From the Department of Pathology, University of
Southern California, Los Angeles, California 90033 and the
§ Department of Cellular and Molecular Pharmacology,
University of California, San Francisco, California 94143
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ABSTRACT |
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Transcriptional coactivators of the p160 family
(SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors
(NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160
proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino
acids 1011-1121), called the auxiliary NID (NIDaux), is required
in vitro and in vivo for efficient interaction
with a subset of NRs, including the glucocorticoid receptor (GR),
androgen receptor, and retinoic acid receptor The nuclear receptor
(NR)1 superfamily is a large
group of structurally homologous transcription factors that includes
the receptors for the five steroid hormones (Class I receptors);
receptors for thyroid hormone, vitamin D, and retinoic acid (Class II
receptors); and a group called orphan receptors in which cognate
ligands are unknown or unnecessary (1-4). NRs play critical roles in
cellular responsiveness to many types of internal and external signals during development and adult life of all higher eukaryotes. NRs are
typically composed of three major functional domains (5-7); many, but
not all, NRs have an N-terminal activation domain (AD) called AF-1 that
carries no sequence homology between different NRs. A highly conserved
DNA-binding domain (DBD) in the central region of the polypeptide
chain, composed of two four-cysteine zinc fingers, is the hallmark of
the NR family. The C-terminal hormone-binding domain (HBD), which
carries a lower degree of sequence homology among diverse NRs, is
responsible for hormone binding in Class I and II NRs and also carries
a second important transactivation domain called AF-2. Hormone binding
controls the biological activity of the Class I and II NRs. NRs
regulate the expression of specific genes by interacting directly or
indirectly with specific enhancer elements in the promoters of the
target genes (2, 8, 9). The promoter-bound NRs help to open the
chromatin structure around the enhancer element (10, 11) and also are
proposed to help recruit a transcription initiation complex to the
promoter by binding to components of the transcription complex (2).
Class I NRs bind DNA as homodimers, whereas Class II NRs generally form
heterodimers with the retinoid X receptors (3, 4).
The most well documented mechanism of gene activation by NRs involves
direct binding of an NR dimer, through its DBDs, to specific enhancer
elements. In addition, many NRs have been shown to modulate the
activities of other types of transcription factors (e.g.
AP1) indirectly, by binding to the other factors directly, or perhaps
through associated coactivator proteins; this modulation can involve
DNA binding by the NR (12) or no DNA binding (8, 9, 13). In all cases,
the ability of the NRs to activate gene transcription depends on the
actions of the AF-1 and AF-2 ADs. These two ADs often contribute
synergistically to transcriptional activation and probably have
different mechanisms of activation. These mechanisms may involve both
direct and indirect (through adaptor or coactivator proteins) contacts
with components of the basal transcription machinery and chromatin (2,
14). Understanding of the mechanism of AF-2 function has been recently
enhanced by the discovery of a family of 160-kDa proteins that bind in
a hormone-dependent manner to the HBDs of Class I and II
NRs and thereby enhance transcriptional activation (14, 15). This
family consists of three genetically distinct but structurally and
functionally related proteins: SRC-1 (steroid
receptor coactivator 1) (9, 16),
GRIP1 (glucocorticoid receptor-interacting protein
1) (17, 18) (also known as TIF2 (transcriptional intermediary
factor 2) (19)), and p/CIP
(p300/CBP-interacting protein) (13) (also known as ACTR (activator
of the thyroid and retinoic acid receptors)
(20), RAC3 (receptor-associated coactivator 3) (21), AIB1
(amplified in breast cancer
1) (22), and TRAM-1 (thyroid hormone
receptor activator molecule
1(23)). Members of the p160 family share ~40% sequence
identity with regions of high and low homology interspersed across
their 1400-amino acid length. These transcriptional coactivators do not
enhance the expression of genes by themselves because they do not bind to DNA and are not components of the basal transcription machinery. Rather, they are recruited to the promoters through their contacts with
the NRs. The p160 coactivators can bind, and may exist in a complex
with, two other types of coactivators: CBP or its homologue p300 and
p/CAF (p300/CBP-associated
factor) (9, 20, 24). All three families of coactivators
help to mediate the activities of NRs, and all three contain histone
acetyltransferase activities that may contribute to their functions as
coactivators by locally affecting chromatin conformation (20, 25-27).
CBP can also bind to components of the basal transcription machinery
(28).
The ability of p160 coactivators to interact with NR HBDs is essential
for their coactivator function (29), which is presumably accomplished
by serving as adaptors that link AF-2 AD in NR HBD with components of
chromatin and the transcription machinery. Mutational analyses of the
AF-2 domains of several NRs have provided a strong correlation between
loss of transactivation activity and binding to p160 coactivators (16,
18, 19). The p160 coactivators interact efficiently with the HBDs of
all Class I and II NRs (14, 29) and also with some orphan receptors
(30).2 To recognize the
diverse group of NR HBDs, the p160 coactivators use multiple mechanisms
of interaction (31). Each p160 coactivator molecule contains three
leucine-rich motifs (LXXLL, where X is any amino
acid), called NR boxes, clustered in the central region of the
polypeptide chain (13, 29, 32, 33), called the NR interaction domain
(NID). Each NR box motif is capable of binding specifically and in a
hormone-dependent manner to a diverse group of NR HBDs, but
the NR binding preferences of each NR box are somewhat different (13,
29, 31, 33). Thus, although no single NR box motif has a universal
ability to bind all NR HBDs efficiently, the multiple motifs present in
the p160 proteins collectively contribute to the broad NR binding specificity.
Here we report an additional mechanism that enables the GRIP1
coactivator to interact efficiently with some of the NR HBDs. We found
that GRIP1 NID was not sufficient by itself to support an efficient
interaction with some NR HBDs. This led to identification of a new
region of GRIP1 required for efficient interaction with a subset of NR HBDs.
Plasmids--
The yeast expression vector pGAD424.GRIP1/fl for
the fusion protein of Gal4 AD and full-length GRIP1 was described
previously (18). The yeast expression vectors for the fusion proteins
of Gal4 AD and different GRIP1 fragments were made by subcloning PCR-amplified GRIP1 cDNA fragments into the
EcoRI/BamHI sites of pGAD424
(CLONTECH), which has a leu2 marker
gene, or into pHGAD, which was made by inserting a PCR-amplified
fragment (containing the adh1 promoter, Gal4 AD, and
adh1 transcription termination signal from pGAD424) into the
XhoI/SacI sites of pH2, which has a 2µ
replication origin and a his3 selection marker and was
derived from pRS423 as described (34). The yeast expression vectors for
fusion proteins of Gal4 DBD and the HBDs of the mouse glucocorticoid receptor (GR), human androgen receptor (AR), human progesterone receptor (PR), human retinoic acid receptor Yeast Two-hybrid Assays--
Yeast two-hybrid assays for the
interaction between GRIP1 fragments and NR HBDs were performed as
described previously (29). Where indicated, the following hormone
concentrations were added to yeast cultures for 14-16 h before
harvest: 10 µM deoxycorticosterone for GR, 100 nM dihydrotestosterone for AR, 500 nM
progesterone for PR, 10 µM all-trans-retinoic
acid for the retinoic acid receptor, 10 µM
9-cis-retinoic acid for the retinoid X receptor, 10 µM 3,5,5'-triiodo-L-thyronine for TR, and 1 µM 1,25-dihydroxyvitamin D3 for the vitamin D
receptor. GST-dependent Protein-Protein Interaction
Assays--
Expression of GST-GRIP1-(563-1121)-His6 or
GST-GRIP1-(563-767)-His6 in Escherichia coli,
purification of fusion proteins, loading of measured amounts of fusion
proteins on glutathione-agarose beads, incubation of in
vitro synthesized TR and GR with the beads, and analysis of bound
TR and GR were performed as described previously (31). Eluted
bead-bound proteins and input labeled proteins were subjected to
SDS-polyacrylamide gel electrophoresis. The fraction of input NR that
was bound to beads was determined by PhosphorImager analysis of the
gel. For peptide competition assays, 35S-labeled in
vitro translated GR was incubated with 1.6 µM
glutathione-agarose-bound GST-GRIP1-(563-767)-His6 or
GST-GRIP1-(563-1121)-His6 in the presence of increasing
amounts of peptide KENALLRYLLDKDD (synthesized by the University of
California at San Francisco Biomolecular Resource Center). The rest of
the procedure was performed as described above. The amount of bound GR
was calculated relative to the amount of retained receptor in the
absence of peptide.
The Minimum Fragment of GRIP1 Required to Bind GR HBD--
The
importance of the three clustered NR box or LXXLL motifs in
the NID of the p160 coactivator family for binding to the HBDs of all
NRs has been established (13, 29, 32). However, although short
polypeptides containing one or more of the NR box motifs are sufficient
for binding some NR HBDs (32), our preliminary experiments had
indicated that the requirements for GR HBD binding to GRIP1 were more
complex. To define the minimum fragment of GRIP1 required for binding
GR HBD, we tested the interactions of GR HBD with various GRIP1
fragments in the yeast two-hybrid system (38). In these assays, GR HBD
was expressed as a fusion protein with Gal4 DBD, and GRIP1 fragments
were fused with Gal4 AD. Interactions between the HBD and a GRIP1
fragment result in the activation of a A Specific Region of GRIP1 Separate from NID Is Important for the
Interaction with Both GR HBD and p300--
Since GRIP1-(730-1121)
exhibited full activity to interact with GR HBD, this fragment was used
as a starting point for a further deletion analysis in the yeast
two-hybrid assays. Deletion of 20 N-terminal amino acids, including NR
box III, resulted in the complete loss of interaction with GR and PR
HBDs (Fig. 2, fragment D); thus, in this context, where NR boxes I and II were
missing, NR box III was absolutely required for the interaction of
GRIP1-(730-1121) with NR HBDs. Deletion of amino acids 1020-1121 or
934-1121 from GRIP1-(730-1121) (Fig. 2, fragments
B and C) also resulted in dramatically reduced
interaction with GR HBD, but had only minor effects on binding of PR
HBD. Thus, GRIP1-(730-1121) is the minimum single fragment of GRIP1
that maintains the ability to interact with GR HBD, although it extends
far downstream from NID. In contrast, efficient binding of PR HBD
requires NID, but not the auxiliary downstream sequences.
Internal deletions of amino acids 869-938 (which correspond to a
natural splicing variant of
GRIP1)4 or amino acids
868-1010 did not affect the interaction between GRIP1 and GR or PR HBD
(Fig. 2, fragments H and I).
Therefore, GRIP1 amino acids 1011-1121, which we have named the
auxiliary NID (NIDaux), constitute a separate region required for
efficient binding to GR HBD, but not PR HBD. GRIP1-(883-1121),
GRIP1-(939-1121), and GRIP1-(1011-1121) (fragments
E-G) alone did not exhibit any ability to bind GR or PR
HBD, but they all bound to p300. Thus, NIDaux alone did not bind GR
HBD, but functioned in the context of one or more of the upstream NR
boxes I-III. GRIP1 amino acids 1011-1121 not only functioned as an
auxiliary domain for binding of GR HBD, but also were necessary and
sufficient for the interaction with p300 (fragments
G and I). p300 was previously shown to bind to
this region of GRIP1 and the related p160 coactivators (20, 24,
29).
To study the mechanism for collaboration between NID and NIDaux in the
binding of GR HBD, we tested whether these two regions of GRIP1 must
work in cis (present in the same protein fragment) or can
work in trans (as separate protein fragments). Efficient binding of GR HBD occurred only when NID and NIDaux were expressed as a
fused protein in the yeast two-hybrid system (Fig.
3). All of the GRIP1 fragments in this
experiment were functionally expressed since fragments that included
amino acids 939-1121 or 1011-1121 interacted with p300 (Fig. 2), and
GRIP1-(730-868) could interact with PR HBD (data not shown) in the
yeast two-hybrid system.
A Subset of NR HBDs Requires the Auxiliary HBD-binding Domain of
GRIP1--
The different requirements of GR and PR HBDs for binding to
GRIP1 suggest that there are two different types of interactions between NR HBDs and GRIP1 (Figs. 1 and 2). We therefore used the yeast
two-hybrid system to test several other Class I and II NR HBDs for
their dependence on the NIDaux of GRIP1. Since different NR HBDs
exhibit different preferences for the three NR boxes (13, 29), we used
GRIP1 fragments that included all three NR box motifs of GRIP1 in these
experiments. Inclusion of all three LXXLL motifs also
presents a more physiological context for the interactions. One
GRIP1 fragment, GRIP1-(563-1121), contained NID and NIDaux; the
second fragment, GRIP1-(563-1019), contained only NID. GR, AR, and
retinoic acid receptor Enhancement of GR Binding in Vitro by NIDaux--
The ability of
two different GRIP1 fragments to interact with full-length GR and
TR
To quantify the effect of GRIP1 NIDaux on GR and TR binding, different
quantities of the two purified GST-GRIP1 fragments were loaded onto
glutathione-agarose beads and incubated with labeled GR or TR. These
titration curves demonstrated a difference of 50-fold in the
concentrations of the two different GST-GRIP1 fragments required to
achieve 50% retention of labeled GR, but only a 7-fold difference for
binding TR (Fig. 5b). The results suggest that in this
in vitro assay, the shorter GRIP1 fragment binds all NR HBDs
less efficiently than the longer GRIP1 fragment; in addition, the fact
that the difference is much more pronounced for GR than for TR
indicates that efficient GR binding requires NIDaux. In a peptide
competition assay, peptide KENALLRYLLDKDD, which is derived from GRIP1
NR box III and its surrounding sequence, inhibited the interaction of
GR with GRIP1-(563-767) more efficiently than with GRIP1-(563-1121),
indicating a stronger interaction between GR and GRIP1-(563-1121)
(data not shown). Thus, the presence of NIDaux dramatically enhanced
the affinity of GR binding in vitro and in
vivo.
GRIP1 NIDaux Enhances the Ability of SRC-1 NID to Interact with GR
and AR HBDs--
SRC-1 and GRIP1 exhibit extensive co-linear sequence
homology (18); both have a highly conserved central NID that contains three LXXLL motifs. SRC-1a, one of the alternative splicing
forms of SRC-1, also contains a fourth LXXLL motif at its
extreme C terminus. Binding of GR and AR HBDs by SRC-1a is almost
entirely due to NR box IV (29). Truncation of SRC-1a immediately after the p300-binding domain dramatically reduced its binding to GR and AR
HBDs, but did not affect its binding to TR HBD in the yeast two-hybrid
system (Fig. 6, SRC-1-(1-977)). The same
result was obtained with SRC-1a-(1-1433), which lacked only the last
eight amino acids including NR box IV (data not shown). One possible reason for the poor binding of GR and AR HBDs by the NIDs of
SRC-1a-(1-977) and SRC-1a-(1-1433) could be the lack of a functional
NIDaux, like that in GRIP1. Fusing the NIDaux of GRIP1 (contained in
amino acids 750-1121) to the central NID of SRC-1 (contained in amino acids 1-753) restored the ability to bind GR and AR HBDs. Thus, although SRC-1 lacks a functional counterpart to GRIP1 NIDaux, the
GRIP1 NIDaux can cooperate with the SRC-1 NID to restore binding of GR
and AR HBDs.
The p300 Binding Activity of GRIP1 Is Genetically Separable from
the NIDaux Function--
Since the GRIP1-(1011-1121) region is
involved in the interaction with both GR HBD and p300, we tested
whether the two activities were mechanistically related,
e.g. whether GR HBD binding by GRIP1 requires an intact p300
binding activity. Protein sequence comparisons within this region of
the three non-allelic members (GRIP1/AIB1/SRC-1) of the p160
coactivator family identified two conserved segments that partially
resemble LXXLL motifs (Fig.
7). These motifs were previously named
LCD4 and LCD5 (leucine charged
domain) (13). LL-to-AA substitutions in each motif were
used to test their potential involvement in GR HBD and/or p300
interactions. In yeast two-hybrid assays, the LCD4 mutation
L1063A+L1064A (Fig. 7, Mut1) in GRIP1 had no effect on the
interaction of GRIP1-(730-1121) with either GR HBD or p300. In
contrast, the LCD5 mutation L1079A+L1080A (Fig. 7,
Mut2) resulted in a 70% loss of p300 binding activity, but had no effect on the interaction with GR HBD. Combining the four amino
acid substitutions (Fig. 7, Mut1 + Mut2) had effects similar to Mut2 alone. Although these results did not identify the
residues of NIDaux required for efficient GR HBD binding, they helped
to localize the p300-binding site and demonstrated that the NIDaux and
p300 binding functions of GRIP1 are genetically separable.
For GRIP1 and the other p160 coactivators to mediate
transcriptional activation by NRs, the NRs must bind the p160
coactivators (13, 29, 32, 33); this interaction is presumably
responsible for recruiting the p160 coactivator, and perhaps a complex
of associated coactivators including CBP or p300 (9, 20, 24), to the
promoter, where the coactivator complex presumably helps to remodel
chromatin and/or to recruit a transcription initiation complex. This
NR-coactivator binding is primarily due to interaction of the
coactivators with the highly conserved AF-2 domain within the HBDs of
most NRs (16, 18, 19, 31, 37). Whereas the p160 coactivators can also
interact with the AF-1 domains of at least a few steroid receptors (39,
40), the binding of these coactivators to the AF-2 domain in the HBDs
of NRs is stronger (39) and appears at this time to be universal, at
least for all NRs that function as transcriptional activators. This
universality is rather remarkable in light of the fact that the degree
of amino acid sequence homology among the NR HBDs that interact with
p160 coactivators can be as low as 10% (41). Nevertheless, the
three-dimensional architecture of the HBD among a diverse group of
Class I and II NRs is highly conserved (42-46). The almost universal
interaction between p160 coactivators and NR HBDs suggests that this
interaction must depend on highly conserved residues and surface
features of the HBD. In fact, a recent mutational analysis (37) and an x-ray crystallographic analysis (31) confirmed that many of the TR From previous studies (29) and the one reported here, we now can
understand in more detail how the p160 coactivators accomplish the task
of interacting with a broad spectrum of NR HBDs with diverse sequences.
They do so by using a variety of interaction sites to make contact with
the NR HBDs. These include three NR box motifs (or four for SRC-1a) and
the auxiliary GRIP1 site described here. Each NR box can bind a broad
spectrum of NR HBDs, but interacts inefficiently with a subset of NR
HBDs. The subset of NR HBDs bound efficiently is different for each of
the NR boxes (29). Interaction affinity and specificity of each NR box
depend on the sequence of the core LXXLL motif and on the
flanking sequences. The relative importance of core and flanking
sequences varies for different NR box-NR HBD combinations (31). Among
the cluster of three NR boxes located in the central NID of the p160
polypeptide chain, NR box I appears to play a relatively minor role
since, in GRIP1/TIF2, this motif binds weakly to most NR HBDs (29, 33).
But NR boxes II and III, which are conserved in all three p160 family
members, and NR box IV, which is found only at the extreme C terminus
of SRC-1a, all contribute unique HBD binding specificities (29). Thus,
although no single NR box can bind efficiently to all NR HBDs, the
combination of the multiple NR boxes in each p160 coactivator
recognizes an extremely broad spectrum of NR HBDs. However, by itself,
the NID of GRIP1 or SRC-1 with three NR box motifs still binds some NR
HBDs inefficiently, including those of GR, AR, and retinoic acid
receptor How does GRIP1 NIDaux promote better binding of GR and AR HBDs? One
possible model is that both NID and NIDaux of GRIP1 make direct contact
with GR and AR HBDs. The binding interaction of the HBD with each
individual region is weak, but the two interactions together provide
sufficient binding energy to form a stable complex. In another possible
model, GRIP1 NIDaux could function through intramolecular interactions
that stabilize a conformation of NID that is favorable for binding GR
and AR HBDs. Although our data do not discriminate between these two
models, they indicate that NID and NIDaux must function in
cis to bind GR HBD and that 144 amino acids between the two
GRIP1 regions can be deleted without destroying their ability to
cooperate in binding GR HBD. Although GRIP1 NIDaux enhances the
affinity for binding some NR HBDs, it apparently does not influence the
specificity of NR box preferences since the preference of GR HBD for NR
box III over NR box II is evident even with GRIP1 NID fragments and
small NR box peptides that lack NIDaux (29, 31).
The GRIP1-(1011-1121) region has dual functions as an auxiliary
HBD-binding region and as the primary CBP/p300-binding domain. The
minimum CBP/p300-binding region determined here is consistent with
those reported previously for other members of the p160 coactivator family (9, 20, 33). Although we have not yet identified the specific
structural features in this region that are responsible for the NIDaux
function, we were able to rule out two obvious possibilities. Two
leucine-rich motifs, previously named LCD4 and LCD5 (13), that
partially resemble LXXLL motifs were not important for
binding GR HBD. LCD4 is only partially conserved among the three
different p160 family members, whereas LCD5 is more highly conserved.
The LCD4 mutation also had no effect on GRIP1 binding to p300, but the
LCD5 mutation substantially reduced p300 binding. Voegel et
al. (33) found that a similar mutation in TIF2, the human
orthologue to GRIP1, also reduced CBP/p300 binding. Since the LCD5
mutation reduced p300 binding without any effect on GR HBD binding,
these two functions are genetically separable, i.e. the
NIDaux function does not depend on p300 binding.
. A second group of
NRs, which includes the progesterone receptor, retinoid X receptor
,
thyroid hormone receptor
1, and vitamin D receptor, required only
NID for efficient interaction. For binding to GR, the NID and NIDaux of
GRIP1 must act in cis, but deletion of up to 144 amino
acids between the two regions did not reduce binding efficiency. Amino
acids 1011-1121 of GRIP1 also contain a p300 interaction domain, but
mutational analysis indicated that the p300 interaction function within
this region is separable from the ability to contribute to GR
hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
, human retinoid X
receptor
, human thyroid receptor
1 (TR
1), and human vitamin D
receptor were described previously (17, 18, 29). The yeast expression
vector for the Gal4 DBD-p300 fusion protein was made by inserting a
PCR-amplified p300 (35) cDNA fragment (coding for amino acids
1856-2414) into the SmaI/SalI sites of pGBT9
(CLONTECH). Internal deletions and point mutations
of the GRIP1 sequence were made by four-primer PCR as described
previously (36), and the resulting DNA sequences were subcloned into
the EcoRI/BamHI sites of pGAD424. The SRC-1-GRIP1
fusion gene was also constructed by four-primer PCR, and the resulting
DNA sequence was subcloned into the SalI/BglII
sites of pGAD424.3 The
bacterial expression vectors for the fusion proteins of GST with GRIP1
fragments 563-767 and 563-1121,
pGEX-4T1.GRIP1563-767His6 and
pGEX-4T1.GRIP1563-1121His6, were made by
inserting PCR-amplified GRIP1 cDNA fragments into the
BamHI/XhoI sites of pGEX-4T1.His6 (31). For in vitro transcription and translation of GR and
TR, pSG5.rGR (31) and pSG5.hTR
1 (37) were described previously.
-Galactosidase activities are shown as the mean ± S.D. from three independent yeast transformants.
RESULTS
-galactosidase reporter gene
controlled by a Gal4 enhancer. Among the GRIP1 fragments tested, only
GRIP1-(730-1121) exhibited the ability to interact with GR HBD in the
yeast two-hybrid assay (Fig. 1,
left panel). Note that GRIP1-(730-1121) contains only the
third of the three LXXLL motifs (NR box III), but it interacted with GR HBD as strongly as full-length GRIP1 (1462 amino
acids). We have previously shown that NR box III is the most important
NR box for binding GR HBD (29). However, GRIP1-(5-765), which contains
all three LXXLL motifs, and GRIP1-(1121-1462), which has no
NR box motifs, did not interact with GR HBD. All the tested GRIP1
fragments were expressed and could interact with control proteins in
the yeast two-hybrid assays (Fig. 1, right panel).
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Fig. 1.
Localization of the GR HBD interaction region
in GRIP1 by yeast two-hybrid assays. Gal4 AD fusion proteins of
GRIP1 fragments (indicated by amino acid numbers) and Gal4 DBD fusion
proteins of GR HBD, PR HBD, p300, or estrogen receptor (ER)
AF-1 were expressed from stably maintained expression plasmids in yeast
strain SFY526 in the presence of appropriate hormones (see
"Experimental Procedures"). Interaction of the fusion proteins
resulted in the activation of a chromosomal -galactosidase reporter
gene controlled by a Gal4 enhancer element; the resulting units
(u) of
-galactosidase (
-Gal) activity are
shown. Each fusion protein was inactive when expressed alone (data not
shown). AD, Gal4 activation domain; DBD, Gal4
DNA-binding domain. The vertical solid bars in
the diagrams represent NR box or LXXLL motifs. *, the
-galactosidase activity for interaction of Gal4
AD-GRIP1-(1121-1462) with Gal4 DBD-estrogen receptor AF-1 was 4 units,
and that for Gal4 AD with Gal4 DBD-estrogen receptor AF-1 was 0.2 units. Similar results were previously published (39).
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Fig. 2.
Localization of an auxiliary region of GRIP1,
separate from NID, that is required for binding GR HBD. Different
GRIP1 fragments, fused with Gal4 AD, were coexpressed with Gal4 DBD
fusion proteins of GR HBD, PR HBD, or p300-(1856-2414) in yeast strain
SFY526 in the presence of appropriate hormones. The resulting
-galactosidase (
-Gal) activities are shown. Each
fusion protein was inactive when expressed alone (data not shown).
u, units.
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Fig. 3.
Collaboration of two different GRIP1 regions
in the interaction with GR HBD. Fragments representing different
domains of GRIP1 were either linked together and expressed as a Gal4 AD
fusion protein or separately fused with Gal4 AD in yeast expression
vectors with a leu2 marker (GRIP1-(730-868)) or with a
his3 marker (GRIP1-(939-1121) and GRIP1-(1011-1121)).
These two different markers allowed different Gal4 AD-GRIP1 fusion
proteins to be coexpressed in yeast strain SFY526 along with the Gal4
DBD-GR HBD fusion protein in the presence of 10 µM
deoxycorticosterone. The resulting -galactosidase
(
-Gal) activities are shown. u, units.
belong to a subset of NRs that interacted
efficiently with GRIP1 NID only when NIDaux was also present; in
contrast, PR, retinoid X receptor
, TR
1, and vitamin D receptor
belong to another group of NRs that did not require NIDaux for
efficient interaction (Fig. 4).
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Fig. 4.
Requirement by different NR HBDs for the
auxiliary HBD-binding region of GRIP1. Two different GRIP1
fragments were expressed as Gal4 AD fusion proteins together with Gal4
DBD fusion proteins of NR HBDs in yeast strain SFY526 in the presence
of appropriate hormones. The resulting -galactosidase
(
-Gal) activities are shown. u, units;
RAR, retinoic acid receptor; RXR, retinoid X
receptor; VDR, vitamin D receptor.
1 was tested in vitro. GRIP1-(563-767), which contains
NID, and GRIP1-(563-1121), which contains NID and NIDaux, were
expressed in E. coli as fusion proteins with GST and
purified by affinity chromatography. 35S-Labeled TR and GR
were synthesized in vitro in the presence or absence of the
appropriate hormone and incubated with GST-GRIP1 proteins bound to
glutathione-agarose beads. Bound material was eluted and analyzed by
SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. TR
interacted well with GRIP1-(563-767) and GRIP1-(563-1121) (Fig.
5a), indicating that the NID
of GRIP1 is sufficient for efficient interaction with TR. However,
GRIP1-(563-767) bound GR poorly when compared with the longer
GRIP1-(563-1121); the shorter GRIP1 fragment also bound GR less
efficiently than TR, whereas the longer GRIP1 fragment bound GR and TR
equally. All interactions were hormone-dependent, and the
antagonist RU486 could not substitute for dexamethasone to induce
GR-GRIP1 interaction. These data were consistent with our observations
in the yeast two-hybrid assays indicating that GRIP1 NID by itself was
not sufficient for efficient binding of GR HBD.
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Fig. 5.
Interaction of GRIP1 fragments with
GR and TR in vitro. a,
35S-labeled in vitro synthesized full-length
TR 1 and GR were incubated with 1.6 µM GST,
GST-GRIP1-(563-1121)-His6, or
GST-GRIP1-(563-767)-His6 bound on agarose beads. TR
1
and GR were synthesized in the absence (
) or presence of 10 µM 3,5,5'-triiodo-L-thyronine (+),
dexamethasone (+), or RU486 (±). Bound material, expressed as percent
of input, was eluted and analyzed as indicated under "Experimental
Procedures." b, 35S-labeled GR or TR
1 was
synthesized in vitro in the presence of 10 µM
dexamethasone or 3,5,5'-triiodo-L-thyronine, respectively,
and then incubated with different amounts of
GST-GRIP1-(563-1121)-His6 or
GST-GRIP1-(563-767)-His6 bound on agarose beads. Bound
material was measured and is presented as described for a.
EC50 values were as follows: for GST-GRIP1-(563-1121), 0.2 µM with GR and 0.3 µM with TR; and for
GST-GRIP1-(563-767), 10 µM with GR and 2 µM with TR.
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Fig. 6.
Interaction of an SRC-1-GRIP1 fusion protein
with NR HBDs. A fusion protein containing the central NID of SRC-1
and the NIDaux of GRIP1 was compared with full-length SRC-1a and a
C-terminally truncated form of SRC-1a for binding to three different NR
HBDs in yeast two-hybrid assays. Each coactivator protein was fused to
Gal4 AD and coexpressed with Gal4 DBD-NR HBD fusion proteins in yeast
strain SFY526 in the presence of appropriate hormones. The resulting
-galactosidase (
-Gal) activities are shown.
u, units. Numbers above each protein
diagram represent SRC-1a amino acids, whereas numbers below
each diagram represent GRIP1 amino acids. NR boxes (NRB) are
represented by vertical bars. CID, CBP/p300
interaction domain.
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Fig. 7.
Within the GRIP1-(1011-1121)
region, the auxiliary GR HBD binding activity and p300 binding activity
are genetically separable. GRIP1-(730-1121) with either wild-type
(wt) sequence or the indicated mutations was expressed as a
fusion protein with Gal4 AD and coexpressed with a fusion protein of
Gal4 DBD with either GR HBD or p300 in yeast strain SFY526 in the
presence of 10 µM deoxycorticosterone (for GR) or in the
absence of hormone (for p300). The resulting -galactosidase
(
-Gal) activities are shown. u, units.
DISCUSSION
1
HBD residues that bind GRIP1 are highly conserved among the NR
superfamily. These residues are found in
-helices 3, 4, 5, and 12. These helices form a hydrophobic groove surrounded by a rim of charged
residues on the surface of the HBD; the groove accommodates the core
leucine residues of the NR box motif (31). However, although highly
conserved residues form the core of the HBD site that interacts with
GRIP1, the 10% sequence identity among the HBDs of diverse members of
the NR family suggests that p160 coactivators may encounter
considerable structural diversity around the core interaction sites of
various NR HBDs. In fact, amino acid sequences flanking the
LXXLL motif of NR box II interact directly with surface
residues from the C-terminal region of the TR HBD that are not
conserved among the NR family; alterations in these TR residues affect
the NR box binding preference of TR (31).
. GRIP1 and SRC-1 use different strategies to solve this
problem. GRIP1 has an auxiliary region (NIDaux), located between amino
acids 1011 and 1121, that cannot by itself bind NR HBDs, but cooperates
with NID to accomplish efficient binding of GR and AR HBDs. SRC-1 lacks
an auxiliary HBD binding function comparable to that found in GRIP1.
Instead, SRC-1a contains an additional NR box IV located at the extreme C terminus of the protein, which binds GR and AR HBDs independently of
the central NID (NR boxes I-III) (29). Another isoform of SRC-1,
called SRC-1e, is produced by alternative splicing of the same
transcript; SRC-1e lacks the C-terminal NR box IV (9, 47, 48) and, as a
result, binds GR and AR
poorly.5
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ACKNOWLEDGEMENTS |
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We thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) for the SRC-1a cDNA and Dr. David Livingston (Dana-Farber Cancer Institute, Boston, MA) for the p300 cDNA.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants DK43093 (to M. R. S.) and CA20535 (to K. R. Y.) from the National Institutes of Health and by National Science Foundation Grant MCB9604938 (to K. R. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by postdoctoral fellowships from the European Molecular Biology Organization and the Helen Hay Whitney Foundation.
Supported by a predoctoral traineeship from the University of
California Breast Cancer Research Program.
** To whom correspondence should be addressed: Dept. of Pathology, HMR 301, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90033. Tel.: 323-442-1289; Fax: 323-442-3049; E-mail: mstallcu{at}zygote.hsc.usc.edu.
The abbreviations used are: NR, nuclear receptor; AD, activation domain; DBD, DNA-binding domain; HBD, hormone-binding domain; CBP, cAMP-responsive element-binding protein-binding protein; NID, nuclear receptor interaction domain; PCR, polymerase chain reaction; GR, glucocorticoid receptor; AR, androgen receptor; PR, progesterone receptor; TR, thyroid hormone receptor; GST, glutathione S-transferase; NIDaux, auxiliary nuclear receptor interaction domain.
2 F. M. Sladek, M. D. Ruse, Jr., L. Nepomuceno, S.-M. Huang, Y. Maeda, and M. R. Stallcup, submitted for publication.
3 SRC-1a sequence is from GenBankTM accession number U90661.
4 H. Hong and M. R. Stallcup, unpublished results.
5 H. Ma and M. R. Stallcup, manuscript in preparation.
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REFERENCES |
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