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INTRODUCTION |
Coactivators are molecules that have recently been found to be
important components of the transactivation activity of steroid hormone
receptors (1-3). Among the first discovered, and the most widely
studied, are the p160 coactivators of SRC-1 (4), TIF2/GRIP1 (5, 6), and
AIB1 (7). The coactivators were originally defined as factors that
increase the total amount of induced gene product with saturating, or
pharmacological, concentrations of hormone (4-7). For the p160
coactivators this action is thought to involve domains with intrinsic
transactivation activity (8-10), called activation domains
(ADs).1 For many of the
coactivators, their intrinsic transactivation activity may be related
to their histone acetyltransferase (HAT) activity (1, 3, 11). In
addition, and for those coactivators without HAT activity like
TIF2/GRIP1 (8), coactivator recruitment of comodulators with HAT
activity, such as CBP (8, 12), p300 (12, 13), and PCAF (14), is
considered important. In general, the comodulators are thought to bind
only to the p160 coactivators (13, 15). However, PCAF, which was
initially identified as a binder of p300/CBP (16), has also been
reported to bind to glucocorticoid receptors in pull-down assays in a
ligand-independent fashion (14). Furthermore, the
ligand-dependent association of PCAF with retinoic acid
receptors is different from its association with SRC-1 or p/CIP (=AIB1)
(17). The multiprotein complexes called DRIP/TRAP/Mediator are thought
to be recruited by the above coactivator-comodulator complexes to relay
the original signal from the steroid receptors (18, 19). These
complexes are proposed then to modify other components of the
transcriptional complex to give the final transcriptional response.
We have recently shown that another property of the p160 coactivators
is to modulate both the dose-response curve of agonists, i.e. the concentration of steroid required for half-maximal
induction (EC50), and the partial agonist activity of
antagonists. This behavior has been seen for glucocorticoid receptors
(GR) (20-22) and progesterone receptors (23). This modulation
of the dose-response curve (or EC50) and of the partial
agonist activity of antisteroids has important theoretical and clinical
consequences. The EC50 for induction of most genes is near
the physiological levels of hormone. Therefore, changes in the
EC50 of one steroid-responsive gene versus
another will both cause different levels of induction from the two
genes by endogenous steroids and provide a sensitive mechanism for the
differential control of expression of these two genes during
development, differentiation, and homeostasis. Antagonists are
extremely useful in endocrine therapies to inhibit the actions of
endogenous hormones in situations such as inflammation (24), conception
(25), and hormone-dependent cancers (26, 27) but often
produce undesirable side effects. If one could increase the partial
agonist activity for genes other than the one to be suppressed, this
would greatly reduce the number of side effects that result from the
indiscriminate repression of all responsive genes.
Interestingly, the overexpression of corepressors (SMRT and NCoR) (28,
29) often produces a response opposite to that of increased
coactivators. Thus, corepressors can cause both a right shift in the
dose-response curve to higher EC50 values and a decrease in
the partial agonist activity of antisteroids for GR (20, 30) and
progesterone receptors (23, 30). Furthermore, the ability of added SMRT
to antagonize the actions of overexpressed TIF2 suggests that the final
responses are determined by an equilibrium reaction with receptors that
depends upon the ratio of coactivators to corepressors (20).
Despite the major quantitative effects on steroid receptor gene
transactivation that are caused by varying the intracellular levels of
coactivators, only general features of the mechanism by which
coactivators modulate steroid receptor induction properties are known.
The ability of coactivators to augment the total amount of induced gene
transcripts requires coactivator binding to the agonist-bound receptors
and may result from more efficient binding/recruitment of transcription
complex core factors (31, 32). In contrast, the conventional model of
steroid hormone action is silent on how coactivators might change
either the EC50, and the position of the dose-response
curve, or the partial agonist activity of antisteroids. In fact, the
current thinking is that coactivators do not interact with
antagonist-bound receptors (1, 3, 11), in which case the modulation of
the partial agonist activity of antisteroid complexes by coactivators
would have to involve indirect interactions.
Whereas the ability of coactivators to increase the total levels of
transactivation with receptor-agonist complexes proceeds via binding to
receptors, it is not known if similar interactions are required for the
modulation of the EC50, and the dose-response curve, of
receptor-agonist complexes. One indication that separate pathways are
involved comes from studies with TIF2, which does not contain the
intrinsic HAT activity that is associated with many coactivators (1, 3,
11). TIF2 binds to CBP (8), although, which does have HAT activity
(12). However, the ability of TIF2 to modulate GR activities was found
to be independent of the HAT activity of CBP (21), which can itself
modulate GR transactivation properties (20). A second indication of
separate pathways is the observation that several factors can alter the dose-response curve and partial agonist activity of GR complexes in a
manner that is independent of their effects on total transcriptional activity of GR (20, 30, 33-35). Finally, a recent report indicates that TIF2 acts via a rate-limiting intermediate or step that does not
regulate the total levels of receptor-mediated gene induction (21).
The purpose of this study, therefore, was to examine in greater detail
whether the intrinsic ability of a coactivator to increase the total
transactivation of GR is required for its ability to modulate the
EC50, or dose-response curve, of GR-agonist complexes and
the partial agonist activity of GR-antagonist complexes. We took the
approach of asking if the minimal domains of coactivators that are
required for GR modulation are the same or different from those needed
for conventional coactivator activity. At the same time, we inquired if
the effects of this minimal modulatory domain could be replaced by any
other proteins that are thought to interact with the p160 coactivators.
The ability of receptor-antagonist complexes to interact with
coactivators was also determined. From these studies, we conclude that
the modulation of GR transactivation activities involves direct
interactions of coactivators with both GR-agonist and -antagonist
complexes in a manner that is mediated by parameters that are unrelated
to both the conventional coactivator domains and known
coactivator-associated proteins.
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MATERIALS AND METHODS |
Unless otherwise indicated, all operations were performed at
0 °C.
Chemicals--
Dexamethasone (Dex) purchased from Sigma while
promegestone (R5020) was from PerkinElmer Life Sciences.
Dex-21-mesylate (Dex-Mes) (36) and Dex-oxetanone (Dex-Ox) (37) were
synthesized as described. RU486 was a gift from Etienne Baulieu (Paris,
France). Restriction enzymes and DNA polymerase were from New England
Biolabs, Amersham Biosciences, or Promega.
Antibodies--
The monoclonal mouse anti-GAL DBD antibody
(sc-510) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids--
The Renilla null luciferase reporter was purchased
from Promega (Madison, WI). (UAS)5tkLuc was from
Clontech (Palo Alto, CA), whereas
pBSK+, pSG5, and pCR3.1 were from Stratagene (La Jolla,
CA). GREtkLUC has been previously described (38). pSVLGR and pRBAL-GR
(Keith Yamamoto, University of California, San Francisco,
CA), Rsv-GAL4-PCAF (Xiang-Jiao Yang, McGill University Health
Center, Montreal, Quebec), GAL4-p300 and GAL-CBP (George Chrousos,
NICHD, National Institutes of Health), SRC-1a (Bert
O'Malley, Baylor College of Medicine, Houston, TX), SRC-1
(1-399), SRC-1 (361-1441), SRC-1 (361-1139), and SRC-1
(1139-1441) (Sergio Oñate, University of Pittsburg, PA), DRIP150
and DRIP205 (Leonard P. Freedman, Memorial Sloan-Kettering Cancer
Center, New York, NY), TIF2, TIF2m123, TIF2.0, TIF2.1, TIF2.2, TIF2.3,
and TIF2.4 (Hinrich Gronemeyer, IGBMC, Strasbourg), and GRIP1,
GRIP
1095-1106, GRIP1057-1109, and GRIP(5-1121)AD1 (Michael
Stallcup, University of Southern California, Los Angeles) were received
as gifts.
pSG5/TIF2.37 and pSG5/TIF2.38 were both made by PCR, using pSG5/TIF2 as
the template. For pSG5/TIF2.37, the primers were
5'-CGGGATCCACCATGAAAGATGATACTAAAGATATTGGTTTACC-3' and
5'-CGGGATCCCTACCCTATATTCATGACCTGAG-3'. For pSG5/TIF2.38, the primers
were 5'-CGGGATCCACCATGAAAGATGATACTAAAGATATTGGTTTACC-3' and
5'-CGGGATCCCTAAGCTCCCTCATCACTCGGAG-3'. In both cases, the amplified DNA
was then cut with BamHI and inserted into the
BamHI site of pSG5.
GAL/TIF2.4 was prepared by first amplifying the TIF2.4 fragment by PCR
from pSG5-TIF2.4 using 5'-GGAATTCGAGAGAGCTGACGGGCAGAGC-3' and
5'-CGGGATCCCTACCCTATATTCATGACCTGAG-3' as primers. The PCR product was
cut with BamHI and EcoRI and inserted into the
BamHI and EcoRI sites of the GAL plasmid (pm,
Clontech). GAL/TIF2.4m123 was constructed by the
same method except that pSG5-TIF2.4m123 was used as the template.
For VP16/TIF2.4, the TIF2.4 fragment was cut from GAL/TIF2.4 and
inserted into the BamHI and EcoRI sites of the
VP16 plasmid (Clontech). For VP16/ TIF2.3, the
TIF2.3 fragment was PCR amplified from pSG5-TIF2.3 with
5'-GGAATTCGAGAGAGCTGACGGGCAGAGC-3' and 5'-CTGTCTCATCATTTTGGC-3',
cut with BamHI and EcoRI, and inserted into the
BamHI and EcoRI sites of VP16 plasmid.
PGEX-TIF2.4 and GAL/TIF2.4m123 were prepared by excising the TIF2.4 (or
TIF2.4m123) fragment from GAL/TIF2.4 (or GAL/TIF2.4m123) and inserting
it into the EcoRI and SalI sites of pGEX-6p-1
(Amersham Biosciences).
Cell Culture and Transient Transfection--
Monolayer cultures
of CV-1 cells were grown in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% fetal calf serum (Biofluids
Inc., Rockville, MD) at 37 °C in a humidified incubator (5%
CO2). Transient transfections were achieved using calcium
phosphate precipitation (39) or 1.0 µl of LipofectAMINE Reagent
(Invitrogen) or 0.5 µl of FuGENE 6 (Roche Molecular Diagnostics) in
0.3 ml of Opti-MEM I (Invitrogen) without serum in 24-well plates
(10-fold more in 60-mm dishes). As a control for the transfected cofactors, equimolar amounts of the empty plasmid vector were added.
The total transfected DNA was adjusted to 300 ng/well of a 24-well
plate (or 3 µg/60-mm dish) with pBluescriptII SK+
(Stratagene). Renilla TK or TS (5-10 ng/well of a 24-well plate) was
included as an internal control. Cells were incubated with plasmid DNA,
Opti-MEM I, and LipofectAMINE or FuGENE 6 reagent for 24 h, after
which this mixture was replaced by the normal media (10% fetal calf
serum, Dulbecco's modified Eagle's medium). The cells were induced
with steroids for 20-24 h and then harvested. The cells were lysed and
assayed for reporter gene activity using the Dual Luciferase Assay
reagents according to manufacturer's instruction (Promega, Madison,
WI). Luciferase activity was measured by an EG&G Berthhold's
luminometer (Microlumat LB 96P). The data were normalized either for
total protein or Renilla null luciferase activity.
Mammalian Two-hybrid Assays--
The recommended procedure for
the Mammalian Matchmaker two-hybrid assay kit
(Clontech) was modified slightly by changing from a
chloramphenicol acetyltransferase reporter to a Luciferase Reporter pFRLuc (Stratagene), which is under the control of five repeats of the
upstream activating sequence for the binding of GAL4.
Bacterial Expression of Proteins--
pGEX series of plasmids
were transformed into Escherichia coli (BL21; Amersham
Biosciences) according to the manufacturer's procedure. A single
colony was picked and inoculated into 3 ml of LB broth with 100 ng/ml
ampicillin. After overnight culture, 1 ml of bacterial culture was
diluted into 50 ml of LB broth containing 100 ng/ml ampicillin, shaken
at 37 °C for 2 h, adjusted to 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside, and shaken at
37 °C for another 3 h. The cells were harvested by
centrifugation, washed once with phosphate-buffered saline, resuspended
in 10 ml of phosphate-buffered saline, and sonicated for 30 cycles at
30% of maximum power. The supernatant was collected for use after
centrifugation (5000 × g for 20 min).
In Vitro Transcription and Translation Assays--
pRBAL-GR (1 µl), 40 µl of Promega TNT (SP6) quick coupled
transcription/translation system master mixture, and 2 µl of
[35S]methionine (Amersham Biosciences) were brought up to
a total volume of 50 µl with H2O and incubated at
30 °C for 90 min according to the manufacturer's (Promega)
recommendations. Different steroid hormones (Dex, Dex-Mes, Dex-Ox,
progesterone, and RU486) were added into the transcription-translation
reaction during the 30 °C incubation to both increase the stability
of the synthesized GR and to cause activation of the GR-steroid complex.
Pull-down Assays--
Sonicated bacterial lysate (0.5 ml)
containing overexpressed GST, GAL/TIF2.4, or GAL/TIF2.4m123 was
incubated with 20 µl of glutathione-Sepharose 4B beads for 1 h
at 0 °C. The mixture was centrifuged (12,000 × g), the
supernatant was discarded, and the pellet was washed with
phosphate-buffered saline (3 × 1 ml). Each 20-µl sample of
immobilized GST or GST-chimera was then incubated overnight with 10 µl of hormone prebound, activated, 35S-labeled GR
translation product. The matrix was washed with phosphate-buffered saline (4× 1 ml). The immobilized proteins were removed from the beads
by heating at 90 °C for 5 min in 20 µl of 2× SDS loading buffer.
The proteins were then separated on 10% SDS-PAGE gels and the bound GR
was located by autoradiography.
Statistics--
Unless otherwise noted, all experiments were
performed in triplicate several times. The error bars in
graphs of individual experiments correspond to the S.D. of the
triplicate values. KaleidaGraph 3.5 (Synergy Software, Reading, PA) was
used to determine a least-squares best fit (R2
was almost always 
0.95) of the experimental data to the theoretical dose-response curve, which is given by the equation derived from Michaelis-Menten kinetics of y = [free
steroid]/([free steroid] + Kd) (where the
concentration of total steroid is approximately equal to the
concentration of free steroid because only a small portion is bound),
to yield a single EC50 value. The values of n
independent experiments are then analyzed for statistical significance by the two-tailed Student's t test using the program
"InStat 2.03" for Macintosh (GraphPad Software, San Diego, CA). In
paired analyses, all comparisons in a given figure are performed at the
same time with the same seeding of cells and then analyzed for
n different experiments.
| |
RESULTS |
Minimum Domain of Coactivator TIF2/GRIP1 for Modulation of GR
Properties--
TIF2/GRIP1 is one of the three p160 coactivators that
increase the total induced levels of gene product with steroid
receptors (5, 6). Of the various domains of TIF2/GRIP1 that have been identified (Fig. 1A), the
receptor interaction domains (RIDs) and the two activation domains (AD1
and AD2) have been found to be required for full coactivator activity
(8). Amino acids 1057-1109 of TIF2/GRIP1 are within the AD1 domain and
are required for CBP/p300 binding (8, 40, 41). This binding of CBP/p300 is thought to be particularly important for the coactivator activity of
TIF2/GRIP1 because TIF2/GRIP1 does not possess any intrinsic HAT
activity (8) but rather relies on the binding of CBP/p300 to provide
HAT activity.
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Fig. 1.
GRIP1 sequences required for modulation of GR
transcription properties and coactivator activity. A,
cartoon of the various domains of GRIP1/TIF2. The amino acid positions
of the various domains are from the literature (8, 41) and are given
below the figure of the protein. The abbreviations for the
domains are above the figure (bHLH, basic
helix-loop-helix; PAS, Per-Arnt-Sim;
RIDaux, auxiliary RID; Q-rich,
glutamine-rich). B, modulatory activity of GRIP1 constructs.
Triplicate samples of CV-1 cells in 60-mm dishes were transiently
transfected with the indicated GRIP1 plasmids, GREtkLUC reporter, and
Renilla control plasmid, and then induced with EtOH ± 1 nM Dex, 1 µM Dex, or 1 µM
Dex-Mes. The luciferase activities, normalized to the internal Renilla
control values, were then expressed as percent of the maximal response
with 1 µM Dex as described under "Materials and
Methods" for the abbreviated assay. The plotted data are the average
of two independent experiments ± range. C, coactivator
activity of GRIP1 constructs. Triplicate samples of CV-1 cells were
transiently transfected as in B and treated with EtOH ± 1 µM Dex. The ratio of the total luciferase activity
(±GRIP1 construct) in the presence of 1 µM Dex,
normalized to the internal Renilla control values, was determined. The
data represent the average ± S.E. of four independent
experiments. The asterisk (*) indicates a statistically
significant difference between the coactivator activity of
GRIP1057-1109 and GRIP1 (paired p = 0.020).
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We previously demonstrated that TIF2 causes a left shift in the
dose-response curve for agonist complexes, and increased partial agonist activity of antagonist complexes, of both GR and progesterone receptors (20, 21, 23). To identify the domain(s) of TIF2/GRIP1 that
are required for this modulation, we now use an abbreviated assay in
which increases in the activity of a subsaturating concentration of
steroid, expressed as percent of maximal induction by saturating concentrations of agonist, have been shown to be diagnostic of a left
shift in the dose-response curve to lower EC50 values (20, 21, 42, 43). The main advantage of this abbreviated assay is that it
requires many fewer samples to reach the same conclusion. The changes
in partial agonist activity of the antisteroid were assessed by the
normal method of expressing the activity of 1 µM
antagonist as percent of maximal induction by saturating concentrations of agonist (20, 21, 23, 30, 33).
Using this abbreviated assay, we assessed the modulatory activity of
GRIP1 mutants lacking the activation domains AD2 and/or segments of AD1
(Fig. 1B). GRIP1095-1106 is missing a portion of AD1
that is involved in binding to p300 (40). However, the ability of this
mutation to cause a left shift of the GR dose-response curve, as
determined from the increased activity of 1 nM Dex relative to that of the samples without added GRIP, is indistinguishable from
the full-length GRIP. This mutation also displays an undiminished capacity to increase the partial agonist activity of the
antiglucocorticoid Dex-Mes. Similarly, GRIP1057-1109, which lacks
almost all of the AD1 domain (8, 40, 41), and GRIP(5-1121)AD1,
which is missing both AD1 and AD2 domains, display the same amount of modulatory activity as does the intact GRIP1. We, therefore, conclude that none of these deleted residues are required for the modulatory activity of GRIP1.
The conventional definition of a coactivator is a molecule that
increases the total level of steroid receptor-induced gene product in
the presence of saturating concentrations of an agonist (1, 3, 11). We,
therefore, asked whether the coactivator activity segregates with the
modulatory activity of the above GRIP1 mutants. As shown in Fig.
1C (average data of n = 4), deletion of the
AD1 domain reduces the coactivator activity of GRIP1 (paired p = 0.020 for 1057 versus GRIP1).
Interestingly, deletion of both AD1 and AD2 to give
GRIP-(5-1121)AD1 did not further decrease the coactivator activity.
Nevertheless, when compared with Fig. 1B, it appears that
there is no correlation between the modulatory activity and coactivator
activity of GRIP1 with the various deletions.
These questions were examined in greater detail using a larger set of
deletions in TIF2, which is the human ortholog of the mouse GRIP1. TIF2
is 94% identical to GRIP1 (44) and has no known different properties
from GRIP1. All of the TIF2 modulatory activity was found to be
contained in TIF2.1 (624-1287) and TIF2.3 (624-1179) (Fig.
2A, top and
middle panels, respectively). These results suggest that
neither the amino-terminal half of TIF2, nor the AD2 domain, are
required for the activity of TIF2 in our assays. TIF2m123 contains a
LXXLL to LXXAA mutation in each of the
three RIDs (Fig. 1A) and has been described to no longer
bind to steroid receptors (8, 45). Thus, the inactivity of TIF2m123 in
our assay (top panel) indicates that TIF2 binding to GR is required for the modulation of GR activities. Not surprisingly, the
TIF2 AD2 domain by itself (TIF2.2) is inactive (top panel). Further removal of AD1 and the auxiliary RID domain (41) from TIF2.3
gives TIF2.4, which still retains almost all of the activity of the
wild type TIF2 (bottom panel). In experiments examining multipoint dose-response curves directly, the average left shift is
2.41 ± 0.17-fold (S.E., n = 16, p < 0.0001) for TIF2 and 1.80 ± 0.09-fold (S.E., n = 13, p < 0.0001) for TIF2.4 (data not shown). For
comparison, the -fold left shift in the dose-response curve with
TIF2m123, which is unable to interact with GR, is 1.09 ± 0.08 (S.E., n = 6). Thus, TIF2.4 is the smallest fragment of
TIF2 that we have found with nearly the full modulatory activity of the
intact TIF2.
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Fig. 2.
Minimum TIF2 sequences required for
modulation of GR transcription properties and coactivator activity.
A, modulatory domain of TIF2. Triplicate samples of CV-1
cells in 24-well plates were transiently transfected with the indicated
TIF2 plasmids, GREtkLUC reporter, and Renilla control plasmid, and then
induced with EtOH ± 1 nM Dex, 1 µM Dex,
or 1 µM Dex-Mes. The luciferase activities were
determined as in Fig. 1B and plotted as the average ± S.E. of multiple experiments (top, n = 12-16 except for TIF2.2 and TIF2.0, where n = 2;
middle, n = 3; bottom,
n = 11). B, coactivator domain of TIF2.
Triplicate samples of CV-1 cells were transiently transfected and
treated with EtOH ± 1 µM Dex as in Fig.
1B. The ratio of the total luciferase activity (± TIF2
construct) in the presence of 1 µM Dex, normalized to the
internal Renilla control values, was determined. The data represent the
average ± S.E. of 11 independent experiments. The
asterisks (***) indicate a statistically significant
difference between the coactivator activity of TIF2.4 and TIF2 (paired
p = <0.0001).
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We next asked how much coactivator activity is preserved in TIF2.3 and
TIF2.4. As shown in Fig. 2B, TIF2.3 is just as effective as
the full-length TIF2 in augmenting the total level of gene induction by
GR with saturating concentrations of the agonist Dex. In contrast,
TIF2.4 has <40% of the activity of TIF2 (Fig. 2B; average
-fold increase is 1.61 ± 0.16 for TIF2.4 versus
2.69 ± 0.22 for TIF2 (±S.E., n = 11, paired
p < 0.0001)). Thus, whereas TIF2.4 retains most of the
modulatory activity of TIF2, it has lost more than half of the
coactivator activity. Collectively, the data with TIF2 and GRIP1
mutants suggest that the modulation of GR properties is independent of
the coactivator activity of TIF2/GRIP1. This implies that the two
activities of TIF2, coactivator and modulator, are expressed by
different mechanisms.
The active mutant TIF2.3 has been divided in half to give TIF2.5 (amino
acids 624-869) and TIF2.7 (amino acids 870-1179) (8). TIF2.7
(870-1179) functions as a dominant suppressor in the coactivator and
modulator assays (21), whereas TIF2.5 has weak activity in both assays
(data not shown). When equal molar amounts of TIF2.5 and TIF2.7
plasmids are cotransfected, the suppressor activity of TIF2.7 is again
dominant (data not shown). Thus, a simple mixture of the two halves of
the TIF2.3 protein is not sufficient to reconstitute the activity of
the intact TIF2.3.
Region of Coactivator SRC-1 Retaining Modulatory Activity of GR
Properties--
SRC-1 is another p160 coactivator (4) that modulates
GR activities (20). Whereas generally similar in organization to TIF2/GRIP1, notable differences of SRC-1 include an activation domain
at the amino-terminal end (AD1), a fourth RID in the region of amino
acids 1139-1441 (9), and a repressive or negative domain in the
COOH-terminal 56 amino acids (46) of SRC-1 (Fig. 3A). Interestingly, the two
activation domains (AD1 and AD2), the three central RIDs, and the
Q-rich domain all make minimal contributions the bulk of the modulatory
activity of SRC-1 (Fig. 3A). A more detailed analysis of
SRC-1 versus SRC-1/1139-1441 (Fig. 3B) confirms
that this activity of SRC-1 is largely contained in the COOH-terminal
300 amino acids. This region is known to contain a RID that shows very
strong binding to GRs (47).
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Fig. 3.
Minimum SRC-1 sequences required for
modulation of GR transcription properties. A, modulatory
domain of SRC-1. Triplicate samples of CV-1 cells in 60-mm dishes were
transiently transfected with the indicated SRC-1 plasmids, GREtkLUC
reporter, and Renilla control plasmid, and then induced with EtOH ± 1 nM Dex, 1 µM Dex, or 1 µM
Dex-Mes. The luciferase activities were determined as in Fig.
1B and plotted as the average ± S.E. of three to six
experiments. B, dose-response curve for Dex induction ± SRC-1 domains. Triplicate samples of CV-1 cells in 60-mm dishes were
transiently transfected with the indicated SRC-1 plasmids, GREtkLUC
reporter, and Renilla control plasmid, and then induced with EtOH ± various concentrations of Dex or 1 µM Dex-Mes as in
Fig. 1B. The data were expressed -fold induction over basal
(EtOH) activity and the average ± S.D. was plotted as percent of
the maximal -fold induction by 1 µM Dex. Curve fitting
was performed by KaleidaGraph as described under "Materials and
Methods."
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SRC-1/1139-1441 has no ability to increase the total activity of
GR-Dex complexes (data not shown). As previously reported, SRC-1
displays very little conventional coactivator activity with GR (4, 20).
Therefore, we cannot say whether SRC-1/1139-1441 has less coactivator
activity than SRC-1. However, it is clear that the modulatory activity
of SRC-1 can be restricted to a domain (amino acids 1139-1441) that is
devoid of conventional coactivator activity. In this respect, the
modulatory activity of SRC-1, like that of TIF2, is independent of any
changes by SRC-1 in the total activity of saturating concentrations of agonist.
PCAF Modulates GR Properties but Does Not Synergize with
SRC-1--
Amino acids 1139-1250 of SRC-1 have been reported to
interact with PCAF (48). Because these sequences are contained
within the COOH-terminal modulatory fragment of SRC-1 (Fig. 3), PCAF is
a potential downstream target for SRC-1 in its modulation of GR
activity. PCAF is also considered to be a target of CBP and other
coactivators, such as AIB1, although there is no report of interactions
of PCAF with TIF2/GRIP1 (14, 16, 17). CBP has recently been reported to
have little to no direct binding to receptors (15, 49) but does bind to
PCAF, which directly interacts with various steroid receptors including
GR (14). Collectively, these data suggest that the modulatory effects
of changing concentrations of GR, and of coactivators and corepressors, could be mediated by PCAF. This hypothesis was tested directly by
looking at the effects on GR transactivation properties of cotransfected PCAF. As seen in Fig.
4A, PCAF causes an ~2.8-fold left shift in the GR dose-response curve and an increase in the partial
agonist activity of DM. At the same time, the added PCAF produces a
slight increase in total activity (1.35 ± 0.29-fold increase) and
a greater increase in -fold induction (2.57 ± 0.77-fold increase, ± S.E., n = 3).
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Fig. 4.
Modulatory activity of PCAF. A,
ability of PCAF to modify GR transcription properties. Triplicate
samples of CV-1 cells in 60-mm dishes were transiently transfected with
40 ng of GR plasmid without or with 120 ng of PCAF plasmid, GREtkLUC
reporter, and Renilla control plasmid, and then induced with EtOH ± various concentrations of Dex or 1 µM Dex-Mes as
described in the legend to Fig. 1B. B, triplicate
samples of CV-1 cells in 24-well plates were transiently transfected
without or with GR (6 ng) and 1 (56 ng) or 0.5 units (28 ng) of
SRC-1/1139-1441, without or with 0.5 units (60 ng) of PCAF plasmids,
plus GREtkLUC reporter and Renilla control plasmid, and then induced
with EtOH ± various concentrations of Dex or 1 µM
Dex-Mes as described in the legend to Fig. 1B. In both
experiments, the data were expressed as -fold induction over basal
(EtOH) activity and the average ± S.D. was plotted as percent of
the maximal -fold induction by 1 µM Dex. Curve fitting
was performed by KaleidaGraph as described under "Materials and
Methods." Similar results were obtained in two (A) or one
(B) additional independent experiments.
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The ability of PCAF to alter GR transactivation properties, coupled
with its binding to the COOH-terminal active region of SRC-1
(SRC-1/1139-1441) suggests that PCAF could be a necessary component of
SRC-1 modulatory activity. To test this hypothesis, we asked if the
combination of PCAF and SRC-1/1139-1441 afforded any additional, or
synergistic, response over the sum of the individual components. To
maximize our ability to observe any combined response, we used
submaximal amounts of each cofactor. The addition of 0.5 units of
SRC-1/1139-1441 (Fig. 4B) and PCAF (data not shown) causes less shift in the dose-response curve, and change in the partial agonist activity of Dex-Mes, than does 1 unit of each factor. Importantly, the combination of 0.5 units of SRC-1/1139-1441 and PCAF
is not significantly more effective than 0.5 units of SRC-1/1139-1441 alone (Fig. 4B). Because PCAF does not increase, either
synergistically or even additively, the effects of SRC-1/1139-1441 on
the GR transactivation properties, we conclude that PCAF does not
participate in the modulatory activity of SRC-1/1139-1441.
Modulatory Activity of TIF2.4 Does Not Involve Binding to CBP,
p300, or PCAF--
While the abilities of SRC-1 and TIF2 to modulate
GR properties appear to be identical (20), the mechanistic details
could be different. Therefore, we inquired whether PCAF might
contribute to the actions of TIF2. There is no report of PCAF binding
to TIF2/GRIP1 (14, 16, 17). This question was pursued by asking if PCAF
can interact in a mammalian two-hybrid assay with those fragments of
TIF2 that possess modulatory activity (TIF2.3 and TIF2.4 of Fig.
2A). Under conditions where a chimera of PCAF fused to the
GAL4 DNA-binding domain (GAL/) interacts with a VP16/GR chimera bound
with Dex, there was no interaction of GAL/PCAF with either TIF2.3 or
TIF2.4 fused to VP16 (Fig.
5A). Therefore, we conclude
that PCAF does not interact with TIF2.4, which is the modulatory domain
of TIF2, and is not involved in TIF2 modifications of GR
properties.
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Fig. 5.
Interaction of TIF2.4 with candidate binding
proteins in mammalian two-hybrid assays. A, comparison of
interaction of PCAF with TIF2 sequences versus GR.
Triplicate samples of CV-1 cells in 24-well plates were transiently
transfected as in Fig. 1B with GAL-DBD and VP16-AD plasmids
containing the indicated fused proteins along with the GAL4-regulated
pFRLuc reporter. Dex (1 µM) was added for the samples
containing VP16/GR. The relative luciferase activity, normalized for
the activity of the internal Renilla control, was determined and
plotted as the average ± S.D. Similar results were obtained in a
second experiment. B, comparison of interaction of CBP and
p300 with TIF2 sequences. Triplicate samples of CV-1 cells were
transiently transfected with plasmids for the indicated chimeras and
plotted as in A. Similar results were obtained in a second
experiment.
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The transactivation activity of TIF2 is thought to require the
activation domains, AD1 and AD2 (Fig. 1A), with CBP and p300 binding to TIF2 being localized to the AD1 domain (8, 40, 41). The
observation in Fig. 2A that TIF2.4, which lacks both AD1 and
AD2, still augments the total activity of GR-Dex complexes suggests
that there might be some weak binding of CBP or p300 to TIF2.4. Using
the same mammalian two-hybrid assay as in Fig. 5A, we
investigated the interactions of CBP, and p300, with TIF2 fragments
containing (TIF2.3) and lacking (TIF2.4) the AD1 domain. Strong
interactions are seen between CBP or p300 and TIF2.3 (Fig. 5B). However, no interaction of CBP or p300 with TIF2.4 is
observed. Therefore, we conclude that the modulatory activity of TIF2.4 does not involve PCAF, CBP, or p300.
DRIP150 and DRIP205 Do Not Modulate GR Transcriptional
Properties--
Gene induction by steroid/nuclear receptors has been
proposed to proceed via sequential complexes, one of which is the DRIP (19) or TRAP (18) complex. Two components of the DRIP/TRAP complex that are of particular interest are DRIP205 and DRIP150. DRIP205 may have an effect on the dose-response curve of some vitamin D
analogs (50) and interacts, with the help of DRIP150, with GR (51).
Furthermore, DRIP150 has been shown to interact with the AF-1 domain of
GR (51). Finally, DRIP150 is a component of mediator-like
complexes. Another component of mediator complexes is hSur2 (52),
which we have recently found is also capable of modulating GR
activities (22). Despite these many interesting associations, the
cotransfection of neither DRIP150 nor DRIP205 has any modulatory
activity on the transactivation properties of GR (data not shown). Even
when high concentrations of GR are used, thereby allowing us to see the
involvement of species downstream of the previously identified
rate-limiting step X (21) that are "kinetically invisible" at low
concentrations of GR (35), there is no effect of DRIP150 or DRIP205
(data not shown). Thus, DRIP150 and DRIP205 are not limiting factors
for the modulation of GR transcription properties under our conditions
in CV-1 cells.
Whereas the DRIPs are inactive by themselves, they might synergize with
TIF2. To address this possibility, we asked if the co-addition of
DRIP150 could significantly increase the effects of TIF2. We found that
the addition of 70 ng of DRIP150 to 10 ng of TIF2 was no more effective
in changing the GR dose-response curve and amount of partial agonist
activity than was 20 ng of TIF2 (average ± S.E.
(n = 3) values for EC50 and percent agonist activity are for control (13.2 ± 1.7 nM; 21.7 ± 0.4%), 10 ng of TIF2 (8.5 ± 1.2 nM; 31.8 ± 1.9%), 10 ng of TIF2 + 70 ng of DRIP150 (6.7 ± 1.7 nM, 41.8 ± 2.5%), and 20 ng of TIF2 (6.9 ± 1.8 nM; 38.3 ± 2.5%)). Therefore, we conclude that
DRIP150 does not synergize with TIF2 to modify GR transcriptional properties.
TIF2 Modulatory Activity Requires the Binding of Additional
Factors--
The modulatory activity of TIF2.4, and TIF2, is presumed
to involve TIF2 as a physical intermediate that connects GR to some other factor. This connection is broken with TIF2m123, which is inactive (Fig. 2A). However, the above experiments suggest
that none of the potential binders of TIF2.4 are involved because they are not able to augment the activity of endogenous TIF2 and other coactivators (53). In fact, no proteins are known to bind to TIF2.4
other than GR and the other steroid/nuclear receptors. To obtain more
direct evidence for a possible novel binder to TIF2.4, we asked if
TIF2.4 fragments lacking the three RIDs (Fig. 2A), and thus
do not bind GR, can competitively inhibit the actions of TIF2.4 by
titrating out our putative TIF2-binding factor. The fragments that we
prepared are TIF2.37, which is TIF2.4 minus the RIDs, and TIF2.38,
which is a 68-amino acid extension of TIF2.37 and contains the
auxiliary domain but not the AD1 transactivation domain (Fig.
6A) (41). As shown in Fig.
6B, added TIF2.37 and TIF2.38 each block the modulatory
activity of TIF2.4. Thus, TIF2.37 and TIF2.38 prevent the average
1.90 ± 0.26-fold left shift (S.E., n = 9, p = 0.0080) in the GR dose-response curve by TIF2.4.
Similarly, both TIF2.37 and TIF2.38 reverse the 1.59 ± 0.08-fold
(S.E., n = 9, p = 0.0001) increase in
Dex-Mes partial agonist activity by TIF2.4. TIF2.37 and TIF2.38 by
themselves are unable to shift the dose-response curve to the left, or
to increase the partial agonist activity of Dex-Mes, and may even
reduce the activities of GR, consistent with TIF2.37 and TIF2.38
antagonizing endogenous coactivators (data not shown). We, therefore,
conclude that TIF2.37 and TIF2.38 competitively bind a factor(s) that
is required for TIF2.4 to modulate GR transactivation properties.
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Fig. 6.
Ability of added TIF2 fragments to inhibit
the modulatory activity of TIF2.4. A, cartoon showing the
sequences of TIF2.4 versus TIF2.37 and TIF2.38. Solid
bars represent RIDs. Shaded area denotes the auxiliary
domain and the amino-terminal end of AD1. B, dose-response
curve for Dex induction, and partial agonist activity of Dex-Mes, with
TIF2.4 ± TIF2.37 or TIF2.38. Triplicate samples of CV-1 cells in
60-mm dishes were transiently transfected with the indicated TIF2
plasmids, GREtkLUC reporter, and Renilla control plasmid, and then
induced with EtOH ± various concentrations of Dex or 1 µM Dex-Mes as described in the legend to Fig.
1B. The data of a representative experiment were expressed
as -fold induction over basal (EtOH) activity and the average ± S.D. was plotted as percent of the maximal -fold induction by 1 µM Dex. Curve fitting was performed by KaleidaGraph as
described under "Materials and Methods."
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TIF2 Modulatory Activity Proceeds via Binding of TIF2 to Agonist-
and Antagonist-bound GRs--
A distinguishing characteristic of the
modulation of GR activities by coactivators is that the properties of
both GR-agonist and GR-antagonist complexes are affected (20, 21). In
general, coactivators are not thought to interact with antagonist-bound receptors (1, 3, 11). However, this question has not yet been examined
directly with GR. We, therefore, used a mammalian two-hybrid assay to
assess the ability of TIF2 to associate in intact cells with GR
complexes of the agonist Dex and various antiglucocorticoids,
i.e. Dex-Mes, progesterone, Dex-Ox, and RU486. Each of these
antiglucocorticoids are effective antisteroids and display low amounts
of partial agonist activity with the GREtkLUC reporter in CV-1 cells
(20-22, 33, 38). A large, Dex-dependent increase in
luciferase activity is seen with the chimeras GAL/GRIP1 and VP16/GR
that is not observed with GAL plus VP16/GR, and thus is specific for
the presence of GRIP1 (Fig.
7A). A similar, strong GRIP1-specific increase in reporter activity is obtained for GR bound
by the antiglucocorticoids Dex-Mes, Dex-Ox, and progesterone. The
response with RU486 is usually somewhat weaker.
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Fig. 7.
Effect of added steroid on interaction of GR
with GRIP1/TIF2. A, interaction of agonist- and
antagonist-bound GR with GRIP1/TIF2 in mammalian two-hybrid assays.
Triplicate samples of CV-1 cells were transiently transfected with
plasmids for the indicated chimeras, treated with 1 µM of
the designated steroids, and plotted as described in the legend to Fig.
5A. Similar results were obtained in at least one additional
experiment. Inset, Western blot of overexpressed GAL/TIF2.4
and GAL/TIF2.4m123. Both GAL chimeras were overexpressed in transiently
transfected CV-1 cells and then detected by Western blotting as
described under "Materials and Methods." B, binding of
agonist- and antagonist-bound GR to TIF2 in pull-down assays.
Bacterially expressed GST, GST/TIF2.4, and GST/TIF2.4m123 that had been
immobilized by matrix-bound anti-GST antibody was treated with in
vitro translated GR that had been bound by agonist
(Dex), antiglucocorticoid (Dex-Mes,
Dex-Ox, Prog, or RU486), or left unbound. GR complexes
that were retained on the matrix by binding to the GST chimeras were
separated on SDS-PAGE gels and visualized by autoradiography. Specific
binding is seen as the GR binding to GST/TIF2.4 that is in excess of
the nonspecific binding to GST alone. The binding of GR complexes to
GST/TIF2.4 that is in excess of that to GST/TIF2.4m123 represents
binding that requires the presence of the RIDs of TIF2. Similar results
were obtained in a second experiment.
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We next used this assay to examine the properties of TIF2.4, which is
the smallest segment of TIF2 to show strong modulatory activity (see
Fig. 2A). As a control, we used GAL/TIF2.4m123, where each
of the three LXXLL sequences of the RIDs have been mutated
to eliminate GR binding to TIF2 (8, 45). Compared with TIF2.4m123, Dex
and most of the antiglucocorticoids elicit a strong interaction between
GR and TIF2.4. The only antiglucocorticoid that is unable to promote an
interaction between GR and TIF2.4 is RU486. The insert shows that the
reduced signals with GAL/TIF2.4m123 are not because of lower amounts of
expressed chimeric protein.
Cell-free pull-down assays were performed with GST/TIF2.4 and
[35S]methionine-labeled, in vitro translated
full-length GR in an effort to obtain additional evidence for the
binding of TIF2 to the various GR-steroid complexes. Significantly
increased binding of GR to GST/TIF2.4 over that seen to GST and
GST/TIF2.4m123 controls is seen for Dex and the antiglucocorticoids
Dex-Mes, Dex-Ox, and progesterone (Fig. 7B). No binding to
TIF2.4 is seen with RU486-bound GR. These results are very similar to
those of the mammalian two-hybrid data (Fig. 7A).
Collectively, the above data argue that amino acids 624-1010 of TIF2
are sufficient to mediate the modulatory activity of TIF2/GRIP1 on GR
transcriptional properties via a mechanism that involves the direct
binding of TIF2/GRIP1 to both GR-agonist and GR-antagonist complexes.
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DISCUSSION |
The ability of p160 coactivators to modulate both the position of
the dose-response curve of GR-agonist complexes, and the partial
agonist activity of GR-antagonist complexes, is a relatively new
function of p160 coactivators (20, 21). The originally described
function of coactivators is to increase the total levels of induced
gene product by receptor-steroid complexes. We have now used a variety
of truncated forms of TIF2/GRIP1 and SRC-1 to show that the modulatory
activity of these p160 coactivators is separable from their coactivator
activity (Figs. 1-3). For both TIF2/GRIP1 and SRC-1, the modulatory
activity can be restricted to a small region of each coactivator that
does not include any of the activation domains (AD1 and AD2).
Furthermore, the minimum sequence that retains high levels of
modulatory activity has a significantly reduced capacity to augment the
total amounts of GR-mediated transactivation (Fig. 2B).
These results are consistent with our earlier conclusions that various
factors can modulate the dose-response curve and partial agonist
activity of GR complexes in a manner that is independent from their
effects on the total levels of induced gene activity (20, 21, 33-35,
54).
The fact that coactivators can alter the partial agonist activity of
GR-antagonist complexes is particularly interesting. We had first
established a functional consequence of coactivators on the biological
properties of GR-antagonist complexes in intact cells (20, 21). The
current general model proposes that coactivator binding to steroid
receptors is seen only in the presence of agonist steroids (1, 11).
However, this model is based largely on the observations of nuclear
receptors, which display significant mechanistic differences with GR.
For example, ligand-free receptors are nuclear and DNA-bound for the
nuclear receptors versus cytoplasmic for GR, binding of
hsp90 is seen for GR and rarely for nuclear receptors (55), only the
nuclear receptors heterodimerize with RXR, and the amino
acids beyond helix 12 are required for ligand binding to GR (56) but
not most nuclear receptors, which lack long amino acid sequences beyond
helix 12. Estrogen receptors (ERs) are more like the nuclear receptors
in several respects: nuclear localization of ligand-free receptors,
diminished importance of hsp90 binding (57), and dispensability of
COOH-terminal residues for steroid binding (58, 59). Nevertheless,
reports exist suggesting an interaction of coactivators with
antagonist-bound ERs (60). SRC-1 binding to ER is seen with all forms
of ER but is promoted by agonists and is the least with antagonists
(60, 61). Also, the binding of coactivators to antagonist-bound ER may
be responsible for their partial agonist activity as microinjection of
anticoactivator antibodies into transfected cells reduces the amount of
partial agonist activity (62). We now establish that for GR, the
coactivator TIF2/GRIP1 binds to GR complexed with several different
antiglucocorticoids. These interactions are seen both in intact cells
in a mammalian two-hybrid assay and in vitro in a pull-down
assay (Fig. 7). In both assays, the interactions of all antagonists
except RU486 were similar in magnitude with that seen with the agonist
Dex. RU486-bound GR displayed a more robust interaction with TIF2/GRIP1
when the full-length GR is used, as opposed to a fragment of
TIF2/GRIP1. The significance of this result is not yet known. However,
the data clearly show that coactivators can bind to antisteroid
complexes of GR, even those that normally display negligible amounts of
partial agonist activity (33, 38, 63). These results also provide
biochemical support for our model of an equilibrium interaction of
coactivators and corepressors binding to antagonist-bound GR (20) and
progesterone receptors (23).
The p160 coactivators contain numerous functional domains. However, the
only known functional domains that are involved in receptor modulatory
activity are the RIDs, which are required for the binding of receptors.
The effects of coactivators on the total levels of steroid
receptor-mediated gene induction are mediated by the AD1 domain, which
binds CBP/p300 (8, 41, 46, 64). An auxiliary domain for the binding of
GR has been identified at residues 1011-1056 of GRIP1 (41). A
COOH-terminal domain is needed for synergism between the activation
sequences of receptors (65). However, none of these domains are present
in TIF2.4, which is the minimally active fragment of TIF2.
The COOH-terminal active segment of SRC-1, corresponding to amino acids
1139-1441 (Fig. 3), is devoid of activation domains but does contain a
HAT activity domain and a RID (9, 48). This segment also displays a
dominant negative repressor activity (4, 46, 66), which appears to be
due simply to its ability to bind tightly to the receptor LBD, thereby
blocking the binding of other coactivators (48). Interestingly, we see
little if any dominant negative activity for this SRC-1 fragment with
GR (data not shown). Finally, this sequence of SRC-1 does bind PCAF (48). The binding of PCAF to the small active segment of SRC-1, coupled
with the ability of PCAF to modulate the dose-response curve, and
partial agonist activity, of GR complexes (Fig. 4), initially made PCAF
an attractive mediator of the modulatory activity of coactivators.
However, the lack of any additive or synergistic response of PCAF with
SRC-1/1139-1441 (Fig. 6) argues that the modulatory activity of SRC-1
does not involve the binding of PCAF.
Several reports suggest that the coactivator activities of SRC-1 and
TIF2 are largely redundant. SRC-1 knockout mice appear to compensate
for the lack of SRC-1 by increasing the levels of TIF2 (67) and recent
data argue that TIF2 and SRC-1 are not functionally distinct (68).
Thus, while we have not established that the modulatory activities of
SRC-1 and TIF2 are expressed by identical mechanisms, we currently
suspect that they are. It should be noted that there are no reports of
PCAF binding to TIF2/GRIP1 (16) and we are unable to detect any
interaction of PCAF with the active TIF2 fragments (Fig.
5A). Therefore, as for SRC-1, the available evidence argue
against an important role of PCAF in the actions of TIF2.
Several other factors that are known to bind to coactivators are shown
not to be involved in the modulation of GR transactivation properties.
CBP/p300 does not interact with TIF2.4 (Fig. 5B) whereas the
p300 binding site in SRC-1 is reported to be at amino acids 913-977
(13), which is outside of the COOH-terminal domain of SRC-1 modulatory
activity. The DRIP/TRAP complex has been found to play an important
role in the expression of receptor transactivation activity (69-71).
DRIP150 and DRIP205 bind to receptors and increase the total amounts of
receptor transactivation (51, 72) but are inactive in our assays, while
DRIP150 does not synergize with TIF2. We cannot presently rule out,
however, that some untested combination of factors might synergize with
SRC-1 or TIF2. No common motif could be recognized in the short
modulatory regions of TIF2 and SRC-1 that might bind some other
transcription cofactor. Therefore, we conclude that a currently
unidentified factor(s) interacts with the above identified unique
domains of TIF2 and SRC-1 to contribute to the modulation of several
transactivation properties of GR. This conclusion is supported by the
ability of a fragment of TIF2 lacking the RIDs to be able to
competitively inhibit the actions of TIF2.4 (Fig. 6), presumably by
titrating out the unidentified factor(s).
In summary, we are assembling a model by which the p160 coactivators
exert additional, unique effects on the transactivation properties of
GRs complexed with both agonist and antagonist steroids. These
properties, which include the ability of coactivators to associate with
GR-antisteroid complexes, are physically separable from the initially
discovered properties of augmenting the total levels of gene induction
by receptor-agonist complexes. The domain of TIF2/GRIP1 that is
sufficient for this new activity is not known to bind any protein and
appears to interact with a currently unidentified factor(s). As the
transcriptional properties of other steroid receptors are similarly
modulated (23), we anticipate that this putative new factor(s) will
similarly affect the dose-response curve of agonists (or
EC50), and partial agonist activity of antisteroids, bound
to other steroid receptors.
,
TOP
ABSTRACT
INTRODUCTION
Both authors contributed equally and should be considered as first authors.
