Hormone-Dependent Interaction between the Amino- and Carboxyl-Terminal Domains of Progesterone Receptor in Vitro and in Vivo
Marc J. Tetel1,2,
Paloma H. Giangrande2,
Susan A. Leonhardt,
Donald P. McDonnell and
Dean P. Edwards
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
|
---|
Full transcriptional activation by steroid hormone
receptors requires functional synergy between two transcriptional
activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2)
terminal regions. One possible mechanism for achieving this functional
synergy is a physical intramolecular association between amino (N-) and
carboxyl (C-) domains of the receptor. Human progesterone receptor (PR)
is expressed in two forms that have distinct functional activities:
full-length PR-B and the amino-terminally truncated PR-A. PR-B is
generally a stronger activator than PR-A, whereas under certain
conditions PR-A can act as a repressor in trans of other
steroid receptors. We have analyzed whether separately expressed N-
(PR-A and PR-B) and C-domains [hinge plus ligand-binding domain
(hLBD)] of PR can functionally interact within cells by mammalian
two-hybrid assay and whether this involves direct protein contact as
determined in vitro with purified expressed domains of PR.
A hormone agonist-dependent interaction between N-domains and the hLBD
was observed functionally by mammalian two-hybrid assay and by direct
protein-protein interaction assay in vitro. With both
experimental approaches, N-C domain interactions were not induced by
the progestin antagonist RU486. However, in the presence of the
progestin agonist R5020, the N-domain of PR-B interacted more
efficiently with the hLBD than the N-domain of PR-A. Coexpression of
steroid receptor coactivator-1 (SRC-1) and the CREB binding protein
(CBP), enhanced functional interaction between N- and C-domains by
mammalian two-hybrid assay. However, addition of SRC-1 and CBP in
vitro had no influence on direct interaction between purified N-
and C-domains. These results suggest that the interaction between N-
and C-domains of PR is direct and requires a hormone agonist-induced
conformational change in the LBD that is not allowed by antagonists.
Additionally, coactivators are not required for physical association
between the N- and C-domains but are capable of enhancing a
functionally productive interaction. In addition, the more efficient
interaction of the hLBD with the N-domain of PR-B, compared with that
of PR-A, suggests that distinct interactions between N- and
C-terminal regions contribute to functional differences between PR-A
and PR-B.
 |
INTRODUCTION
|
---|
The human progesterone receptor (PR) is a member of the nuclear
receptor superfamily of transcriptional activators that regulates
development, differentiation, and homeostasis of various reproductive
functions (1, 2). PR is expressed as two distinct molecular forms from
a single gene: full-length PR-B and truncated PR-A that lacks the first
164 amino acids of the amino terminus (3). PR, as well as other steroid
receptors, has a conserved structural and functional organization that
has been well characterized (1, 2). Both forms of PR are identical in
their centrally located DNA-binding domain (DBD) and carboxyl (C-)
terminal ligand-binding domain (LBD). PR-A and PR-B also contain two
independent transcriptional activation domains (AF): a constitutive
AF-1 in the amino terminus and a hormone-dependent AF-2 in the LBD (4, 5). A third transcriptional modulatory domain has been defined in the
amino (N-) terminal segment unique to PR-B that requires interaction
with other regions of the receptor (5, 6). Under certain cell and
promoter contexts, PR-B is a stronger transcriptional activator than
PR-A (7, 8, 9, 10). This difference in activity is most likely due to
conformational or other structural differences between the N termini of
the two-receptor isoforms (3, 11, 12). Under conditions in which PR-A
is not an activator, it can functionally repress the transcriptional
activity of other steroid receptors (7, 8, 9, 10). While the mechanism for
this repression by PR-A is not fully understood, a discrete
transcriptional inhibitory region has been identified in human PR-A
that may allow it to interact with factors that do not interact with
PR-B (11, 12).
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
|
---|
Hormone-Agonist Dependent Interaction in Vitro between
Amino- and Carboxyl-Terminal Domain Polypeptides of PR
Protein-protein interaction in vitro between separately
expressed N- and C-domains of PR was analyzed initially by a
polyhistidine-tagged protein pull-down assay with the polypeptides
shown schematically in Fig. 1
. The PR
fragments included polyhistidine-tagged N-domains of PR-A (ANhis; aa
165535) and PR-B (BNhis; aa 1535) and nontagged C-domains
containing either the entire LBD (aa 688933) or the LBD plus hinge
region (hLBD; aa 634933). 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).

View larger version (62K):
[in this window]
[in a new window]
|
Figure 1. PR Amino-Carboxyl Terminal Interactions Detected
in Vitro by Polyhistidine Pull-Down Assay
Schematic of PR domains expressed in baculovirus (upper
panel): The amino termini of PR-B (BNhis, aa 1535) and PR-A
(ANhis, aa 165535) were expressed with a 6x polyhistidine tag. The
hinge region (h) and ligand binding domain (hLBD, aa 634933) and the
LBD alone (aa 688933) were expressed and prepared as nonfusion
proteins. Full-length PR-B (aa 1933) is shown for alignment of all
the receptor domains. Six sequential N-terminal histidine residues
(his). Whole-cell extracts of infected Sf9 cells containing
polyhistidine-tagged N-domains of PR-B (BNhis) or PR-A (ANhis) were
mixed with equal amounts (determined by Western blot and
steroid-binding analysis) of C-terminal domains (LBD or hLBD) and
incubated with metal ion affinity resins (Talon). The LBD and hLBD were
bound to the synthetic progestin R5020 during expression in Sf9 cells.
After washes of the resin, bound proteins were eluted with 2% SDS and
analyzed by Western blot with a mixture of MAbs that recognize epitopes
in either the N-domain or the LBD of PR (AB-52 and C-262,
respectively). Assay input (10%) of polyhistidine-tagged N-domains
(ANhis and BNhis), and the carboxyl-terminal, LBD and hLBD, are shown
in lanes 14. Lanes 57 are the LBD incubated with metal resins
(Talon) in the absence (nonspecific binding control, lane 5) or
presence of ANhis (lane 6) or BNhis (lane 7). Lanes 810 are the hLBD
incubated with metal resins in the absence (lane 8) or presence of
ANhis (lane 9) or BNhis (lane 10). The Western blot detection method
was 35S-labeled protein A and autoradiography.
|
|
By this pulldown assay, no specific association was detected between
the LBD and the N-domains of either form of PR (Fig. 1
, lanes 57).
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 810). 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.
To determine whether interaction between N-domains and the hLBD is
dependent on ligand binding, similar polyhistidine-tagged protein
pull-down experiments were performed with the hLBD prepared in the
unliganded state, or bound to R5020 or the progesterone antagonist
RU486. The hLBD did not physically associate with the N-domains of PR-A
or PR-B in the absence of ligand (Fig. 2
, lanes 46) or when bound to RU486 (Fig. 2
, lanes 79). The hLBD
efficiently interacted with the N-domains of PR (A or B form) only when
bound to R5020 (Fig. 2
, lanes 1012). 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.

View larger version (44K):
[in this window]
[in a new window]
|
Figure 2. Interactions of Amino-Carboxyl Domains in
Vitro are Hormone Agonist Dependent
Whole-cell extracts of Sf9 cells containing the PR domains shown in the
schematic were mixed, and association between the N-domains and the
hLBD was detected by polyhistidine-tagged pull-down assay as described
in Fig. 1 . The hLBD was either unliganded (lanes 46) or was bound to
RU486 (lanes 78) or R5020 (lanes 1012). Proteins bound to Talon
resins were eluted and analyzed by Western blot with a mixture of MAbs
(AB-52 and C-262) that together detect the N-domains and the hLBD.
Inputs (10% of total) of the hLBD and the polyhistidine-tagged
N-domain (ANhis and BNhis) are shown in lanes 13.
|
|
Interaction between Amino and Carboxyl Domains of PR Is Direct
between Purified PR Fragments and Does Not Require Other Proteins
The in vitro protein interaction experiments depicted
in Figs. 1
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
).

View larger version (58K):
[in this window]
[in a new window]
|
Figure 3. Purification of PR Domain Polypeptides
Recombinant PR hLBD-GST purified by glutathione Sepharose 4B affinity
chromatography, and the N-domains of PR-A (ANhis) and PR-B (BNhis)
purified by Ni-NTA affinity chromatography, were analyzed by SDS-PAGE
and silver staining (panel A) and by Western blot (panel B) with a
mixture of MAbs that recognize epitopes in the N-domain common to PR-A
and PR-B (AB-52) and in the LBD (C262).
|
|
Approximately equal amounts (determined from silver-stained SDS
gels) of purified N-domains and hLBD-GST were mixed together in GST
pull-down assays. The hLBD-GST and a baculovirus-expressed GST as a
control for nonspecific binding were preimmobilized to
glutathione-Sepharose resins. The hLBD-GST, GST, and blank resins were
then incubated with purified N-domain polypeptides, and after washing
of the resins in buffer with 125 mM NaCl, bound proteins
were eluted and analyzed by Western blot. Detection of specifically
associated N-domains was by use of a MAb (1294) that recognizes an
epitope in the N-terminal region of human PR that is common to both A
and B isoforms (Fig. 4
). 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.

View larger version (42K):
[in this window]
[in a new window]
|
Figure 4. Direct Interaction in Vitro between
purified N- and C-Domains of PR
Purified hLBD-GST, or GST as a control, were preimmobilized to
glutathione Sepharose. Equal amounts (determined by Western blot) of
purified N-domains of PR-A (panel A) or PR-B (panel B) were incubated
with the hLBD-GST, GST, or blank resins for 1 h at 4 C. After
washing of resins, bound proteins were eluted and analyzed by Western
blot with an MAb (1294) that detects an epitope in the N-domain common
to PR-A and PR-B. Assay input (10%) of the N terminus of PR-A
(AN) or PR-B (BN) for each GST-pull down assay is indicated.
|
|
Functional Hormone-Agonist Dependent Interaction between the Amino-
and Carboxyl-Terminal Domains of PR by Mammalian Two-Hybrid Assay
A mammalian two-hybrid assay was used to determine whether
the N-terminal regions of PR-A and PR-B can functionally interact with
the C-terminal hLBD within cells. The hybrid protein constructs
depicted in Fig. 5
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.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 5. Mammalian Two-Hybrid Assay Constructs
Schematic diagram of the fusion constructs and reporter gene used in
the mammalian two-hybrid assay. The VP16 acidic activation domain (aa
411455) was fused to the N terminus of PR-B (aa 1550, BN-VP16), the
N terminus of PR-A (aa 165550, AN-VP16), or the SV40 large T antigen
(T-VP16). The Gal4 DBD (aa 1147) was fused to the PR LBD plus hinge
sequences (aa 634933) to yield hLBD-Gal4. Interaction between the
hLBD-Gal4 and N-domain VP16 fusion proteins was measured as an
induction of expression of the luciferase reporter gene under the
regulation of five Gal4 DNA-binding sites (5x Gal4-RE-LUC).
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Figure 6. Hormone Agonist-Dependent Functional Interaction
between Amino and Carboxyl Domains of PR
HepG2 cells (A) or HeLa cells (B) were transiently cotransfected with
hLBD-Gal4 and VP16 fusion constructs containing the N-domains of PR-B
(BN-VP16), PR-A (AN-VP16), or the SV40 large T antigen (T-VP16).
Transcriptional activity of the luciferase gene was assayed on the 5x
Gal4-RE-LUC reporter as a measure of functional interaction between
Gal4 and VP16 fusion proteins. Transcriptional activity was measured in
the absence of ligand or in the presence of progesterone
(10-7 M) or the progesterone antagonist RU486
(10-7 M). The data are represented as fold
induction over the control interaction between hLBD-Gal4 and T-VP16 for
each ligand treatment group that was normalized to 1.0.
Bars are the mean ± SEM from three
independent experiments.
|
|
In the absence of ligand, hLBD-Gal4 did not functionally interact
in either HepG2 cells (Fig. 6A
) 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).

View larger version (31K):
[in this window]
[in a new window]
|
Figure 7. Coexpression of Coactivators Enhances Functional
Interaction between Amino and Carboxyl Domains of PR
HepG2 cells were transiently transfected with hLBD-Gal4 and the VP16
fusion constructs expressing SV40 large T antigen (T), or the N-domains
of PR-B (BN) or PR-A (AN), as in Fig. 6 , except in the absence (empty
pCR3.1 vector) or presence of expression vectors for SRC-1, CBP, or
both coactivators. The data are calculated as fold inductions over the
control interaction between hLBD-Gal4 and T-VP16 for each group and are
the mean of triplicate determinations (±SEM) from a single
representative experiment.
|
|
To test the extent to which coactivators are essential for functional
interaction between N- and C-domains of PR, the activities of
endogenous SRC-1 and CBP were inhibited in the mammalian two-hybrid
assay. To inhibit SRC-1, cells were cotransfected with a
dominant-negative form of SRC-1 (0.8) that contains the
C-terminal nuclear receptor-binding site enabling it to bind to PR, but
lacks the centrally located nuclear receptor-binding sites and both
transcriptional activation domains (16, 28). Coexpression of SRC-1
(0.8) in the mammalian two-hybrid assay effectively inhibited
progesterone-dependent interaction between hLBD-Gal4 and the N-domains
of either PR isoform (Fig. 8A
). 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
|
---|
Full transcriptional activity of steroid receptors requires
functional synergy between activation functions located in the amino
and carboxyl domains of receptor (4, 5, 32, 33, 34, 35, 36, 37, 38, 39). Previous studies with
ER (40) and AR (30, 41, 42, 43, 44) using a standard or modified two-hybrid
assay, suggest that this functional synergy involves a ligand-dependent
association between the amino and carboxyl domains of receptor. Using a
mammalian two-hybrid interaction system, we have also observed a
hormone-agonist dependent functional interaction between N-terminal
domains and the hLBD of human PR, suggesting that N-C interdomain
interaction is a common mechanism for all steroid hormone receptors. An
unresolved question from previous two-hybrid results is whether the
observed functional interaction represents direct protein contacts or
is indirectly mediated by other proteins that associate with either N-
or C-domains of the receptor. To resolve this question we have
investigated the ability of N- and C-domain polypeptides of PR to
interact directly in vitro. When expressed as recombinant
polypeptides in Sf9 cells and prepared as whole-cell extracts, the
N-domains of both PR isoforms interacted efficiently with the
C-terminal hLBD of PR. Furthermore, this in vitro
interaction was dependent on the hormone agonist R5020 and was not
detected in the absence of ligand (Figs. 1
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.

View larger version (37K):
[in this window]
[in a new window]
|
Figure 9. Model of Hormone Agonist-Dependent Intramolecular
Association of Amino and Carboxyl Domains of PR
The three major domains of PR-A and PR-B are indicated schematically:
the amino-terminal domain containing AF-1, the DBD, and the
carboxyl-terminal LBD containing AF-2. The model depicts the hormone
agonist-activated PR with the stippled region
representing the N-terminal extended segment unique to PR-B. The
dashed lines represent direct contacts between N- and
C-domains, and the coactivator complexes associated with PR-A and PR-B
contain distinct subunit compositions.
|
|
 |
MATERIALS AND METHODS
|
---|
Materials
Unlabeled progesterone and ovalbumin were purchased from
Sigma Chemical Co. (St. Louis, MO). Unlabeled RU486
(Mifepristone, 17-hydroxy-11 [4-dimethlyaminophenyl]
17-propynyl-estra-4, 5-diene-3-one) was a gift from Roussel-UCLAF
(Romainville, France) or Ligand Pharmaceuticals, Inc. (San
Diego, CA). Nickel-NTA (Ni-NTA) and metal ion affinity resins (Talon)
were obtained from Qiagen (Valencia, CA) and
CLONTECH Laboratories, Inc. (Palo Alto, CA), respectively.
Mouse IgG1 MAbs generated against human PR include AB-52 and 1294,
which recognize epitopes in the amino terminus common to PR-A and PR-B
(Ref. 46 and B. Spaulding, L. Sherman, and D. P. Edwards, unpublished
data), B-30, which recognizes only PR-B (46), and C-262, which
is directed against the last 14 amino acids of the carboxyl-terminal
end of PR (47). A polyclonal antibody raised against PR-A (B13-TK) was
a gift from Nancy Weigel (Baylor College of Medicine, Houston, TX). A
MAb generated against the polyhistidine tag and enterokinase cleavage
site fusion sequences (mouse IgG clone 1162/F6) contained in the
pBlueBacHis2 baculovirus transfer plasmid (Invitrogen) was
used for Western blot detection of baculovirus polyhistidine-tagged
proteins and a MAb produced to GST (mouse IgG clone 794/H12) was used
for Western blot detection of GST-fusion proteins (D. P. Edwards
and S. Anderson, unpublished data). Secondary antibodies, Hybond-C
Extra (nitrocellulose) transfer membrane, and x-ray developing film
were obtained from Amersham (Arlington Heights, IL). DNA
restriction and modification enzymes were obtained from Promega Corp. (Madison, WI), Boehringer Mannheim
(Indianapolis, IN), or New England Biolabs, Inc. (Beverly,
MA). PCR reagents were obtained from Perkin-Elmer Corp.
(Norwalk, CT) or Promega Corp.
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 1535), the N-terminus of PR-A (ANhis, aa
165535), and the hLBD (aa 634933). The LBD alone (aa 688933) 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 17792671 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 1535. 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. OMalley, Baylor College of Medicine,
Houston, TX) as the template cDNA (50). The PCR amplification
product resulted in aa 634933 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. OMalley, 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. OMalley (Baylor College of
Medicine).
Spodoptera frugiperda (Sf9) insect cells were grown in
spinner vessels (150500 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 04 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 634933) was cloned as a fusion protein at the
amino terminus with Gal4-DBD (aa 1147) into the pBK-CMV mammalian
expression vector (Stratagene, La Jolla, CA) as described
previously (11). The amino terminus of PR-A (aa 165550) and PR-B (aa
1550) 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. OMalley
(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 Students 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
|
---|
The authors acknowledge the expert technical assistance of Kurt
Christensen, Suzanne Meizner, and Kristen Cullen for expression of
recombinant proteins in the baculovirus insect cell system, the
technical assistance of Neal Van Hoeven with polyhistidine-tagged
pull-down assay, Lori Sherman for purification of baculovirus-expressed
proteins, and Vida Melvin for assistance with computer graphics.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dean P. Edwards, Ph.D., Department of Pathology, B216, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail:
Dean.Edwards{at}UCHSC.edu
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
|
---|
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P,
Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P,
Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer
H, Chambon P 1990 Two distinct estrogen-regulated promoters generate
transcripts encoding the two functionally different human progesterone
receptor forms A and B. EMBO J 9:16031614[Abstract]
-
Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer
H 1990 Agonistic and antagonistic activities of RU486 on the functions
of the human progesterone receptor. EMBO J 9:39233932[Abstract]
-
Meyer ME, Quirin-Stricker C, Lerouge T, Bocquel MT,
Gronemeyer H 1992 A limiting factor mediates the differential
activation of promoters by the human progesterone receptor isoforms.
J Biol Chem 267:1088210887[Abstract/Free Full Text]
-
Sartorius CA, Melville MY, Hovland AR, Tung L, Takimoto GS,
Horwitz KB 1994 A third transactivation function (AF3) of human
progesterone receptors located in the unique N-terminal segment of the
B-isoform. Mol Endocrinol 8:13471360[Abstract]
-
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, OMalley BW,
McDonnell DP 1993 Human progesterone receptor A form is a cell- and
promoter-specific repressor of human progesterone receptor B function.
Mol Endocrinol 7:12441255[Abstract]
-
McDonnell DP, Goldman ME 1994 RU486 exerts antiestrogenic
activities through a novel progesterone receptor A form-mediated
mechanism. J Biol Chem 269:1194511949[Abstract/Free Full Text]
-
Tung L, Kamel Mohamed M, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate
transcription without binding to progesterone response elements and are
dominantly inhibited by A-receptors. Mol Endocrinol 7:12561265[Abstract]
-
Wen DX, Xu YF, Mais DE, Goldman ME, McDonnell DP 1994 The A
and B isoforms of the human progesterone receptor operate through
distinct signaling pathways within target cells. Mol Cell Biol 14:83568364[Abstract]
-
Giangrande PH, Pollio G, McDonnell DP 1997 Mapping and
characterization of the functional domains responsible for the
differential activity of the A and B isoforms of the human progesterone
receptor. J Biol Chem 272:3288932900[Abstract/Free Full Text]
-
Hovland AR, Powell RL, Takimoto GS, Tung L, Horwitz KB 1998 An
N-terminal inhibitory function, IF, suppresses transcription by the
A-isoform but not the B-isoform of human progesterone receptors. J
Biol Chem 273:54555460[Abstract/Free Full Text]
-
Smith DF, Toft DO 1993 Steroid receptors and their associated
proteins. Mol Endocrinol 7:411[Medline]
-
Truss M, Beato M 1993 Steroid hormone receptors: interaction
with deoxyribonucleic acid and transcription factors. Endocr Rev 14:459479[Abstract]
-
DeMarzo A, Beck CA, Oñate SA, Edwards DP 1991 Dimerization of mammalian progesterone receptors occurs in the absence
of DNA and is related to the release of the 90-kDa heat shock protein.
Proc Natl Acad Sci USA 88:7276[Abstract]
-
Oñate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Edwards DP 1999 Role of coregulatory proteins in nuclear
receptor action. Vitam Horm 55:165218[Medline]
-
Janknecht R, Hunter T 1996 A growing coactivator network.
Nature 383:2223[CrossRef][Medline]
-
Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B,
Kurokawa R, Brown M 1996 p300 is a component of an estrogen receptor
coactivator complex. Proc Natl Acad Sci USA 93:1154011545[Abstract/Free Full Text]
-
Hibata H, Spencer TE, Oñate SA, Jenster G, Tsai SY, Tsai
MJ, OMalley BW 1997 Role of coactivators and corepressors in the
mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141165[Medline]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Xu J, Qiu Y, Demayo FJ, Tsai SY, Tsai MJ, OMalley BW 1998 Partial hormone resistance in mice with disruption of the steroid
receptor coactivator-1 (SRC-1) gene. Science 279:19221925[Abstract/Free Full Text]
-
Jenster G, Spencer TE, Burcin MM, Tsai SY, Tsai MJ, OMalley
BW 1997 Steroid receptor induction of gene transcription: a two step
model. Proc Natl Acad Sci USA 94:78797884[Abstract/Free Full Text]
-
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA,
McKenna NJ, Oñate SA, Tsai SY, Tsai MJ, OMalley BW 1997 Steroid
receptor coactivator-1 is a histone acetyltransferase. Nature 389:194197[CrossRef][Medline]
-
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney
EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription
factor-specific requirements for coactivators and their
acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
-
Oñate SA, Boonyaratanakornkit V, Spencer TE, Tsai SY,
Tsai MJ, Edwards DP, OMalley BW 1998 The steroid receptor
coactivator-1 contains multiple receptor interacting and activation
domains that cooperatively enhance the activation function 1 (AF1) and
AF2 domains of steroid receptors. J Biol Chem 273:1210112108[Abstract/Free Full Text]
-
Smith CL, Nawaz Z, OMalley BW 1997 Coactivator and
corepressor regulation of the agonist/antagonist activity of the mixed
antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657666[Abstract/Free Full Text]
-
Ikonen T, Palvimo JJ, Janne OA 1997 Interaction between the
amino- and carboxyl-terminal regions of the rat androgen receptor
modulates transcriptional activity and is influenced by nuclear
receptor coactivators. J Biol Chem 272:2982129828[Abstract/Free Full Text]
-
Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP,
Chen D, Huang S-M, Subramanian S, McKinerney E, Katzenellenbogen
BS, Stallcup MR, Kushner PJ 1998 Estrogen receptor activation function
1 works by binding p160 coactivator proteins. Mol Endocrinol 12:16051618[Abstract/Free Full Text]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
non-acidic transcriptional activation functions. Cell 59:477487[Medline]
-
Lees JA, Fawell SE, Parker MG 1989 Identification of two
transactivation domains in the mouse oestrogen receptor. Nucleic Acid
Res 17:54775487[Abstract]
-
Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization
of the amino-terminal transcriptional activation function of the human
estrogen receptor in animal and yeast cells. J Biol Chem 270:95359542[Abstract/Free Full Text]
-
Bocquel MT, Kumar V, Stricker C, Chambon P, Gronemeyer H 1989 The contribution of the N- and C-terminal regions of steroid receptors
to activation of transcription is both receptor- and cell-specific.
Nucleic Acid Res 17:10071019
-
Tasset D, Tora L, Fromental C, Scheer E, Chambon P 1990 Distinct classes of transcriptional activating domains function by
different mechanisms. Cell 62:11771187[Medline]
-
Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]
-
Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG,
Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor
transactivational capacity is determined by both cellular and promoter
context and mediated by two functionally distinct intramolecular
regions. Mol Endocrinol 8:2130[Abstract]
-
Pham TA, Hwung YP, Santisomere D, McDonnell DP, OMalley BW 1992 Ligand-dependent and ligand-independent function of the
transactivation regions of the human estrogen receptor in yeast. Mol
Endocrinol 6:10431050[Abstract]
-
Kraus WL, McInerney EM, Katzenellenbogen BS 1995 Ligand-dependent, transcriptionally productive association of the
amino- and carboxyl-terminal regions of a steroid hormone nuclear
receptor. Proc Natl Acad Sci USA 92:1231412318[Abstract]
-
Langley E, Zhou Z, Wilson EM 1995 Evidence for an
anti-parallel orientation of the ligand-activated human androgen
receptor dimer. J Biol Chem 270:2998329990[Abstract/Free Full Text]
-
Doesburg P, Kuil CW, Berrevoets CA, Steketee K, Faber PW,
Mulder E, Brinkmann AO, Trapman J 1997 Functional in vivo
interaction between the amino-terminal, transactivation domain and the
ligand binding domain of the androgen receptor. Biochemistry 36:10521064[CrossRef][Medline]
-
Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO 1998 Functional interactions of the AF-2 activation domain core region
of the human androgen receptor with the amino-terminal domain and with
the transcriptional coactivator TIF2 (transcriptional intermediary
factor 2). Mol Endocrinol 12:11721183[Abstract/Free Full Text]
-
McInerney EM, Tsai MJ, OMalley BW, Katzenellenbogen BS 1996 Analysis of estrogen receptor transcriptional enhancement by a
nuclear hormone receptor coactivator. Proc Natl Acad Sci USA 93:1006910073[Abstract/Free Full Text]
-
Tetel MJ, Jung S, Carbajo P, Ladtkow T, Skafar DF, Edwards DP 1997 Hinge and amino-terminal sequences contribute to solution
dimerization of human progesterone receptor. Mol Endocrinol 11:11141128[Abstract/Free Full Text]
-
Estes PA, Suba EJ, Lawler-Heavner J, Elashry-Stowers D, Wei
LL, Toft DO, Sullivan WP, Horwitz KB, Edwards DP 1987 Immunological
analysis of human breast cancer progesterone receptors. 1.
Immunoaffinity purification of transformed receptors and production of
monoclonal antibodies. Biochemistry 26:62506262[Medline]
-
Weigel NL, Beck CA, Estes PA, Prendergast P, Altmann M,
Christensen K, Edwards DP 1992 Ligands induce conformational changes in
the carboxyl-terminus of progesterone receptors which are detected by a
site-directed antipeptide monoclonal antibody. Mol Endocrinol 6:15851597[Abstract]
-
Zhang Y, Beck CA, Poletti A, Edwards DP, Weigel NL 1994 Identification of phosphorylation sites unique to the B form of human
progesterone receptor. In vitro phosphorylation by casein
kinase II. J Biol Chem 269:3103431040[Abstract/Free Full Text]
-
Lundblad JR, Kwok RPS, Laurance ME, Harter ML, Goodman RH 1995 Adenoviral E1A-associated protein p300 as a functional homologue of the
transcriptional co-activator CBP. Nature 374:8588[CrossRef][Medline]
-
Allan GF, Leng X, Tsai SY, Wiegel NL, Edwards DP, Tsai MJ,
OMalley BW 1992 Hormone and antihormone induce distinct
conformational changes which are central to steroid receptor
activation. J Biol Chem 267:1951319520[Abstract/Free Full Text]
-
Christensen K, Estes PA, Oñate SA, Beck CA, DeMarzo A,
Altmann M, Lieberman BA, St.John J, Nordeen SK, Edwards DP 1991 Characterization and functional properties of the A and B forms of
human progesterone receptors synthesized in a baculovirus system. Mol
Endocrinol 5:17551770[Abstract]
-
Beekman JM, Allan GF, Tsai SY, Tsai MJ, OMalley BW 1993 Transcriptional activation by the estrogen receptor requires a
conformational change in the ligand binding domain. Mol Endocrinol 7:12661274[Abstract]
-
Kallio PJ, Janne OA, Palvimo JJ 1994 Agonists, but not
antagonists, alter the conformation of the hormone-binding domain of
androgen receptor. Endocrinology 134:9981001[Abstract]
-
Langley E, Kemppainen JA, Wilson EM 1998 Intermolecular
NH2-/carboxyl-terminal interactions in androgen receptor dimerization
revealed by mutations that cause androgen insensitivity. J Biol
Chem 273:92101[Abstract/Free Full Text]
-
Cohen-Solac K, Bailly A, Ranch C, Quense M, Milgrom E Specific
binding of progesterone receptor to progesterone response elements does
not require prior dimerization. Eur J Biochem 214:189195
-
Mohamed MK, Tung L, Takimoto GS, Horwitz KB 1994 The leucine
zippers of c-fos and c-jun for progesterone receptor dimerization:
A-dominance in the A/B heterodimer. J Steroid Biochem Mol Biol 51:241250[CrossRef][Medline]
-
Williams SP, Sigler PB 1998 Atomic structure of progesterone
complexed with its receptor. Nature 393:392396[CrossRef][Medline]
-
Tetel MJ, Beck CA, Ladtkow T, Christensen K, Weigel NL,
Edwards DP 1997 Functional properties and post-translational
modification of steroid hormone receptors in the baculovirus expression
system. In: Maramorosch K, Mitsuhashi J (eds) Invertebrate Cell
Culture. Science Publishers, Enfield, NH, pp 201210
-
Boonyaratanakornkit V, Melvin V, Prendergast P, Altmann M,
Ronfani L, Bianchi ME, Taraseviciene L, Nordeen SK, Allegretto EA,
Edwards DP 1998 High-mobility group chromatin proteins 1 and 2
functionally interact with steroid hormone receptors to enhance their
DNA binding in vitro and transcriptional activity in
mammalian cells. Mol Cell Biol 18:44714487[Abstract/Free Full Text]
-
Kwok RPS, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP,
Brennan RG, Roberts SGE, Green MR, Goodman RH 1994 Nuclear protein CBP
is a coactivator for the transcription factor CREB. Nature 370:223229[CrossRef][Medline]
-
MacGregor GR, Caskey CT 1989 Construction of plasmids that
express E. coli ß-galactosidase in mammalian cells.
Nucleic Acid Res 17:2365[Medline]
-
Norris J, Fan D, Aleman C, Marks JR, Futreal PA, Wiseman RW,
Iglehart JD, Deininger PL McDonnell DP 1995 Identification of a new
subclass of alu repeats which can function as estrogen
receptor-dependent transcriptional enhancers. J Biol Chem 270:2277722782[Abstract/Free Full Text]