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Transactivation Function 1 (AF-1) and AF-2 Mediated by Steroid Receptor Coactivator Protein-1: Requirement for the AF-1 -Helical Core and for a Direct Interaction Between the N- and C-Terminal Domains
Equipe d’Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, Université de Rennes I, 35042 Rennes Cedex, France
Address all correspondence and requests for reprints to: Farzad Pakdel, Equipe d’Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, Université de Rennes I, Rennes Cedex, France. E-mail: farzad.pakdel{at}univ-rennes1.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(or NR3A1) after binding of
ligand is mediated through synergistic action between activation
functions (AFs) AF-1 and AF-2 and the transcriptional machinery. This
is functionally achieved by bridging coactivators such as CEBP binding
protein/p300 and members of the p160 subfamily such as steroid
receptor coactivator protein-1 (SRC-1). We previously identified a
conserved potential -helical structure within the AF-1 functional
core, and by evaluating point mutants of human ER (hER) within
this region, we show that in transfection experiments this structure is
required for synergism between SRC-1 and hER. We report that the
transcriptional synergism between AF-1 mutants and SRC-1 was abolished
in AF-1-sensitive cells such as HepG2, whereas it was reduced by 50%
in CHO-K1 cells, which have a mixed context that is sensitive to both
the AF-1 and AF-2 regions of hER.
Glutathione-S-transferase pulldown assays demonstrate
that the AF-1 core is able and sufficient for the hER N-terminal
region to interact with SRC-1. Interestingly, an enhancement of this
recruitment in the presence of the hER ligand-binding domain was
observed, which was found to be dependent on a direct interaction
between the N-terminal B domain and the ligand-binding domain. Another
functional consequence of this physical interaction, which is promoted
by both partial and full agonists of hER, was an increase in the
phosphorylation state of the N-terminal domain. Binding of
4-hydroxytamoxifen (OHT) to the hER C-terminal region induced a
functional AF-1 conformation in vitro through this N-
and C-terminal interaction. The involvement of an SRC-1-mediated
pathway in transactivation mediated by hER AF-1 was further
substantiated by transfection experiments using the OHTresponsive human
C3 promoter, which showed that OHT-induced hER AF-1 activity was
enhanced by SRC-1 and required the AF-1 -helical structure. In
conclusion, we demonstrate that the synergism between AF-1 and AF-2 is
mediated in part by a cooperative recruitment of SRC-1 by both the AF-1
-helical core and AF-2 regions and that it is stabilized by a direct
interaction between the B and C-terminal domains. This interaction of
SRC-1 with the AF-1 -helical core is essential for both
E2- and OHT-induced ER activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß (NR3A1 and NR3A2, respectively)
(5). Both are ligand-inducible transcription factors
(6, 7). ERs belong to the highly diverse superfamily of
nuclear receptors (NRs), which are structurally organized into six
independent domains, termed A to F, each of which encodes specific
functions (8, 9, 10 ; Fig. 1A
).
The NR signature motif is the cysteine-rich, zinc-coordinated structure
of the C domain, which mediates binding to DNA (11, 12, 13).
The C-terminal region, also termed the ligand-binding domain (LBD), is
organized into 10–13 -helices (14, 15). ERs have two
major transcriptional regulatory functions, activation function 1
(AF-1) at the N terminus and AF-2 at the C terminus. AF-2 can be
further subdivided into AF-2 core and AF-2a transactivating regions
(16, 17, 18, 19 ; Fig. 1A). These AFs exhibit distinct
transactivation properties that depend on both cell and promoter
contexts (20, 21). The full transcriptional activity of
the ERs is thought to proceed through a synergism between these AFs
(11, 17, 18, 19, 20, 21).
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AF-1 CoAs have been identified to date,
the p68 and p72 RNA helicases (29, 30). However, recent
evidence indicates that some CoAs that are known to mediate AF-2
activity could also interact with the N-terminal region of ERs to
mediate AF-1 activity (Fig. 1A
). Indeed, both ER and ERß are able
to recruit SRC-1 or TIF-2 through their N-terminal regions
(31, 32, 33). Interactions involved in this process seem not
to require the CoA NR boxes, in contrast to CoA recruitment by AF-2
(Refs. 31 and 32 ; Fig. 1B). Moreover, in the
case of ERß, SRC-1 targeting is modulated by phosphorylation on
N-terminal serines that are involved in AF-1 regulation
(33). Thus, members of the CBP/p300, SRC-1, TIF-2 CoA
subclass are able to interact with both the N- and C-terminal regions
of ERs. This led several groups to propose the concept of AF-bridging
proteins, allowing for transcriptional synergism between the N- and
C-terminal AFs (31, 32, 33, 34, 35, 36, 37). For some NRs, such as the ARs and
PRs (NR3C4 and NR3C3, respectively), AF-bridging CoA recruitment might
be facilitated by the existence of direct contacts established between
the N- and C-terminal domains of these receptors
(38, 39, 40, 41, 42, 43).
We previously identified a conserved potential helix at the
beginning of the B domain of ER as the AF-1 functional core
(44 ; Fig. 1A). This putative -helix was required for
100% and for 30–50% of the AF-1 activity detected for the rainbow
trout (rt) and human (h) ER, respectively. The present study was
undertaken to determine if this -helix could participate in the
synergism between both of the ER AFs. Our data demonstrate that this
structure is necessary for ER total activity and that it is directly
involved in recruiting SRC-1 but not in recruiting the p68 RNA
helicase. Moreover, we show that the E2-induced functional synergism
between both ER AFs, which occurs in cells sensitive to both AFs,
relies on a direct physical interaction between the N- and C-terminal
regions that does not require the AF-1 core -helix. This process has
two consequences: first, phosphorylation of the N-terminal region can
take place, and second, cooperative recruitment of the SRC-1 CoA by the
AF-1 helical core and AF-2 can occur. Induction of AF-1 activity by the
selective agonist 4-hydroxytamoxifen (OHT) was also found to require
both the interaction of SRC-1 with the hER AF-1 helical core and the
C-terminal-mediated activation of AF-1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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AF-1 -Helical Core Is Required for SRC-1-Mediated
hER Transcriptional Activity AF-1 and AF-2 (31, 37). The identification
of a potential -helix in the ER AF-1 core (Ref. 44
and Fig. 1) led us to evaluate if this structure could be involved in
synergism between AF-1 and AF-2. Transient transfection experiments
were carried out by expressing hER, hER C to F domains [the hER
46-kDa isoform (45)], or hER point mutants in which
the AF-1 helical core region is rendered functionally inactive [hER
L39P or hER Y43P, because the mutation of the L39 or Y43 residue to
a proline destroys the -helical structure (44)]
together with SRC-1 or p68. Estrogen-responsive
element-thymidine kinase-luciferase (ERE-TK-Luc) was used as
an E2-responsive reporter gene. Transfections were performed in three
cell lines exhibiting differential sensitivity to ER AFs: HeLa
cells, which are predominantly sensitive to ER AF-2; HepG2 cells,
which are almost entirely sensitive to AF-1; and CHO-K1 cells, in which
ER transactivation is mediated by both the AF-1 and AF-2 regions
(18, 20, 44, 46). This experimental design was used to
discriminate between processes caused by either AF acting on its own
and those caused by the synergism between AF-1 and AF-2. Corresponding
histograms are shown in Fig. 2.
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-induced transactivation was
enhanced 2-fold by SRC-1 but not by the specific AF-1 CoA p68. This was
also observed with the hER CF mutant and both hER L39P and hER
Y43P mutants, although they lack either the whole N-terminal region or
the AF-1 -helix (Fig. 2A). These data reflect and confirm the
AF-2-sensitive context of HeLa cells. Conversely, hER
transactivation ability was increased approximately 2.5-fold by both
SRC-1 and p68 RNA helicase in HepG2 cells (Table 1). As expected in these cells, deletion
of the N-terminal region (hER CF) suppressed almost all hER
transactivation, and coexpression of p68 or SRC-1 with this mutant did
not increase hER transactivation (Fig. 2B). Similarly, replacement
of the L39 or Y43 residue with proline gave rise to a 2-fold weaker
induction of the reporter gene compared with that of wild-type hER.
The transactivation ability of these two hER point mutants, however,
was still enhanced by p68 but not by SRC-1. These results indicate that
p68 coactivation does not require the presence of a functional AF-1
-helical core structure. However, SRC-1-mediated enhancement of
hER activity absolutely requires this structure in an AF-1-sensitive
cell context. Moreover, the strongest induction of hER activity by
SRC-1 was observed in CHO-K1 cells, in which both hER AF-1 and AF-2
are functional. Indeed, as shown in Fig. 2C and Table 1, hER
transactivation was increased 5.6-fold by SRC-1, whereas an enhancement
of 2.4-fold was obtained with the AF-1-specific CoA p68 RNA helicase.
Deletion of the hER A and B domains (mutant hER CF) reduced by
2-fold the hER activity and totally abolished p68 coactivation.
SRC-1 functioned as a CoA for this mutant hER CF, but resulted in
only 2-fold induction. Finally, destruction of the AF-1 -helical
core structure in hER (hER L39P and hER Y43P) did not reduce
the p68 effect, although it decreased the SRC-1 enhancement of ER
activity 2-fold (Fig. 2C). These data clearly demonstrate that the AF-1
-helical core is absolutely required for SRC-1 to mediate ER AF-1
transcriptional activity in cells sensitive to AF-1 as well as in cells
in which synergism occurs between AF-1 and AF-2.
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-Helical Core Physically Interacts with SRC-1 but Not
with p68 RNA Helicase AF-1 activity results from a
physical interaction between the AF-1 -helical core region and
SRC-1, glutathione-S-transferase (GST) pull-down assays were
performed. The hER AF-1 -helical core or a point mutant version
that possesses no intrinsic transactivation ability, as assessed in
yeast by fusion to the Gal4DBD (Fig. 3A),
was fused to GST. The hER AB domains or B domain alone, containing
point mutations within the AF-1 -helix core or at residues S104,
S106, S118, and S167, known to be target sites for phosphorylation
events (47, 48, 49, 50), were also fused to the GST. These fusion
proteins were correctly expressed, as indicated by Coomassie blue
staining (Fig. 3B) of SDS-PAGE gels containing the purified fusion
proteins. Representative interactions of these GST fusion proteins with
the SRC-1 and p68 CoAs are shown in Fig. 3, C–F. As expected
(51), SRC-1 interacted with the C-terminal region of
hER in an E2-dependent manner, validating the pull-down conditions
(Fig. 3C). Likewise, consistent with previous studies (28, 29), SRC-1 and p68 were able to directly interact with the
hER B domain; moreover, the presence of the A domain did not affect
these interactions (Fig. 3, C and D). Whereas point mutations of the
phosphorylation target serine residues had no effect on the interaction
with SRC-1, substitution of S118 by alanine reduced contact with p68
(Fig. 3E). Strikingly, point mutations within the -helical core of
hER AF-1 in the B domain markedly diminished (8-fold) the
interaction of the B domain with SRC-1 but not with p68 (hER B L39P
and hER B Y43P; Fig. 3E).
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-helical structure in the
physical interaction between SRC-1 and the hER N-terminal domain,
GST pull-down experiments were performed with the wild-type or
mutant AF-1 -helix core region (Fig. 3B). Although p68 did not
interact with these GST fusion proteins (Fig. 3F,
bottom), SRC-1 was specifically retained on beads coupled to
the wild-type form of the AF-1 -helical core region (Fig. 3F, top). A point mutation in this sequence, which abolished
both structure and transactivation property (43 ; Fig. 3A),
also totally inhibited the SRC-1 interaction with the GST fusion
proteins. Therefore, these data demonstrate that the hER AF-1 core
region spatial organization is required for SRC-1 interaction.
SRC-1 Recruitment Is Enhanced When Both hER N- and C-Terminal
Domains Are Present
The SRC-1 protein was shown to mediate synergism between AF-1 and
AF-2 in cells sensitive to both hER AFs, such as CHO-K1 cells (Fig. 2C). Therefore, we determined whether this synergism could be
correlated with an increased recruitment of SRC-1 in the presence of
both the hER N- and C-terminal domains. Competition experiments in
GST pull-down assays were performed using as bait either hER AB (or
B) (Fig. 4A) or DF (Fig. 4B) domains
fused to GST. As anticipated, complexes were formed between hER AB
(or B) or DF fusion proteins with labeled SRC-1, and these were
competed by increasing amounts of AD or liganded CF regions,
respectively (Fig. 4, A and B, left). In the same way,
full-length hER was able to decrease N- and C-terminal interactions
with SRC-1 in the presence of estrogens (Fig. 4, A and B,
middle). In contrast, increasing the amounts of hER
regions complementary to those fused to GST enhanced the recruitment of
SRC-1 (Fig. 4, A and B, right). Similar experiments
performed with p68 RNA helicase showed that recruitment of this CoA by
the N-terminal region of hER was not affected by the presence of the
hER C-terminal domain (data not shown). Therefore, the presence of
both N- and C-terminal domains of hER results in a specific increase
in SRC-1 CoA recruitment, which might explain hER AF-1/AF-2
synergism obtained in cells in which both AFs are functional (Fig. 2
and Table 1).
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N- and C-Terminal Regions. First, SRC-1 protein complexed with one ER AF domain might
result in a higher affinity for the other AF domain. Second, additional
interactions involving surfaces different from those involved in the
initial recruitment of SRC-1 may also lead to an increase in SRC-1
binding. Such additional contacts might imply a direct interaction
between the N- and C-terminal regions of ER. To determine whether
such physical contacts between these modules of hER could occur,
in vivo experiments using yeast two-hybrid assays were
performed. However, a positive result in these assays could reflect
either the existence of a direct interaction or the occurrence of a
ternary complex composed of hER B and C-terminal domains bridged by
a third protein such as a CoA. Therefore, it was critical to ensure
that any functional interactions detected between ER N- and
C-terminal regions were not caused by the establishment of a
CoA-mediated bridge between the AF domains. We decided to use mutated
or deleted ER N-terminal regions that are unable to transactivate ER
element reporter constructs in yeast and thus incapable of interacting
with CoAs.
As illustrated in Fig. 5A, the AF-1
-helical core is not the only AF-1 CoA contacting region within
hER (30, 44), because hER 
1–44 and hER
1–38 L39P receptors exhibit residual AF-1 activity. However,
deletion of this structure within the rtER abolishes AF-1
transactivation in yeast (Fig. 5B). For this reason, the yeast
two-hybrid approach was used with rtER/Gal4AD (activation domain)
fusion proteins. Moreover, the fact that the rtER AF-1 -helical
core is able to replace the hER core (44) legitimizes
this comparative approach. Fusions of the rtER DF and rtER B
domains either with or without the AF-1 helical core (B and B8–17)
were generated (Fig. 5C). Y190 yeast cells were transformed with
different combinations of these constructs and, after auxotrophy
selection, treated for 4 h with ethanol (EtOH) as control,
10-8 M E2, or
10-5 M of the anti-estrogens OHT and
ICI164,384 (ICI). Interestingly, the
results indicated that a functional interaction between the N- and
C-terminal regions of rtER occurred whether or not the B domain
contained the AF-1 helical region (Fig. 5C). Moreover, this functional
association was enhanced 5-fold in the presence of full or partial
agonists (E2 or OHT, respectively), but not in the presence of
ICI164,384. Furthermore, we observed the
functional dimerization of two rtER C-terminal domains, which was
strictly ligand dependent, as anticipated (7, 52). These
two-hybrid data indicate that the ER B domain is able to interact
with the ER C-terminal region. This is most likely to occur without
a CoA bridge, because an ER B domain devoid of CoA interaction
surface could contact the C-terminal domain. Thus, the association
detected in the yeast two-hybrid experiments between the N- and
C-terminal regions of ER is probably the result of physical contact
between the two regions.
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C-Terminal Region Physically Interacts with the B Domain
and Induces Its Hyperphosphorylation that was disrupted in the presence of ligands (44).
Therefore, we wished to discriminate between interactions mediated by
the B domain and those involving the A domain. As illustrated in Fig. 6A, the hER B domain fused to the GST
was able to interact directly with the hER C-terminal (DF) region
under pull-down conditions. This interaction occurred in a
ligand-dependent manner, reproducing previous yeast two-hybrid results
(Refs. 34, 36 , and 37 and our unpublished
results). E2 and OHT but not ICI164,384 were able
to promote induction of this interaction, although a weak but
significant physical contact was also detected in the absence of
ligand. Furthermore, results obtained with the A domain fused to the
GST were in agreement with those previously published
(44). Finally, compared with the A or B domain alone, the
combined AB domains had better interaction with the hER C-terminal
region. Already significant in the absence of ligand, this association
was enhanced 3- to 4-fold in the presence of ligands, although
ICI164,384 actively repressed this hER N- and
C-terminal interaction. These data provide evidence that in the
full-length ER context, the interactions of the A and B domains with
ER C-terminal regions occur in an integrated manner. However, the
AF-1 -helical core region was not involved in these interactions
(Fig. 6B). Indeed, point mutations within the AF-1 helical structure
did not abrogate the hER N- and C-terminal interaction, and a GST
fusion protein that included only the AF-1 -helix was unable to
retain the C-terminal region of hER on the beads (Fig. 6C).
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N-terminal phosphorylation on
ligand binding to the C-terminal LBD could be through intramolecular
signaling between these two regions, as for PPAR (55).
Therefore, to unequivocally illustrate the occurrence and crucial
importance of this interaction in hER functions, we determined
whether the hER N- and C-terminal interaction had further functional
importance than the recruitment of SRC-1. We developed a new in
vitro phosphorylation assay in which protein complexes from GST
pull-down experiments were incubated with whole-cell extracts. The cell
extract was used to provide appropriate kinase activity(ies). As
illustrated on Fig. 6D, rabbit reticulocyte lysate had no effect on the
phosphorylation status of a GST/hER AB, GST/hER B, or GST/hER
BL39P fusion protein. However, the
ligand-dependent interaction of the hER C-terminal region with these
N-terminal domains increased the incorporation of
32P. Because this also occurred with GST/hER
BL39P, this increase in phosphorylation reflects
the physical interaction between the hER N- and C-terminal
regions.
Ligand Determinants in the Direct ER N- and C-Terminal Domain
Physical Interaction and Links to SRC-1-Mediated Synergism
Throughout our experiments, OHT was able to mimic E2 action in the
interaction between the N and C termini of hER in two ways: it
promoted dissociation of the A domain from the C-terminal region and
enhanced the interaction of the B domain with the C terminus. These
results, in conjunction with OHT being a partial hER agonist whose
activity depends on hER AF-1 (19, 20), led us to assess
the role of the interaction between the hER N and C termini in OHT
agonism. We first performed transient transfection experiments with the
human complement C3 promoter region (-307 to +58), a well
characterized OHTresponsive promoter (56, 57). As
shown in the response curves (Fig. 7A),
the maximum hER activity under our transfection conditions was
obtained with high doses of OHT (i.e. 5 x
10-6 M). Control
experiments using the ERE-TK-Luc reporter (Fig. 7B) demonstrate that
5 x 10-6 M OHT alone
cannot induce hER activity on this reporter construct, indicating
that the human C3 activation by OHT-liganded hER is specific.
Moreover, this dose was the most efficient one competing for E2-induced
hER activity (Fig. 7B). Therefore, the involvement of SRC-1 and the
N- and C-terminal interaction in AF-1 activation by OHT was determined
using these conditions and was evaluated in HeLa, HepG2, and CHO-K1
cell lines (Fig. 7, C, D, and E, respectively). Transient transfection
experiments were carried out using constructs expressing hER, hER
CF, or the hER L39P and hER Y43P point mutants, together with
SRC-1 or p68, which was used as a control. In HeLa cells, hER and
all mutants used were able to induce the human C3 (-307/+58)-Luc
reporter only when liganded to E2, demonstrating that OHT is unable to
activate AF-2 (20, 46). Furthermore, in the presence of
E2, only SRC-1 and not the specific AF-1 CoA p68 was able to coactivate
hER transcriptional activity, albeit to a lesser extent than on the
ERE-TK-Luc reporter (1.5-fold vs. 2-fold; Fig. 7C and Tables 1 and 2).
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activity on the human C3 (-307/+58)-Luc
reporter by 2-fold (Fig. 7D and Table 2). In the presence of OHT, SRC-1
but not p68 was able to coactivate wild-type hER protein activity,
again by 2-fold. This indicates that p68 is not involved in
AF-1-mediated activity when hER is complexed with this ligand.
SRC-1-mediated coactivation of OHT-liganded hER on this reporter
construct depends on AF-1, because neither SRC-1 nor p68 was able to
induce transactivation activity from hER CF protein in the presence
of OHT. Consistent with the data presented in Fig. 1, SRC-1 was unable
to increase the activity of the hER L39P and hER Y43P point
mutant proteins in HepG2 cells, illustrating the absolute requirement
of the AF-1 helical motif in transactivation by E2-liganded,
hER-mediated coactivation by SRC-1. This is a similar pattern of
coactivation to that obtained with the ERE-TK-Luc reporter, but we
demonstrate, for the first time, that the AF-1 helical core is also
required for AF-1 activation by OHT, because SRC-1 was unable to
enhance the activity of the two point mutant proteins when they were
liganded to OHT. As previously seen, CHO-K1 cells have a cell context
sensitive to both hER AFs. However, the pattern of activation
obtained in these cells was similar to that observed in HepG2 cells.
This demonstrates the predominance of AF-1-mediated transactivation in
hER activation of the human C3 promoter. From all of these results,
we conclude that the hER AF-1 helical core region is required for
the agonistic activities of OHT, which mostly rely on AF-1
(20). Moreover, this structure is necessary for SRC-1 to
enhance hER activity when it is bound to this molecule.
The hER C-terminal region, when associated with OHT, was shown to
exhibit an altered conformation that could not recruit p160 CoAs
(14, 58 ; Fig. 8A). Because
these transfection results could also reflect altered SRC-1 recruitment
by the C-terminal region, we performed, as described previously, GST
pull-down assays with increasing amounts of either hER full-length
or hER CF domains in the presence of OHT or
ICI164,384. Interestingly, increasing amounts of
OHT-liganded hER resulted in a decrease in the amount of SRC-1
retained by the GST/hER AB fusion protein (Fig. 8B, top).
However, increasing the amount of hER CF protein, either unliganded
(-OHT) or liganded to either of the two anti-estrogens (+OHT, +ICI),
did not affect the interaction of SRC-1 with the hER AB domain (Fig. 8B, bottom). These results are likely to indicate that the
spatial organization of the OHT-liganded hER C-terminal region is
unable to support a recruitment of SRC-1 (Fig. 8A) and is thus also
unable to cooperatively recruit this CoA through interaction with the
hER N- and C-terminal regions. More importantly, these experiments
demonstrate that within hER, OHT is able to induce a functional
conformation that favors SRC-1 recruitment, because full-length hER
is able to compete for this process. Furthermore, the fact that the
isolated hER C-terminal region is unable to reproduce this decrease
in SRC-1 interaction with the hER AB domain demonstrates that this
is caused by competition for SRC-1 recruitment to the same hER
region (i.e. the B domain). Thus, OHT binding in the context
of hER mimics E2 action by inducing an interaction between the B
domain and the C-terminal regions, which in turn allows the B domain to
recruit SRC-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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AF-1 core as a short segment that
has been conserved during evolution, likely organized as an -helix
and that is positioned within the beginning of the B domain (amino
acids 39–45 in hER) (Ref. 44 and Fig. 1A). Helical
structures are involved in many protein-protein contacts, including
those established between NR AF-2 regions and their coregulators
(23, 24, 25). In this study, we wished to characterize the
role of the AF-1 -helical structure in hER function, particularly
in the recruitment of p160 subfamily CoA proteins such as SRC-1. A
previous report had shown that the major surface of the hER
N-terminal region interacting with p160 lies between amino acids 38 and
64, thereby overlapping the AF-1 -helical sequence (31 ;
Fig. 1A). Transfection experiments in cell lines that exhibit
differential responses to hER AF-1 and AF-2 revealed that this
structure was absolutely required for an enhancement of hER AF-1
activity by SRC-1. Moreover, in cells responsive to both hER AF-1
and AF-2, such as CHO-K1 cells, we demonstrate that this AF-1 structure
is essential for synergism between hER AFs. Furthermore, this
structure was found to recruit SRC-1 and to be responsible for
approximately 70% in the interaction between SRC-1 and the hER
N-terminal region. The length and sequences of NR N-terminal regions
are highly divergent among individual AF-1 structures depending on the
receptor. Moreover, despite data describing secondary structures within
the N-terminal region of NRs, there may not be a general rule for all
of the superfamily members, because SF-1 and VDR possess very short AB
domains (59). In the case of GR, three -helices reside
within its 
1 core region (60), allowing interactions
with some TATA box protein- associated factors
(TAFIIs) and other general transcription
factors (61). AR also contains a structural unit within
its AF-1a that is organized around a ß-turn and an -helix
(62), as does the short AF-1 of HNF-4, which also has an
-helix (63). These structures could contact general
transcription factors (64, 65). However, this report
provides the first example to our knowledge of an interaction between
an AF-1 structure and a p160 CoA protein.
SRC-1, and more generally p160 members, use different regions to
interact with the N- and C-terminal regions of ER. EF domains make
contact through the NR boxes, whereas AB domains use the glutamine-rich
region (Refs. 31 and 32 and Fig. 1B). This
could signify that the interaction between the hER AF-1 helical core
and SRC-1 could require both structural and charge components.
Interestingly, the proposed AF-1 -helix contains positively charged
residues. However, disrupting the structure through proline
substitution abolished the interaction with SRC-1. This likely reflects
a critical role for the structure of the hER AF-1 core. In contrast
to SRC-1, p68 RNA helicase is not involved in a synergistic process
between hER AFs. This is likely to occur, because p68 is not
recruited to the hER N terminus through the hER AF-1 helical
structure but through a region encompassing the phosphorylated S118
residue (29). In consequence, we observed that the
recruitment of SRC-1 and p68 by hER N-terminal region is not
mutually exclusive (data not shown), indicating that these two proteins
may be recruited concomitantly.
hER, like other NRs, exhibits modular properties. Functions have
been ascribed to individual domains, but some data are now consistent
with a more flexible organization, because some functional aspects
require the entire receptor. Indeed, binding to DNA results in
allosteric control of NR function (66) and was recently
shown to affect N-terminal region structures in both PR-A and GR
(67, 68) or the overall ER helical content
(69). Such interrelations between different domains of NR
were considered possible between the N- and C-terminal regions of ER.
Indeed, in the absence of ligand, AF-1 ligand-independent activity is
repressed by C-terminal domains in full-length protein (6, 19, 70). The synergism between hER AFs was also ascribed to a
functional interrelation between these two regions of hER, and it
was proposed that this was mediated through CoA recruitment (36, 37). However, it was not clear if hER N- and C-terminal
domains were able to interact directly with each other. Indeed, a
bridging CoA, able to interact with both regions, was hypothesized to
be necessary for an indirect interaction between hER N and C
termini. However, pull-down competition assays unexpectedly indicated
that SRC-1 interaction with one region was enhanced by the other. This
observation did not reflect a bridging property of SRC-1, because in
this case either no changes in the amounts of retained SRC-1 or a
reduction in these amounts attributable to competition for labeled
SRC-1 should have occurred. This enhancement in SRC-1 recruitment
provided evidence for a third interaction surface. We then went on to
prove, using two different approaches, that a direct interaction
between the hER N- and C-terminal regions occurs and that this does
not require the AF-1 -helical core. This is similar to situations
already described for other NRs such as the PR and the AR
(38, 39, 40, 41, 42, 43). Our data provide evidence for a functional
importance of this direct interaction between the N- and C-terminal
regions of hER at two levels. First, a cooperative recruitment of
SRC-1 can occur, and second, hyperphosphorylation of the receptor
N-terminal region can occur. This could be a general rule for other
members of the NR superfamily, because N-terminal domain
phosphorylation of some NRs was shown to be modulated by their
C-terminal regions (54, 55, 71).
Our results are in sharp contrast with those of two recent reports,
which failed to detect any direct interaction between the hER N and
C termini. Consequently, bridging properties were ascribed to NCoA-2
and CBP/p300 (34, 35). These conflicting results may arise
from different conditions used in pull-down assays, particularly
because Kobayashi et al. (35) were unable to
find a recruitment of p160 proteins to hER or hERß N-terminal
domains, which has been demonstrated by several other groups
(31, 32, 33). These two reports were also compromised by
results obtained in a mammalian two-hybrid system, which is affected by
the endogenous expression of NCoA-2 and CBP/p300 proteins. Finding a
direct interaction between the N- and C-terminal domains would resolve
and simplify these indirect observations. We propose that the physical
interaction between isolated N- and C-terminal domains in the presence
of ligand occurs and results in an enhanced recruitment of endogenous
CoAs such as p160 or CBP/p300. The resulting transcriptional activation
using isolated hER N- and C-terminal domains in combination, as
previously reported (36), reflects AF-1/AF-2 synergism. In
the study by Benecke et al. (34), however, this
transcriptional activity was not observed, perhaps because they used a
less sensitive system. Because a significant promoter activation
required increasing amounts of p160 to be detected, these authors
suggested that the p160s acted as a bridging protein, because
detectable synergism between the hER N and C termini was observed
only when they were present. In the report by Kobayashi et
al. (35), transcriptional activity attributable to
interaction between the N and C termini was observed under basal
conditions, reproducing previous results (36, 37). We
propose that, in this case, the p160 coactivation of hER was
maximal, as a result of the saturation of transcription factors
involved in hER recruitment of the transcription machinery. This may
be attributable to the CBP/p300 integrator, which is limiting for
transcriptional activation in cells (72). Increasing the
amounts of available CBP/p300 by transfection would relieve this
process and allow greater stimulation of the reporter gene construct.
In this case, CBP/p300 was considered as a bridging factor between the
hER N- and C-terminal regions mediating synergism between AF-1 and
AF-2. Finally, our proposals imply that physical interaction between
the B and C-terminal domains could involve different regions than those
determined for the hER B domain interrelations with CBP/p300 and
p160s.
We also show that, in addition to interacting with the B domain, the
hER C-terminal region interacts with the A domain, and this physical
contact is reduced in the presence of agonists (44). A
complex network of interrelations occurs between hER N- and
C-terminal domains, because this interaction is also responsible in
part for the inhibition of the hER AF-1 in the absence of ligand.
Two contrasting effects can occur after the interaction of the AB
domain with the C-terminal part of hER, a suppression of activity
mediated by the A domain that occurs in absence of ligand, and the
ligand-induced synergism between AF-1 and AF-2 caused by the B domain.
These two mechanisms appear to be independent of each other because the
A domain has no influence on the cooperative recruitment of SRC-1 by
the B domain and the C-terminal region. This indicates that precise
molecular determinants discriminate between the influences of two
adjacent regions. The binding of hER agonists is central in this
process, because they are able to switch the negative effect of a
direct interaction (A domain/C-terminal region) to an interaction
between the B domain and the C-terminal region that results in
transactivation. Thus, it is interesting that a proline-rich stretch or
a glycine/alanine-rich sequence is present at the end of all known
ER A domains. These sequences may provide a hinge region between the
A and B domains and result in the structural involvement of this region
in the agonist-mediated activation of hER. Structural changes in
this hinge region geometry may cause a reorientation of hER AF-1
with AF-2, resulting in SRC-1-mediated synergism in the presence of
ligand.
One of the most interesting features of AF-1 is its involvement in OHT
agonism, because AF-1 seems to be the only hER AF to be activated
when hER binds OHT (19, 20). This is supported by our
observation of a strong correlation between OHT agonism and the ability
of this compound to mimic E2 action in the interaction between the
hER N- and C-terminal regions. Both E2 and OHT binding inhibit the
interaction of the C-terminal region with the A domain, thereby
promoting an interaction between C-terminal domains and the B domain.
We thus hypothesized that OHT-mediated activation of AF-1 might involve
an interaction between SRC-1 and the N- and C-terminal regions of
hER. Using the human C3 promoter (-307/+58 region) as an
OHT-responsive promoter (56, 57), we show that the
interaction of SRC-1 with the AF-1 helical core is required for an
enhancement of hER activity in the presence of OHT. This highlights
the importance of this structure in AF-1 function and in the
induction of transactivation by agonists and also the involvement of
SRC-1/N-terminal/C-terminal interrelations in OHT agonism.
Correspondingly, within the full-length receptor, AF-1 is a
ligand-induced transactivation domain. Binding of E2 or OHT to the LBD
results in an activation of the "far" N-terminal AF-1, likely as a
result of a direct interaction between these regions. OHT was unable to
promote a coactivation of hER activity by the p68 RNA helicase,
indicating that the B domain conformation and/or the S118
phosphorylation (29) is inadequate for such a process
within OHT-complexed hER. In vitro pull-down experiments
demonstrated that OHT is able to induce a specific conformation within
full-length hER, allowing AF-1 to function through the recruitment
of SRC-1. Conversely, in the presence of OHT, there was no cooperative
recruitment of SRC-1 by the hER B and C-terminal domains, resulting
in a 2-fold enhancement of AF-1 activity by this CoA in cells
responsive to AF-1. Therefore, it seems that although the C-terminal
domain interacts with the B domain in the presence of OHT, this
physical contact does not provide a conformation that can mediate a
synergistic effect of SRC-1 by both hER AFs, reflecting the
inability of SRC-1 to interact with the C-terminal region when hER
is complexed with OHT.
Our data show that E2- and OHT-bound hER are able to recruit SRC-1
mostly through the AF-1 helical core. This implies that the different
conformations induced by E2 and OHT result in the activation of AF-1
but that OHT acts solely on AF-1. This demonstrates that the activation
of AF-1 by partial or full agonists relies on common structural
features within the known crystal structures of the hER C-terminal
region when complexed with E2 or OHT. During the course of these
experiments, we unexpectedly found that OHT was able to induce hER
CF activity to 35% of the full-length protein activity in cell types
sensitive to AF-1, such as CHO-K1, HepG2, or yeast (data not shown).
However, this transactivation ability could not be related to the AF-2
core, which is unable to generate cofactor-binding surfaces upon
binding OHT (58). However, the AF-2a subdomain was
reported to function in AF-1-sensitive cells (18).
Therefore, we suggest that OHT is able to induce this AF-2a function in
such cells. This extends the potential agonist properties of OHT, which
is considered a strict antagonist of the C-terminal functions of hER
(73).
We provide evidence of a recruitment of SRC-1 to hER that depends on
the AF-1 helical core. Moreover, SRC-1 is cooperatively recruited to
full-length hER through AF-1 and AF-2, and this is caused by a
direct association of the B domain with the C-terminal region of
hER. A precise understanding of the stoichiometry of the
hER/SRC-1 complex is now required. It was recently shown that one
SRC-1-derived protein is complexed with each hER dimer
(74). As a consequence, the SRC-1 monomer interacting with
the hER C-terminal dimer could also interact with the hER B
domain, or the B domain could recruit another SRC-1 monomer. Other
CoAs, such as CBP/p300, which could be recruited to an hER/p160
subfamily CoA complex, were found to interact directly with both hER
AFs, but to another sequence/structure within the N-terminal domain.
This leads to an emerging concept in which multiple combinatorial
protein-protein interactions are required for full NR activity
(26). Other proteins, however, cannot be excluded, such as
the DRIP/TRAP complexes that may bridge the N- and C-terminal domains.
Indeed, one component (DRIP150) of these Mediator-like complexes was
shown to be recruited by GR 1, presumably through its potential
structures (75). Other CoA complexes not yet identified
may be required to fulfill hER AF-1 function and to dictate the cell
specificity of hER function. Indeed, although considerable advances
have been gained in understanding the mechanisms underlying hER AF-2
function by isolating many CoA complexes, no clear scheme is emerging
to describe the differential sensitivity of different cell types to
AF-1 or AF-2.
|
MATERIALS AND METHODS |
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|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
L39P, pSG5/hER Y43P,
pSG5/hER AD, YEPrtERS, YEPrtER CF,
YEPrtER8–17, YEPhER CF, YEPhER1–37, and
YEPhER1–37L39P vectors, the yeast reporter pLG178/3EREc
construct, and the plasmids encoding the GST/hA, hER35–47/Gal4DBD,
hER35–47L39P/Gal4DBD, and hER35–47Y43P/Gal4DBD fusion proteins
have been described (44, 76). The diverse Gal4DBD and
Gal4AD fusion proteins were obtained by subcloning the corresponding
inserts in either pAS2-1 or pACT-2 vectors (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. The GST/hER
DF protein was expressed using the pGEX2TK/hER282–595 plasmid, a
gift from Dr. B.S. Katzenellenbogen (77). GST/hER AB,
GST/hER B, GST/hER B L39P, GST/hER B Y43P, GST/hER B S118A,
GST/hER B S167A, and GST/hER B S104A,S106A were expressed using
pGEX2T (Pharmacia, Little Chalfont, Buckinghamshire, UK)
constructs containing the corresponding PCR inserts. The serine point
mutant used as a PCR matrix [hER S118A (HE457)] was a gift from
Prof. P. Chambon (78), whereas the hER S167A and hER
S104A,S106A were kindly provided by Dr. B. S. Katzenellenbogen
(47). The GST fusion proteins with the hER AF-1
-helical core sequence were obtained by subcloning double-stranded
oligonucleotides within the SmaI-EcoRI sites of
the pGEX2T vector. All of these fusions were verified by sequencing.
For transient expression in mammalian cells and in vitro
expression in rabbit reticulocyte lysate, we used the pSG5/hER and
pSG5/hER p46 (hER CF) vectors and the ERE-TK-Luc reporter gene,
which were gifts from Prof. F. Gannon (45). The human C3
(-307/+58)-Luc reporter was obtained by nested PCR on human genomic
DNA (CLONTECH Laboratories, Inc.) using
5'-AACTGGGGATGAGGTCCAAGACATC-3' and 5'-TCCATGGTGCTGGGACAGTGCAGGG-3' as
forward and reverse primers, respectively. These oligonucleotides were
designed as referred to the human complement C3 promoter sequence (Ref.
79 ; accession no. X62904). The second round of
amplification was performed with internal primers possessing a
SmaI or BglII restriction site in 5' and 3',
respectively. This SmaI-BglII insert was inserted
within the corresponding sites of the pGL2-Basic vector (Promega Corp., Madison, WI). Plasmids encoding the SRC-1 and p68 RNA
helicase CoAs were gifts from Dr. M. J. Tsai (51) and
Dr. S. Kato (29), respectively.
In Vitro Translation in Rabbit Reticulocyte
Lysate
Proteins were in vitro synthesized using the T7
RNA polymerase in the rabbit reticulocyte-coupled
transcription/translation kit (Promega Corp.), as
recommended by the manufacturer. Labeled protein expression was
monitored by estimating the relative amounts of protein on SDS-PAGE
gels. To study the ligand effects, lysates were submitted to EtOH, 50
µM E2 (Sigma, St.
Quentin-Fallavier, France), 500 µM OHT or
ICI164,384 (from Dr. A. Wakeling, ICI Pharmaceuticals, Macclesfield, Cheshire, UK) treatments for
1 h in TEG buffer (50 mM Tris-HCl, pH 7.4,
1.5 mM EDTA, 50 mM NaCl,
10% glycerol, 5 mM MgCl2,
and 1 mM dithiothreitol).
Host Strains
The bacteria strain used for subcloning and protein expression
was Escherichia coli DH5
[supE44lacU169 (Ø80
lacZM15) hsdR17 Rec A1 end A1 gyrA96
thi-1 relA1]. The yeast strains used in this study were Y190
(CLONTECH Laboratories, Inc.; MAT, ura 3-52,
his 3-200, ade 2-201, lys 2-801,
trp 1-901, leu 2-3, 112, gal 4,
gal 80, cyhr 2,
LYS2::GAL1UAS-HIS3TATA-HIS3,
URA3::GAL1UAS-GAL1TATA-
LacZ) and BJ2168 (Yeast Genetic Stock Center, Berkeley, CA;
a leu2 trp1 ura3-52 prb 1-1122 pep4-3 prc1-407 gal2) for the
transactivation assay. Yeast cells were transformed using a modified
lithium acetate method (76). Selection of stable
transformants and liquid ß-galactosidase assays were performed as
previously described (44, 76).
Bacterial Production of GST Fusion Proteins and GST Pull-Down
Assay
After 4 h of expression with 0.1 mM isopropyl
ß-D-thiogalactoside, E. coli DH5 cells were
harvested by centrifugation, resuspended in NETN buffer [100
mM NaCl, 20 mM Tris, pH 8,
1 mM EDTA, 0.5% Nonidet, 1
mM phenylmethylsulfonyl fluoride (PMSF), and the
protease inhibitors leupeptin, pepstatin, and aprotinin at a final
concentration of 10 µg/ml], and then lysed by sonication. Lysates
were clarified by centrifugation and immediately placed in contact with
a 50% suspension of glutathione-agarose beads (Sigma) in
NETN buffer. Incubation was performed overnight under rotation. Washed
fusion proteins bound to the beads were resuspended in binding buffer
(50 mM NaCl, 50 mM Tris, pH
8, 0.02% BSA, 0.02% Tween-20, 1 mM PMSF, and 10
µg/ml of the proteases inhibitors). For quantification, Bradford
assay was performed, together with analysis by SDS-PAGE,
allowing an evaluation of the stability of the fusion protein and the
amounts of protein recovered. GST fusion proteins (250 ng) bound to the
beads were incubated for 3 h with equal amounts of
[35S]methionine-labeled proteins (near
0.5–2 µl of the hormone- or EtOH-treated lysates) in the presence or
absence of 50 µM E2 and 500
µM OHT or ICI164,384 in a
total volume of 300 µl of binding buffer. Beads were collected by
centrifugation and washed five times in 400 µl of washing buffer 300
(WB300; 50 mM Tris, pH 8, 300
mM NaCl, 1 mM PMSF, 0.05%
Tween-20, and 10 µg/ml of the protease inhibitors). Washed beads were
denatured at 100 C for 5 min, and the supernatant was subsequently
submitted to SDS-PAGE. 35S-Labeled proteins were
visualized after autoradiography using Kodak (Rochester,
NY) BioMax films. All experiments were performed at least three
times.
Cell Culture, Transient Transfection Experiments, and Whole-Cell
Extracts
CHO-K1 and HeLa cells were routinely maintained in DMEM-F12
(Sigma) supplemented with 5% FCS (Sigma),
whereas HepG2 cells were grown in DMEM/5% FCS. All of the cell lines
were cultured at 37 C and 5% CO2, and all media
contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml
Amphotericin (Sigma). One day before
transfection, cells were dispatched in 6-well plates in the case of
HeLa and HepG2 cell lines and 24-well plates for CHO-K1 cells. The
three cell lines were transfected at 60–70% confluence with a classic
calcium phosphate/DNA precipitation protocol after replacing the media
1 h before transfection with DMEM-F12/4% charcoal/dextran-treated
(desteroided) FCS. Transfection in 6-well plates was performed using 3
µg of total DNA per well and containing 250 ng of expression vector,
500 ng of ERE-TK-Luc or human C3 (-307/+58)-Luc reporter plasmids, and
1.5 µg of the internal control pCH110. For CHO-K1 cells, 1 µg of
total DNA per well was used, containing 25 ng of expression vector, 50
ng of ERE-TK-Luc or human C3 (-307/+58)-Luc reporter, and 150 ng of
pCH110. In all cases, the total amount of DNA was maintained constant
by adding Bluescript plasmid. Cells were kept in contact with the
precipitate overnight at 37 C with 2% CO2,
washed once with PBS, and replaced in fresh DMEM-F12/8% desteroided
FCS medium supplemented with EtOH, or increasing concentrations of E2
or OHT, or a mix of 10-8 M E2 and
increasing concentrations of OHT. After 36 h of transient
expression, cells were harvested and 10% of the cellular extract was
used to measure luciferase activity. Half of the remaining extract was
used for the ß-galactosidase assay. Luciferase activities were
normalized for transfection efficiency with the ß-galactosidase
activity and expressed as the E2 fold induction vs. the
activity obtained with the promoter alone. A value of 100% was
ascribed to the hER activity. All transfections were performed in
triplicate at least three times. Whole-cell extracts from untransfected
cells were obtained by harvesting confluent cells, and lysis in
whole-cell extract buffer (20 mM HEPES, pH 7.9,
400 mM KCl, 2 mM
dithiothreitol, and 20% glycerol) was obtained by three
thawing/freezing cycles performed at -80 and 37 C. After lysis,
soluble proteins were recovered in the supernatant by
centrifugation.
In Vitro Phosphorylation
Two hundred fifty nanograms of purified bacterially expressed
GST/hER AB or B (wild type or point mutant) fusion proteins linked
to glutathione-agarose beads were incubated for 3 h with 1.5 µl
of in vitro labeled and E2- or EtOH-treated hER DF. Beads
were then washed four times in pull-down WB300 before being resuspended
in binding buffer and submitted to the phosphorylation reaction. This
was performed for 30 min at 30 C with 10 µCi of
[
-32P]ATP in phosphorylation buffer [40
mM Tris-HCl, pH 7.4, 50 mM
MgCl2, 5 mM dithiothreitol,
and 250 µg ATP plus phosphatase inhibitors (1
µM okadaic acid and 200
µM sodium orthovanadate)] with 10 µg
of whole-cell extract. The total volume of whole-cell extract buffer
was maintained constant, and the final reaction volume was 50 µl.
Beads were then washed two times in WB300, resuspended in 15 µl of
SDS-PAGE loading buffer, boiled for 5 min, and centrifuged, and the
supernatant was fractionated on SDS-PAGE gels. After Coomassie blue
staining and drying, gels were submitted to autoradiography for 4–16 h
with Kodak BioMax films. Experiments were carried out at
least two times.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Current address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany. 
Abbreviations: AF, Activation function; CBP, CEBP binding
protein; CEBP, cAMP response element binding protein; CoA, coactivator;
ERE-TK-Luc, estrogen-responsive element-thymidine kinase-luciferase;
EtOH, ethanol; GST, glutathione-S-transferase; hER,
human ER; LBD, ligand-binding domain; NR, nuclear receptor; OHT,
4-hydroxytamoxifen; PMSF, phenylmethylsulfonyl fluoride; rtER,
rainbow trout ER; SRC-1, steroid receptor coactivator protein-1;
TAFIIs, TATA box binding protein-associated factors;
TIF-2, transcriptional intermediary factor 2; WB300, washing buffer
300.
Received for publication January 22, 2001. Accepted for publication August 1, 2001.
|
REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Q. Wu, R. Burghardt, and S. Safe Vitamin D-interacting Protein 205 (DRIP205) Coactivation of Estrogen Receptor {alpha} (ER{alpha}) Involves Multiple Domains of Both Proteins J. Biol. Chem., December 17, 2004; 279(51): 53602 - 53612. [Abstract] [Full Text] [PDF]
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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]
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T. L. Ramsey, K. E. Risinger, S. C. Jernigan, K. A. Mattingly, and C. M. Klinge Estrogen Receptor {beta} Isoforms Exhibit Differences in Ligand-Activated Transcriptional Activity in an Estrogen Response Element Sequence-Dependent Manner Endocrinology, January 1, 2004; 145(1): 149 - 160. [Abstract] [Full Text] [PDF]
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M. Dutertre and C. L. Smith Ligand-Independent Interactions of p160/Steroid Receptor Coactivators and CREB-Binding Protein (CBP) with Estrogen Receptor-{alpha}: Regulation by Phosphorylation Sites in the A/B Region Depends on Other Receptor Domains Mol. Endocrinol., July 1, 2003; 17(7): 1296 - 1314. [Abstract] [Full Text] [PDF]
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K. D. S. A. Wansa, J. M. Harris, G. Yan, P. Ordentlich, and G. E. O. Muscat The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine J. Biol. Chem., June 27, 2003; 278(27): 24776 - 24790. [Abstract] [Full Text] [PDF]
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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]
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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]
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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]
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K. D. S. A. Wansa, J. M. Harris, and G. E. O. Muscat The Activation Function-1 Domain of Nur77/NR4A1 Mediates Trans-activation, Cell Specificity, and Coactivator Recruitment J. Biol. Chem., August 30, 2002; 277(36): 33001 - 33011. [Abstract] [Full Text] [PDF]
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G. Flouriot, H. Brand, B. Seraphin, and F. Gannon Natural Trans-spliced mRNAs Are Generated from the Human Estrogen Receptor-alpha (hERalpha ) Gene J. Biol. Chem., July 12, 2002; 277(29): 26244 - 26251. [Abstract] [Full Text] [PDF]
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