a
flow rate of 5 µl/min over the anti-FLAG antibody-coated surface.
After three injections (total extract volume of 120 µl), 1126 RU of
SRC-1 was immobilized. Each extract injection was followed by three
injections of 10 µl of 0.05% SDS. The tables next to the
sensograms show the flow cell number in which injection takes place
(column 1), time after the sensogram started (column 2); relative
response, the difference between the refractive index before and after
injection (column 3); and injected substance
(column 4). C, immobilization of
TIF2/SRC-2 in flow cell 2. A single injection of 150 µl of
TIF2/SRC-2-programmed oocyte extract over the flow cell was sufficient
to immobilize 830 RU of TIF2/SRC-2. D, immobilization of
RAC3/SRC-3 in flow cell 3. Three injections (120 µl each) of
RAC3/SRC-3-programmed oocyte extract were sufficient to immobilize 971 RU of RAC3/SRC-3.
and ER
to
anti-FLAG chips primed with unprogrammed Xenopus extract was consistently undetectable (data not shown).
and ER
Interact with Immobilized Full-length
SRC/p160s in a Ligand-dependent Manner--
The
ability of NR agonists to promote the interaction of NRs with
coactivators and the ability of antagonists to block this interaction
have been firmly established by previous studies. X-ray
crystallographic analysis of ER
(23), in addition to receptors for thyroid hormone (9) and 9-cis-retinoic acid
(24), as well as peroxisome proliferator-activated receptor-
(25), have shown this phenomenon to involve agonist- or antagonist-induced realignments of helix 12, respectively potentiating or attenuating AF-2-mediated functions. Accordingly, we first wished to confirm that
immobilized SRC/p160s interacted in a ligand-dependent
fashion with ER
and ER
under the conditions of our assay. Fig.
2 shows overlaid sensograms of injection
of ER
(120 nM) (Fig. 2A) and ER
(240 nM) (Fig. 2B) in the absence of ligand or the
presence of 17
-estradiol, 4(OH)-tamoxifen, raloxifene, and
ICI-182,780 over immobilized SRC-1. In their unliganded forms, both
receptors interacted with SRC/p160 coactivators. As anticipated, the
presence of 17
-estradiol significantly enhanced the binding of both
ERs to SRC-1. Similar results were obtained with respect to the
interaction of ER
and ER
with TIF2/SRC-2 and RAC3/SRC-3.
Surprisingly, whereas the abrogation of the interaction of ER
with
SRC/p160s in the presence of ICI-182,780, raloxifene, and tamoxifen was
essentially complete (Fig. 2B), residual ER
binding in
the presence of 4(OH)-tamoxifen (Fig. 2A) was consistently
observed. We have previously described the effects of these ligands on
ER
and ER
binding to the NR interaction domain fragments of
SRC/p160s (13). In this case, ICI-182,780, raloxifene, and
4(OH)-tamoxifen also reduced binding of both receptors to the
coactivator fragments but, as with the full-length SRC/p160s, did not
completely abolish this binding.
View larger version (19K):
[in a new window]
Fig. 2.
Full-length ER and
ER
interact in a ligand-dependent
manner with intact SRC-1. Overlaid sensograms of injections of
ER
(A) and ER
(B) over immobilized SRC-1.
Prior to the injection, receptors (250 nM for ER
and 360 nM for ER
) were preincubated with corresponding ligand
(1 µM) for 1 h at room temperature.
and ER
in the presence of 17
-estradiol (1 µM) at receptor
concentrations ranging from 1.12 to 360.5 nM for ER
(Fig. 3A) and from 3.12 to
420.0 nM for ER
(Fig. 3B). Saturable
interaction was detected for all three SRC/p160 family members,
indicating specific binding of ERs to the coactivators. Saturation was
also reached for the ER
-SRC1 interaction.
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[in a new window]
Fig. 3.
ER and
ER
interact in a dose-dependent
manner with SRC/p160 family members. A, overlaid
sensograms of injections of ER
at increasing concentrations (from
bottom, 1.12, 4.50, 13.40, 40.0, 120.0, and 360.5 nM) over immobilized SRC-1, TIF2/SRC-2, and RAC3/SRC-3.
Prior to each injection, receptor was incubated with 1 µM
17
-estradiol at room temperature for 1 h. B,
overlaid sensograms of injections of ER
at increasing receptor
concentrations (from bottom, 3.1, 15.6, 46.8, 140.2, and
420.0 nM) over immobilized SRC-1, TIF2/SRC-2, and
RAC3/SRC-3. Prior to each injection, receptor was incubated with 1 µM 17
-estradiol at room temperature for 1 h.
injection over SRC-1. As can
be seen, the obtained data do not fit adequately into a simple Langmuir
interaction model (A + B
AB; Fig. 4A) but fit well into
a model that describes a two-state reaction (A + B
AB*
AB; Fig.
4B). Kinetic analysis indicates that rapid initial ER
binding (kon1 = 2-7 e5,
M
1 s
1) leads to formation of an
unstable transitional intermediate ER-SRC/p160 complex
(koff1 = 0.03-0.05 s
1), which
then assumes a more stable conformation
(koff2 = 2-5 e
4
s
1) at a significantly slower rate (0.02- 0.05 s
1). Similar kinetics, consistent with a bipartite
reaction model, were observed for all other ER-SRC/p160 combinations
(data not shown).
View larger version (25K):
[in a new window]
Fig. 4.
The interaction of ER
and ER
with SRC-1 fits a two-step
reaction model. The interaction between ER
and SRC-1 was
evaluated using global fitting analysis with BIAevaluation 3.1 software
assuming Langmuir (A; A + B
AB) and two-state
(B; A + B
AB*
AB) interaction models. The
theoretical fit and the experimental data are provided for ER
injections over SRC-1. For each receptor concentration, two lines are
presented, one experimental and one theoretical, corresponding to the
progress of a reaction following "ideal" reaction kinetics.
and ER
--
Based on the two-step interaction
model, affinity constants (Ka) were calculated for
interactions between all ER-SRC/p160 combinations. Distinct
affinities of ER
and ER
for individual SRC/p160s were observed,
indicating an interaction hierarchy for ER
of RAC3/SRC-3 > SRC-1 > TIF2/SRC-2 and for ER
of SRC-1 > TIF2/SRC-2 > RAC3/SRC-3. This order of preference recapitulates that determined
previously for the interactions between ERs and the NR interaction
domain peptides of SRC/p160 family members (15). Significantly,
however, full-length SRC/p160 family members exhibit affinities
3-5-fold in excess of those determined for the NR interaction domains
of the corresponding SRC/p160s (see Table
I).
Affinity constants (Ka) of ER interactions with full-length
molecules (left column) and individual NR interaction domains (NRID,
right column) for each SRC/p160 family member
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
ER
. Our results provide evidence that a well defined hierarchy governs interactions between ER isoforms and SRC/p160 family members, such that RAC3/SRC-3 is the partner of preference for ER
, and SRC-1
is the preferred interaction partner for ER
. Factors such as the
relative expression levels of coactivators, their subcellular localization, and their post-translational modification status probably
modulate the inherent affinities of individual receptors and
coactivators along a given signaling axis. These considerations notwithstanding, however, the fact that ER isoforms exhibit
hierarchical affinities for potential binding partners is an intriguing
facet of their pharmacology and one that will have an important bearing on the development of ER isoform-specific selective ER modulators. It
remains to be seen whether such striking discrimination among coactivators is characteristic of other members of the NR superfamily and whether, on the basis of these data, accurate predictions can be
made concerning the interactions of these molecules in cells.
-estradiol
enhanced the affinity of receptor-NR box interaction, whereas
4(OH)-tamoxifen, raloxifene, and ICI-182,780 inhibited the interaction
(15). Intriguingly, the current study showed that this inhibition was
much more complete in the context of the full-length coactivators
compared with the NR interaction domains. Moreover, 4(OH)-tamoxifen,
when bound to ER
, was a less competent inhibitor of ER-SRC/p160
interactions compared with raloxifene and ICI-182,780.
homodimer remains to be
determined, but it can be envisaged that the slower phase we observed
might correspond to the sterically induced binding of a second NR box. Significantly, this model admits a role for AF-1 in participating in
coactivator binding, an event suggested by previous studies of several
NRs (27, 28).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health U19 Award DK62434-01.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.
§ These authors contributed equally to this work.
Recipient of Department of Defense Postdoctoral Breast Cancer
Fellowship DAMD17-98-1-8026.
** Present address: Metabolex, Inc., 3876 Bay Center Pl., Hayward, CA 94545.
To whom correspondence should be addressed: Dept. of Molecular
and Cellular Biology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030. Tel.: 713-798-6205; Fax: 713-798-5599; E-mail:
berto@bcm.tmc.edu.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M211031200
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ABBREVIATIONS |
---|
The abbreviations used are: NR, nuclear receptor; AF-1 and -2, activation function-1 and -2, respectively; CREB, cAMP-response element-binding protein; ER, estrogen receptor; RAC3/SRC-3, receptor-associated coactivator-3, a member of the SRC/p160 family; RU, relative units; SRC/p160, member of the steroid receptor coactivator/p160 family; SRC-1, steroid receptor coactivator-1, a member of the SRC/p160 family; TIF2/SRC-2, transcription intermediary factor-2, a member of the SRC/p160 family.
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