Nuclear Receptor-Binding Sites of Coactivators Glucocorticoid Receptor Interacting Protein 1 (GRIP1) and Steroid Receptor Coactivator 1 (SRC-1): Multiple Motifs with Different Binding Specificities
Xiu Fen Ding1,
Carol M. Anderson1,
Han Ma,
Heng Hong,
Rosalie M. Uht,
Peter J. Kushner1 and
Michael R. Stallcup1
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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The activity of the AF-2 transcriptional
activation function of nuclear receptors (NR) is mediated by the
partially homologous transcriptional coactivators, glucocorticoid
receptor interacting protein 1 (GRIP1)/transcriptional intermediary
factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1). GRIP1 and
SRC-1 bound nine different NRs and exhibited similar, but not
identical, NR binding preferences. The most striking difference was
seen with the androgen receptor, which bound well to GRIP1 but poorly
to SRC-1. GRIP1 and SRC-1 contain three copies of the NR binding motif
LXXLL (called an NR Box) in their central regions. Mutation of both NR
Box II and NR Box III in GRIP1 almost completely eliminated functional
and binding interactions with NRs, indicating that these two sites are
crucial for most of GRIP1s NR binding activity. Interactions of GRIP1
with the estrogen receptor were more strongly affected by mutations in
NR Box II, whereas interactions with the androgen receptor and
glucocorticoid receptor were more strongly affected by NR Box III
mutations. One isoform of SRC-1 has an additional NR Box (NR Box IV) at
its extreme C terminus with an NR-binding preference somewhat different
from that of the central NR-binding domain of SRC-1. GRIP1 has no NR
Box in its C-terminal region and therefore no C-terminal NR-binding
function. In summary, GRIP1 and SRC-1 have overlapping NR-binding
preferences, but specific NRs display both coactivator and NR Box
preferences that may contribute to the specificity of hormonal
responses.
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INTRODUCTION
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Gene transcription is activated by transcriptional activator
proteins that bind to specific regulatory DNA sequences, called
enhancer elements, which are associated with the transcriptional
promoters of genes (1, 2, 3). In the past few years the mechanism by which
the binding of such proteins to DNA activates transcription has been
further elucidated by the discovery of a class of proteins known as
transcriptional coactivators. Although they generally do not bind DNA
directly, these coactivators associate with DNA-bound activator
proteins and are required for the transcriptional activation process to
occur (4, 5, 6, 7). Coactivators mediate the effects of transcriptional
activator proteins, presumably by helping to recruit a complex of RNA
polymerase II and associated basal transcription factors (a
preinitiation complex) to the promoter or by activating a preinitiation
complex that has already been assembled on the promoter. Some
coactivators, such as the two related proteins CREB (cAMP-response
element binding protein)-binding protein (CBP) and p300, mediate the
effects of diverse groups of transcription factors (8, 9, 10, 11, 12); other
coactivators, like the recently discovered class of coactivators for
the nuclear hormone receptors (NRs) (13, 14), are functionally more
specific (10, 15).
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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A Comparison of the NR-Binding Specificities of Full-Length SRC-1a
and GRIP1
Full-length SRC-1a and GRIP1 were tested for their relative
interaction specificities with the following NRs in a yeast two-hybrid
assay: class I (steroid hormone) receptors, including glucocorticoid
receptor (GR), estrogen receptor (ER), androgen receptor (AR),
mineralocorticoid receptor (MR), and progesterone receptor (PR); and
class II receptors, including vitamin D receptor (VDR), retinoic acid
receptor (RAR)
, 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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Figure 1. Interaction of SRC-1a and GRIP1 with NR HBDs in the
Yeast Two-Hybrid System
Yeast containing an integrated ß-galactosidase reporter gene
controlled by a GAL4-binding site were transformed with expression
plasmids coding for a GAL4 DBD-NR HBD fusion protein and a second
fusion protein of GAL4 AD with full-length SRC-1a or GRIP1 (see Fig. 2 , A and C). After transformation, yeast were grown with the appropriate
hormone for 15 h, and the resulting cell extracts were assayed for
ß-gal activity. Hormones and concentrations used are specified in
Materials and Methods. A, ß-gal activity from yeast
two-hybrid assays represents the level of interaction between the
indicated NR HBD and SRC-1a (left panel) or GRIP1
(right panel) (striped bars). Activity of
the NR fusion protein in the absence of a coactivator is also shown as
a control (black bars). B, Immunoblots, performed with
an antibody against GAL4 DBD, were used to determine relative
expression levels of the various GAL4 DBD-NR HBD fusion proteins.
Top panel, Expression in yeast containing SRC-1a fused
with GAL4 AD; bottom panel, expression in yeast
containing GRIP1 fused with GAL4 AD; NO, extract from yeast lacking a
GAL4 DBD-NR HBD fusion protein.
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While all of the NRs interacted with SRC-1a and GRIP1, there were some
differences in the relative NR preferences of the two coactivators.
Most notable was that AR, relative to the other NRs, bound weakly to
SRC-1a but bound strongly to GRIP1. Immunoblot experiments using an
antibody against the GAL4 DBD indicated that the relative expression
levels of the various GAL4 DBD-NR HBD fusion proteins were very similar
(Fig. 1B
). 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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Figure 2. Interaction of Central and C-Terminal Fragments of
SRC-1a and GRIP1 with Various NR HBDs in Yeast Two-Hybrid Assays
A, The diagram shows the fusion proteins used in the yeast two-hybrid
system. NR HBDs were fused with the GAL4 DBD (white
box). hSRC-1a fragments were fused with the GAL4 AD
(ovals). Numbers associated with coactivator fragments
indicate amino acid positions. Vertical black bars with
Roman numerals indicate NR Box motifs ( LXXLL
consensus); the associated amino acid number corresponds to the first L
in the consensus motif. Black regions indicate the
CBP/p300 binding site. B, ß-gal activity from yeast two-hybrid assays
represents the level of interaction between hSRC-1a fragments and the specific NR HBD indicated on the x-axis;
activity is expressed as percent of the activity obtained for the same
NR HBD interacting with full-length hSRC-1a (Fig. 1A ). C, The diagram
shows the fusion proteins used in the yeast two- hybrid assays for the
interaction of NR HBDs with GRIP1 fragments. Symbols and amino acid
numbering are as in panel A. D, ß-gal activity from yeast two-hybrid
assays represents the level of interaction between GRIP1 fragments and
various NR HBDs, as in panel C.
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The presence of an NR-binding domain in the central region of the GRIP1
polypeptide chain has been established previously (24, 25). The
NR-binding strength and preference of the central GRIP1 fragment
(GRIP1320-1121) were essentially the same as those of
full-length GRIP1 (Fig. 2D
). 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 18562414) 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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Figure 3. Yeast Two-Hybrid Assays of NR HBDs Binding to GRIP1
Containing Mutations in NR Box Motifs
A, NR Box motifs from TIF1, RIP140, GRIP1, and hSRC-1a, with the
consensus sequence below; * indicates C terminus. B, Mutant NR Box II
and NR Box III motifs of GRIP1 with the amino acid substitutions shown
in larger type. C, Yeast two-hybrid assays were used to
compare binding of NR HBDs to full-length GRIP1 with a wild-type
sequence or mutations in NR Box II, NR Box III, or both. The GAL4
DBD-NR HBD fusion proteins and the GAL4 AD-GRIP1.FL fusion proteins are
as in Fig. 2C . Yeast were incubated with the appropriate hormone before
preparing cell extracts for ß-gal assays. Amino acid substitutions:
NRBIIm, L693A+L694A; NRBIIIm, L748A+L749A. ß-gal activity is
expressed as percent of the activity obtained for the same NR HBD
interacting with wild-type GRIP1 (Fig. 1A ).
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To investigate whether NR Box II and NR Box III were
important for NR binding by GRIP1, we changed the last two leucines in
each of these
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 (7080%) 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
1030% 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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Figure 4. Role of NR Box IV in NR Binding by the C-Terminal
SRC-1a Domain
A, Diagram of GAL4 fusion proteins used to assess interaction of NR
HBDs with hSRC-1a fragments in yeast two-hybrid assays. Symbols and
amino acid numbering are as in Fig. 2A . B, Yeast two-hybrid assays for
interaction of various NR HBDs or the C-terminal
p3001856-2414 fragment with C-terminal fragments of
SRC-1a containing or lacking NR Box IV.
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Effects of NR Box II and III Mutations and of Competing NR Box II
and III Peptides on Binding of ER, GR, and TR to GRIP1 in
Vitro
The yeast two-hybrid system data indicated that NR Box II is more
important than NR Box III for ER binding but that the opposite is true
for GR binding to GRIP1 (Fig. 3
). 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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Figure 5. Effect of NR Box Mutations on Interaction of GRIP1
with ER HBD in Vitro
Full-length, radiolabeled GRIP1 (wild-type or containing mutations in
NR Box sites) was synthesized in vitro and then
incubated with Sepharose beads containing immobilized GST, GST-ERHBD
(with or without bound estradiol, E2), or
GST-CBP2041-2240; bound GRIP1 was eluted and observed by
SDS-PAGE and autoradiography. The GST fusion proteins and the affinity
binding assays are described in Materials and Methods;
the amino acid substitutions in NR Box II (NRBIIm) and NR Box III
(NRBIIIm) are described in Fig. 3 .
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Peptides of 13 amino acids representing NR Box II or NR Box III were
next used as competitors in similar NR-GRIP1-binding assays in
vitro. ER HBD translated in vitro bound to a
GST-GRIP1730-1121 fusion protein in an estradiol-dependent
manner (Fig. 6
, 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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Figure 6. NR Box II and NR Box III Peptides as Competitors
for GRIP1 Binding to ER, GR, and TR HBDs in Vitro
Radiolabeled ER HBD (top panel), GR DBD-HBD
(middle panel), or TRß1 (bottom
panel) were synthesized in vitro and incubated
with Sepharose-immobilized GST or GST-GRIP1730-1121 in the
presence of the indicated amounts of NR Box II peptide (KHKILHRLLQDSS)
or NR Box III peptide (ENALLRYLLDKDD). Where indicated, ER contained
bound estradiol, GR contained bound dexamethasone, and TR contained
bound T3. Bound NR proteins or fragments were eluted and
observed as in Fig. 5 .
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The relatively weak effect of the competitor peptides on GR binding to
GRIP1 is presumably because GR binds more strongly than ER or TR to the
specific GST-GRIP1 fusion protein used. The GST-GRIP1 protein contained
only amino acids 730-1121 of GRIP1 and thus contained NR Box III but
not NR Box I or II. As shown above (Fig. 3
) 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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Figure 7. Effect of NR Box II and NR Box III Mutations on
GRIP1 Coactivator Function in Mammalian Cells
HeLa cells were transiently transfected as described in
Materials and Methods with the indicated expression
vector for an NR or the GAL4-CBP fusion protein, a suitable reporter
gene (5 µg) for each NR or for GAL4, and a GRIP1 expression vector (1
µg) coding for full-length GRIP1 with a wild-type (wt) sequence or
with mutations in NR Box II (NRBIIm), NR Box III (NRBIIIm), or both.
Transfected cells were grown without (white bars) or
with (black bars) the appropriate hormone, and cell
extracts were tested for reporter gene activity, i.e.
luciferase or ß-gal enzyme activity. NR Box mutations were as
described in Fig. 3B . NR expression vectors and reporter genes were
used as follows: A, Full-length hER with the G400V mutation (0.5 µg)
and MMTV-ERE-luc; B, MMTV-CAT was activated by the endogenous GR in
HeLa cells; C, full-length hTRß1 (0.2 µg) and
MMTV-TREpal-luc. D, GAL4 DBD-CBP2060-2174 fusion protein
(1 µg) and (GALRE)5-e1b-luc reporter gene.
|
|
As a control for the integrity of the mutant GRIP1 species we took
advantage of the ability of CBP and GRIP1 to enhance or trigger each
others transcriptional activation function (P. Webb, P. J.
Kushner, et al., submitted). Although the mechanism of
coactivation by GRIP1 and SRC-1 is still not understood, these
unpublished results suggest that the association of a p160 coactivator
(i.e. GRIP1 or SRC-1) and CBP or p300 with a DNA-bound NR at
the promoter serves two important functions: first, it serves to
recruit the coactivators to the promoter; and second, the interactions
between NRs, p160 coactivators, and CBP/p300 are necessary to trigger
the full activity of the transcriptional activation functions of each
of these proteins. In our control experiment (Fig. 7D
) 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
|
---|
NR-Binding Preferences of Full-Length GRIP1 and SRC-1a and Their
Central and C-Terminal Fragments
GRIP1 and SRC-1 are related proteins that both serve as
coactivators for nuclear hormone receptors (9, 15, 24, 25, 26). Although
the specific protein isoforms expressed by the GRIP1 and SRC-1 genes in
various cell types have not been determined, it appears that most
tissues express both of these genes (15, 24, 25). The reason for
coexpression of both of these coactivator genes in most tissues is not
known. The two proteins could have redundant functions or they could
serve as coactivators for different or overlapping subsets of nuclear
receptors. We found that GRIP1 and SRC-1a both interact with all class
I and class II NRs tested and exhibited only minor differences in their
relative preferences to bind NRs. Perhaps the only major difference was
that AR bound poorly to SRC-1a relative to other NRs, whereas GRIP1
bound AR just as avidly as most other NRs. In terms of NR binding
preferences, these results suggest that GRIP1 and SRC-1a may have
largely redundant functions. Whether other aspects of the coactivator
functions of GRIP1 and SRC-1a (e.g. how they interact with
the transcription machinery) are the same or different remains to be
studied.
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. GRIP1s 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 GRIP1s 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 GRIP1s 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.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Yeast expression plasmids coding for fusion proteins of GAL4 DBD
and the HBDs of GR, ER, PR, AR, MR, and VDR were described previously
(25). Similar yeast expression plasmids for GAL4 DBD fused to the HBDs
of TRß1, RAR
, 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
202461; hRAR
, amino acids 155462; hRXR
, amino acids 200462.
The yeast expression vector coding for fusion proteins of GAL4 AD and
full-length GRIP1 (amino acids 51462), 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 20602174 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 20412240 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 12 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 Coons 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 12 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
|
---|
We thank Drs. Hinrich Gronemeyer and Pierre Chambon
(Université Louis Pasteur, Paris, France) for generously
communicating their unpublished results on the NR Boxes of TIF2. In
this manuscript we have adopted their NR Box naming and numbering
system. We thank Dr. Ming-Jer Tsai (Baylor College of Medicine,
Houston, TX) for the SRC-1a cDNA; Dr. David Livingston (Dana-Farber
Cancer Institute, Boston, MA) for the p300 cDNA; Dr. John Chrivia
(Saint Louis University, St. Louis, MO) for the GAL4-CBP expression
vector; Dr. Yoshihiro Nakatani (NIH, Bethesda, MD) for the GST-CBP
expression vector; Dr. Keith Yamamoto (University of California, San
Francisco, CA) for GR expression vectors; Dr. Brian West (University of
California, San Francisco, CA) for hTRß1 expression
vector; Ms. Jeanette Shinsako (University of California, San Francisco,
CA) for expert technical assistance; and Dr. Paul Webb and Dr. Weijun
Feng (University of California, San Francisco, CA) for helpful
discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael R. Stallcup, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90033.
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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