(Received for publication, November 15, 1996, and in revised form, April 17, 1997)
From the Promoter interference assay was employed to
examine in intact cells the roles of the functional domains of androgen
receptor (AR) and the ligand for specific DNA interactions using a
cytomegalovirus-(androgen response element)-chloramphenicol
acetyltransferase reporter (pCMV-ARE2-CAT). Native
rat and human ARs interfered with pCMV-ARE2-CAT
expression in a hormone-dependent fashion. Low steroid-independent
interference seemed to occur because of the ligand binding domain
(LBD), which was transcriptionally inhibitory also in a heterologous
context. AR devoid of LBD (rAR Androgen receptor (AR)1 belongs to the
nuclear receptor superfamily of ligand-regulated transcription factors
(1-4). Although the interaction of AR with specific hormone-responsive
DNA elements is usually required for androgen-dependent
transcriptional activation, binding of AR to specific DNA motifs is not
always necessary for the receptor's ability to down-regulate gene
expression (5-7). With regard to the ligand requirement for the
recognition of specific DNA elements by AR, in vitro
electrophoretic mobility shift assays have yielded conflicting results.
AR protein expressed in reticulocyte lysate or produced insect cells is
capable of binding to specific androgen response elements (AREs)
in vitro even in the absence of androgen (8, 9); however,
the ligand requirement for the binding of hAR to AREs in
vitro has also been reported (10). Very limited information is
available on ARE occupancy by the receptor protein in intact cells.
In vivo footprinting of an androgen-dependent enhancer of the mouse slp gene failed to reveal clear
protection of hormone response elements by AR (11). While this work was in progress, Kuil and Mulder (12) reported that AR interaction with an
"idealized" consensus ARE derived from polymerase chain reaction
selection experiments is ligand-dependent in cultured cells.
The promoter interference assay developed by Hu and
Davidson (13) is based on competition of DNA-binding
proteins, such as nuclear receptors, for binding with essential
transcription factors driving a constitutively active heterologous
promoter. The interference is achieved by inserting an attachment
sequence between the TATA box and the start site of transcription in
the promoter. A promoter interference assay has been employed in a few
reports to examine the interaction of estrogen receptor (ER) with its
cognate response elements in mammalian and frog cells (14, 15) and that
of AR with AREs in Chinese hamster ovary cells (12). Because of the use
of transiently transfected cells, the physiological chromatin
environment is not achieved under these conditions. However, using a
pCMV-PRE2-CAT construct stably integrated into T47D breast
cancer cells, Gass et al. (16) recently reported that
progesterone receptor (PR) is able to interfere with expression of this
reporter also in the context of stable chromatin conformation, albeit
less well than in transiently transfected cells.
In our previous studies, we constructed several
NH2-terminal deletion mutants of AR to define domains
crucial for the activation functions of this protein (5, 8, 9). One of
the mutants, rAR In the present work we have used promoter interference assays to
investigate whether binding of AR to specific DNA sequences is
ligand-dependent in intact CV-1 cells. We have also
examined which functional domains of the wild-type receptor, in
addition to the DNA binding domain (DBD), are mandatory for specific
DNA binding in vivo. To assess the role of the ligand,
several non-steroidal and steroidal anti-androgens alone or together
with an androgen agonist were used. Finally, immunocytochemical
analysis of cultured cells was employed to rule out the possibility
that some of the results were compromised by altered subcellular
distribution of receptor proteins.
Chemicals were obtained from Sigma, Bio-Rad, and
Qiagen GmbH (Hilden, Germany). Testosterone was purchased from Makor
Chemicals (Jerusalem, Israel). [3H]Acetyl-coenzyme A was
obtained from DuPont NEN. DNA-modifying enzymes were purchased from
Pharmacia Biotech Inc. and Promega Corp. (Madison, WI).
Oligonucleotides were synthesized using Gene Assembler Plus
(Pharmacia). Non-steroidal anti-androgens casodex ((2RS)-4 pCMV-ARE2-CAT
reporter was constructed from pCMV-0-CAT (a gift from Dr. B. Katzenellenbogen, University of Illinois, Urbana) by inserting a
double-stranded 45-base pair oligonucleotide,
5 CV-1 and COS-1 cells were
obtained from American Type Culture Collection (Rockville, MD) and
maintained in Dulbecco's modified Eagle's medium containing
penicillin (25 units/ml) and streptomycin (25 units/ml) supplemented
with 10% (v/v) fetal bovine serum (Life Technologies, Inc.).
Twenty-six h before transfection, 1.5 × 106 cells
were seeded on 10-cm plates. The medium was replaced 2-4 h before
transfection with Dulbecco's modified Eagle's medium containing 10%
charcoal-stripped fetal bovine serum. The calcium phosphate
coprecipitation method was used for transfections as described
previously (8). The total amount of DNA was 10 µg/10-cm plate and
included the following components: 0.5-2.5 µg of receptor expression
vector DNA (pSGrAR, pSGhAR, pSGrARC562G, pSGhARM807R, pSGrAR Table I.
The ability of different steroidal and nonsteroidal anti-androgens to
confer ARE binding ability on AR as determined by promoter interference
assay
Institute of Biomedicine,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
641-902) decreased
pCMV-ARE2-CAT activity by 50%. The rAR
46-408 mutant
devoid of the NH2-terminal transcription activation region
exhibited ligand-dependent promoter interference of a
similar magnitude. Ligand and DNA binding-deficient mutants (hARM807R
and rARC562G, respectively) did not influence pCMV-ARE2-CAT expression, although hARM807R binds to ARE in vitro.
Non-steroidal anti-androgens casodex and hydroxyflutamide antagonized
agonist-dependent promoter interference, whereas
cyproterone acetate, RU 56187, RU 57073, and RU 59063 were partial
agonists/antagonists. Collectively, interaction of ARs with ARE in
intact cells does not require the presence of the COOH-terminal or
NH2-terminal domain and/or their interaction. In the
context of native AR, however, the androgen-induced conformational
change in LBD is mandatory for generation of a transcriptionally
competent receptor that binds to DNA in intact cells.
46-408, devoid of a region mandatory for
transcriptional activation (8), bound to ARE in vitro with
somewhat lower affinity than the wild-type AR (9) but behaved as a
dominant negative regulator of the native protein, possibly through
forming transcriptionally inactive heterodimers with the latter (8, 9).
The ability of rAR
46-408, or comparable mutants of the
glucocorticoid receptor subfamily of nuclear receptors, to interact
with specific DNA sequences in intact cells has, however, not been
addressed previously. The COOH-terminal ligand binding domain (LBD)
provides the means to regulate the function of native AR in a
steroid-dependent fashion. Removal of this region generates
a constitutively active receptor form (17, 18), whose activity is
dependent on the promoter context.2
However, in vivo DNA binding characteristics of
LBD-deficient AR forms or those of other members of the glucocorticoid
receptor subfamily have not been examined.
Materials
-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3
-(trifuoromethyl)propionanilide) and hydroxyflutamide
(4-hydroxy-
,
,
-trifluoro-2-methyl-4
-nitro-m-propionotoluidide) were obtained from Zeneca Pharmaceuticals (Macclesfield, U. K.) and
Schering Corp. (Bloomfield, NJ), respectively. Cyproterone acetate
(6-chloro-1,2-methylene-17
-hydroxy-4,6-pregnadiene-3,20-dione acetate) was from Schering AG (Berlin, Germany), and RU 56187 (4-(5-oxo-2-thioxo-3,4,4-tri-methyl-1-imidazolidinyl)-2-trifluoromethylbenzonitrile), RU 57073
(4-[4,4-dimethyl-3-(2-hydroxyethyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-trifluoro-methylbenzonitrile) and RU 59063
(4-[4,4-dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-trifluoromethylbenzonitrile) were donated by Dr. G. Teutsch (Roussel UCLAF, Romainville,
France).
-CATAGTACGTGATGTTCTAGGCCTAGTACGTGATGTTCTCGAGCT-3
, containing duplicated high affinity AREs (half-sites underlined) of the
C3(1) gene of prostatic binding protein (9, 19) into the
SacI site between the TATA box and the transcription start site of pCMV-0-CAT. pCMV-ERE2-CAT was constructed in the
same way except that the inserted 45-base pair oligomer contained two estrogen response elements in lieu of AREs (14). For transcriptional activation experiments, the reporters pARE2-tk-CAT and
pMMTV-CAT were used (20). Expression vectors pSGrAR, pSGhAR,
pSGrARC562G, pSGhARM807R, pSGrAR
46-408, pSGrAR
46-408/C562G, and
pSGrAR
641-9022 have been described previously (5,
8, 21, 22). pG5SV40CAT containing five GAL4-binding elements upstream
of the SV40 promoter and pSV40CAT control vector were gifts from Dr. N. Lehming (Max-Delbrück Laboratory, Max-Plack Institute, Cologne,
Germany). GAL4-LBD and GAL-Nterm contained amino acids 641-902 and
5-538 of rAR, respectively, fused in-frame to Saccharomyces
cerevisiae GAL4-DBD.2
46-408,
pSGrAR
46-408/C562G, and pSGrAR
641-902), 2 µg of
pCMV-ARE2-CAT reporter (or pCMV-ERE2-CAT when
indicated), 3 µg of
-galactosidase expression vector (pCH110,
Pharmacia) as an internal control, and empty pSG5 DNA as needed. Ten
µg of pGEM-3Z DNA was used in mock transfections. In transfections
with pG5SV40CAT or pSV40CAT reporter (1 µg/10-cm plate), 5 µg of
GAL4-DBD-LBD, GAL4-DBD-Nterm, or GAL4-DBD expression vector was used,
and the amount of total DNA was adjusted to 10 µg with pBluescript II SK DNA (Stratagene, La Jolla, CA). Eighteen h after transfection, the
cells were switched into fresh medium containing androgens and
anti-androgens at concentrations specified in the legends to the
figures and Table I. The cells were harvested 30 h later, and CAT
and
-galactosidase activities were measured and normalized as
described previously (23, 24).
46-408, 2 µg of
pCMV-ARE2-CAT, 3 µg of pCH110 (
-galactosidase), and 4 µg of pSG5 vector DNA. Eighteen h after transfection, the cells received fresh medium with 10 nM testosterone or 1 µM
anti-androgen and were collected 30 h later for the measurement of
CAT and
-galactosidase activities. The values (mean ± S.E.,
n = 3) are expressed relative to that in CV-1 cells
transfected without pSGrAR
46-408.
Compound
pCMV-ARE2-CAT activity
%
Vehicle
85.5 ± 2.9
Testosterone (10 nM)
46.0 ± 3.5
Casodex (1 µM)
77.0 ± 3.1
Hydroxyflutamide (1 µM)
99.5 ± 6.1
RU 56187 (1 µM)
43.5 ± 4.0
RU 57073 (1 µM)
48.0 ± 1.5
RU 59063 (1 µM)
42.5 ± 1.9
Cyproterone acetate (1 µM)
40.0 ± 1.6
CV-1 cells were seeded on glass slips
on 10-cm plastic plates and were transfected with 1 or 2.5 µg of
pSG5r/hAR DNA, 2 µg of pCMV-ARE2-CAT, 3 µg of pCH110
and filled to 10 µg of total DNA with empty pSG5. Replacement of
medium and exposure to steroids were conducted as in the promoter
interference assay. Cells were fixed in methanol at 20 °C for 10 min and washed three times with phosphate-buffered saline (150 mM NaCl, 20 mM Na2HPO4,
pH 7.3) at 22 °C for 10 min. The primary antibody K183 (diluted
1:200) is a polyclonal rabbit antiserum raised against full-length rAR purified to homogeneity using preparative polyacrylamide gel
electrophoresis under denaturing conditions from Sf9 insect cells
infected with a recombinant baculovirus encoding rAR (25). In short,
the purification procedure was as follows. Two × 107
cells were lysed in 2 ml of electrophoresis sample buffer (26), passed
five times through a 25-gauge needle, and soluble proteins were
separated using Bio-Rad model 491 Prep Cell apparatus with a 37-mm
(inner diameter) tube. Electrophoresis was carried out at 50 mA,
proteins were eluted with 50 mM Tris/HCl, 5 mM
-mercaptoethanol, 0.02% (v/v) Nonidet P-40 (pH 8.0) at 0.45 ml/min,
and 4.5-ml fractions were collected. Fractions containing rAR were
pooled and concentrated using Amicon P-10 membrane (Amicon, Beverly,
MA). Rabbits were injected with 50 µg of rAR protein at each
immunization. Diluted K183 antiserum (1:200) was incubated on cells for
1 h at 22 °C, and fluorescein isothiocyanate-conjugated goat
anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories,
Philadelphia) was used (1:200 dilution, 30 min at 22 °C) to detect
the antigen.
Conditions for immunoblotting were as described previously (8), except that antiserum K183 (diluted 1:1,500) was used as the primary antibody.
An
AR-dependent promoter interference reporter plasmid,
pCMV-ARE2-CAT, was constructed in a fashion similar to that
described for ER (14) by inserting a duplicated high affinity ARE
sequence between the TATA box and transcription start site of
pCMV-0-CAT (Fig. 1A). Two AREs were inserted,
as the presence of more than one palindromic response element
facilitates AR-ARE interactions in vitro (9). pCMV-0-CAT
devoid of ARE sequences was initially used as the control reporter, but
its basal activity in CV-1 cells was several times higher than that of
pCMV-ARE2-CAT. This result was in agreement with a previous
report (13) that insertion of sequences into a similar location
(between the TATA box and the start site of transcription) decreases
CAT expression. For this reason, we used pCMV-ERE2-CAT
reporter (Fig. 1A) as control to assess the specificity of
AR-ARE interaction in intact cells. CV-1 cells were cotransfected with
various AR expression plasmids (Fig. 1B) and
pCMV-ARE2-CAT (or pCMV-ERE2-CAT) reporter
driven by the constitutively active CMV promoter.
Specific Promoter Interference by the Native AR Protein Requires Intact Ligand and DNA Binding Domains
Native rAR and hAR
interfered with pCMV-ARE2-CAT expression in such a way that
both ligand-dependent and ligand-independent inhibition of
the reporter activity was seen (Fig. 2, A and
B). rAR showed somewhat higher ligand-independent
promoter-interfering activity than hAR. Since expression levels of rAR
and hAR proteins were not compared in these experiments, the reason for
this species difference remains to be elucidated. Both ARs also
elicited some inhibition of pCMV-ERE2-CAT expression which
was, however, not increased by androgen (Fig. 2, C and
D). Even though the weak ligand-independent activity of AR
was not specific for the hormone response element inserted in the
promoter interference construct, it was specific for the promoter
interference assay itself, as a similar androgen-independent activity
was not observed in transactivation experiments. It is also worth
pointing out that maximal promoter interference and transcriptional
activation were achieved with comparable amounts of rAR or hAR
expression plasmid (1 µg DNA/plate).3
However, ligand-occupied hAR was twice as active as rAR in these latter
experiments (110-fold versus 50-fold increase over control, respectively3), which is in contrast to the behavior of
apo-hAR and apo-rAR in promoter interference assays (see Fig. 2,
A and B).
AR forms incapable of DNA or hormone binding (rARC562G and hARM807R,
respectively) were used to assess the importance of these regions for
specific AR-ARE interaction in vivo. rARC562G with a Cys Gly substitution at codon 562 does not bind to DNA in vitro
and is transcriptionally inactive (22). hARM807R is incapable of
hormone binding because of a Met
Arg substitution at codon 807 (21). In electrophoretic mobility shift assays under cell-free conditions, hARM807R bound to ARE with approximately the same affinity
as the wild-type receptor in the absence of androgen (21), but exposure
to androgen did not elicit a conformational change in the LBD of
hARM807R in a fashion similar to that in the wild-type LBD (21,
27).
rARC562G and hARM807R mutants did not interfere with
pCMV-ARE2-CAT activity in a steroid-dependent
fashion, but they exhibited weak hormone-independent interference,
essentially indistinguishable from that of the wild-type apo-AR (Fig.
3, A and B). The fact that
rARC562G behaved like apo-AR both in the presence and absence of
testosterone implies that specific binding of the wild-type AR to DNA
in intact cells is totally dependent on the integrity of the first zinc
finger and that the conformational change elicited by an active
androgen in the LBD does not abolish the ability of this region to
interfere weakly with pCMV-ARE2-CAT expression.
Influence of COOH-terminal and NH2-terminal Regions of AR on Receptor-ARE Interaction in Intact Cells
Previous studies
have shown that AR forms devoid of the entire LBD behave as
constitutively active transactivators and are capable of binding to
specific DNA elements in vitro (17, 18).2 The
rAR641-902 mutant that lacks the LBD reduced expression of
pCMV-ARE2-CAT by 50% but did not influence that of
pCMV-ERE2-CAT (Fig. 4A). It is of
note that maximal promoter interference was achieved with the lowest
amount of pSGrAR
641-902 expression vector used (0.5 µg of
DNA/10-cm plate), and no additional increase in the interference
occurred when the amount of plasmid was augmented 5-fold (Fig.
4A) or 10-fold (data not shown). Interaction of
rAR
641-902 with DNA was, as expected, independent of the presence
of ligand, whether an agonist (Fig. 4A) or an antagonist
(54 ± 5% of control in cells transfected with 0.5 µg of
pSGrAR
641-902 in the presence of 1 µM casodex).
To examine the role of the NH2-terminal region,
rAR46-408 and rAR
46-408/C562G mutants were used. rAR
46-408
expressed in insect cells binds to specific DNA sequences in
vitro, albeit with a somewhat lower affinity than the wild-type AR
(8, 9). When expressed in CV-1 cells, rAR
46-408 elicited promoter
interference in an androgen-dependent fashion (Fig.
4B). This interference required the presence of ARE
sequences, as a similar steroid-dependent inhibition of
pCMV-ERE2-CAT expression failed to occur under the same
conditions (Fig. 4B). Likewise, an intact DBD of the
receptor was required, as judged by the inability of
rAR
46-408/C562G to interfere with pCMV-ARE2-CAT
activity.2 Some hormone-independent inhibition of
pCMV-ARE2-CAT activity was observed with rAR
46-408 when
higher amounts of expression plasmid were used in transfections (Fig.
4B).
Taken together, interaction of AR with specific DNA sequences in intact
cells can take place in the absence of LBD or most of the
NH2-terminal region. DNA binding of the receptor, even in
the presence of LBD and steroid, is not sufficient to elicit proper
physiological responses, as the rAR46-408 mutant is
transcriptionally inactive (8). Comparison of the dose-response curves
between rAR
641-902 and rAR
46-408 suggests that LBD is
responsible for the weak hormone-independent interference of AR with
pCMV-ARE2-CAT or pCMV-ERE2-CAT expression (Fig.
4, A and B). The results with full-length AR
forms (Fig. 2) were in general agreement with those obtained with
rAR
641-902 and rAR
46-408 mutants.
The ability of LBD to interfere with transcription was
examined by additional experiments, in which pG5SV40CAT reporter
containing five GAL4 binding sites upstream of the SV40 promoter was
used. CV-1 cells were cotransfected using this reporter or an
appropriate control reporter (pSV40CAT) and expression vectors encoding
GAL4-DBD, GAL4-LBD, or GAL4-Nterm fusion proteins. The
NH2-terminal region of AR (residues 5-538) fused to
GAL4-DBD did not influence the activity of the latter; however, when
the fusion partner was LBD (residues 641-902), repression of
transcriptional activity took place (Fig.
5A). It is of note that the ability of
ligand-free LBD to repress transcription at a distance exceeded that of
the ligand-occupied LBD in the GAL4-DNA-binding construct (Fig.
5A). The repressive action of LBD-containing fusion protein
was dependent on its tethering to DNA, as no inhibition was detected in
the absence of GAL4-binding elements (Fig. 5B).
Antagonistic and Agonistic Activities of Anti-androgens
Several anti-androgens (casodex, hydroxyflutamide,
RU 56187, RU 57073, RU 59063, and cyproterone acetate) were examined
by promoter interference assays using rAR46-408, as differentiation between ligand-dependent and -independent activities was
more clear-cut with this mutant than with native ARs. All other
anti-androgens examined but hydroxyflutamide were able to confer some
promoter-interfering activity upon rAR
46-408, with cyproterone
acetate and the RU compounds being as potent as an agonist (10 nM testosterone) at the concentration used (1 µM) (Table I). Additional studies with casodex revealed
that its low agonist-like activity in the promoter interference assay
was also detectable with wild-type rAR and at varying receptor levels
(Fig. 6A). When present concomitantly with
androgen, casodex inhibited the agonist-induced promoter interference
significantly but not completely (Fig. 6B). Previous studies
have shown that casodex and hydroxyflutamide are completely devoid of
inherent transactivation ability (22, 28), whereas cyproterone acetate
is a partial agonist/antagonist (29).
Three N-substituted arylthiohydantoin anti-androgens
(RU 56187, RU 7073, and RU 59063) were as effective as testosterone
in conferring promoter-interfering activity on rAR46-408 (Table I). This is in agreement with binding affinities of
these compounds for rat and human ARs in vitro, which are
92-300% of that of testosterone (30). When CV-1 cells cotransfected
with pSGrAR and pMMTV-CAT vectors were exposed to 1 µM of
each RU anti-androgen alone, CAT activity was 50% with RU 56187, 28%
with RU 57073, and 35% with RU 59063 of that with 10 nM
testosterone. Concomitant exposure of the cells to 10 nM
testosterone and 1 µM RU anti-androgens resulted in
reporter gene activities that were 39-66% of those achieved with
androgen alone, indicating that the three RU compounds were partial
agonists/antagonists under these conditions. As the RU compounds
promoted AR-ARE interaction in CV-1 cells as well as testosterone
(Table I), conformational requirements on LBD for transcriptional
activation and recognition of specific DNA sequences (promoter
interference) are not identical in intact cells.
The polyclonal antiserum (K183) used in these
experiments was specific for AR proteins, as no extra bands were
observed in mock-transfected cells, and only the receptor protein was
detected in immunoblots of COS-1 cells transfected with some of the AR expression constructs used in promoter interference assays (Fig. 7). The antiserum recognized rAR46-408 and
rAR
641-902 mutants as efficiently as the native receptor protein
and, therefore, was useful for immunological studies of different AR
forms in cultured cells.
CV-1 cells transfected with 1 µg of pSGrAR DNA showed some
immunoreactivity in the cytoplasm in the absence of androgen, whereas unliganded hAR was more clearly nuclear in its localization under the
same conditions (Fig. 8, A and C).
Both receptors became solely nuclear upon hormone exposure (Fig. 8,
B and D). rARC562G and hARM807R exhibited both
cytoplasmic and nuclear localization in the absence of testosterone
(Fig. 8, E and F). The AR form devoid of most of
the NH2-terminal region (rAR46-408) showed mainly nuclear localization even in the absence of androgen (Fig.
8G), and it was strictly nuclear after exposure to
testosterone (data not shown). Likewise, the LBD-deficient mutant
(rAR
641-902) was nuclear in the absence of the hormone (Fig.
8H). CV-1 cells transfected with pSGhAR expression vector
and exposed to casodex or hydroxyflutamide exhibited only nuclear
localization of the receptor protein (Fig. 8, I and
J). Taken together, immunocytochemical studies of
transfected CV-1 cells expressing wild-type and mutant AR proteins
revealed that the receptor forms were mainly nuclear even in the
absence of ligand, indicating that differences in subcellular
localization do not explain their dissimilar behavior in the promoter
interference assay.
The results of this work show that sequence-specific DNA binding
of the native AR in cultured CV-1 cells requires the presence of
ligand. In this respect, our data agree with a recent report by Kuil
and Mulder (12), who also used the promoter interference assay to
examine the interaction of AR with a synthetic consensus ARE in Chinese
hamster ovary cells. Our data show further that neither LBD nor a major
part of the NH2-terminal transactivation region of the
receptor is needed for its interaction with specific DNA sequences
in vivo. LBD is not only dispensable but also appears to be
responsible for the ability of apo-AR to interfere weakly with
pCMV-ARE2-CAT expression. In addition, unoccupied LBD fused to GAL4-DBD was capable of repressing transcriptional activity of a
heterologous SV40 promoter at a distance. Should AR bind to AREs as a
homodimer in intact cells, as it does in vitro (8, 9), the
results with rAR641-902 illustrate that this dimerization utilizes
sequences other than those residing in LBD. Moreover, the interaction
between COOH- and NH2-terminal regions of AR, which has
been shown to occur in mammalian cells (31),2 is not
mandatory for the ability of the receptor to recognize specific DNA
elements in vivo, as shown by appropriate deletion mutants
in promoter interference assay. Studies with rAR
46-408 that lacks
most of the NH2-terminal region and with anti-androgens also provided compelling evidence that mere DNA binding in
vivo is not sufficient for the AR to become a competent
transcriptional activator.
The hARM807R mutant is incapable of hormone binding, totally inactive in transactivation assays, and the reason for complete androgen insensitivity of a patient (21). It could be speculated that inappropriate folding of this mutant's LBD may render it free of intracellular chaperones such as heat shock proteins (32), thereby permitting its binding to AREs even in vivo. However, our present data and those of Kuil and Mulder (12) demonstrate that specific DNA binding in living cells is not achieved without a ligand-induced allosteric change in the LBD. The findings that hARM807R binds well to AREs in vitro (21) and is localized in nuclei of transfected cells (this study) are interpreted to mean that the steroid-induced change in LBD conformation is primarily required for the release of AR from associated inhibitory protein complexes, rather than for generation of a receptor form capable of ARE recognition.
The mutant rAR46-408, which has a region mandatory for
transcriptional activation deleted (8), recognized specific DNA sequences in CV-1 cells in a fashion indistinguishable from that of the
wild-type receptor, implying that the NH2 terminus is not essential for this interaction. This result is in agreement with our
previous studies on AR-ARE interaction in vitro, in that
NH2-terminal internal deletions compromised the affinity of
rAR for AREs only to a moderate extent; the KD of
interaction increased from 0.5-1.0 nM to 3.0 nM (8, 9). AR protein produced in insect cells has been
suggested to require intracellular hormone exposure to overcome the
inhibition imposed by the NH2-terminal domain on
dimerization and DNA binding of the aporeceptor (10). The present
experiments showed, however, that unliganded rAR
46-408 was
incapable of sequence-specific DNA binding in CV-1 cells (Fig. 4B), implying that a major part of the
NH2-terminal region is not critical for maintaining the
receptor in a non-DNA-binding form.
Anti-androgens that were examined in this work can be divided into two groups on basis of their ability to modulate the binding of AR to AREs. The first group is not capable of converting the receptor to a form that binds to specific DNA sequences in CV-1 cells, or it does this poorly. When present concomitantly with an agonist, these compounds inhibit the agonist-dependent promoter interference. Non-steroidal anti-androgens casodex and hydroxyflutamide belong to this group. The second group includes the steroidal anti-androgen cyproterone acetate and three N-substituted arylthiohydantoin anti-androgens (RU 56187, RU 57073 and RU 59063). These compounds, at least at the concentrations used, converted AR to a form capable of ARE binding in CV-1 cells. Moreover, the compounds of the second group behaved as partial agonists/antagonists in transactivation experiments. Kuil and Mulder (12) have also reported that in Chinese hamster ovary cells, casodex and hydroxyflutamide inhibited DNA binding of AR, but cyproterone acetate failed to do so.
Ligand-free human and rat ARs exhibited weak promoter-interfering activity. In this respect, our results on AR differed form those reported by Reese and Katzenellenbogen (14) and Xing and Shapiro (15) on human and frog ERs, respectively. In these latter cases, unoccupied wild-type ER showed moderate to high interfering activity, and some ligand-free ER mutants were even more potent than the estrogen-bound wild-type ER (14, 15). Likewise, apo-PR elicited strong interference with pCMV-PRE-CAT expression, which was almost 50% of that of the progestin-bound PR (16). The observed differences in the behavior of nuclear receptors in promoter interference assays could result, at least in part, from dissimilar affinities of their LBDs for auxiliary proteins communicating with transcription machinery. A COOH-terminal region of PR has recently been reported to contain a transcriptional repressor domain that functions through a putative corepressor (33); however, a similar function has not so far been described for the COOH terminus of AR. The use of a heterologous GAL4 system supported the notion that LBD is responsible for the weak ligand-independent promoter-interfering activity of AR. The activity of this LBD in the context of GAL4 fusion protein was not dependent on the ligand-induced conformational change; rather, ligand-free GAL4-LBD bound to elements upstream of SV40 promoter exhibited stronger repressive action than the ligand-occupied form, suggesting that apo-LBD is the favored conformation in recruitment of putative repressor proteins.
In conclusion, the results of this work indicate that interaction of AR with specific DNA sequences in CV-1 cells does not involve COOH-terminal or NH2-terminal regions of the receptor, even though the NH2-terminal domain contains sequences required for transcriptional activation. In the context of wild-type AR, the androgen-induced conformational change in the COOH terminus is, however, mandatory for generation of a transcriptionally competent receptor protein that binds to ARE sequences in intact cells, even though mere DNA binding in vivo is not sufficient for the AR to acquire competence in transcriptional activation. And finally, LBD appears to be responsible for the weak promoter-interfering activity of apo-AR.
We thank Dr. Ismo Virtanen for help in immunocytochemistry and Drs. Benita Katzenellenbogen and Norbert Lehming for plasmids.