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)
Cor A. Berrevoets1,
Paul Doesburg1,
Karine Steketee,
Jan Trapman and
Albert O. Brinkmann
Departments of Endocrinology and Reproduction (C.A.B., A.O.B.) and
Pathology (P.D., K.S., J.T.) Erasmus University 3000 DR
Rotterdam, The Netherlands
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ABSTRACT
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Previous studies in yeast and mammalian cells
showed a functional interaction between the amino-terminal domain
and the carboxy-terminal, ligand-binding domain (LBD) of the human
androgen receptor (AR). In the present study, the AR subdomains
involved in this in vivo interaction were determined in
more detail. Cotransfection experiments in Chinese hamster ovary (CHO)
cells and two-hybrid experiments in yeast revealed that two regions in
the NH2-terminal domain are involved in the
functional interaction with the LBD: an interacting domain at the very
NH2 terminus, located between amino acid
residues 3 and 36, and a second domain, essential for transactivation,
located between residues 370 and 494. Substitution of glutamic acid by
glutamine at position 888 (E888Q) in the AF-2 activation domain (AD)
core region in the LBD, markedly decreased the interaction with the
NH2-terminal domain. This mutation neither
influenced hormone binding nor LBD homodimerization, suggesting a role
of the AF-2 AD core region in the functional interaction between the
NH2-terminal domain and the LBD. The AF-2 AD
core region was also involved in the interaction with the coactivator
TIF2 (transcriptional intermediary factor 2), as the E888Q mutation
decreased the stimulatory effect of TIF2 on AR AF-2 activity.
Cotransfection of TIF2 and the AR NH2-terminal
domain expression vectors did not result in synergy between both
factors in the induction of AR AF-2 activity. TIF2 highly induced AR
AF-2 activity on a complex promoter [mouse mammary tumor virus
(MMTV)], but it was hardly active on a minimal promoter (GRE-TATA). In
contrast, the AR NH2-terminal domain induced AR
AF-2 activity on both promoter constructs. These data indicate that
both the AR NH2-terminal domain and the
coactivator TIF2 functionally interact, either directly or indirectly,
with the AF-2 AD core region in the AR-LBD, but the level of
transcriptional response induced by TIF2 depends on the promoter
context.
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INTRODUCTION
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The human androgen receptor (hAR) mediates physiological effects
of testosterone and dihydrotestosterone (DHT), which are essential for
development and functional maintenance of male reproductive and
accessory sex tissues. As a ligand-dependent transcription factor, its
structural organization shows a high level of molecular identity to
other members of the nuclear receptor superfamily. For these receptors,
separate functional domains have been characterized. The variable
NH2-terminal domain is involved in transcription activation
and contains the transactivation function AF-1 (see Ref. 1 for review).
The centrally located, highly conserved DNA-binding domain (DBD)
mediates the interaction with hormone-response elements on the
DNA (2). The carboxy-terminal (C-terminal) region contains the ligand
binding domain (LBD), which is involved in receptor dimerization
(3, 4, 5, 6), and can functionally interact with transcriptional intermediary
factors (TIFs) (for review see Ref. 7).
For the hAR, two separate transcription activation units (TAUs) in the
NH2-terminal domain were defined (8). One of these
transactivation units [TAU-1; amino acids (aa) 100370] is only
active in the full-length ligand-activated AR. In a truncated hAR,
which lacks the LBD, another region (TAU-5, aa 360485) functions as a
constitutively active transactivation domain. The role of TAU-5 in the
full-length AR is not yet clear.
The LBDs of various nuclear receptors contain a ligand-dependent
transactivation function, AF-2 (9, 10, 11). An autonomous activating domain
(AD) in this AF-2 region, AF-2 AD, is conserved among many nuclear
receptors and is located in the C-terminal part of the LBD. A core
region in the AF-2 AD,
-helix 12, appeared to be important for
transcriptional activity (9, 10, 12, 13) and the hormone-dependent
interaction with TIFs. These TIFs or coactivators can modulate the
transcriptional activity of a broad range of nuclear receptors
(14, 15, 16, 17). Mutations in the AF-2 AD core abolish the in vitro
association of the receptor with these coactivators. The recent finding
that a coactivator displays histone acetyltransferase activity
has provided further insights into the molecular events occurring at
the chromatin level during transcription activation (18). Wurtz
et al. (19) proposed a general mechanism for nuclear
receptor activation, in which the AF-2 AD core, present in helix 12,
plays a central role in the generation of an interaction surface,
allowing binding of TIFs to the LBD. The functional role of the AF-2 AD
core in the AR is not yet well understood.
Recently, for the estrogen receptor (ER) a functional,
ligand-dependent, in vivo association between the
NH2-terminal domain and the LBD was described (20). Also
for the AR a functional in vivo interaction between the
NH2-terminal domain and the LBD was demonstrated (21, 22).
In the study presented here, we determined in more detail AR subdomains
involved in the functional interaction of the LBD with the
NH2-terminal domain. The data indicated that two
NH2-terminal regions, together with the AF-2 AD core region
in the LBD, are important for a functional in vivo
interaction. Induction of AR AF-2 activity by the transcriptional
coactivator TIF2 also required an intact AF-2 AD core. This enhancement
of AR AF-2 activity by TIF2 appeared to be promoter dependent.
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RESULTS
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Two Regions in the AR NH2-Terminal Domain
Are Involved in the Functional Interaction with the AR-LBD
Studies in CHO Cells
A functional in vivo association between the
NH2-terminal domain and LBD of the hAR was found previously
(22). Experiments performed in yeast and mammalian cells showed that
this interaction was hormone dependent and could be blocked by
antiandrogens. The present study focused on AR subdomains involved in
this NH2-terminal domain/LBD protein-protein
interaction.
To determine regions in the NH2-terminal domain involved in
the functional interaction with the AR-LBD, different deletion mutants
of the NH2-terminal domain were constructed, as shown in
Fig. 1A
(AR.N1-AR.N7). The deletion
mutants were transiently transfected into CHO cells, and the
appropriate expression of the proteins was assessed by immunoaffinity
purification and Western blot analysis of cytosolic fractions (Fig. 1B
, lanes 17). Constructs (AR.N1- AR.N6) were expressed as proteins of
expected molecular mass and at comparable expression levels. The
expression level of AR.N7 could not be determined because this protein
lacks an epitope for the available antibodies.

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Figure 1. Structure and Protein Expression Patterns of AR
Mutants Used in the Transfection Studies in CHO Cells
A, The full-length hAR is shown with its NH2-terminal
domain (N-TERM), DBD, and LBD. The total number of amino acid residues
in the full-length hAR is 910 and is based on amino acid stretches of
20 Gln and 16 Gly residues. Synthetic peptide sequences in the
AR to which antibodies were raised and that were used in this study are
depicted (SP). The various NH2-terminal constructs (AR.N1
to AR.N7) and C-terminal constructs [AR.C and AR.C(EQ)] are shown. B,
The AR proteins were expressed in CHO cells and immunopurified from
cytosolic fractions, as described in Materials and
Methods. The proteins were visualized by immunoblotting with
polyclonal antibodies against SP197 (lanes 14), SP061 (lanes 57),
or SP066 (lanes 810). The molecular mass marker values are indicated
on the left of each blot.
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The ability of these mutants to induce transcription activation through
interaction with the AR-LBD (Fig. 1A
; AR.C), using mouse mammary tumor
virus (MMTV)-LUC as a reporter gene, is shown in Fig. 2
. As shown previously (22), neither
AR.N1 nor AR.C could, when expressed separately in CHO cells, activate
transcription from the reporter in response to androgens. The lack of
activity of AR.C is not due to the experimental setup, because a
similar glucocorticoid receptor (GR) fragment, GR.C (aa 369777),
highly induced luciferase activity in CHO cells (24-fold) upon addition
of 10 nM dexamethasone (data not shown). Coexpression of
AR.N1 and AR.C resulted in an androgen-dependent induction (18-fold) of
luciferase expression (Fig. 2
; AR.N1), which is comparable to
full-length AR activity. Deletion mutants AR.N2 and AR.N3 showed
similar responses as seen with the intact NH2-terminal
domain. However, deletion of amino acid residues 371494 markedly
reduced the androgen-induced LUC-activity (AR.N4). Deletions in the
first part of the NH2-terminal domain (AR.N5, AR.N6, AR.N7)
almost completely abolished the R1881-mediated response. These data
indicate that, for a functional interaction of the
NH2-terminal domain with the LBD of the AR in CHO cells,
amino acid residues 151 and 371494 are important. This suggests
that the TAU-5 domain, located in the latter region, is involved in
this functional in vivo interaction. The region between
residues 51 and 360, harboring TAU-1, appears not to be involved.

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Figure 2. Transcriptional Activities of AR
NH2-Terminal Deletion Mutants Cotransfected with AR.C in
CHO Cells
Transcriptional activities were determined by transfecting full-length
AR (3 ng/well) or by cotransfection of AR.C (50 ng/well) with each of
the NH2-terminal constructs (100 ng/well), together with a
MMTV-LUC reporter plasmid (200 ng/well), into CHO cells. Cells were
incubated either with vehicle (open bars) or with 10
nM R1881 (closed bars). Luciferase activity
was determined as described in Materials and Methods.
The interexperimental variation of the mean maximal luciferase
activities ranged between 2,000 and 9,000 light units. For each
experiment the mean AR.N1 activity was set at 100%, and all individual
points were calculated relative to this value. Each bar
represents the mean (± SEM) luciferase activity for three
experiments. Fold induction is shown at the top of each
bar and represents the ratio of activity determined in the
presence or absence of R1881.
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Studies in Yeast
To extend the CHO cell experiments, functional NH2-terminal
domain/LBD interactions were studied in yeast, using a two-hybrid
system. The high sensitivity of the yeast two-hybrid assay made it also
possible to detect weak associations. Furthermore, because of the
presence of the Gal4 transactivation domain, the in vivo
interaction does not solely depend on an intact AR-transactivating
domain.
Truncated forms of the AR NH2-terminal domain were fused to
the transactivation domain of GAL4 (Gal4-TAD), in the high-expression
vector pACT2 (Fig. 3A
). AR-LBD was fused
to the DBD of GAL4 (Gal4-DBD). Figure 3B
shows the protein expression
in yeast Y190 cells, as assessed by immunoblot analysis using specific
antibodies directed against either the AR (lanes 14) or Gal4-TAD
(lanes 510). The immunoblot shows expression of most fusion proteins
of the appropriate length. However, the expression of GalAD-AR.N9 (lane
2) appeared to be somewhat lower. GalAD-AR.N8 became visible only at an
extended exposure time (lane 1a), indicating a very low protein
expression level.

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Figure 3. Structure and Expression Patterns of AR-Gal4 Fusion
Proteins Used in the Two-Hybrid Studies in Yeast
A, Shown are AR NH2-terminal mutants fused to the Gal4
transactivating domain (TAD) and the LBD fused to the Gal4 DBD. B, The
appropriate expression of the AR constructs in yeast was determined by
immunoblotting of lysates of transformed S. cerevisiae
Y190 cells with specific antibodies against the AR (SP197, lanes 14
and lanes 1116) or against Gal4-TAD (lanes 510). Lane 1a represents
GalAD-AR.N8 at a 5-fold longer exposure time of the immunoblot during
chemiluminescence detection. The molecular mass marker values are
indicated on the left side of each blot.
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Activation of the integrated UASGAL1-lacZ reporter gene,
indicative for the functional interaction of GalAD-AR.N with the
promoter-bound GalDBD-AR.C, was assessed in a ß-galactosidase
(ß-GAL) assay (Fig. 4A
). The observed
reporter gene activity for GalAD-AR.N8 was lower as compared with
GalAD-AR.N9, GalAD-AR.N10, and GalAD-AR.N12, most likely due to the
very low protein expression level of GalAD-AR.N8. The constructs
showing a high ß-GAL activity (GalAD-AR.N8, -N9, -N10, and -N12) all
share one common region, spanning residues 336. Deletion of this
region from constructs GalAD-AR.N12 and GalAD-AR.N15, resulting in
constructs GalAD-AR.N13 and GalAD-AR.N16, respectively, caused an
intense drop in ß-GAL activity. Therefore, in accordance with the CHO
results, the data imply an important role of NH2-terminal
amino acid residues 336 in the functional interaction with the
AR-LBD.

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Figure 4. Two-Hybrid Assay of AR NH2-Terminal
Deletion Mutants and GalDBD-AR.C in Yeast
Yeast Y190 cells, containing the integrated UASGal1-lacZ
reporter gene, were transformed with the expression plasmid
GalDBD-AR.C, together with each of the Gal4-TAD fusion proteins
(GalAD-AR.N8 to GalAD-AR.N16) (A), or with the AR proteins without the
Gal4-TAD part (AR.N8 to AR.N12 and AR.N15) (B). ß-GAL activity was
determined after incubation of the cells in the absence (open
bars, not visible) or presence (solid bars) of 1
µM DHT. Fold induction is shown at the top of each
bar and represents the ratio of activity determined in the
presence and absence of DHT. ß-GAL values represent the mean
(±SEM) of three experiments.
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Mutant GalAD-AR.N11, missing residues 245494, was reduced in its
activity as compared with the intact NH2-terminal domain. A
decrease in activity was also seen when residues 330494 were deleted
from GalAD-AR.N12, resulting in GalAD-AR.N15. This implies that a
second region, between residues 244 and 494, is involved in the
functional interaction with the LBD. More detailed mapping within this
region did not clearly reveal distinctive interaction sites, because
neither deletion of residues 245360 (GalAD-AR.N9), nor deletion of
residues 371494 (GalAD-AR.N10), resulted in a distinct decrease of
ß-GAL activity. However, interpretation of these data is complicated
because of the difference in expression levels of the different
constructs (Fig. 3B
).
As reported previously (22), the measured ß-GAL activities are partly
due to Gal4-TAD transactivity, but could also be influenced by the
intrinsic transactivating activity of the AR NH2-terminal
domain. The contribution of the intrinsic AR activity to the total
measured ß-GAL activity could vary between different constructs and
thereby affect the mapping of interacting regions. To discriminate
between AR transactivity and AR interaction potential, the Gal4-TAD
part was deleted from these GalAD-AR.N constructs that showed a
considerable interaction with GalDBD-AR.C (as depicted in Fig. 4A
).
This resulted in constructs AR.N8-AR.N12 and AR.N15. Protein expression
of these constructs, as determined by immunoblot analysis, is shown in
Fig. 3B
(lanes 1116).
The activities of the AR.N constructs, in the two-hybrid assay with
GalDBD-AR.C, are shown in Fig. 4B
. AR.N11 and AR.N15 did not induce a
functional interaction, although the corresponding Gal4-TAD chimeric
constructs, as shown in Fig. 4A
, were active. This indicates that
AR.N11 and AR.N15 only contain interaction potential and not
transactivity. These constructs have the 336 fragment in common.
Deletion of the region between residues 244 and 360 (AR.N9) did not
reduce ß-GAL activity; AR.N12 (missing residues 37243) also showed
a clear response. This indicates that both these proteins still have
transactivating activity and interaction potential. However, by
deleting residues 371494 (AR.N10), ß-GAL activity dropped by
approximately 80%, a decrease comparable to the data obtained in CHO
cells (Fig. 2
; AR.N4). These results narrow the second interacting
region (aa 244494) to involvement of residues 371494 as a
major transactivation domain in the functional interaction with the
LBD.
Taken together, the yeast two-hybrid data imply a prominent role of
amino acid residues 336 of the AR NH2-terminal domain in
the interaction with the LBD. A second region seems to be mainly
constrained to residues 371494 and further supports the involvement
of TAU-5, as a transactivating region, in the functional in
vivo interaction with the LBD.
A Mutation in the LBD Affects the Functional Interaction with the
NH2-Terminal Domain, but not LBD
Dimerization
To determine regions in the AR-LBD involved in the functional
interaction with the NH2-terminal domain, mutational
analysis was constrained to the conserved amphipathic helix 12 in the
LBD. This helix constitutes the AF-2 activation domain (AD) core
region, of which the charged residues might generate a protein-protein
interacting surface (19). A single amino acid substitution in the AF-2
AD core was introduced: glutamic acid at position 888, which is highly
conserved among nuclear receptors, was replaced by glutamine
(E888Q).
Both the wild-type and the mutated LBD construct (Fig. 1A
; AR.C and
AR.C(EQ), respectively) were transfected into CHO cells. The
appropriate expression of both proteins is shown in Fig. 1B
(lanes 8
and 9). To determine whether the AF-2 AD mutation had an effect on
hormone binding, an in vivo androgen binding assay in
transfected CHO cells was performed. Scatchard analysis of the data
revealed that both proteins had similar Kd values for
R1881: AR.C, 0.37 nM, and AR.C(EQ), 0.33 nM,
respectively, indicating that the E888Q mutation did not affect hormone
binding.
The effect of the E888Q mutation on the functional in vivo
interaction of the LBD with the NH2-terminal domain was
investigated by cotransfecting AR.N1 with increasing amounts of either
AR.C or AR.C(EQ) and the MMTV-LUC reporter into CHO cells (Fig. 5A
). As expected, no response to 1
nM R1881 was observed in the absence of the LBD constructs.
Coexpression of AR.C and AR.N1 resulted in a high induction (to
24-fold) of luciferase activity. The E888Q mutation strongly reduced
the response to R1881 (to maximally a 7-fold induction) as compared
with the wild-type LBD.

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Figure 5. Differential Effects of the AF-2 AD Core Mutation
E888Q on Functional in Vivo NH2-Terminal
Domain/LBD and LBD/LBD Interactions
A, CHO cells were transfected with AR.N1 (100 ng/well) and increasing
amounts of AR.C or AR.C(EQ) together with the MMTV-LUC reporter
plasmid. Cells were incubated with vehicle (open bars)
or 1 nM R1881 (solid bars). LUC activity was
determined as described in Materials and Methods. The
interexperimental variation of the mean maximal luciferase activities
ranged between 8,000 and 13,000 light units. For each experiment the
mean AR.C activity (5 ng/well) was set at 100%, and all individual
points were calculated relatively to this value. Each
bar represents the mean (±SEM) luciferase
activity for three experiments. Fold induction represents the ratio of
activity determined in the presence and absence of R1881. B, Yeast Y190
cells, containing the integrated UASGal1-lacZ reporter
gene, were transformed with the expression plasmid GalAD-AR.N8 together
with GalDBD-AR.C or GalDBD-AR.C(EQ). ß-GAL activity was
determined after incubation of the cells in the absence (open
bars) or presence (solid bars) of 1
µM DHT. Fold induction represents the ratio of activity
determined in the presence and absence of DHT. ß-GAL values represent
the mean (±SEM) of three experiments. C, Yeast Y190 cells,
containing the integrated UASGal1-lacZ reporter gene, were
transformed with GalDBD-AR.C (low) and GalAD-AR.C (high) or
GalDBD-AR.C(EQ) (low) and GalAD-AR.C(EQ) (high). ß-GAL activity was
determined as described in panel B.
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The effect of the E888Q mutation on the functional interaction
between the two AR domains was also studied in yeast, using the
two-hybrid system. Both C-terminal domains (wild-type and mutant),
fused to Gal4-DBD, were transformed together with GalAD-AR.N8 into Y190
(Fig. 5B
; NH2/LBD). The effect of the E888Q mutation
appeared to be even more pronounced than in CHO cells, as ß-GAL
activity measured for the interaction of both AR domains (set at 100%
for the intact LBD) dropped more than 90% upon introduction of the
E888Q mutation. The data from CHO cells and from yeast indicate that
the AF-2 AD core region in the AR-LBD is important for a functional
interaction with the NH2-terminal domain.
Previously, homodimerization of the AR-LBD was demonstrated in yeast
(22, 23). To determine whether the E888Q mutation had an effect on LBD
dimerization, both LBDs (wild-type and mutant) were coupled to Gal4-TAD
and transformed together with the corresponding LBD domains coupled to
Gal4-DBD (Fig. 5C
). In this LBD/LBD interaction assay, the wild-type
AR.C and the mutant AR.C(EQ) showed similar ß-GAL levels, indicating
that the E888Q mutation had no effect on LBD dimerization.
TIF2-Induced Activity of AR AF-2 in CHO Cells Is Affected by the
E888Q Mutation
The functional role of the AR AF-2 AD core region was further
characterized by comparing the functional in vivo
interaction of the AR-LBD and the AR NH2-terminal domain
with the interaction of the AR-LBD and the coactivator TIF2. The
coactivator TIF2 is able to enhance the AF-2 activity of several
steroid receptors including the AR (17). The involvement of the AR AF-2
AD core region in the functional in vivo interaction of TIF2
and the AR-LBD was investigated by examining the effect of the E888Q
mutation.
TIF2 was cotransfected with the MMTV-LUC reporter and increasing
amounts of the wild-type (AR.C) or the mutant LBD (AR.C(EQ)) into CHO
cells (Fig. 6
). In the absence of the LBD
constructs, no response to 1 nM R1881 was observed. As
expected, coexpression of TIF2 and AR.C highly induced luciferase
activity (up to 26-fold). The E888Q mutation reduced luciferase
activity by approximately 70%, compared with AR.C. This decrease in
functional interaction of AR-LBD with TIF2, caused by the E888Q
mutation, was comparable to the negative effect of the mutation on the
interaction with the NH2-terminal domain (as shown in Fig. 5A
).

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Figure 6. E888Q Mutation Affects TIF2-Enhanced Activation of
AR.C in CHO Cells
TIF2 (100 ng/well) was cotransfected into CHO cells with increasing
amounts of AR.C or AR.C(EQ) and MMTV-LUC as a reporter. Cells were
incubated with vehicle (open bars) or 1 nM
R1881 (solid bars). LUC activity was determined as
described in Materials and Methods. The
interexperimental variation of the mean maximal luciferase activities
ranged between 2,800 and 5,900 light units. For each experiment the
mean AR.C activity (15 ng/well) was set at 100%, and all individual
points were calculated relative to this value. Each bar
represents the mean (±SEM) luciferase activity of three
experiments. Fold induction represents the ratio of activity determined
in the presence and absence of R1881.
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TIF2 Does Not Promote the Functional Interaction between the AR
NH2-Terminal Domain and the LBD
To determine whether TIF2 enhances the functional interaction
between the NH2-terminal and C-terminal domains of the AR,
coexpression studies with different concentrations of TIF2 and the
NH2-terminal domain were performed (Fig. 7
). The luciferase activities obtained
were compared with the transactivating activities measured after
separate transfection of the different plasmids. Individual expression
of TIF2 and the NH2-terminal domain induced the
transcriptional activity of AR.C on the MMTV promoter up to 24-fold
(panels a and d, respectively). When coexpressed with 50 ng/well AR.N1
(panel b), TIF2 slightly enhanced the luciferase activity induced by
the functional interaction of AR.N1 and AR.C (up to 34-fold). When
higher amounts of AR.N1 were transfected, no additional effects of TIF2
could be observed (panels c and d). These data most likely exclude
synergy between TIF2 and the NH2-terminal domain for the
interaction with the AR-LBD, indicating that TIF2 is not necessary for
the functional interaction between the AR domains.

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Figure 7. The Effect of Cotransfecting TIF2 on the Functional
Interaction of AR.N1 and AR.C
CHO cells were transfected with AR.C (25 ng/well) together with
increasing amounts of either TIF2 (50400 ng/well, panel a) or AR.N1
(50400 ng/well, represented by - in panels bd;) and with
combinations of both (panels b, c, and d). MMTV-LUC was used as a
reporter. Cells were incubated with vehicle (open bars)
or 1 nM R1881 (solid bars). LUC activity was
determined as described in Materials and Methods. The
interexperimental variation of the mean maximal luciferase activities
ranged between 120,000 and 375,000 light units. For each experiment the
mean AR.N1 activity (400 ng/well) was set at 100%, and all individual
points were calculated relative to this value. Each bar
represents the mean (±SEM) luciferase activity of three
experiments. Fold induction represents the ratio of activity determined
in the presence and absence of R1881.
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TIF2-Induced Activity of AR AF-2 in CHO Cells Is Promoter
Dependent
To further examine the induction of AR AF-2 activity by the
NH2-terminal domain and by TIF2, in vivo
interaction assays in CHO cells were performed using different promoter
constructs (Fig. 8
). AR.C was
cotransfected into CHO cells with increasing amounts of either AR.N1
(Fig. 8
, A and C) or TIF2 (Fig. 8
, B and D), together with either
MMTV-LUC (Fig. 8
, A and B) or GRE-TATA-LUC (Fig. 8
, C and D) as
reporter constructs. In the absence of either AR.N1 or TIF2, no
significant response to 1 nM R1881 could be detected. AR.N1
as well as TIF2 induced AF-2 activity on the MMTV-LUC promoter
(maximally 39- and 44-fold, respectively). Luciferase activity was also
highly induced by AR N.1 on the minimal, GRE-TATA, promoter (Fig. 8C
).
In contrast, the TIF2-induced luciferase activity appeared to be much
more affected on this promoter (Fig. 8D
). This indicates that the level
of transcriptional response induced by TIF2 depends on the promoter
context.

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Figure 8. Enhancement of the Transcriptional Response by TIF2
in CHO Cells Is Promoter-Dependent
CHO cells were transfected with AR.C (50 ng/well) and increasing
amounts of AR.N1 (panels A and C) or TIF2 (panels B and D) together
with the reporter plasmids MMTV-LUC (panels A and B) or GRE-TATA-LUC
(panels C and D). Cells were incubated with vehicle (open
bars) or 1 nM R1881 (solid bars).
LUC activity was determined as described in Materials and
Methods. The interexperimental variation of the mean maximal
luciferase activities ranged between 4,800 and 6,400 light units for
panels A and B, and between 11,500 and 13,500 light units for panels C
and D. For each experiment the mean AR.N1 activity (1000 ng/well) was
set at 100%, and all individual points were calculated relative to
this value. Each bar represents the mean
(±SEM) luciferase activity of three experiments. Fold
induction represents the ratio of activity determined in the presence
and absence of R1881.
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DISCUSSION
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Upon hormone binding, steroid receptors undergo conformational
changes that result in a cascade of events leading toward DNA binding
and transcription activation. Protein-protein interactions play a
prominent role in these processes. After releasing associated heatshock
proteins, the receptor homodimerizes, and subsequently interacts with
TIFs and basal transcription factors, to induce transcription of
specific target genes (3, 4, 5, 6, 7). For the hAR, two separate transcription
activation units in the NH2-terminal domain were defined:
TAU-1 and TAU-5 (8). TAU-1 is active in the full-length, hormone-bound
AR, whereas TAU-5 is active in a constitutive receptor that lacks the
LBD. This shift in TAU in the NH2-terminal domain,
depending on the absence or presence of the LBD, suggested a functional
interaction between the LBD and the NH2-terminal
domain.
It was described recently, for the ER and AR, that functional in
vivo interactions between the separate NH2-terminal
domain and the LBD can occur (20, 21, 22). Whether this association is
direct or requires additional factors is unknown. Neither is it clear
which regions in these AR domains are involved in this functional
interaction. For a better understanding of AR action, we determined the
subdomains in the AR involved in the functional interaction between the
NH2-terminal domain and the LBD.
Cotransfection experiments of the AR-LBD with NH2-terminal
deletion mutants in CHO cells and in yeast imply a major role for amino
acid residues 336 of the NH2-terminal domain in the
functional in vivo interaction with the LBD. A previous
report by Langley et al. (21) indicated residues 14150 to
be involved, suggesting that the interaction domain
might be constrained to residues 1436. In this respect it is
interesting that region 1636 is potentially capable of forming an
amphipathic
-helix, if projected on a helical wheel diagram.
Experiments in CHO cells revealed a second region, amino acid residues
371494, important for a functional in vivo interaction
with the AR-LBD. The involvement of this NH2-terminal
region was confirmed by data obtained with the two-hybrid system in
yeast. This region harbors TAU-5 (8), postulating a role for this
transcription activation unit in the functional interaction with the
LBD. A recent report by McEwan and Gustafsson (24) described the
interaction of the AR NH2-terminal domain (aa 142485)
with the general transcription factor TFIIF. It remained unclear which
of the transactivation units (TAU-1 or TAU-5) in the AR
NH2-terminal domain is involved in the interaction with
TFIIF, as both TAUs are present in the AR domain used in this study.
Therefore, the role of TAU-5 in the interaction with the AR-LBD might
be, at least in part, by recruiting the transcriptional machinery to
the target promoter.
The in vivo experiments described here imply an important
role for the AR AF-2 AD core in the functional interaction with the
NH2-terminal domain. The E888Q mutation markedly decreased
the interaction between the two AR domains, without altering hormone
binding. As the E888Q mutation did not completely reduce the
interaction, it is likely that also other residues or regions in the
LBD are involved in the formation of a proper interacting surface.
Wurtz et al. (19) proposed a general mechanism for nuclear
receptor activation, in which hormone-induced conformational changes
within the LBD result in a close contact of helix 12 and helix 4,
thereby creating an interaction surface that allows binding of
coactivators to the AF-2 activation domain. As the formation of this
interaction surface might also be important for the functional
interaction of the LBD with the NH2-terminal domain, this
would suggest that helix 4 is involved. Two-hybrid experiments in yeast
showed that the E888Q mutation affected the functional interaction with
the NH2-terminal domain but did not influence LBD
homodimerization. This implies that different regions in the AR-LBD are
involved in the interaction with the NH2-terminal domain
and in LBD dimerization.
The E888Q mutation also decreased the stimulatory effect of the
coactivator TIF2 on AR AF-2 activity. This suggests that the AF-2 AD
core region of the hAR is involved in interacting with both the
NH2-terminal domain and the coactivator TIF2. A possible
mechanism of TIF2 action could be as a bridging factor in the process
of transcription activation by the AR. McInerney et al. (25)
demonstrated that the steroid receptor coactivator SRC-1 enhanced the
functional interaction between the NH2-terminal and
C-terminal domain of the ER. From these data it was suggested that
SRC-1 may act as an adaptor protein that promotes the integration of
NH2-terminal and C-terminal ER domains. However,
cotransfection experiments with TIF2 and the AR
NH2-terminal domain in CHO cells did not show synergy
between TIF2 and the AR NH2-terminal domain in the
induction of AR AF-2 activity (Fig. 7
). Therefore, it is unlikely that
TIF2 functions as a bridging factor for the functional interaction of
the NH2-terminal domain and the LBD of the AR.
The AR appears to be a unique member in the nuclear receptor
superfamily, because the AR C-terminal domain, containing the AF-2
AD core region, is inactive in the absence of the
NH2-terminal domain or TIF2. This lack of activity is not
due to the experimental setup or the cell type used (CHO), because a
similar C-terminal construct of the GR is active. The most likely
explanation of this difference is that interactions of the AR-AF2 AD
with endogenous coactivators are very weak and therefore undetectable.
Overexpression of coactivators (e.g. TIF2) might enhance the
interaction and consequently transcriptional activity. Another
explanation for the absence of AR AF-2 activity in CHO cells is that
endogenous levels of TIF2 are not sufficient for activation of the MMTV
promoter in the absence of AR.N1.
A signature motif in transcriptional coactivators that mediated binding
to liganded nuclear receptors was described recently (26, 27, 28). Most
coactivators, including TIF2, harbor several of these leucine-rich
motifs (LXXLL). Two-hybrid experiments in yeast revealed that the
interaction of the ER-LBD with LXXLL motifs was abolished by mutating
the AF-2 AD core region in the LBD (26). Despite the demonstration that
the NH2-terminal domain of the AR also interacts with the
AF-2 AD, it does not harbor such a motif, suggesting a different type
of cooperation between the two AR domains.
The induction of AF-2 activity by TIF2 appeared to be promoter
dependent. TIF2 strongly stimulated AR AF-2 activity on a MMTV-LUC
reporter but much less on a minimal GRE-TATA promoter. The AR
NH2-terminal domain could, in contrast to TIF2, induce
AF-2 activity on both promoters. Possibly, TIF2 not only affects the AR
but also interacts with other transcription factors that bind to the
complex MMTV promoter and not to the minimal promoter.
From the data presented here, together with previously obtained results
(22), the following model for functional interactions between AR
domains is proposed. Androgen binding induces a conformational change
in the receptor that facilitates the AF-2 AD core in the LBD to
interact with the NH2-terminal domain. This interaction
might be either direct or indirect, requiring additional factors, and
results in AR transcription activation. Enhancement of AR activity by
the coactivator TIF2 also involves the AF-2 AD core. In addition to a
functional NH2-terminal domain/LBD interaction, a LBD/LBD
interaction is proposed that requires a region different from the AF-2
AD core.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
Plasmid constructions were performed according to standard
methods (29) and where denoted, rendered blunt ended with Klenow. All
blunt-ended fusion constructs, and constructs including a PCR
amplification step for preparation, were sequenced to verify the
correct reading frame and the absence of random mutations. AR.N1 and
AR.C were described previously as pSVAR(TAD1-494)(22) and
pSVAR-104 (8), respectively. AR.N2, AR.N3, AR.N5, AR.N6, and AR.N7 were
obtained by modification of AR
51211 and AR
244360 (30),
AR124, AR127, and AR106 (8) respectively : plasmids were digested with
KpnI and EcoRI and religated by a
KpnI-EcoRI linker. AR.N4 was obtained by
insertion of a RsrII-EcoRI linker into AR.N1.
pSVAR(EQ) was constructed by site-directed mutagenesis of pSVAR (31),
using the following oligonucleotides (the modified codon is shown
underlined):
AR/EQ1: 5'-ACAGCCAGTGTGTCCGAATG-3';
AR/EQ2: 5'-CTTGCACAGAGATGATTTGTGCCATC-3';
AR/EQ3: 5'-GATGGCACAAATCATCTCTGTGCAAG-3';
AR/EQ4: 5'-CAAGGGGCTTCATGATGTCC-3'.
AR.C(EQ) was prepared by digestion of pSVAR(EQ) with
EcoRI and ligating the 465-bp fragment into AR.C. MMTV-LUC
reporter plasmid was kindly provided by Dr. Dijkema (Organon, Oss, The
Netherlands) and was described previously (32). GRE-TATA-LUC,
containing the TATA-box and a Sp1-site derived from the Oct-6 gene
promoter, has been described previously as
pJH4-(ARE)2-OCT-LUC (33). GR.C (aa 369777) was
constructed by digestion of RshGR
(34) with BstXI and
religation with a compatible DNA fragment obtained by annealing the
following oligos: 5'-AGCCCGGGACCATGGGAT-3' and
5'-CATGGTCCCGGGCTATCC-3'. The obtained plasmid was digested with
SmaI and DraI, and the 1074-bp fragment,
containing the GR DBD and LBD, was ligated into
SmaI-digested pSV328A (35).
The GAL4(DBD1-147) two-hybrid cloning vector pGBT9 and the
parental GAL4(TAD768-881) cloning vector pGAD424 (low
expression), or the high-level expression derivative pACT2 (all from
Clontech, Palo Alto, CA), were used to generate all yeast fusion
protein constructs. GalAD-AR.N8 (low) was described previously as
pGAL4(TAD)AR(TAD) (22). GalAD-AR.N11 (low) was generated by digesting
GalAD-AR.N8 (low) with NcoI and SalI, followed by
religation of the blunted vector. The previously described pTZ19NAR
(22), containing an additional BamHI site by addition of a
BamHI linker to the blunt-ended EcoRI site in the
polylinker, was digested with SmaI and NcoI, and
the resulting vector was blunt ended and religated, yielding
pTZ19AR.N12. The AR fragment was excised with BamHI and
cloned in frame into the corresponding site in pGAD424, yielding
GalAD-AR.N12 (low). The 0.4-kb EcoRI-Acc651
(blunt ended) fragment from the previously described pSVAR-123 (8),
containing an additional EcoRI site by addition of an
EcoRI linker to the blunt-ended XbaI site, was
cloned in frame into pGAD424 via EcoRI and SmaI
compatible ends, yielding GalAD-AR.N14 (low). GalDBD-AR.N8 (low)
[previously described as pGAL4(DBD)-AR(TAD) (22)] was digested with
RsrII and Acc651, and the blunted vector was
religated, yielding GalDBD-AR.N10 (low). The AR fragment was excised
with BamHI and cloned in frame into the corresponding site
in pGAD424, yielding GalAD-AR.N10 (low). GalAD-AR.N15 (low) was
generated by deletion of the internal PstI fragment from
GalAD-AR.N12 (low). GalAD-AR.N16 (low) was constructed by digesting
GalAD-AR.N15 (low) with EcoRI and NcoI, followed
by religation of the blunted vector. GalAD-AR.N13 (low) was prepared by
integration of the 0.5-kb GalAD-AR.N12 (low) PstI fragment
into the homologous GalAD-AR.N16 (low) site. AR.N8, containing the AR
fragment cloned in frame to the SV40 large T antigen nuclear
localization signal in pGAD424, was described previously as
pAR(TAD3-494) (22). AR.N12 (low) was generated by
exchanging the Acc651 (blunt ended)-EcoRI
fragment of pGAD424 with the 0.9-kb BamHI (blunt
ended)-EcoRI fragment of pTZ19AR.N12. AR.N15 (low) was
prepared by digesting GalAD-AR.N15 (low) with Acc651 and
BamHI, followed by religation of the blunt-ended vector. All
high expression derivatives were constructed by exchanging the internal
HindIII fragments with the internal pACT2 HindIII
fragment. AR.N9 (high) was generated by exchanging the internal 0.9-kb
StuI-EcoRI fragment of AR.N8 (high) with the
0.5-kb StuI-EcoRI fragment of AR.N3 (described
above). AR.N11 (high) was generated by digesting AR.N8 (high) with
NcoI and EcoRI, followed by religation of the
blunt-ended vector. Similarly, the high expression level AR.N10 was
generated by digesting AR.N8 (high) with RsrII and
EcoRI, followed by religation of the blunt-ended vector.
Only the high expression AR.N constructs were used in the two-hybrid
assays. The expression vectors GalDBD-AR.C (low expression) and
GalAD-AR.C (high expression) were previously described as
pGAL4(DBD)AR(LBD) and pGAL4(TAD)AR(LBD), respectively (22).
GalDBD-AR.C(EQ) was prepared by integration of the 0.7-kb
StuI-SalI fragment of pSVAR(EQ) (as described
above) into the homologous GalDBD-AR.C sites. Similarly, the
high-expression GalAD-AR.C(EQ) derivative was generated by integration
of the 0.45-kb EcoRI fragment of pSVAR(EQ) into the
homologous GalAD-AR.C site.
CHO Cell Culture, Transfection, and LUC Assay
Chinese hamster ovary (CHO) cells were maintained in DMEM/F12
culture medium, supplemented with 5% dextran-coated charcoal-treated
FCS (Life Technologies, Gaithersburg, MD). For transcription activation
experiments, CHO cells were plated in 12-well plates at a density of
0.6 x 105 cells per well (7 cm2) and
grown overnight. Cells were transfected using the calcium phosphate
precipitation method as described previously (36), with AR expression
plasmids and where indicated with TIF2, reporter plasmids (200
ng/well), and pTZ19 carrier plasmid to a total DNA concentration of 2
µg/well. After an overnight incubation, the cells were washed
and R1881 (methyltrienolone; New England Nuclear, Boston, MA) or
vehicle (0.1% ethanol) was added. After overnight incubation the cells
were harvested for the luciferase (LUC) assay, as described previously
(37). For the GR experiments, GR.C (50 ng/well) was cotransfected with
MMTV-LUC and pTZ19 carrier plasmid as described above. After overnight
incubation, dexamethasone (10 nM) was added, and luciferase
activity was measured the next day. For the coexpression studies of
TIF2 and AR.N1, the total amount of vector, added to each well, was
equalized by the addition of empty vector, to a total concentration of
800 ng/well. In addition, pTZ19 carrier plasmid was added to a total
concentration of 2 µg DNA/well.
Yeast Growth and Methods
All yeast studies were performed in strain Y190 (MATa,
ura352, his3-
200, ade2101, trp1901, leu23, leu2112,
GAL4
, GAL80
, URA3::GAL-lacZ,
cyhr, LYS2::GAL-HIS3), which was
purchased from CLONTECH. Yeast cells were grown in the appropriate
selective minimal medium [0.67% (wt/vol) yeast nitrogen base without
amino acids and 2% (wt/vol) dextrose, pH 5.8] supplemented to the
nutritional requirements of the yeast transformants. Yeast
transformations were carried out according to the lithium acetate
method (38).
Quantitative ß-GAL activity assays, indicative of AR domain
interactions, were performed as described previously (22).
Immunoprecipitation and Immunoblotting
CHO cells were plated in 80-cm2 culture flasks
(1 x 106 cells per flask), grown overnight, and
transfected with 4 µg AR-plasmid and 16 µg pTZ19 as carrier
plasmid. The next day, medium was refreshed and the cells were grown
for another day. Cytosol of the transfected cells was prepared as
described before (22). Immunoprecipitation was performed as described
previously (39), using monoclonal antibodies F112.1.1 [directed
against synthetic peptide SP197; AR aa 120 (40)], F39.4.1 [directed
against SP061; aa 301320; (41)], or F52.24.4 [directed against
SP063; aa 593612; (39)]. Subsequent to SDS-PAGE and Western
blotting, the membrane was blocked with 5% nonfat dry milk and
incubated with polyclonal antisera against SP197, SP061, or SP066
[directed against AR aa 892910 (40)]. The proteins were visualized
by chemiluminescence detection.
AR proteins in yeast were isolated as described previously (22).
Yeast-lysate samples (2.5 µl) were run on SDS-polyacrylamide
gels, after which the gels were electroblotted under semidry
conditions. The blots were blocked overnight with 5% nonfat dry
milk and incubated with GAL4AD monoclonal antibody (CLONTECH) or
the polyclonal AR-antibody SP197. Proteins were visualized by
chemiluminescence detection.
In Vivo Hormone Binding Assay
CHO cells were plated in six-well plates at a density of 1
x 105 cells per well, grown overnight, and transfected as
described above with AR-C or AR-C(EQ) (0.75 µg/well). The whole-cell
binding assay was performed 48 h later. Cells were washed once
with PBS and incubated with various [3H]R1881 (New
England Nuclear) concentrations (0.0130 nM) in the
presence or absence of a 200-fold molar excess of unlabeled R1881 in
DMEM/F12 for 1 h at 37 C. The cells were washed four times with
PBS, collected in PBS, and transferred to a centrifuge tube. After
centrifugation (10 min, 800 x g) the pellet was lysed
in 0.5 M NaOH (15 min at 56 C), and radioactivity was
determined by liquid scintillation counting.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Gronemeyer and Dr. Chambon for providing the TIF2
construct, Dr. Dijkema for providing MMTV-LUC, Dr. Evans for the
RshGR
plasmid, and Dr. Kuil for making the GR.C construct.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. A.O. Brinkmann, Department of Endocrinology and Reproduction, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail:
brinkmann{at}endov.fgg.eur.nl
1 These authors contributed equally to this study. 
Received for publication November 6, 1997.
Revision received April 13, 1998.
Accepted for publication May 1, 1998.
 |
REFERENCES
|
---|
-
Gronemeyer H 1992 Control of transcription activation by
steroid hormone receptors. FASEB J 6:25242529[Abstract/Free Full Text]
-
Glass CK 1994 Differential recognition of target genes by
nuclear receptor monomers, dimers and heterodimers. Endocr Rev 15:391407[Medline]
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344[Medline]
-
Green S, Chambon P 1988 Nuclear receptors enhance our
understanding of transcription regulation. Trends Genet 4:309314[CrossRef][Medline]
-
Parker MG 1993 Steroid and related receptors. Curr Opin Cell
Biol 5:499504[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Jenster G, van der Korput HA, Trapman J, Brinkmann AO 1995 Identification of two transcription activation units in the N-terminal
domain of the human androgen receptor. J Biol Chem 270:73417346[Abstract/Free Full Text]
-
Danielian PS, White R, Lees JA, Parker MG 1992 Identification
of a conserved region required for hormone dependent transcriptional
activation by steroid hormone receptors. EMBO J 11:10251033[Abstract]
-
Barettino D, Vivanco Ruiz MM, Stunnenberg HG 1994 Characterization of the ligand-dependent transactivation domain of
thyroid hormone receptor. EMBO J 13:30393049[Abstract]
-
Durand B, Saunders M, Gaudon C, Roy B, Losson R, Chambon P 1994 Activation function 2 (AF-2) of retinoic acid receptor and 9-cis
retinoic acid receptor: presence of a conserved autonomous constitutive
activating domain and influence of the nature of the response element
on AF-2 activity. EMBO J 13:53705382[Abstract]
-
Lanz RB, Rusconi S 1994 A conserved carboxy-terminal subdomain
is important for ligand interpretation and transactivation by nuclear
receptors. Endocrinology 135:21832195[Abstract]
-
Montano MM, Ekena K, Krueger KD, Keller AL, Katzenellenbogen
BS 1996 Human estrogen receptor ligand activity inversion mutants:
receptors that interpret antiestrogens as estrogens and discriminate
among different antiestrogens. Mol Endocrinol 10:230242[Abstract]
-
Cavailles V, Dauvois S, LHorset F, Lopez G, Hoare S, Kushner
PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional
activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
LeDouarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat P,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein
T18. EMBO J 14:20202033[Abstract]
-
Vom Baur E, Zechel C, Heery D, Heine MJ, Garnier JM, Vivat V,
Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential
ligand-dependent interactions between the AF-2 activating domain of
nuclear receptors and the putative transcriptional intermediary factors
mSUG1 and TIF1. EMBO J 15:110124[Abstract]
-
Voegel JJ, Heine MJ, Zechel C, Chambon P, Grone-meyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Jenster G, Spencer TE, Burcin MM, Tsai SY, Tsai M-J, OMalley
BW 1997 Steroid receptor induction of gene transcription: a two-step
model. Proc Natl Acad Sci USA 94:78797884[Abstract/Free Full Text]
-
Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D 1996 A canonical structure for the ligand-binding domain of nuclear
receptors. Nature Struct Biol 3:8794[Medline]
-
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 ZX, 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, Brinkman 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]
-
Nemoto T, Ohara-Nemoto Y, Shimazaki S, Ota M 1994 Dimerization
characteristics of the DNA- and steroid-binding domains of the androgen
receptor. J Steroid Biochem Mol Biol 50:225233[CrossRef][Medline]
-
McEwan IJ, Gustafsson J-Å 1997 Interaction of the human
androgen receptor transactivation function with the general
transcription factor TFIIF. Proc Natl Acad Sci USA 94:84858490[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]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid
receptor-associated coactivator that is related to SRC-1 and TIF2. Proc
Natl Acad Sci USA 94:84798484[Abstract/Free Full Text]
-
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]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
-
Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast
TH, Trapman J, Brinkmann AO 1991 Domains of the human androgen receptor
involved in steroid binding, transcriptional activation, and
subcellular localization. Mol Endocrinol 5:13961404[Abstract]
-
Brinkmann AO, Faber PW, van Rooij HC, Kuiper GG, Ris C,
Klaassen P, van der Korput JA, Voorhorst MM, van Laar JH, Mulder E,
Trapman J 1989 The human androgen receptor: domain structure,
genomic organization and regulation of expression. J Steroid Biochem 34:307310[CrossRef][Medline]
-
de Ruiter PE, Teuwen R, Trapman J, Dijkema R, Brinkmann AO 1995 Synergism between androgens and protein kinase-C on
androgen-regulated gene expression. Mol Cell Endocrinol 110:R16
-
Blok LJ, Themmen AP, Peters AH, Trapman J, Baarends WM,
Hoogerbrugge JW, Grootegoed JA 1992 Transcriptional regulation of
androgen receptor gene expression in Sertoli cells and other cell
types. Mol Cell Endocrinol 88:153164[CrossRef][Medline]
-
Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645652[Medline]
-
Van Heuvel M, Bosveld IJ, Mooren ATA, Trapman J, Zwarthoff EC 1986 Properties of natural and hybrid murine alpha interferons. J
Gen Virol 67:22152222[Abstract]
-
Veldscholte J, Berrevoets CA, Ris-Stalpers C, Kuiper GG,
Jenster G, Trapman J, Brinkmann AO, Mulder E 1992 The androgen receptor
in LNCaP cells contains a mutation in the ligand binding domain which
affects steroid binding characteristics and response to antiandrogens.
J Steroid Biochem Mol Biol 41:665669[CrossRef][Medline]
-
Kuil CW, Berrevoets CA, Mulder E 1995 Ligand-induced
conformational alterations of the androgen receptor analyzed by limited
trypsinization. Studies on the mechanism of antiandrogen action. J
Biol Chem 270:2756927576[Abstract/Free Full Text]
-
Gietz D, St. Jean A, Woods RA, Schiestl RH 1992 Improved
method for high efficiency transformation of intact yeast cells.
Nucleic Acids Res 20:1425[Medline]
-
Veldscholte J, Berrevoets CA, Zegers ND, van der Kwast TH,
Grootegoed JA, Mulder E 1992 Hormone-induced dissociation of the
androgen receptor-heat-shock protein complex: use of a new monoclonal
antibody to distinguish transformed from nontransformed receptors.
Biochemistry 31:74227430[Medline]
-
Kuiper GG, de Ruiter PE, Trapman J, Boersma WJ, Grootegoed JA,
Brinkmann AO 1993 Localization and hormonal stimulation of
phosphorylation sites in the LNCaP-cell androgen receptor. Biochem
J 291:95101[Medline]
-
Zegers ND, Claassen E, Neelen C, Mulder E, van Laar JH,
Voorhorst MM, Berrevoets CA, Brinkmann AO, van der Kwast TH, Ruizeveld
de Winter JA, Trapman J, Boersma WJA 1991 Epitope prediction and
confirmation for the human androgen receptor: generation of monoclonal
antibodies for multi-assay performance following the synthetic peptide
strategy. Biochim Biophys Acta 1073:2332[Medline]