| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Pathology and Molecular Biology Program (M.J.T.,
S.A.L., D.P.E.) University of Colorado Health Sciences Center
Denver, Colorado 80262
Department of Pharmacology and Cancer
Biology (P.H.G., D.P.M.) Duke University Medical Center Durham,
North Carolina 27710
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Steroid receptors, including PR, are latent transcription factors that are inactive in the absence of hormone and undergo a multistep activation process upon binding ligand. Receptor activation includes the steps of ligand-induced conformational change, dissociation from an inactive oligomeric complex composed of heat shock proteins and immunophilins, dimerization, and binding to specific DNA sequences of steroid-responsive genes to thereby alter rates of gene transcription (2, 13, 14, 15). The identification of coactivators that interact directly with a broad range of nuclear receptors in a hormone- and AF-2-dependent manner has provided important insights into the mechanism by which receptor-DNA interaction modulates gene transcription. The p160 family of coactivators and the CREB binding protein (CBP) family of coactivators (16, 17, 18, 19, 20) have been shown to enhance the transcriptional activity of nuclear receptors and to be essential for maximal hormonal responses in vivo (16, 21, 22, 23). Nuclear receptor coactivators appear to act as bridging proteins between the receptor and general transcription factors, thereby facilitating recruitment of the preinitiation complex. Coactivators are also believed to be involved in targeted remodeling of chromatin due to their intrinsic histone acetyltransferase activity (24, 25, 26, 27). The coactivators identified so far primarily interact with and mediate the function of AF-2; AF-1-specific coactivators have not been identified. However, the p160 coactivators such as steroid receptor coactivator SRC-1 and glucocorticoid receptor-interacting protein GRIP-1 have been recently shown to directly interact with amino-terminal sequences of PR or ER, albeit less efficiently than they interact with AF-2, and to be capable of mediating coactivation function through the amino terminus (28, 29, 30, 31).
Under certain cell and promoter contexts, both AF-1 and AF-2 can function independently. However, under most conditions, functional synergy between AF-1 and AF-2 is required for full transcriptional activity (4, 5, 32, 33, 34, 35, 36, 37, 38, 39). Studies with estrogen receptor (ER) and androgen receptor (AR) have suggested that an intramolecular association between the amino- and carboxyl-terminal regions of receptor contributes to the functional synergy between AF-1 and AF-2. In a modified mammalian cell two-hybrid interaction assay, separately expressed amino- and carboxyl-terminal domains of ER were observed to functionally interact in vivo in a hormone agonist-dependent manner (40). Using both yeast and mammalian two-hybrid interaction assays, several groups have also observed a hormone-agonist dependent interaction between amino- and carboxyl-terminal domains of AR (30, 41, 42, 43). It is not clear from these two-hybrid interaction experiments whether amino-carboxyl domain interactions are direct or indirectly mediated by coactivators or other proteins that associate with either domain of the receptor. Functional interactions in a two-hybrid assay could be the result of either direct or indirect binding. Conflicting results have been reported for the effect of nuclear coactivators on functional interactions between N- and C-domains as detected by two-hybrid assays. It was reported that SRC-1 enhances ER N-C domain interactions (44), both SRC-1 and CBP enhanced interactions between the N- and C-domains of AR, while a truncated form of SRC-1 was observed to inhibit these interactions in AR (30). In another study, the transcriptional intermediary factor TIF-2 had no effect on the functional interaction between the N- and C-domains of AR (43). Direct in vitro interaction between purified N- and C-domains of steroid receptors has not been reported.
In the present study we have investigated whether the N- and C-domains of human PR are capable of interacting in a hormone agonist-dependent manner. To resolve the question of whether these interdomain interactions are direct or indirect, they were analyzed by direct protein-protein interaction assays in vitro with purified N- and C-domain polypeptides of PR and by a mammalian two-hybrid assay. We also investigated whether the N-domains of the A and B forms of PR interact the same or differently with the C-terminal LBD as a possible contributing factor to the different functional activities of the two receptor forms.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
. The PR
fragments included polyhistidine-tagged N-domains of PR-A (ANhis; aa
165–535) and PR-B (BNhis; aa 1–535) and nontagged C-domains
containing either the entire LBD (aa 688–933) or the LBD plus hinge
region (hLBD; aa 634–933). It should be noted that the expressed N-
and C-domains both lack the DBD and thus share no overlapping sequences
(Fig. 1) that might contribute to protein-protein interaction through
homodimerization. Because the baculovirus PR domain vectors contain
polyhistidine tags, it was necessary to cleave the tag from one of the
paired PR fragments, which was done with the C-domain polypeptides by
treatment with enterokinase as described previously (45). Each domain
polypeptide was expressed from baculovirus vectors in Sf9 insect cells,
and the C-domains were bound to the synthetic progestin R5020 during
expression before cell lysis. Whole-cell extracts containing N- or
C-domains were mixed and incubated for 30 min at 4 C before
immobilization to metal affinity resins (Talon) through the
polyhistidine-tagged N-domain polypeptide. After washing the Talon
resins with 15 mM imidazole and 100 mM NaCl to
remove nonspecific proteins, bound proteins were eluted and analyzed by
Western blot with a mixture of monoclonal antibodies (MAbs) that
recognize epitopes in the N terminus (AB-52) and the C terminus of PR
(C262) (46, 47). To determine the nonspecific binding of C-domain
polypeptides that lack polyhistidine tags, the LBD and hLBD were
incubated with Talon resins in the absence of the polyhistidine tagged
N-domains of PR (Fig. 1, lanes 5 and 8).
|
, lanes 5–7).
However, a significant amount of hLBD associated in a specific manner
with the N-domain of either PR-A or PR-B (Fig. 1, lanes 8–10). It
should be noted that the triplet bands of the N-domain of PR-B are due
to phosphorylation sites in the unique N terminus of PR-B (48). The
ratio of the C-domain polypeptide specifically associated with
Talon-immobilized polyhistidine-tagged N-domains was determined by
Phosphorimager analysis from multiple pull-down experiments, and the
results are summarized in Table 1. These
quantitative analyses confirmed there was no detectable specific
interaction between the LBD and the N-domains of PR-A (LBD/ANhis
ratio = 0.02 ± 0.01, n = 3) or PR-B (LBD/BNhis
ratio = 0.01 ± 0.01, n = 3). In contrast, a substantial
amount of the hLBD specifically associated with the N-domains of either
PR-A or PR-B. We also observed a significantly higher ratio of hLBD
interaction with the N-domain of PR-B (ratio of hLBD/BNhis =
0.27 ± 0.04) than with the N-domain of PR-A (ratio of
hLBD/Anhis = 0.14 ± 0.03; P < 0.05), (Table 1, R5020 column). Taken together, these results suggest a direct
protein interaction between N- and C-domains of PR that requires both
the hinge plus LBD as the minimal C-terminal region and that there is a
more efficient interaction of the hLBD with the N terminus of PR-B than
with the N-domain of PR-A.
|
, lanes 4–6) or when bound to RU486 (Fig. 2, lanes 7–9). The hLBD
efficiently interacted with the N-domains of PR (A or B form) only when
bound to R5020 (Fig. 2, lanes 10–12). Results of quantitative analysis
by Phosphorimaging of multiple pull-down experiments are summarized in
Table 1 and confirm that interaction between the N- and C-domains of PR
in vitro is dependent on hormone agonist binding to the hLBD
and is not allowed by the antagonist RU486.
|
and 2 and summarized in Table 1 were performed with PR
domain polypeptides present in crude extracts of Sf9 insect cells. To
determine whether these interactions are direct or require other
proteins, similar pull-down experiments were done using purified PR
domain polypeptides. Baculovirus expressed N-domains of PR (ANhis and
BNhis) were purified as described in Materials and Methods
by affinity chromatography on nickel chelation resins using imidazole
to elute the proteins under nondenaturing conditions. Because we
encountered problems with low yields of purified polyhistidine-tagged
hLBD from nickel resin, followed by enterokinase cleavage necessary to
generate nontagged hLBD for polyhistidine pull-down assays, we used a
baculovirus-expressed glutathione S-transferase (GST)-tagged
hLBD and GST-pull down assays for experiments with purified PR
fragments. The hLBD-GST was bound to R5020 during expression in Sf9
insect cells and was purified by affinity chromatography with
glutathione-Sepharose resins as described in Materials and
Methods using reduced glutathione to elute the hLBD under
nondenaturing conditions. Silver-stained SDS-gels and Western blot to
confirm the identity of the PR domain polypeptides shows that the
N-domains of PR-A (AN) and PR-B (BN) and the GST-hLBD were purified to
greater than 90% (Fig. 3).
|
). To confirm equal
loading and binding of hLBD-GST to the glutathione-Sepharose resins,
separate Western blots were performed with the C-262 MAb that
recognizes an epitope in the LBD (not shown). A significant
fraction of the N-domain of PR-A (AN) (Fig. 4A) and the N-domain of
PR-B (BN) (Fig. 4B) specifically associated with hLBD-GST above the
little to no binding of the N-domains to GST, or to blank
glutathione-Sepharose resins. Quantitative Phosphorimager analysis from
multiple GST pull-down assays similar to that in Fig. 4 revealed that,
on average, 5.7% (SEM ± 1.12%, n = 9) of the assay
input of the N-domain of PR-B and 6.51% (SEM ± 0.801,
n = 9) of the input of the N-domain of PR-A specifically
associated with immobilized hLBD-GST. Thus, the more efficient
interaction of the hLBD with the N-domain of PR-B, as compared with the
N-domain of PR-A that was detected with PR domain polypeptides prepared
as crude cell extracts, was not detected by GST pull-down assay with
highly purified PR fragments. Whether these different results are due
to the use of different assay methods (GST vs. polyhistidine
pull-down assays) or to the presence of other bridging proteins that
facilitate interaction between the N-domain of PR-B and the hLBD is not
known. To investigate this question further we analyzed the influence
of SRC-1 and CBP on the interaction between purified N- and C-domain PR
fragments. SRC-1 and CBP were each expressed as full-length proteins
with polyhistidine tags in the baculovirus system and were purified by
nickel chelation affinity chromatography. As a control for the general
effect of other proteins on the stability of highly purified PR
fragments, ovalbumin (10 µg) was added and was observed to have no
effect on these in vitro interactions (not shown). Addition
of SRC-1, CBP, or both proteins together also had no influence on the
interactions detected by GST pull-down assay between purified N-domains
of PR-A or PR-B with the hLBD (not shown). Thus, we conclude that the
N-domains of PR-A and PR-B can make direct protein contact with the
hLBD in a manner that does not require SRC-1 or CBP. These results with
purified PR fragments also suggest that the more efficient interaction
of the hLBD with the N-domain of PR-B, as compared with the N-domain of
PR-A observed in whole-cell extracts, is likely due to proteins other
than SRC-1/CBP, or to a coactivator complex consisting of SRC-1 and CBP
plus additional factors.
|
included the hLBD
fused to the DBD of Gal4 (hLBD-Gal4) and the N-domains of PR-A
(AN-VP16) and PR-B (BN-VP16) fused to the VP16 transcriptional
activation domain. An SV40 large T antigen fused to VP16 (T-VP16) was
used as a control for nonspecific interaction of the hLBD with an
unrelated protein. A luciferase gene inserted downstream of five Gal4
DNA-binding sites (5x Gal4-RE-LUC) was used as the reporter for
detection of functional interaction between hLBD-Gal4 and the VP16
fusion proteins (Fig. 5). Human hepatoma (HepG2) or human cervical
carcinoma (HeLa) cells were cotransfected with hLBD-Gal4 and one of the
three VP16-fusion constructs, and the cells were treated without and
with PR ligands for 48 h before harvest and measurement of
luciferase activity. Western blot analysis confirmed that the fusion
products were expressed as correctly sized proteins and at levels
similar to full-length transfected wild-type PR (data not shown). For
the experiments in Fig. 6, the
nonspecific luciferase expression resulting from interaction between
SV40-VP16 and hLBD-Gal4 for each ligand treatment group was normalized
to a value of 1.0, and the specific luciferase expression, dependent on
both hLBD-Gal4 and PR N-domain VP16 fusion constructs, was calculated
as the fold induction over the nonspecific expression of
luciferase.
|
|
) or HeLa cells (Fig. 6B) with the
N-domain-VP16 fusions of either PR-A (AN-VP16) or PR-B (BN-VP16) above
that of the background interaction with SV40-VP16 (T-VP16). However,
progesterone addition to both cell types induced a significant
functional interaction between hLBD-Gal4 and either PR N-domain VP16
construct (Fig. 6). Additionally, hLBD-Gal4 interacted more efficiently
with the N terminus of PR-B (5.2 ± 0.1 fold induction in HepG2
and 6.3 ± 0.7 in HeLa cells) than the N-domain of PR-A (2.7
± .01 in HepG2 and 4.0 ± 0.3 in HeLa cells; P <
0.05) in both cell lines (Fig. 6). In agreement with the in
vitro protein-protein interaction results, RU486 failed to induce
a functional interaction between hLBD-Gal4 and either PR isoform
N-domain construct in HepG2 cells (Fig. 6A) and with the N-domain of
PR-A in HeLa cells (Fig. 6B). However, in HeLa cells a small but
significant RU486 stimulation (1.9 ± 0.3-fold over the T-VP16
control, P < .01) of hLBD-Gal4 interaction with the
N-domain of PR-B was observed, which was considerably less than that
stimulated by progesterone (Fig. 6B). Thus, functional interaction
between the C- and N-domains of PR within mammalian cells is hormone
agonist-dependent and is either not allowed or greatly reduced (hLBD
interaction with the N-domain of PR-B in HeLa cells) by the antagonist
RU486.
Coactivators Are Involved in Functional Interaction between Amino-
and Carboxyl-Terminal Domains of PR within Whole Cells
To investigate the role of transcriptional coactivators in the
functional interaction between the N- and C-domains of PR, we analyzed
whether coexpression of full-length SRC-1, CBP, or both proteins would
influence these interdomain interactions in the mammalian two-hybrid
assay. Separate cotransfections with either SRC-1 or CBP in HepG2 (Fig. 7) or HeLa cells (data not shown) had
minimal effect on progesterone-dependent interaction between hLDB-Gal4
and the N-domain VP16 constructs of PR-A and PR-B. However,
cotransfection with SRC-1 and CBP together resulted in a
significant stimulation of progesterone-dependent functional
interaction between hLBD-Gal4 and the N-domains of PR-A or PR-B (Fig. 7). In HepG2 cells, cotransfected SRC-1 and CBP together
increased hLBD-Gal4 interaction with PR-B N-domain from a 4.6- to a
13-fold induction (2.8x) and hLBD-Gal4 interaction with PR-A N-domain
from a 3.2 to a 6.6 fold induction (2.06x) (Fig. 7). A similar
enhancement of functional interaction between hLBD and the N terminus
of PR-B (3.37-fold increase) and the N-terminus of PR-A (4.41-fold
increase) was observed by cotransfecting HeLa cells with SRC-1 and CBP
(not shown). Enhancement by SRC-1 and CBP is largely PR specific and
does not appear to be due to a coactivation effect on general
transcription. Coexpression of SRC-1 and CBP together resulted
in only a 1.4- to 1.5-fold stimulation of Gal4-VP16 transactivation of
the Gal4-RE-LUC reporter gene in both HeLa and HepG2 cells, indicating
that SRC-1 and CBP are not affecting transcription activation in
general (not shown).
|
). To
inactivate endogenous CBP, cells were cotransfected with an expression
plasmid for the adenovirus protein 12S E1A (E1A), which binds to the
third zinc finger motif of CBP and inactivates its coactivator function
(49). E1A cotransfection in the mammalian two-hybrid assay effectively
inhibited progesterone induction of the functional interaction between
hLDB-Gal4 and the N-domains of either PR isoform (Fig. 8B). As a
control for effects on general transcription activation, the dominant
negative SRC-1 (0.8) and E1A did not affect the constitutive
transactivation of the GAL4-RE-LUC reporter mediated by a Gal4-VP16
activator (not shown). These mammalian two-hybrid results, taken
together with the in vitro protein-protein interaction data,
suggest that SRC-1 and CBP are essential for functional
hormone-dependent interaction between the amino- and carboxyl-terminal
domains of PR, but are not required as bridging, or adaptor, proteins
for association between the N- and C-domains, which occurs by direct
contact in the absence of other proteins.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and 2). When the N- and
C-domain polypeptides were purified to more than 90% from Sf9
whole-cell extracts, they continued to interact by pull-down assay in a
specific manner indicating that the N- and C-domains of PR are capable
of making direct protein contacts and do not require other proteins to
physically associate.
While the interaction between the amino- and carboxyl domains of PR
in vitro (Fig. 2 and Table 1), and within cells by mammalian
two-hybrid assay, was observed to be hormone agonist dependent (Fig. 6), little or no interaction was detected by either experimental
approach in the presence of the progesterone antagonist RU486. Androgen
antagonists were similarly reported to diminish functional interaction
between the N- and C-domains of AR in a mammalian two-hybrid assay
(42). However, different results were observed for the effects of the
antiestrogen trans-hydroxytamoxifen (TOT), on ER N-C domain
interactions. Separately expressed ER polypeptides containing the amino
terminus linked to the DBD and the LBD were observed to functionally
interact on an estrogen response element (ERE)-controlled reporter gene
in response to estradiol, but not to TOT. In contrast, TOT was observed
to induce a strong functional interaction between the N-terminal DBD
construct and the LBD on the ERE-responsive reporter gene when the LBD
was fused to VP16 (40). Because TOT only induced a response between the
N- and C-domains when the LBD was expressed as a fusion protein with
VP16, it has been suggested that TOT produces a nonproductive
interaction between the N- and C-domains (40). A different conclusion
must be drawn from the present studies for the influence of RU486 on PR
N-C domain interactions, since RU486 failed to induce an interaction
between the N-domains and the hLBD of PR in vitro (Fig. 2)
and functionally inhibited hLBD interaction with N-domain VP16 fusion
construct in whole cells by mammalian two-hybrid assay (Fig. 6). Thus,
we conclude that RU486 fails to induce, or impairs, a physical
association between the N- and C-domains of PR, rather than promoting
an interaction that is transcriptionally nonproductive as reported for
the effect of TOT on ER N-C domain interaction (40). The reason for the
apparent difference between RU486 and TOT is not known. This could be
due to differences in assay methods, or to RU486 antagonism of PR
operating by a different mechanism than TOT antagonism of ER. Indeed,
TOT is well known to exhibit partial agonist effects that are both cell
type and promoter dependent, suggesting this difference between TOT and
RU486 may reflect the partial agonist effects of TOT. In this regard,
RU486 exhibits cell- and promoter-specific partial agonist effects that
are mediated solely by the B isoform of PR (4, 7, 10). RU486 stimulated
a weak functional interaction between the N terminus of PR-B and the
hLBD in HeLa cells that was not observed in HepG2 cells (Fig. 6). This
weak RU486 stimulation of N-C interaction correlates with the
previously reported weak agonist activity of RU486 mediated by
full-length PR-B in HeLa cells on selected promoters (4, 9). Many
studies have revealed that agonists and antagonists induce distinct
conformational changes in the LBD of steroid receptors and that these
conformations are central to whether receptor is transcriptionally
active or inactive (50, 51, 52, 53). Therefore, an altered conformation in the
LBD of PR induced by RU486 may contribute to inactivation of receptor
by not permitting an efficient physical association between the amino
and carboxyl domains.
The p160 family of nuclear receptor coactivators was initially
identified as AF-2-interacting proteins and has been shown to interact
with AF-2 as a complex of coactivators consisting minimally of p160 as
the direct binding component, CBP, and pCAF (CBP-associated factor)
(17, 18, 19, 20, 21, 22). The p160 proteins, SRC-1 and GRIP1, have also been found to
be capable of interacting with and mediating coactivation effects
through N-terminal regions of ER and PR (28, 29, 30, 31). Interestingly,
separate regions of p160 proteins interact with N- and C-domains of
receptors, suggesting that p160 proteins are capable of mediating, or
bridging, an association between the N- and C-domains of the receptor
(Ref. 31 and V. Boonyaratanakornkit and D. P. Edwards,
unpublished). To address the role of coactivators in terms of
N-C-domain interactions of PR, the present study analyzed the influence
of SRC-1 and CBP on direct N-C domain binding in vitro with
purified PR fragments and functionally by mammalian two-hybrid assay.
Addition of SRC-1, CBP, or both proteins together had no effect on the
direct interactions between purified N- and C-domains of PR. However,
when cells were cotransfected with SRC-1 and CBP expression plasmids
together, functional hormone-dependent interaction between the
N- and C-domains of PR in the mammalian two-hybrid assay was enhanced
(Fig. 7). Additionally, inactivation of endogenous SRC-1 by
transfecting cells with a dominant negative mutant form of SRC-1 (16),
or inactivation of CBP with EIA (49), effectively inhibited functional
interaction between the N- and C-domains (Fig. 8). The influence of the
dominant negative SRC-1 does not preclude other closely related nuclear
receptor coactivators from having a role in mediating a functional N-C
domain interaction. The dominant negative SRC-1 may compete with other
coactivators containing the same nuclear receptor interaction box
sequences (LXXLL motif) that bind AF-2 in the LBD. These direct
in vitro binding and functional two-hybrid results, taken
together, are consistent with the conclusion that the N- and C-domains
of PR are capable of making direct protein contact without the aid of
coactivators, but that transcriptionally productive interactions
require both SRC-1 (or closely related coactivators) and CBP.
Although SRC-1, CBP, or both proteins had no influence on interactions between purified N- and C-domain PR fragments, we observed that CBP addition to the PR domain polypeptides in crude extracts of Sf9 cells increased N-C domain interactions (not shown). Since coactivators appear to exist as preformed multiprotein complexes containing SRC-1, CBP, pCAF, and other factors (17), this result suggests the possibility that CBP, as a component of a larger protein complex, can facilitate or stabilize direct associations between the C and N terminii of PR.
When comparing the interaction of the hLBD with the N-domains of the two forms of PR, the N-domain of PR-B was found to interact more efficiently than the N-domain of PR-A. This differential interaction was detected functionally by mammalian two-hybrid assay and in vitro by pull-down assays with PR domain polypeptides prepared as whole-cell extracts of Sf9 cells. However, this differential was not observed in vitro with highly purified PR domain polypeptides, suggesting that the more efficient interaction of the N-domain of PR-B with the hLBD is dependent on other proteins, most likely coactivator complexes containing SRC-1, CBP, and other components. Additionally, the more efficient interaction observed between the hLBD and the N terminus of PR-B, as compared with the N terminus of PR-A, could be due to 1) additional protein contact sites provided by the extended N-terminal segment unique to PR-B; 2) a different overall conformation conferred by the unique N terminus of PR-B on sites that are common to the N-domains of PR-A and PR-B; or 3) the three phosphorylation sites that are located in the N-terminal segment unique to PR-B (48). Further studies are required to distinguish between these possibilities. The more efficient interaction of the hLBD with the N terminus of PR-B, compared with the N-terminus of PR-A, under the conditions observed in this study, correlates with PR-B functioning as a generally stronger transcriptional activator than PR-A (7, 8, 9, 10, 11, 12). These results support the notion that a differential association between the C-terminal hLBD and the N terminus of PR-A and PR-B contributes to the experimentally observed differences in transcriptional activities of the two PR isoforms.
Because the N- and C-domains of PR were expressed as separate polypeptides, the present results cannot distinguish between an intramolecular association between the N and C termini in the full-length receptor and an intermolecular interaction resulting from antiparallel dimerization as suggested by studies with AR (41, 54). Several lines of evidence indicate that PR homodimerization occurs in a parallel fashion, thus supporting the notion that the observed N-C domain interactions reflect an intramolecular association. For example, we and others have shown that the C-terminal hLBD of PR is capable of mediating homodimerization in the absence of N-terminal sequences (45, 55). Furthermore, fusion of the leucine zipper of c-fos or c-jun to the C terminus of full-length PR forced parallel dimers that were transcriptionally active (56). However, whether fos/jun-forced antiparallel dimers are also active was not tested. Additionally, the recently published three-dimensional structure of the LBD of PR bound to agonist revealed the presence of a dimer interface that mediates parallel interactions through the C terminus (57). As a further suggestion that interactions between isolated N- and C-domains detected in this study in vitro and in vivo by mammalian two-hybrid assay reflect an intramolecular interaction within the holoreceptor, the N- and C-domains of PR coexpressed in mammalian cells attached to their own DBD were observed to reconstitute a functional transcriptional response in trans on a progesterone response element-containing reporter gene (28). Furthermore, cotransfection with SRC-1, or the closely related TIF-2, markedly enhanced this transcriptional response.
The hLBD was capable of interacting with the N-domain of PR in
vitro, while the LBD was not (Fig. 1), suggesting the hinge region
is involved in N-C domain interactions. Whether hinge sequences are
directly involved in protein interaction with N-domain fragments has
not been investigated. Although a direct involvement remains a
possibility, we favor the idea that the hinge exerts an effect on the
conformation of the LBD enabling it to make protein contacts with
N-domains. Although studies to show directly whether the hinge confers
structural stability on the PR LBD have not been performed, indirect
functional studies comparing the LBD and hLBD fragments are consistent
with this role for the hinge. We have shown previously that the
expressed LBD alone is not capable of mediating homodimerization and
binds ligand with an affinity that is 3- to 4-fold lower than the
affinity of full-length receptor. The LBD with additional hinge
sequences is the minimum region of PR capable of binding ligand with
wild-type affinity and mediating homodimerization (45).
In Fig. 9 we have modeled our findings in
the context of full-length PR. We propose that a fully active receptor
requires assembly of AF-1 and AF-2 from different regions of the same
PR polypeptide. Receptor bound to agonist undergoes a conformational
change that allows a direct intramolecular association between the N-
and C-domains (dashed lines). The p160 subunit of the
transcriptional coactivator complex is capable of simultaneously
binding with amino (AF-1) and carboxyl (AF-2) regions of receptor, and
this complex is required for a transcriptionally productive interaction
between the N- and C-domains. The N terminus of PR-B interacts more
efficiently with the hLBD than the N terminus of PR-A, suggesting that
differential N-C domain interactions contribute to the distinct
functional activities of PR-A and PR-B. This differential interaction
appears to be facilitated by protein components (checkered
symbol) of a coactivator complex through the extended N-terminal
segment of PR-B. Direct N-C domain interactions are markedly inhibited
in the presence of RU486, suggesting that failure to induce an
association between the N- and C-domains contributes to the mechanism
by which antagonists inactivate the receptor.
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Expression of PR Fragments and Coactivators in the Baculovirus
Insect Cell System
Recombinant baculovirus vectors expressing different domains of
PR with N-terminal polyhistidine tags (6x) (Fig. 1) included the N
terminus of PR-B (BNhis, aa 1–535), the N-terminus of PR-A (ANhis, aa
165–535), and the hLBD (aa 634–933). The LBD alone (aa 688–933) was
expressed without polyhistidine tags. These vectors have been described
and used previously (45) except for BNhis, which was constructed by
restriction digestion of PR-B from plasmid pH PR-B (7, 59) by
EcoNI, which dropped out the base pair 1779–2671 fragment
of PR-B cDNA. The EcoNI ends were made blunt by digestion
with Mung Bean nuclease and then religated resulting in a cDNA encoding
a PR fragment, aa 1–535. For expression of the hLBD as a fusion
protein containing an amino-terminal GST tag (hLBD-GST), the hLBD was
generated by PCR with the primers 5'-GATCGGATCCGGCATGGTCCTTG GAGGT and
5'-CTAGAATCCAAAGATGACATTCACTTTTTATG, using the pT7BhPR-A plasmid
(provided by M. Tsai and B. O’Malley, Baylor College of Medicine,
Houston, TX) as the template cDNA (50). The PCR amplification
product resulted in aa 634–933 of PR containing BamHI and
EcoRI restriction sites at the 5'- and 3'-ends,
respectively, which was ligated into the respective restriction sites
of the pAcG2T baculovirus transfer vector (PharMingen, San
Diego, CA).
A recombinant baculovirus transfer vector for steroid receptor coactivator-1 (SRC-1) (16) was constructed by inserting the SRC-1 cDNA excised from pBK-CMVSRC-1 (provided by Sergio Oñate, M.-J. Tsai and B. O’Malley, Baylor College of Medicine) into BamHI and PstI sites of the baculovirus transfer plasmid pBlueBacHis2(C) (Invitrogen). The SRC-1 coding region was inserted in frame with amino-terminal sequences of the plasmid containing an ATG translation start site, six sequential histidine residues, and an enterokinase cleavage site encoding aa 361-1440 of SRC-1 (SRC-1 his). The recombinant virus for expression of full-length mouse CBP as an N-terminally polyhistidine-tagged protein (CBPhis) was provided by N. Weigel and B. O’Malley (Baylor College of Medicine).
Spodoptera frugiperda (Sf9) insect cells were grown in spinner vessels (150–500 ml) in Graces’ insect cell medium supplemented with 10% FBS (HyClone Laboratories, Inc., Logan, UT). Cells were infected with recombinant viruses at a multiplicity of infection of 1.0 for 48 h at 27 C as described previously (51, 58). Insect cell cultures for expression of C-terminal PR fragments were incubated with 200 nM R5020 or RU486, as indicated, for the final 6 h of infection before harvest.
Purification of Baculovirus-Expressed PR Domains and
Coactivators
The N-terminal domains of PR-A and PR-B expressed in baculovirus
with a polyhistidine tag (ANhis and BNhis) were purified by metal ion
affinity chromatography as described previously (58, 59) with minor
modifications. Sf9 cells expressing either ANhis or BNhis were lysed in
20 mM Tris and 10% glycerol (TG) buffer, pH 8.0,
containing 350 mM NaCl, 15 mM imidazole, 1
mM ß-mercaptoethanol, and a mixture of protease
inhibitors (59). All procedures were done at 0–4 C. Cell lysates were
centrifuged at 100,000 x g for 30 min, and the
supernatant was taken as a soluble whole-cell extract. Whole-cell
extracts were bound to nickel affinity resins (1 ml packed Ni-NTA
resins) by resuspension in a 50-ml siliconized tube followed by
incubation for 1 h on an end-over-end rotator. The resins were
then washed four times by centrifugation (1500 rpm) with lysis buffer.
The resins were washed once more in lysis buffer lacking salt and then
transferred to a 2-ml siliconized tube. Bound proteins were eluted from
the resin by suspension in lysis buffer containing 100 mM
imidazole, and the supernatant containing the eluted protein was
collected by centrifugation. Eluates were stored at -80 C in aliquots
and analyzed by Lowry assay for protein concentration, by
silver-stained SDS-PAGE for purity, and by Western blot for
identification of purified products. CBPhis and SRC-1 his were purified
using the same procedure except that the lysis buffer contained 2
mM imidazole.
The hLBD-GST fusion protein was purified by glutathione Sepharose affinity chromatography. Whole-cell extracts were made in cell lysis buffer (10 mM Tris-base, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol, containing 350 mM NaCl) as described above and bound to glutathione Sepharose 4B resins (Pharmacia Biotech, Piscataway, NJ) by resuspension in a 50-ml siliconized tube for 2 h on an end-over-end rotator. The resins were washed four times by centrifugation (1500 rpm) with lysis buffer. The resins were washed once more in lysis buffer lacking salt, and then transferred to a 2-ml siliconized tube. Bound proteins were eluted with 20 mM glutathione and collected by centrifugation. Eluted samples were analyzed as described above.
Pull-Down Assays to Detect PR Domain Interactions in
Vitro
For experiments in crude extracts, Sf9 cells expressing
different PR domains were lysed as above, and whole cell extracts were
dialyzed against lysis buffer lacking salt. PR hLBDhis was treated with
EnterokinaseMax (Invitrogen) to cleave off the N-terminal
polyhistidine tag as described previously (45). Sf9 whole-cell extracts
were added to the hLBD lacking the his-tag, which was then dialyzed
against lysis buffer without salt. The hLBD was analyzed by Western
blot with the PR-specific MAb C-262 and the anti-his tag MAb (1162/F6)
to confirm removal of the his-tag (data not shown). The PR LBD was
expressed as a non-his-tagged protein and prepared as whole-cell
extracts for Sf9 cells. The LBD or hLBD was incubated with
polyhistidine-tagged N-terminal domain polypeptides of PR-A (ANhis),
PR-B (BNhis), or buffer, which served as a control for nonspecific
binding of non-his LBD or hLBD to metal resins, in siliconized
microcentrifuge tubes for 30 min on ice. TG buffer (20 mM
Tris-HCl, pH 8.0, plus 10% glycerol) containing 45 mM
imidazole and 300 mM NaCl was added to bring the final
imidazole concentration to 15 mM imidazole and NaCl to 100
mM. One hundred microliters of a 1:1 suspension of Talon
(CLONTECH Laboratories, Inc.) metal affinity resin or
Ni-NTA resin (Qiagen) were added to each tube. Samples
were then resuspended and incubated in batch at 4 C for 1 h on an
end-over-end rotator followed by washing of the resins four times by
centrifugation in TG buffer containing 15 mM imidazole and
100 mM NaCl. Resins were transferred to a new
microcentrifuge tube and washed twice more. Bound proteins were
extracted with 2% SDS sample buffer and electrophoresed on 10% or
7.5% polyacrylamide SDS gels as previously described (45, 46, 47).
Separated proteins were transferred to nitrocellulose paper and
detected by Western blot assays with a mixture of MAbs including C-262
generated against the C terminus and AB-52 generated against the N
terminus common to PR-A and PR-B (46, 47). [35S]protein A
(Amersham) and autoradiography were used as the detection
methods as described previously (45).
For experiments using purified receptors, a GST pull-down assay was developed that was similar to the polyhistidine pull-down assay except for the following modifications. The purified hLBD-GST was bound to 100 µl of a 1:1 suspension of glutathione Sepharose 4B resin, which had been pretreated with ovalbumin (5 µg/100 µl of resin) for 15 min, on an end-over-end rotator for 1 h at 4 C in TG buffer containing 100 mM NaCl. The resins were washed once by centrifugation with TG buffer containing 100 mM NaCl. Ten micrograms of ovalbumin and either purified ANhis or BNhis were added to the sample. TG buffer containing 300 mM NaCl was added to bring the final concentration of NaCl to 100 mM. Samples were incubated on an end-over-end rotator for 1 h at 4 C and then washed by centrifugation once with TG containing 100 mM NaCl, twice with TG containing 125 mM NaCl, and once more with TG containing 100 mM NaCl. Resins were transferred to a new microcentrifuge tube and washed twice more with TG containing 100 mM NaCl. Bound proteins were eluted and analyzed as described above for polyhistidine pull-down assay.
Mammalian Two-Hybrid Assay
The PR hLBD (aa 634–933) was cloned as a fusion protein at the
amino terminus with Gal4-DBD (aa 1–147) into the pBK-CMV mammalian
expression vector (Stratagene, La Jolla, CA) as described
previously (11). The amino terminus of PR-A (aa 165–550) and PR-B (aa
1–550) were cloned into the pVP16 fusion vector (CLONTECH Laboratories, Inc.) to yield AN-VP16 and BN-VP16, respectively,
as follows: the fusion constructs Gal4-DBD-BN and Gal4-DBD-AN were
digested with EcoRI and XbaI, and the coding
sequences for the respective PR domains were ligated into pVP16,
previously digested with EcoRI and XbaI. A
control vector for nonspecific protein interaction contained the SV40
large T antigen fused to VP16 (T-VP16) and was purchased from
CLONTECH Laboratories, Inc. The luciferase reporter gene
contained a TATA box and five copies of the Gal4 DNA-binding sites (5x
Gal4-TATA-LUC, a gift from X. F. Wang, Duke University, Durham,
NC). Mouse CBP cDNA was excised from pRc/RSV-mCBP-HA-RK (a gift from R.
Goodman, Oregon Health Sciences Center, Portland, OR) (60) by digestion
with HindIII and NotI. The full-length CBP cDNA
was then inserted into the HindIII and NotI
restriction sites of pCR3.1 mammalian expression vector
(Invitrogen) to yield pCR3.1-CBP, which expresses
full-length mouse CBP with an HA (hemagglutinin antigen) tag.
pCR3.1-SRC-1 and SRC-1(0.8) were gifts from B. W. O’Malley
(Baylor College of Medicine). The mammalian expression vector for E1A
(pbcl2-E1A12S) was a gift from J. Nevins (Duke University).
HeLa cells and HepG2 cells were maintained in MEM plus 10% FCS (Life Technologies, Gaithersburg, MD). Cells were plated in 24-well dishes (coated with 0.1% gelatin for HepG2 cells) and allowed to grow 24 h before transfection. DNA was introduced into the cells using Lipofectin (Life Technologies). Briefly, triplicate transfections were performed using 3 µg of total DNA. For standard transfections 50 ng of pBKC-ß-gal (normalization vector) (61), 500 ng of reporter (5x Gal4-TATA-LUC), 1000 ng of hLBD-Gal4, 1000 ng of VP16 fusion constructs, and 450 ng of pCR3.1, 450 ng pCR3.1-hSRC-1, 450 ng pCR3.1-CBP, or a combination of 225 ng of pCR3.1-CBP and 225 ng of pCR3.1-SRC-1 (total of 450 ng of plasmid) were used. Cells were incubated with Lipofectin for 3 h, at which time media were removed and cells were treated with the appropriate hormone diluted in phenol red-free media containing 10% charcoal-stripped FCS (HyClone Laboratories, Inc., Logan, UT). Incubation with hormone continued for 48 h, after which cells were lysed and assayed for luciferase and ß-galactosidase activity as described previously (62).
Data Analysis
Comparisons of results from protein-tagged pull-down and
mammalian two-hybrid assays were done by Student’s t tests
or ANOVA using Excel 5.0 (Microsoft Corp.) to
determine whether there was a significant difference among groups.
Results were considered statistically significant at P
< 0.05.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This research was supported in part by NIH Grant DK-49030 (D.P.E.), NIH Grant DK-50495 (D.P.M.), NIH National Research Service Award Postdoctoral Fellowships DK-09225 (M.J.T.) and DK-09662 (S.A.L.), Linnea Basey Breast Cancer Fellowship (M.J.T.), US Army Medical Research and Materiel Command Predoctoral Fellowship (P.H.G.), and the Tissue Culture CORE facility of the University of Colorado Cancer Center (P30 CA-46934).
1 Present address: Center for Neuroendocrine Studies, Tobin Hall, Box
37720, University of Massachusetts, Amherst, Massachusetts 01003. 
2 Equal contributors to this work and should both be considered as
first authors.
Received for publication February 1, 1999. Revision received March 9, 1999. Accepted for publication March 11, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. B. Pippal, Y. Yao, F. M. Rogerson, and P. J. Fuller Structural and Functional Characterization of the Interdomain Interaction in the Mineralocorticoid Receptor Mol. Endocrinol., September 1, 2009; 23(9): 1360 - 1370. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Abdel-Hafiz, M. L. Dudevoir, and K. B. Horwitz Mechanisms Underlying the Control of Progesterone Receptor Transcriptional Activity by SUMOylation J. Biol. Chem., April 3, 2009; 284(14): 9099 - 9108. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Graham, A. R. Hanson, A. J. Croft, A. H. Fox, and C. L. Clarke Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci FASEB J, February 1, 2009; 23(2): 546 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B Pippal and P. J Fuller Structure-function relationships in the mineralocorticoid receptor J. Mol. Endocrinol., December 1, 2008; 41(6): 405 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Molenda-Figueira, S. D. Murphy, K. L. Shea, N. K. Siegal, Y. Zhao, J. G. Chadwick Jr., L. A. Denner, and M. J. Tetel Steroid Receptor Coactivator-1 from Brain Physically Interacts Differentially with Steroid Receptor Subtypes Endocrinology, October 1, 2008; 149(10): 5272 - 5279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-W. Jeong, K. Y. Lee, S. J. Han, B. J. Aronow, J. P. Lydon, B. W. O'Malley, and F. J. DeMayo The p160 Steroid Receptor Coactivator 2, SRC-2, Regulates Murine Endometrial Function and Regulates Progesterone-Independent and -Dependent Gene Expression Endocrinology, September 1, 2007; 148(9): 4238 - 4250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. P. Madauss, E. T. Grygielko, S.-J. Deng, A. C. Sulpizio, T. B. Stanley, C. Wu, S. A. Short, S. K. Thompson, E. L. Stewart, N. J. Laping, et al. A Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor Modulator Mol. Endocrinol., May 1, 2007; 21(5): 1066 - 1081. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Z. Lu, S. E. Wardell, K. L. Burnstein, D. Defranco, P. J. Fuller, V. Giguere, R. B. Hochberg, L. McKay, J.-M. Renoir, N. L. Weigel, et al. International Union of Pharmacology. LXV. The Pharmacology and Classification of the Nuclear Receptor Superfamily: Glucocorticoid, Mineralocorticoid, Progesterone, and Androgen Receptors Pharmacol. Rev., December 1, 2006; 58(4): 782 - 797. [Full Text] [PDF]
|
|
|
|
|
|
L. Tung, H. Abdel-Hafiz, T. Shen, D. M. E. Harvell, L. K. Nitao, J. K. Richer, C. A. Sartorius, G. S. Takimoto, and K. B. Horwitz Progesterone Receptors (PR)-B and -A Regulate Transcription by Different Mechanisms: AF-3 Exerts Regulatory Control over Coactivator Binding to PR-B Mol. Endocrinol., November 1, 2006; 20(11): 2656 - 2670. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Georgiakaki, N. Chabbert-Buffet, B. Dasen, G. Meduri, S. Wenk, L. Rajhi, L. Amazit, A. Chauchereau, C. W. Burger, L. J. Blok, et al. Ligand-Controlled Interaction of Histone Acetyltransferase Binding to ORC-1 (HBO1) with the N-Terminal Transactivating Domain of Progesterone Receptor Induces Steroid Receptor Coactivator 1-Dependent Coactivation of Transcription Mol. Endocrinol., September 1, 2006; 20(9): 2122 - 2140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tian, M. A. Mahajan, C. T. Wong, I. Habeos, and H. H. Samuels The N-Terminal A/B Domain of the Thyroid Hormone Receptor-{beta}2 Isoform Influences Ligand-Dependent Recruitment of Coactivators to the Ligand-Binding Domain Mol. Endocrinol., September 1, 2006; 20(9): 2036 - 2051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Proia, B. W. Nannenga, L. A. Donehower, and N. L. Weigel Dual Roles for the Phosphatase PPM1D in Regulating Progesterone Receptor Function J. Biol. Chem., March 17, 2006; 281(11): 7089 - 7101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wang and S. S. Simons Jr. Corepressor Binding to Progesterone and Glucocorticoid Receptors Involves the Activation Function-1 Domain and Is Inhibited by Molybdate Mol. Endocrinol., June 1, 2005; 19(6): 1483 - 1500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Narayanan, A. A. Adigun, D. P. Edwards, and N. L. Weigel Cyclin-Dependent Kinase Activity Is Required for Progesterone Receptor Function: Novel Role for Cyclin A/Cdk2 as a Progesterone Receptor Coactivator Mol. Cell. Biol., January 1, 2005; 25(1): 264 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Dubbink, R. Hersmus, C. S. Verma, H. A. G. M. van der Korput, C. A. Berrevoets, J. van Tol, A. C. J. Ziel-van der Made, A. O. Brinkmann, A. C. W. Pike, and J. Trapman Distinct Recognition Modes of FXXLF and LXXLL Motifs by the Androgen Receptor Mol. Endocrinol., September 1, 2004; 18(9): 2132 - 2150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Mulac-Jericevic and O. M Conneely Reproductive tissue selective actions of progesterone receptors Reproduction, August 1, 2004; 128(2): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Merot, R. Metivier, G. Penot, D. Manu, C. Saligaut, F. Gannon, F. Pakdel, O. Kah, and G. Flouriot The Relative Contribution Exerted by AF-1 and AF-2 Transactivation Functions in Estrogen Receptor {alpha} Transcriptional Activity Depends upon the Differentiation Stage of the Cell J. Biol. Chem., June 18, 2004; 279(25): 26184 - 26191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Molenda, C. P. Kilts, R. L. Allen, and M. J. Tetel Nuclear Receptor Coactivator Function in Reproductive Physiology and Behavior Biol Reprod, November 1, 2003; 69(5): 1449 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhang, D. R. Dowd, A. Staal, C. Gu, J. B. Lian, A. J. van Wijnen, G. S. Stein, and P. N. MacDonald Nuclear Coactivator-62 kDa/Ski-interacting Protein Is a Nuclear Matrix-associated Coactivator That May Couple Vitamin D Receptor-mediated Transcription and RNA Splicing J. Biol. Chem., September 12, 2003; 278(37): 35325 - 35336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. U. Agoulnik, W. C. Krause, W. E. Bingman III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel Repressors of Androgen and Progesterone Receptor Action J. Biol. Chem., August 15, 2003; 278(33): 31136 - 31148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Condon, P. Jeyasuria, J. M. Faust, J. W. Wilson, and C. R. Mendelson A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition PNAS, August 5, 2003; 100(16): 9518 - 9523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Hu, L. Cherbas, and P. Cherbas Transcription Activation by the Ecdysone Receptor (EcR/USP): Identification of Activation Functions Mol. Endocrinol., April 1, 2003; 17(4): 716 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Callewaert, G. Verrijdt, V. Christiaens, A. Haelens, and F. Claessens Dual Function of an Amino-terminal Amphipatic Helix in Androgen Receptor-mediated Transactivation through Specific and Nonspecific Response Elements J. Biol. Chem., February 28, 2003; 278(10): 8212 - 8218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kumar and E. B. Thompson Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions Mol. Endocrinol., January 1, 2003; 17(1): 1 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Leonhardt and D. P. Edwards Mechanism of Action of Progesterone Antagonists Experimental Biology and Medicine, December 1, 2002; 227(11): 969 - 980. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kaul, P. J. M. Murphy, J. Chen, L. Brown, W. B. Pratt, and S. S. Simons Jr. Mutations at Positions 547-553 of Rat Glucocorticoid Receptors Reveal That hsp90 Binding Requires the Presence, but Not Defined Composition, of a Seven-amino Acid Sequence at the Amino Terminus of the Ligand Binding Domain J. Biol. Chem., September 20, 2002; 277(39): 36223 - 36232. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Abdel-Hafiz, G. S. Takimoto, L. Tung, and K. B. Horwitz The Inhibitory Function in Human Progesterone Receptor N Termini Binds SUMO-1 Protein to Regulate Autoinhibition and Transrepression J. Biol. Chem., September 6, 2002; 277(37): 33950 - 33956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Wardell, V. Boonyaratanakornkit, J. S. Adelman, A. Aronheim, and D. P. Edwards Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator Mol. Cell. Biol., August 1, 2002; 22(15): 5451 - 5466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. He, L. W. Lee, J. T. Minges, and E. M. Wilson Dependence of Selective Gene Activation on the Androgen Receptor NH2- and COOH-terminal Interaction J. Biol. Chem., July 5, 2002; 277(28): 25631 - 25639. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. He, J. T. Minges, L. W. Lee, and E. M. Wilson The FXXLF Motif Mediates Androgen Receptor-specific Interactions with Coregulators J. Biol. Chem., March 15, 2002; 277(12): 10226 - 10235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. M. Conneely, B. Mulac-Jericevic, F. DeMayo, J. P. Lydon, and B. W. O'Malley Reproductive Functions of Progesterone Receptors Recent Prog. Horm. Res., January 1, 2002; 57(1): 339 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. He, N. T. Bowen, J. T. Minges, and E. M. Wilson Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 Coactivator Recruitment by Activation Function 2 J. Biol. Chem., November 2, 2001; 276(45): 42293 - 42301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Metivier, G. Penot, G. Flouriot, and F. Pakdel Synergism Between ER{alpha} Transactivation Function 1 (AF-1) and AF-2 Mediated by Steroid Receptor Coactivator Protein-1: Requirement for the AF-1 {alpha}-Helical Core and for a Direct Interaction Between the N- and C-Terminal Domains Mol. Endocrinol., November 1, 2001; 15(11): 1953 - 1970. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Tung, T. Shen, M. G. Abel, R. L. Powell, G. S. Takimoto, C. A. Sartorius, and K. B. Horwitz Mapping the Unique Activation Function 3 in the Progesterone B-receptor Upstream Segment. TWO LXXLL MOTIFS AND A TRYPTOPHAN RESIDUE ARE REQUIRED FOR ACTIVITY J. Biol. Chem., October 19, 2001; 276(43): 39843 - 39851. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P A Mote, J F Johnston, T Manninen, P Tuohimaa, and C L Clarke Detection of progesterone receptor forms A and B by immunohistochemical analysis J. Clin. Pathol., August 1, 2001; 54(8): 624 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. M. Conneely Perspective: Female Steroid Hormone Action Endocrinology, June 1, 2001; 142(6): 2194 - 2199. [Full Text] [PDF]
|
|
|
|
|
|
J. G. A. Savory, G. G. Préfontaine, C. Lamprecht, M. Liao, R. F. Walther, Y. A. Lefebvre, and R. J. G. Haché Glucocorticoid Receptor Homodimers and Glucocorticoid-Mineralocorticoid Receptor Heterodimers Form in the Cytoplasm through Alternative Dimerization Interfaces Mol. Cell. Biol., February 1, 2001; 21(3): 781 - 793. [Abstract] [Full Text]
|
|
|
|
|
|
R. Métivier, F. G. Petit, Y. Valotaire, and F. Pakdel Function of N-Terminal Transactivation Domain of the Estrogen Receptor Requires a Potential {alpha}-Helical Structure and Is Negatively Regulated by the A Domain Mol. Endocrinol., November 1, 2000; 14(11): 1849 - 1871. [Abstract] [Full Text]
|
|
|
|
|
|
J. Lim, F. J. Ghadessy, A. A. R. Abdullah, L. Pinsky, M. Trifiro, and E. L. Yong Human Androgen Receptor Mutation Disrupts Ternary Interactions between Ligand, Receptor Domains, and the Coactivator TIF2 (Transcription Intermediary Factor 2) Mol. Endocrinol., August 1, 2000; 14(8): 1187 - 1197. [Abstract] [Full Text]
|
|
|
|
|
|
P. H. Giangrande, E. A. Kimbrel, D. P. Edwards, and D. P. McDonnell The Opposing Transcriptional Activities of the Two Isoforms of the Human Progesterone Receptor Are Due to Differential Cofactor Binding Mol. Cell. Biol., May 1, 2000; 20(9): 3102 - 3115. [Abstract] [Full Text]
|
|
|
|
|
|
B. He, J. A. Kemppainen, J. J. Voegel, H. Gronemeyer, and E. M. Wilson Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain J. Biol. Chem., December 24, 1999; 274(52): 37219 - 37225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. He, J. A. Kemppainen, and E. M. Wilson FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor J. Biol. Chem., July 21, 2000; 275(30): 22986 - 22994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-N. Song, B. Huse, S. Rusconi, and S. S. Simons Jr. Transactivation Specificity of Glucocorticoid Versus Progesterone Receptors. ROLE OF FUNCTIONALLY DIFFERENT INTERACTIONS OF TRANSCRIPTION FACTORS WITH AMINO- AND CARBOXYL-TERMINAL RECEPTOR DOMAINS J. Biol. Chem., June 29, 2001; 276(27): 24806 - 24816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhang, T. A. Baudino, D. R. Dowd, H. Tokumaru, W. Wang, and P. N. MacDonald Ternary Complexes and Cooperative Interplay between NCoA-62/Ski-interacting Protein and Steroid Receptor Coactivators in Vitamin D Receptor-mediated Transcription J. Biol. Chem., October 26, 2001; 276(44): 40614 - 40620. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
