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Departments of Pathology and of Biochemistry and
Molecular Biology (X.F.D., H.M., H.H., M.R.S.) University of
Southern California Los Angeles, California 90033
Metabolic Research Unit (C.M.A., R.M.U., P.J.K.) University
of California San Francisco, California 94143
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The NRs are a class of hormone-regulated transcriptional activator proteins that include the receptors for the five steroid hormones, thyroid hormone, retinoids (vitamin A), and vitamin D, among others (16, 17, 18, 19). These hormone receptors contain two transcriptional activation domains (ADs) (16, 20, 21, 22). AF-2, located within the hormone-binding domain (HBD) near the C terminus of the proteins, is highly conserved among the NRs mentioned above. AF-1 is an N-terminal AD that is not conserved in sequence. Recent studies have uncovered a large number of mammalian proteins that interact with the AF-2 transcriptional ADs of NRs (13, 14). These proteins bind to NRs that are occupied by the appropriate agonist, but generally not to ligand-free NRs or NRs occupied by antagonists (15, 23, 24, 25). Among AF-2-binding proteins are a related group of mammalian proteins of approximately 160 kDa in size, called the p160 coactivators, which have been demonstrated to serve as transcriptional coactivators for NRs in mammalian cells and in yeast (9, 15, 24, 25, 26). Steroid receptor coactivator-1a (SRC-1a) (9) is another isoform of SRC-1 (15), the first NR coactivator discovered; SRC-1a contains an N-terminal PAS domain that is lacking in SRC-1 (9). Glucocorticoid receptor interacting protein 1 (GRIP1) (25, 26) and transcriptional intermediary factor 2 (TIF2) (24) are nearly identical proteins found in mouse and human cells, respectively, which share 43% sequence identity with SRC-1a (25). Several other proteins, including receptor-interacting protein 140 (RIP140) (23) and TIF1 (27, 28), which are unrelated in sequence to GRIP1 and SRC-1, also bind to NR AF-2 domains, but so far their possible roles in transcriptional activation by NRs are unclear.
Transcriptional activation by NRs requires an isoform of either SRC-1 or GRIP1 as a coactivator (10, 25, 26). In addition, the participation of another coactivator, CBP or its partial homolog p300, is required; SRC-1a and GRIP1, as well as the NRs, can bind directly to CBP and p300 (9, 11). The ability of various transcription factors, coactivators, and corepressors to interact with CBP/p300 suggested that these complexes may serve as a mechanism for integrating the input from multiple signaling pathways (9).
In the study reported here we characterized further the NR-binding domains and NR-binding preferences of GRIP1 and SRC-1a. Initial studies of SRC-1a and GRIP1/TIF2 suggested that these partial homologs have similar and possibly overlapping activities as NR coactivators (9, 15, 24, 25). Therefore, we looked for possible differences in the NR-binding preferences of GRIP1 and SRC-1a by conducting a comprehensive analysis of the ability of GRIP1 and SRC-1a to interact with a diverse group of NRs that includes the five steroid hormone receptors (NR class I) and four class II receptors (including representatives of the thyroid hormone, retinoid, and vitamin D receptors). Previously, it was shown that SRC-1a has two separable NR-binding domains, one in the central region of the polypeptide chain and one in the C-terminal region (11). We therefore tested whether these two binding domains of SRC-1a bound the same or different sets of NRs. Like SRC-1a, GRIP1 also has an NR-binding domain in the central region of its polypeptide chain (24, 25); we asked whether GRIP1, like SRC-1a, also has an NR-binding domain in its C-terminal region.
Recently a simple motif, LXXLLL (where L = leucine and X = any amino acid) and called an NR Box, was shown to be necessary and sufficient for binding of some NRs by TIF1 and RIP140 (28). Although these two proteins are unrelated in sequence to GRIP1 and SRC-1, the discovery of NR Boxes in other NR AF-2-binding proteins prompted us to search for similar motifs in GRIP1 and SRC-1a and to make mutations in them to determine whether they are responsible for interactions with NR HBDs. Our study found the NR-binding domains of GRIP1 and SRC-1a to be surprisingly complex; rather than a simple, single binding site, these coactivators employ multiple NR- binding motifs with overlapping but different NR-binding preferences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, retinoid X receptor (RXR) , and thyroid hormone
receptor (TR) ß1. The coactivators were expressed as
fusion proteins with the GAL4 transcriptional AD; and C-terminal NR
fragments containing the complete HBD and most of the hinge region
(which separates the NR DNA-binding domain from the HBD) were expressed
as fusion proteins with the GAL4 DNA-binding domain (DBD). In these
yeast two-hybrid assays, binding of the coactivators with the NR HBDs
leads to expression of a ß-galactosidase (ß-gal) gene controlled by
a GAL4 enhancer element. Both full-length coactivators exhibited
binding to all of the NR HBDs tested in the presence of a suitable
agonist for each NR (Fig. 1A
). RAR and
RXR, but none of the other NRs tested, also exhibited some
coactivator binding in the absence of ligand (data not shown) (25). As
controls, each GAL4 DBD-NR HBD fusion protein was coexpressed with the
GAL4 AD that lacked an attached coactivator. In these tests there was
little or no activity in the presence (Fig. 1A) or absence (not shown)
of the appropriate NR ligand. The GAL4 AD-GRIP1 fusion protein was also
inactive by itself (25, 26).
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). Note that in a previous study (25) the fusion proteins for
the RAR, RXR, and TR HBDs were expressed at higher levels than the
fusion proteins for the steroid receptors and VDR because a different
expression vector was used for the RAR, RXR, and TR fusion proteins.
Our current studies have indicated that the overexpression of the
RAR fusion protein in the yeast two-hybrid assays of the previous
study (25) caused an artifactual reduction (presumably due to a
squelching effect) in the level of ß-gal activity observed in the
presence of retinoic acid; as a result, retinoic acid appeared to
reduce the level of interaction between GRIP1 and RAR in the yeast
two-hybrid system. In the current study, with all NR fusion proteins
expressed at the same low levels, the interaction between GRIP1 and all
NR HBDs, including RAR, was enhanced by agonist binding (data not
shown).
NR Binding Activity and Preferences of the Central and C-Terminal
Domains of SRC-1a and GRIP1
SRC-1a has two functionally separable NR-binding domains, one in
the central region of the polypeptide and the other at the C terminus
(11). We examined the NR-binding preference of each domain in the yeast
two-hybrid system; coactivator fragments were fused with the GAL4 AD,
and NR HBDs were fused with the GAL4 DBD (Fig. 2A). The central and C-terminal domains
of SRC-1a had overlapping but nonidentical NR-binding specificities
(Fig. 2B). ER, PR, VDR, RAR, and TRß1 HBDs bound more
strongly to the central SRC-1a domain than to the C-terminal domain. In
contrast, GR and AR HBDs bound preferentially to the C-terminal domain;
in fact, there was almost no AR binding to the central domain. MR and
RXR HBDs bound with approximately equal strength to both SRC-1a
domains.
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). This region shares partial homology with
the central SRC-1a NR-binding domain (25). Since GRIP1 and SRC-1a also
have extensive partial sequence homology in the C-terminal region (25),
we tested whether GRIP1 also has a C-terminal NR-binding domain
comparable to that of the SRC-1a C-terminal region described above.
Neither GRIP11121-1462 (data not shown) nor a longer
C-terminal GRIP1 fragment GRIP1775-1462 (Fig. 2D) had
any NR-binding activity. The longer C-terminal GRIP1 fragment contains
the CBP/p300-binding site (Fig. 2C), which was previously mapped in
SRC-1a (11). This allowed us to perform a control experiment to
demonstrate that GRIP1775-1462 bound strongly to a
C-terminal p300 fragment (amino acids 1856–2414) in the yeast
two-hybrid assay (Fig. 2D) and was thus stably produced and
structurally intact. Thus, GRIP1 has no C-terminal NR-binding function
comparable to that of SRC-1a.
Identification of Two NR Box Motifs in GRIP1 and Their Relative
Contributions to the NR-Binding Strength and Preference of GRIP1 in
Yeast Two- Hybrid Assays
When the NR Box motif LXXLLL was initially reported as an
NR-binding sequence in TIF1 and RIP140 (28), which are not related to
GRIP1 and SRC-1, we looked for similar motifs in GRIP1. While there
were no perfect matches for this sequence in GRIP1, there were many
sequences that partially matched the TIF1/RIP140 consensus sequence. We
decided to focus on two motifs, NR Box
II2 with the first leucine at
position 690 and NR Box III with the first leucine at position 745,
because they alone met the following three criteria: First, they
conformed to the sequence 
LXXLL (where = hydrophobic, L =
leucine, and X = any amino acid), found in the TIF1 and RIP140 NR
Boxes (Fig. 3A). Second, they were highly
conserved in GRIP1 and SRC-1a; NR Box II and Box III are located in
regions where at least 10 consecutive amino acids are conserved between
GRIP1 and SRC-1 (Fig. 3A). Third, they were located within the minimum
NR-binding domains defined for SRC-1a (11) and GRIP1 (25) (our
unpublished data). Another possible NR Box motif in this region (NR Box
I at position 641) did not fit these criteria well and was therefore
not analyzed in this study.
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LXXLL motifs to alanines, resulting in LXXAA (Fig. 3B). Analogous substitutions were previously shown to eliminate NR
binding by the TIF1 NR Box motif (28). Binding of full-length wild-type
and mutant GRIP1 species to various NR HBDs was tested in yeast
two-hybrid assays in the presence of the appropriate agonist (Fig. 3C).
Altering leucines L693 and L694 to alanines in NR Box II of GRIP1
caused a drastic loss (>90%) of binding to ER, a severe but less
dramatic reduction in binding (70–80%) to AR, and 
50% loss of
binding to the other NRs. In contrast, substituting alanines for
leucines L748 and L749 in NR Box III reduced binding by more than 95%
to AR, reduced binding by more than 70% to GR and ER, but had little
if any effect on binding to the remaining NRs. Altering the two
N-terminal leucines L744 and L745 to alanines in NR Box III produced
virtually the same phenotype as altering L748 and L749 to alanines
(data not shown). When both NR Box II and NR Box III were altered in
the same GRIP1 molecule, binding to most of the NRs was almost
completely eliminated; however, this mutant GRIP1 retained about
10–30% of wild type binding to MR, PR, and RXR (Fig. 3C). None of
these mutations caused any loss of binding of GRIP1 to p300 (Fig. 3C),
indicating that the mutations did not compromise the stability or
overall structural integrity of GRIP1. These mutational studies suggest
that NR Box II and NR Box III can account for most of the NR-binding
activity of GRIP1, with each motif contributing distinct but
overlapping specificities for the various NRs.
The GRIP1320-1121 fragment containing NR Boxes I, II, and
III bound all the NRs as efficiently as full-length GRIP1 (Fig. 2D).
GRIP1730-1121, which contained only NR Box III, bound most
NRs as efficiently as full-length GRIP1 but bound ER very weakly (data
not shown). Thus, both this deletion analysis and the NR Box point
mutations discussed above (Fig. 3) indicated that NR Box II is
primarily responsible for ER interactions with GRIP1.
A Fourth NR Box at the C Terminus of SRC-1a, but Not GRIP1
We investigated why SRC-1a, but not GRIP1, has a C-terminal
NR-binding function. NR Box motifs were responsible for NR binding in
the central binding domain of GRIP1 and, by inference from sequence
homologies, SRC-1a. This finding prompted us to focus on an additional
motif that matched the NR Box consensus sequence and was located within
the defined C-terminal NR-binding region of SRC-1a (Figs. 3A and 4A). This fourth motif, which we call NR
Box IV, was located at the extreme C terminus of SRC-1a in a 50-amino
acid region that has no homologous counterpart in GRIP1. To test the
importance of NR Box IV for the NR-binding function of the C-terminal
SRC-1a domain, we first demonstrated that the C-terminal 206 amino
acids of SRC-1a (SRC-1a1236-1441), as well as the longer
C-terminal fragment SRC-1a789-1441, was sufficient for
strong NR binding (Fig. 4). Deletion of the extreme eight C-terminal
amino acids, including NR Box IV, from the SRC-1a789-1441
fragment essentially eliminated NR binding but left the ability to bind
p300 intact (Fig. 4B). Thus, NR Box IV is essential for NR binding by
the C-terminal NR-binding domain of SRC-1a. As a corollary to this
finding, we conclude that the inability of the GRIP1 C-terminal region
to bind NR is due to the absence of the NR Box IV motif in this region
of GRIP1.
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). As a further test of this different
NR specificity of the two NR Box motifs, binding of GRIP1 with ER was
measured in vitro, using glutathione S-transferase (GST)
fusion proteins. Full-length GRIP1, wild-type or containing NR Box
mutations, was synthesized in vitro and tested for specific,
hormone-dependent binding to an immobilized fusion protein consisting
of GST and the ER HBD (GST-ERHBD). Wild-type GRIP1 bound specifically
to GST-ERHBD in the presence, but not in the absence, of estradiol
(Fig. 5). A mutation in NR Box II caused
a severe but not complete loss of ER binding, while the analogous
mutation in NR Box III caused a more modest reduction in binding.
Mutation of both NR Box II and NR Box III eliminated ER binding. In
contrast, the wild-type GRIP1 and all three mutants bound at similar
levels to a GST-CBP fusion protein, demonstrating that the GRIP1
mutations had specific effects on ER binding rather than general
effects on the structural integrity of the entire GRIP1 protein.
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, top panel).
When NR Box peptides were added as competitors to this reaction, the NR
Box II peptide was a much more effective competitor than the NR Box III
peptide. Similarly, for specific binding of hormone-activated
TRß1 to GST-GRIP1, the NR Box II peptide was a more
effective competitor than the NR Box III peptide (Fig. 6, bottom
panel), although the difference in competitor activity of the two
peptides was less dramatic than with ER. In contrast, when a labeled GR
DBD-HBD fragment was bound to GST-GRIP1 in the presence of
dexamethasone, both peptides were relatively weak competitors, but the
NR Box III peptide was a better competitor than the NR Box II peptide
(Fig. 6, middle panel). Densitometric analysis of the
autoradiogram indicated that 5 µg of NR Box III peptide gave 50%
competition, while there was no competition by 5 µg of NR Box II
peptide. Several controls demonstrated that the inhibitory effect of
these peptides were specific for the NR-GRIP1 interaction. Substitution
of alanines for the two C-terminal leucines or substitution of glutamic
acid for the penultimate leucine in either NR Box peptide eliminated
its ability to compete with GRIP1 for binding to ER, indicating the
importance of the NR Box motif within these peptides (data not shown).
Furthermore, the wild-type NR Box peptides failed to inhibit ER binding
to the TATA-binding protein and also failed to inhibit GRIP1 binding to
CBP in similar GST pull-down assays (data not shown).
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) NR Box II is more important
than NR Box III for strong ER binding activity; GR has the opposite NR
Box preference, and TR binds well to both NR Boxes.
Effects of NR Box Mutations on the Coactivator Function of GRIP1 in
Mammalian Cells
Full-length GRIP1 containing mutations in NR Box II, NR Box III,
or both were tested for their ability to serve as coactivators for ER,
TRß1, and GR in transiently transfected HeLa cells (Fig. 7). Reporter genes controlled by an
appropriate hormone response element were cotransfected with an
expression vector for the corresponding nuclear receptor (except that
the endogenous GR in HeLa cells was used in Fig. 7B) and an expression
vector for mutant or wild-type GRIP1. Cells were then incubated in the
presence or absence of hormone, and reporter gene product was measured
in the resulting cell extracts by using the appropriate enzyme assay.
Wild-type GRIP1 enhanced the hormone- dependent activities of ER, GR,
and TR by several fold (Fig. 7). Mutation of either NR Box II or III
caused moderate to severe loss of GRIP1 coactivator function for all
three NRs, and simultaneous mutation of NR Boxes II and III almost
completely eliminated the ability of GRIP1 to enhance transcriptional
activation by the NRs. The NR Box II mutation caused a more severe loss
of function than the NR Box III mutation when GRIP1 was tested with ER
(Fig. 7A) or TRß1 (Fig. 7C); in contrast, the NR Box III
mutation was more severe with GR (Fig. 7B).
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) the GRIP1
mutants were tested for their ability to enhance transcriptional
activation by a GAL4 DBD-CBP fusion protein, using a reporter gene with
GAL4-binding sites. The NR Box mutations had no effect on the ability
of GRIP1 to activate CBP (Fig. 7D), demonstrating that the NR Box II
and III mutations selectively affected the NR-binding activity of GRIP1
rather than the overall structural integrity or level of expression of
GRIP1 protein. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our results indicate that the ability of SRC-1a and GRIP1 to bind a wide range of NRs is accomplished by similar but not identical structural solutions. SRC-1a has two separable NR-binding domains, one in the central region of the polypeptide chain and the other at the C-terminal end (11). Both NR-binding domains of SRC-1a bound a wide range of NRs, but with some differences in NR specificity. The central domain of SRC-1a failed to bind AR and bound GR poorly, while the C-terminal domain bound ER, VDR, RAR, and TR poorly, relative to the central domain. In contrast to SRC-1a, GRIP1 has only the central NR-binding domain; this domain efficiently bound all of the NRs tested and thus has a somewhat broader NR-binding repertoire than the central domain of SRC-1a.
Roles of NR Boxes in NR Binding by GRIP1 and SRC-1a
Our discovery that NR Box motifs in GRIP1 and SRC-1a are essential
for NR binding by these NR coactivators, combined with the previous
demonstration that these motifs are responsible for NR binding by TIF1
and RIP140 (28), which are unrelated to GRIP1 and SRC-1, indicates that
the NR Box motif is widely used for interaction with the AF-2
transactivation domains of NRs. While this manuscript was in
preparation, two other groups reported the involvement of these motifs
in NR binding by SRC-1a (10, 29). Torchia et al. (10)
designated six sequences in SRC-1a that resembled the NR Box motif and
called them leucine-charged domains (LCD). LCD1, LCD2, and LCD3
correspond to the NR Boxes I, II, and III described here. These three
NR Boxes are conserved among SRC-1a, GRIP1, and the recently discovered
p/CIP, which represents a third genetically distinct member of the p160
coactivator family (10). LCD6 is the same as NR Box IV of SRC-1a. Heery
et al. (29) demonstrated that small SRC-1a fragments
representing each of the NR Boxes I-IV of SRC-1a bound ER HBD. Torchia
et al. (10), using small fragments containing various
combinations of NR Boxes I, II, and III, found that NR Box II was the
most important motif in the central NR-binding domain of SRC-1a for
binding ER and RAR; NR Box IV at the C terminus also bound ER and RAR.
The results of these studies on the role of NR Boxes in SRC-1a binding
of ER and RAR (10, 29) will be compared, below, with our studies on the
role of NR Boxes in GRIP1 binding to a broader spectrum of NRs. It is
important to note that, in spite of the extensive partial homology
between SRC-1a and GRIP1, their overall homology in the region of the
central NR-binding domain is relatively low (25). Therefore, the
relative contributions of the individual NR Box motifs to the binding
of specific NRs may not be exactly the same in these two coactivators,
as exemplified by the nonidentical NR binding preferences exhibited by
the central NR-binding domains of GRIP1 and SRC-1a.
Mutations in NR Boxes II and III of GRIP1 were used to assess the contributions of these motifs to NR-binding function and specificity in the context of the intact coactivator. Mutations in either NR Box II or NR Box III of GRIP1 caused partial loss of NR-binding activity in vitro and in vivo, as well as the ability of GRIP1 to serve as a NR coactivator in mammalian cells. Simultaneous amino acid substitutions in NR Boxes II and III almost completely eliminated these activities, demonstrating that these two NRs are necessary and probably responsible for the majority of the NR-binding activity of GRIP1. The combined data from NR-binding studies in vitro and in vivo and studies to measure coactivator activity in mammalian cells demonstrated that NR Boxes II and III had overlapping but somewhat different NR-binding preferences. GRIP1’s interactions with ER and TR were highly dependent on NR Box II, while interactions with GR and AR depended more on NR Box III. Torchia et al. (10) made similar conclusions about the relative roles of these two NR Boxes for ER binding by SRC-1a.
In our experiments, the individual NR Box mutations caused a severe loss of GRIP1’s coactivator activity for TR in mammalian cells but caused only minor losses of TR binding in yeast two-hybrid assays. The relatively minor effects of the individual NR Box mutations on TR binding in the yeast two-hybrid assays may be due to the extreme sensitivity of the yeast two-hybrid system for detecting even weak interactions or to differences between the yeast and mammalian systems.
Presence of NR Box IV in C-Terminal Region of SRC-1a but Not GRIP1
Explains GRIP1’s Lack of a C-Terminal NR-Binding Function
The recent studies on NR Box motifs in SRC-1a demonstrated that
small fragments containing NR Box IV are sufficient for binding ER and
RAR (10, 29). Here we demonstrated in the context of the intact
C-terminal NR-binding domain of SRC-1a that this motif is essential for
binding a wide range of NRs. Thus, NR Box IV is necessary and
sufficient for the C-terminal NR-binding function of SRC-1a. These
findings provide the basis for understanding why the GRIP1 C-terminal
region cannot bind NR: GRIP1 lacks a NR Box motif in its C-terminal
region. While GRIP1 and SRC-1a share approximately 43% amino acid
sequence identity that extends through most of the length of the
polypeptide chain, there are a few regions in which each coactivator
contains unique sequences not found in the other. The C-terminal 54
amino acids of SRC-1a, which includes NR Box IV, have no homologous
region in GRIP1 (25).
Some isoforms of SRC-1 contain the C-terminal NR Box IV motif and
some do not (Refs. 9, 11, 15, and the GenBank files cited therein),
presumably due to alternative splicing patterns. Our results suggest
that SRC-1 isoforms that lack the C-terminal NR Box will not have a
C-terminal NR-binding function. Since AR and GR bound poorly to the
central NR-binding domain of SRC-1a (Fig. 2B), SRC-1 isoforms lacking
NR Box IV would be predicted to bind AR and GR poorly. Thus, if there
is differential expression of SRC-1 isoforms containing or lacking NR
Box IV in different cell types, it could affect the ability of the
cells to support glucocorticoid and androgen responses.
Roles of Other Sequences in GRIP1 and SRC-1a That Resemble the NR
Box Motif
This study has focused on NR Boxes II and III, which are conserved
in GRIP1 and SRC-1a, and NR Box IV, found only in SRC-1a. In addition,
there are three other sequences that partially or substantially
resemble the NR Box consensus LXXLL and are partially or
substantially conserved in GRIP1 and SRC-1a (10, 25). The NR Box I
sequence KLLQLLTT begins at amino acid 640 of GRIP1. Heery et
al. (29) found that the homologous SRC-1a NR Box I sequence
KLVQLLTTT bound ER. However, Torchia et al. (10) found that
deletion of NR Box I from a small SRC-1a fragment that also contained
NR Box II and/or NR Box III had little if any effect on ER and RAR
binding. In addition, our mutational analyses of NR Boxes II and III
(Fig. 3) and our results with GRIP1 fragments that lack NR Box
I (data not shown) indicated that NR Boxes II and III can account
for most of the NR binding activity of GRIP1 and therefore suggest that
NR Box I may play, at most, a redundant role in NR binding.
Torchia et al. (10) designated two additional sequences that
resemble the NR Box consensus sequence as LCD4 and LCD5 but did not
test their activity; these motifs are within the CBP/p300-binding
region of SRC-1a and GRIP1. While LCD5 is mostly conserved between
GRIP1 and SRC-1a, LCD4 in GRIP1 only partially resembles the consensus
NR Box sequence. Furthermore, our deletion analysis of the C-terminal
domain of SRC-1a demonstrated that the region containing LCD4 and LCD5
is neither necessary nor sufficient for the NR binding activity of the
C-terminal domain of SRC-1a (Fig. 4). Rather, as discussed above, NR
Box IV (LCD6) is necessary and sufficient for the C-terminal NR-binding
activity of SRC-1a. Heery et al. (29) also found LCD4 to be
inactive in ER binding, but they did not test LCD5. Together, these
results suggest that LCD4 and LCD5 are not involved in NR binding.
Why Do NR Coactivators Have Multiple NR-Binding Motifs?
Thus, NR binding by the p160 coactivators GRIP1 and SRC-1a is
accomplished by a surprisingly complex strategy. Rather than relying on
a single structure to bind NRs, GRIP1 and SRC-1a have multiple motifs
that contribute differentially to the binding of different NRs. The
reason for multiple NR-binding motifs rather than a single one remains
to be investigated. Perhaps nature found it difficult to design,
through evolution, a single NR-binding sequence that could efficiently
bind all of the NRs; instead, perhaps the multiple NR Boxes with
overlapping but nonidentical NR preferences solved the problem of how
these coactivators could interact with a broad range of NRs. Another
possible reason for multiple NR Boxes might be to allow each
coactivator molecule to interact with more than one NR monomer. For
example, using multiple NR Boxes, one coactivator molecule could
interact with both members of an NR dimer bound to a hormone response
element; or when tandem hormone response elements occur in a promoter,
one coactivator molecule could conceivably interact with one NR monomer
in each of two different NR dimers bound to the tandem enhancer
elements. The last scenario could conceivably explain some types of
synergism that result when multiple NR dimers bind to tandem hormone
response elements.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and RXR were constructed by inserting
EcoRI-BamHI (for TR and RAR) or
EcoRI-PstI (for RXR) cDNA fragments into pGBT9
(CLONTECH, Palo Alto, CA) as follows: hTRß1, amino acids
202–461; hRAR, amino acids 155–462; hRXR, amino acids 200–462.
The yeast expression vector coding for fusion proteins of GAL4 AD and
full-length GRIP1 (amino acids 5–1462), called pGAD424.GRIP1/FL, was
also described previously (25). Expression vectors coding for fusion
proteins of GAL4 AD with fragments of GRIP1 or with full-length hSRC-1a
or fragments of hSRC-1a were similarly constructed in pGAD424
(CLONTECH). The hSRC-1a sequences and amino acid numbering are
according to Spencer et al. (GenBank accession number
U90661), with one exception. According to the GenBank file, the
C-terminal amino acid sequence of hSRC-1a, including NR Box IV, is
LRQQLLTE. However, we sequenced the cDNA clone, which was kindly
provided by Dr. Ming-Jer Tsai, and found that the true encoded amino
acid sequence is LLQQLLTE, so that the NR Box IV motif is conserved
between hSRC-1a and mSRC-1a (9). For expression of full-length GRIP1 in vitro and in mammalian cells, a 4.7-kb EcoRI fragment containing the entire open reading frame of GRIP1 (25) was subcloned into pSG5 (30), which has both T7 and SV40 promoters. The mammalian expression vectors HE0 coding for full-length hER with a G400V mutation (30) and hTRß1 wt (W. Feng and P. J. Kushner, in preparation) have been described. The GAL4 DBD-CBP expression vector, encoding a fusion of the GAL4 DBD with amino acids 2060–2174 of CBP, has been described (12).
The reporter gene plasmid mouse mammary tumor virus (MMTV)-chloramphenicol acetyltransferase (CAT), containing the MMTV long terminal repeat, has been described (21). In MMTV-thyroid response element (TRE)-luciferase and MMTV-ERE-luciferase (31) the major GREs located between -190 and -88 of the MMTV long terminal repeat have been deleted and replaced with a single palindromic TRE or estrogen response element (ERE). The reporter (GALRE)5, containing five GAL4 response elements upstream of the e1b promoter, will be described elsewhere (P. Webb and P. J. Kushner, in preparation).
Bacterial expression vectors for GST-ERHBD have been described (32). GST-GRIP1 encodes a fusion of GST to amino acids 730-1121 of GRIP1 (26). GST-CBP encodes a fusion of GST to amino acids 2041–2240 of CBP (33). The expression vector for the ER LBD has been described (32). The in vitro transcription/translation vector pSP64/rGR407C (34) contains the SP6 promoter upstream of the rat GR-coding region.
Mutations in the NR Box sites of GRIP1 were introduced into the pGAD424.GRIP1/FL and pSG5.GRIP1/FL vectors using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and verified by sequencing.
Protein-Protein Interaction Assays
Yeast two-hybrid assays for interaction of coactivators with NR
HBDs were performed as described previously (26) except as follows.
Yeast culture and ß-gal assays were performed and quantified in
96-well microtiter dishes with a Dynatech MR4000 plate reader as
described (35) except that o-nitrophenyl
ß-D-galactopyranoside was used as substrate. Where
indicated, yeast cultures were incubated for 15 h before harvest
with various hormones at the following concentrations: 10
µM deoxycorticosterone for GR; 100 nM
estradiol for ER; 100 nM dihydrotestosterone for AR; 500
nM progesterone for PR; 10 µM corticosterone
for MR; 10 µM T3 for TR; 1 µM
1,25-dihydroxy-vitamin D3 for VDR; 10 µM
all-trans-retinoic acid for RAR; 10 µM
9-cis-retinoic acid for RXR. Data shown are the mean and
SD for the results from three independent yeast
transformants and are representative of two or more independent
experiments.
For in vitro binding assays, proteins were translated in vitro in the presence of [35S]methionine, using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). When GR fragments were translated, separate translations were performed in the presence and absence of 10 µM dexamethasone. GST fusion proteins were prepared as described previously (32). For all of the binding assays except those involving GST-GRIP1, a volume of the bead suspension containing 10 µg total protein was incubated with 1–2 µl 35S-labeled in vitro translated protein in buffer IPAB-150 (20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.1% NP-40, 0.1% Triton X-100, and a protease inhibitor cocktail, pH 7.9) supplemented with 20 µg/ml BSA, in the presence of either 100 nM estradiol or vehicle, for a total volume of 150 µl. After incubation for 90 min at 4 C, beads were washed four times in IPAB-150. Beads and input-labeled proteins were then subjected to SDS-PAGE and visualized by fluorography.
The experiments involving GST-GRIP1 binding to ER, GR, or TR in the presence of various doses of peptides were performed essentially as described (26). Briefly, GST-GRIP1 beads containing 10 µg total protein were incubated with 5 µl in vitro translated protein in buffer NETN (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 0.01% NP-40), in the presence of 100 nM estradiol (for ER), 10 µM dexamethasone (for GR), 100 nM T3 (for TR), or vehicle, and 1 µl peptide (amounts specified in each experiment) or vehicle, for a total volume of 50 µl. After incubation for 1 h at 4 C, beads were washed four times in NETN and subjected to SDS-PAGE and fluorography. NR Box peptides were synthesized by the University of California at San Francisco Biomolecular Resource Center.
Immunoblotting
Yeast extracts were prepared by a urea-SDS method (36).
Electrophoresis and blotting methods were described previously (37).
All incubations for blocking and immunostaining were performed at room
temperature. Blots were incubated 3 h in blocking solution
consisting of TBST (10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.1% Tween-20) containing 1% BSA and 0.02%
sodium azide. The primary antibody, mouse monoclonal RK5C1 (Santa Cruz
Biotechnology, Santa Cruz, CA) against GAL4 DBD, was diluted in
blocking solution to 0.1 µg/ml and incubated with the blot for 1
h. The blot was washed three times for 15 min each in TBST, blocked
again for 30 min in TBST plus BSA plus sodium azide, and incubated for
45 min in TBST containing the secondary antibody, goat anti-mouse IgG
coupled to horseradish peroxidase (Promega), at a dilution of 1:2500.
After three more 15-min washes in TBST, the Amersham (Arlington
Heights, IL) enhanced chemiluminescence (ECL) system was used to
visualize the immunostaining pattern.
Cell Culture and Transfections
HeLa cells were maintained in DME H-16/F-12 Coon’s modified
medium without phenol red (Sigma), supplemented with 10%
iron-supplemented calf serum (Sigma). Cells were transfected by
electroporation, using plasmids indicated in each experiment. In each
cuvette 1–2 million cells were suspended in 0.5 ml PBS containing
0.1% glucose and 10 µg/ml Biobrene (Applied Biosystems, Foster City,
CA). Cells were electroporated at 0.24 kV, 960 µFarads in a Bio-Rad
Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA). After
electroporation, cells were resuspended in medium, plated into six-well
dishes, and treated immediately with vehicle or hormones as follows:
100 nM estradiol for ER, 100 nM dexamethasone
for GR, and 100 nM T3 for TR. After 40 h,
cells were washed with PBS and lysed with 200 µl lysis buffer (100
mM Tris-HCl, pH 7.8, 0.1% Triton X-100, 1 mM
dithiothreitol). Luciferase activity was measured using the Luciferase
Assay System (Promega). CAT assays were performed as previously
described (38). CAT activities were defined as the increase in counts
per unit time, corrected for background CAT activity. Both CAT and
luciferase activities were corrected for efficiency of transfection.
Transfection efficiency was monitored by cotransfection with a plasmid
containing ß-gal reporter gene driven by the actin promoter, which
was a gift of Michael Garabedian (New York University). ß-gal
activity was measured using the Galacto-Light Plus chemiluminescent
assay (Tropix, Bedford, MA). Luciferase and CAT activities shown are
the means and SDs of triplicate wells from a single
experiment and are representative of at least three independent
experiments.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by USPHS Grants DK-43093 (to M.R.S.), DK-51083 (to P.J.K.), and K08 DK-02335 (to R.M.U). from the National Institute of Diabetes and Digestive and Kidney Disease, and by AIBS Grant 562 (to P.J.K.) from the U. S. Army Breast Cancer Research Program.
1 These authors contributed equally to the work described. 
2 To promote conformity and clarity in the NR
coactivator field, we have adopted the NR Box numbering system
suggested by Drs. P. Chambon and H. Gronemeyer (28a).
Received for publication August 1, 1997. Revision received October 24, 1997. Accepted for publication November 10, 1997.
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H. J. Dubbink, R. Hersmus, C. S. Verma, H. A. G. M. van der Korput, C. A. Berrevoets, J. van Tol, A. C. J. Ziel-van der Made, A. O. Brinkmann, A. C. W. Pike, and J. Trapman Distinct Recognition Modes of FXXLF and LXXLL Motifs by the Androgen Receptor Mol. Endocrinol., September 1, 2004; 18(9): 2132 - 2150. [Abstract] [Full Text] [PDF]
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Z. Chen, B. S. Katzenellenbogen, J. A. Katzenellenbogen, and H. Zhao Directed Evolution of Human Estrogen Receptor Variants with Significantly Enhanced Androgen Specificity and Affinity J. Biol. Chem., August 6, 2004; 279(32): 33855 - 33864. [Abstract] [Full Text] [PDF]
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M. Powzaniuk, S. McElwee-Witmer, R. L. Vogel, T. Hayami, S. J. Rutledge, F. Chen, S.-i. Harada, A. Schmidt, G. A. Rodan, L. P. Freedman, et al. The LATS2/KPM Tumor Suppressor Is a Negative Regulator of the Androgen Receptor Mol. Endocrinol., August 1, 2004; 18(8): 2011 - 2023. [Abstract] [Full Text] [PDF]
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Q. Wang, J. A. Blackford Jr., L.-N. Song, Y. Huang, S. Cho, and S. S. Simons Jr. Equilibrium Interactions of Corepressors and Coactivators with Agonist and Antagonist Complexes of Glucocorticoid Receptors Mol. Endocrinol., June 1, 2004; 18(6): 1376 - 1395. [Abstract] [Full Text] [PDF]
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K. Duda, Y.-I. Chi, and S. E. Shoelson Structural Basis for HNF-4{alpha} Activation by Ligand and Coactivator Binding J. Biol. Chem., May 28, 2004; 279(22): 23311 - 23316. [Abstract] [Full Text] [PDF]
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Y. Liu, B. O. Kim, C. Kao, C. Jung, J. T. Dalton, and J. J. He Tip110, the Human Immunodeficiency Virus Type 1 (HIV-1) Tat-interacting Protein of 110 kDa as a Negative Regulator of Androgen Receptor (AR) Transcriptional Activation J. Biol. Chem., May 21, 2004; 279(21): 21766 - 21773. [Abstract] [Full Text] [PDF]
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M. A. Iannone, C. A. Simmons, S. H. Kadwell, D. L. Svoboda, D. E. Vanderwall, S.-J. Deng, T. G. Consler, J. Shearin, J. G. Gray, and K. H. Pearce Correlation between in Vitro Peptide Binding Profiles and Cellular Activities for Estrogen Receptor-Modulating Compounds Mol. Endocrinol., May 1, 2004; 18(5): 1064 - 1081. [Abstract] [Full Text] [PDF]
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E. Charmandari, T. Kino, E. Souvatzoglou, A. Vottero, N. Bhattacharyya, and G. P. Chrousos Natural Glucocorticoid Receptor Mutants Causing Generalized Glucocorticoid Resistance: Molecular Genotype, Genetic Transmission, and Clinical Phenotype J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1939 - 1949. [Abstract] [Full Text] [PDF]
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Y.-H. Lee, H. D. Campbell, and M. R. Stallcup Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity Mol. Cell. Biol., March 1, 2004; 24(5): 2103 - 2117. [Abstract] [Full Text] [PDF]
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L. Pascual-Le Tallec, O. Kirsh, M.-C. Lecomte, S. Viengchareun, M.-C. Zennaro, A. Dejean, and M. Lombes Protein Inhibitor of Activated Signal Transducer and Activator of Transcription 1 Interacts with the N-Terminal Domain of Mineralocorticoid Receptor and Represses Its Transcriptional Activity: Implication of Small Ubiquitin-Related Modifier 1 Modification Mol. Endocrinol., December 1, 2003; 17(12): 2529 - 2542. [Abstract] [Full Text] [PDF]
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K. Brown, Y. Chen, T. M. Underhill, J. S. Mymryk, and J. Torchia The Coactivator p/CIP/SRC-3 Facilitates Retinoic Acid Receptor Signaling via Recruitment of GCN5 J. Biol. Chem., October 10, 2003; 278(41): 39402 - 39412. [Abstract] [Full Text] [PDF]
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L. Ding, J. Yan, J. Zhu, H. Zhong, Q. Lu, Z. Wang, C. Huang, and Q. Ye Ligand-independent activation of estrogen receptor {alpha} by XBP-1 Nucleic Acids Res., September 15, 2003; 31(18): 5266 - 5274. [Abstract] [Full Text] [PDF]
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K. Nishimura, H.-J. Ting, Y. Harada, T. Tokizane, N. Nonomura, H.-Y. Kang, H.-C. Chang, S. Yeh, H. Miyamoto, M. Shin, et al. Modulation of Androgen Receptor Transactivation by Gelsolin: A Newly Identified Androgen Receptor Coregulator Cancer Res., August 15, 2003; 63(16): 4888 - 4894. [Abstract] [Full Text] [PDF]
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T. Fujita, Y. Kobayashi, O. Wada, Y. Tateishi, L. Kitada, Y. Yamamoto, H. Takashima, A. Murayama, T. Yano, T. Baba, et al. Full Activation of Estrogen Receptor {alpha} Activation Function-1 Induces Proliferation of Breast Cancer Cells J. Biol. Chem., July 11, 2003; 278(29): 26704 - 26714. [Abstract] [Full Text] [PDF]
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X. Li, J. Wong, S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley Progesterone and Glucocorticoid Receptors Recruit Distinct Coactivator Complexes and Promote Distinct Patterns of Local Chromatin Modification Mol. Cell. Biol., June 1, 2003; 23(11): 3763 - 3773. [Abstract] [Full Text] [PDF]
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F. Liang, P. Webb, A. Marimuthu, S. Zhang, and D. G. Gardner Triiodothyronine Increases Brain Natriuretic Peptide (BNP) Gene Transcription and Amplifies Endothelin-dependent BNP Gene Transcription and Hypertrophy in Neonatal Rat Ventricular Myocytes J. Biol. Chem., April 18, 2003; 278(17): 15073 - 15083. [Abstract] [Full Text] [PDF]
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C. J. Loy, K. S. Sim, and E. L. Yong Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions PNAS, April 15, 2003; 100(8): 4562 - 4567. [Abstract] [Full Text] [PDF]
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Y. Bai and V. Giguere Isoform-Selective Interactions between Estrogen Receptors and Steroid Receptor Coactivators Promoted by Estradiol and ErbB-2 Signaling in Living Cells Mol. Endocrinol., April 1, 2003; 17(4): 589 - 599. [Abstract] [Full Text] [PDF]
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V. H. Coulthard, S. Matsuda, and D. M. Heery An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220 J. Biol. Chem., March 21, 2003; 278(13): 10942 - 10951. [Abstract] [Full Text] [PDF]
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B. He and E. M. Wilson Electrostatic Modulation in Steroid Receptor Recruitment of LXXLL and FXXLF Motifs Mol. Cell. Biol., March 15, 2003; 23(6): 2135 - 2150. [Abstract] [Full Text] [PDF]
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J. Liu, K. S. Knappenberger, H. Kack, G. Andersson, E. Nilsson, C. Dartsch, and C. W. Scott A Homogeneous in Vitro Functional Assay for Estrogen Receptors: Coactivator Recruitment Mol. Endocrinol., March 1, 2003; 17(3): 346 - 355. [Abstract] [Full Text] [PDF]
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Y. Wu, W. W. Chin, Y. Wang, and T. P. Burris Ligand and Coactivator Identity Determines the Requirement of the Charge Clamp for Coactivation of the Peroxisome Proliferator-activated Receptor gamma J. Biol. Chem., February 28, 2003; 278(10): 8637 - 8644. [Abstract] [Full Text] [PDF]
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C. Teyssier, K. Belguise, F. Galtier, V. Cavailles, and D. Chalbos Receptor-Interacting Protein 140 Binds c-Jun and Inhibits Estradiol-Induced Activator Protein-1 Activity by Reversing Glucocorticoid Receptor-Interacting Protein 1 Effect Mol. Endocrinol., February 1, 2003; 17(2): 287 - 299. [Abstract] [Full Text] [PDF]
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T. Kino and G. P. Chrousos Tumor Necrosis Factor alpha Receptor- and Fas-associated FLASH Inhibit Transcriptional Activity of the Glucocorticoid Receptor by Binding to and Interfering with Its Interaction with p160 Type Nuclear Receptor Coactivators J. Biol. Chem., January 24, 2003; 278(5): 3023 - 3029. [Abstract] [Full Text] [PDF]
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I. Rogatsky, H. F. Luecke, D. C. Leitman, and K. R. Yamamoto Alternate surfaces of transcriptional coregulator GRIP1 function in different glucocorticoid receptor activation and repression contexts PNAS, December 24, 2002; 99(26): 16701 - 16706. [Abstract] [Full Text] [PDF]
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V. Christiaens, C. L. Bevan, L. Callewaert, A. Haelens, G. Verrijdt, W. Rombauts, and F. Claessens Characterization of the Two Coactivator-interacting Surfaces of the Androgen Receptor and Their Relative Role in Transcriptional Control* J. Biol. Chem., December 13, 2002; 277(51): 49230 - 49237. [Abstract] [Full Text] [PDF]
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Y. He, D. Szapary, and S. S. Simons Jr. Modulation of Induction Properties of Glucocorticoid Receptor-Agonist and -Antagonist Complexes by Coactivators Involves Binding to Receptors but Is Independent of Ability of Coactivators to Augment Transactivation J. Biol. Chem., December 13, 2002; 277(51): 49256 - 49266. [Abstract] [Full Text] [PDF]
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M. A. Rao, H. Cheng, A. N. Quayle, H. Nishitani, C. C. Nelson, and P. S. Rennie RanBPM, a Nuclear Protein That Interacts with and Regulates Transcriptional Activity of Androgen Receptor and Glucocorticoid Receptor J. Biol. Chem., December 6, 2002; 277(50): 48020 - 48027. [Abstract] [Full Text] [PDF]
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C. Teyssier, D. Chen, and M. R. Stallcup Requirement for Multiple Domains of the Protein Arginine Methyltransferase CARM1 in Its Transcriptional Coactivator Function J. Biol. Chem., November 22, 2002; 277(48): 46066 - 46072. [Abstract] [Full Text] [PDF]
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M. E. Torres-Padilla, F. M. Sladek, and M. C. Weiss Developmentally Regulated N-terminal Variants of the Nuclear Receptor Hepatocyte Nuclear Factor 4alpha Mediate Multiple Interactions through Coactivator and Corepressor-Histone Deacetylase Complexes J. Biol. Chem., November 15, 2002; 277(47): 44677 - 44687. [Abstract] [Full Text] [PDF]
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T. Barkhem, L.-A. Haldosen, J.-A. Gustafsson, and S. Nilsson Transcriptional Synergism on the pS2 Gene Promoter between a p160 Coactivator and Estrogen Receptor-{alpha} Depends on the Coactivator Subtype, the Type of Estrogen Response Element, and the Promoter Context Mol. Endocrinol., November 1, 2002; 16(11): 2571 - 2581. [Abstract] [Full Text] [PDF]
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C. M. Litterst and E. Pfitzner An LXXLL Motif in the Transactivation Domain of STAT6 Mediates Recruitment of NCoA-1/SRC-1 J. Biol. Chem., September 20, 2002; 277(39): 36052 - 36060. [Abstract] [Full Text] [PDF]
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S. Kaul, P. J. M. Murphy, J. Chen, L. Brown, W. B. Pratt, and S. S. Simons Jr. Mutations at Positions 547-553 of Rat Glucocorticoid Receptors Reveal That hsp90 Binding Requires the Presence, but Not Defined Composition, of a Seven-amino Acid Sequence at the Amino Terminus of the Ligand Binding Domain J. Biol. Chem., September 20, 2002; 277(39): 36223 - 36232. [Abstract] [Full Text] [PDF]
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P. Delerive, W. W. Chin, and C. S. Suen Identification of Reverbalpha as a Novel RORalpha Target Gene J. Biol. Chem., September 13, 2002; 277(38): 35013 - 35018. [Abstract] [Full Text] [PDF]
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N. Kotaja, U. Karvonen, O. A. Janne, and J. J. Palvimo The Nuclear Receptor Interaction Domain of GRIP1 Is Modulated by Covalent Attachment of SUMO-1 J. Biol. Chem., August 9, 2002; 277(33): 30283 - 30288. [Abstract] [Full Text] [PDF]
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C. Lethimonier, G. Flouriot, O. Kah, and B. Ducouret The Glucocorticoid Receptor Represses the Positive Autoregulation of the Trout Estrogen Receptor Gene by Preventing the Enhancer Effect of a C/EBP{beta}-Like Protein Endocrinology, August 1, 2002; 143(8): 2961 - 2974. [Abstract] [Full Text] [PDF]
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S. S. Koh, H. Li, Y.-H. Lee, R. B. Widelitz, C.-M. Chuong, and M. R. Stallcup Synergistic Coactivator Function by Coactivator-associated Arginine Methyltransferase (CARM) 1 and beta -Catenin with Two Different Classes of DNA-binding Transcriptional Activators J. Biol. Chem., July 12, 2002; 277(29): 26031 - 26035. [Abstract] [Full Text] [PDF]
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T. Kucera, M. Waltner-Law, D. K. Scott, R. Prasad, and D. K. Granner A Point Mutation of the AF2 Transactivation Domain of the Glucocorticoid Receptor Disrupts Its Interaction with Steroid Receptor Coactivator 1 J. Biol. Chem., July 12, 2002; 277(29): 26098 - 26102. [Abstract] [Full Text] [PDF]
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S. Cheng, S. Brzostek, S. R. Lee, A. N. Hollenberg, and S. P. Balk Inhibition of the Dihydrotestosterone-Activated Androgen Receptor by Nuclear Receptor Corepressor Mol. Endocrinol., July 1, 2002; 16(7): 1492 - 1501. [Abstract] [Full Text] [PDF]
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Y. Maeda, C. Rachez, L. Hawel III, C. V. Byus, L. P. Freedman, and F. M. Sladek Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205 Mol. Endocrinol., July 1, 2002; 16(7): 1502 - 1510. [Abstract] [Full Text] [PDF]
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 B. C. Freeman and K. R. Yamamoto Disassembly of Transcriptional Regulatory Complexes by Molecular Chaperones Science, June 21, 2002; 296(5576): 2232 - 2235. [Abstract] [Full Text] [PDF]
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A. Vottero, T. Kino, H. Combe, P. Lecomte, and G. P. Chrousos A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2658 - 2667. [Abstract] [Full Text] [PDF]
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W. Shao, S. Halachmi, and M. Brown ERAP140, a Conserved Tissue-Specific Nuclear Receptor Coactivator Mol. Cell. Biol., May 15, 2002; 22(10): 3358 - 3372. [Abstract] [Full Text] [PDF]
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N. Kotaja, M. Vihinen, J. J. Palvimo, and O. A. Janne Androgen Receptor-interacting Protein 3 and Other PIAS Proteins Cooperate with Glucocorticoid Receptor-interacting Protein 1 in Steroid Receptor-dependent Signaling J. Biol. Chem., May 10, 2002; 277(20): 17781 - 17788. [Abstract] [Full Text] [PDF]
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M. D. Ruse Jr., M. L. Privalsky, and F. M. Sladek Competitive Cofactor Recruitment by Orphan Receptor Hepatocyte Nuclear Factor 4{alpha}1: Modulation by the F Domain Mol. Cell. Biol., March 15, 2002; 22(6): 1626 - 1638. [Abstract] [Full Text] [PDF]
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B. He, J. T. Minges, L. W. Lee, and E. M. Wilson The FXXLF Motif Mediates Androgen Receptor-specific Interactions with Coregulators J. Biol. Chem., March 15, 2002; 277(12): 10226 - 10235. [Abstract] [Full Text] [PDF]
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H. Liu, W.-C. Park, D. J. Bentrem, K. P. McKian, A. D. L. Reyes, J. A. Loweth, J. M. Schafer, J. W. Zapf, and V. C. Jordan Structure-Function Relationships of the Raloxifene-Estrogen Receptor-alpha Complex for Regulating Transforming Growth Factor-alpha Expression in Breast Cancer Cells J. Biol. Chem., March 8, 2002; 277(11): 9189 - 9198. [Abstract] [Full Text] [PDF]
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J. M. Hall, D. P. McDonnell, and K. S. Korach Allosteric Regulation of Estrogen Receptor Structure, Function, and Coactivator Recruitment by Different Estrogen Response Elements Mol. Endocrinol., March 1, 2002; 16(3): 469 - 486. [Abstract] [Full Text] [PDF]
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R. V. Weatherman, C.-Y. Chang, N. J. Clegg, D. C. Carroll, R. N. Day, J. D. Baxter, D. P. McDonnell, T. S. Scanlan, and F. Schaufele Ligand-Selective Interactions of ER Detected in Living Cells by Fluorescence Resonance Energy Transfer Mol. Endocrinol., March 1, 2002; 16(3): 487 - 496. [Abstract] [Full Text] [PDF]
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S. Fan, Y. X. Ma, C. Wang, R.-Q. Yuan, Q. Meng, J.-A. Wang, M. Erdos, I. D. Goldberg, P. Webb, P. J. Kushner, et al. p300 Modulates the BRCA1 Inhibition of Estrogen Receptor Activity Cancer Res., January 1, 2002; 62(1): 141 - 151. [Abstract] [Full Text] [PDF]
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