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(Received for publication, June 8, 1995; and in revised form, September 20, 1995) From the
Domain interactions of the human androgen receptor (AR) dimer
were investigated using a protein-protein interaction assay in which
the NH
The androgen receptor (AR) ( Steroid receptor dimerization is well documented (16, 17, 18, 19) and apparently does
not have a strict requirement for ligand or DNA binding, particularly
with the estrogen (ER) (20, 21, 22, 23, 24) and
progesterone receptors(25, 26) . The glucocorticoid
receptor forms ligand-dependent homodimers independent of DNA binding (27) . A mutant progesterone receptor lacking the
steroid-binding domain fails to dimerize in solution but activates a
reporter gene, suggesting that receptor dimerization mediated through
the steroid-binding domain is not a requirement for DNA binding and
that dimerization after DNA binding is mediated by the DNA-binding
domain(28) . Direct evidence for receptor dimerization was
revealed in electron micrographs showing dumbbell shaped glucocorticoid
receptor monomers with globular NH Numerous recent
studies have taken advantage of a protein-protein interaction assay
developed originally in yeast (34) and later adapted for
mammalian cells (35) that relies on the coexpression of two
fusion proteins, each containing a protein or protein region coupled to
a transcription factor functional domain. Stable protein-protein
interactions bring together DNA binding and transactivation functions
that regulate a reporter gene. Using this assay, we demonstrate an
androgen-dependent interaction between the AR NH
Androgen
binding was determined in COS and CHO cells using a whole cell binding
assay (38) for vectors comprised of exons D-H (residues
624-919)(42) . COS cells in 12-well (10
Figure 1:
Schematic
representation of GALD-H and VP-A1 chimeric vectors. AR exons D-H
coding for residues 624-919 were inserted 3` into pGALO
containing GAL4 protein DNA-binding domain amino acid residues
1-147. AR DNA sequence coding for residues 1-503 was cloned
into pNLVP 3` of the herpes simplex virus VP16 protein transcriptional
activation sequence coding for residues 411-456. Reporter vector
G5E1bLuc has five GAL4-binding sites, the E1b promoter, and the
luciferase coding sequence.
GALD-H and
VPD-H comprise the GAL4 DNA-binding and VP16 transactivation domains,
respectively, fused NH GAL-A1 and VP-A1
comprise the GAL4 DNA-binding and VP16 transactivation domains,
respectively, fused NH
Figure 2:
Immunoblots of wild-type and deletion
mutant VP-A1 chimeras expressed in CHO and COS cells. Immunoblots were
performed as described under ``Experimental Procedures''
where the VP-A1 expression vectors (5 µg) were transfected into CHO (A) or COS (B) cells. The blot was probed with
antibodies AR32 (40) and AR PG-21 (41) raised against
AR NH
Figure 3:
Luciferase activity induced by the
chimeric vectors. Combinations of the indicated parent and chimeric
expression vectors (1 µg DNA/0.45
Only
a 2-fold induction of luciferase activity was observed using the
reciprocal chimeras GAL-A1 and VPD-H (Fig. 3), suggesting a
preferential orientation for fusion protein interaction. GAL-A1 alone
activated luciferase expression (Fig. 3) presumably resulting
from linking the AR transactivation domain to the GAL-4 DNA-binding
domain, thus increasing background activity in the
NH
Figure 4:
Luciferase activity induced by wild-type
and LNCaP mutant GALD-H and VP-A1 with different androgens and the
antiandrogen hydroxyflutamide. GALD-H or GAL-LNCaPD-H with VP-A1 (1
µg of plasmid DNA each) and the G5E1bLuc reporter vector (5 µg)
were cotransfected into CHO cells and incubated with 1 nM DHT,
R1881, testosterone (T), and/or increasing concentrations of
hydroxyflutamide (OHFL) as indicated. The GAL-LNCaPD-H mutant
contains a Thr-877
Because hydroxyflutamide is a potent
antiandrogen, we investigated whether it would disrupt the
NH
Figure 5:
Effects of estradiol, progesterone, and
cyproterone acetate on the GALD-H and GAL-LNCaPD-H interaction with
VP-A1. VP-A1 and GALD-H containing wild-type or LNCaP mutant AR
sequence were cotransfected into CHO cells with the reporter vector,
G5E1bLuc, as described under ``Experimental Procedures'' and
incubated with 0.5 µM 17
Figure 6:
Effect of AR NH
Figure 7:
Effect of AR NH
The
NH
The objective of the present study was to establish whether a
direct interaction occurs between the NH Although the present data do not rule out
that the ligand-induced NH The two regions of the AR NH The affinity of the NH Human AR and ER differ in
the length of their NH
Volume 270,
Number 50,
Issue of December 15, 1995 pp. 29983-29990
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and carboxyl-terminal regions of human AR were
fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain
and herpes simplex virus VP16 transactivation domain to produce
chimeric proteins. Transcriptional activation of a GAL4 luciferase
reporter vector up to 100-fold was greater than Fos/Jun leucine zipper
binding, indicating stable AR interaction between AR
NH-terminal residues 1-503 and steroid-binding domain
residues 624-919 that was specific for and dependent on androgen
binding to the steroid-binding domain and was inhibited by
anti-androgen binding. Deletion mutagenesis within the
NH-terminal region indicated transactivation domain
residues 142-337 were not required for dimerization, whereas
deletions near the NH terminus (
14-150) or
NH-terminal to the DNA-binding domain (339-499)
reduced or eliminated the AR interaction, respectively. An
NH-/NH-terminal interaction was also observed,
but no interaction was detected between ligand-free or bound
steroid-binding domains. The results indicate that high affinity
androgen binding promotes interactions between the
NH-terminal and steroid-binding domains of human AR,
raising the possibility of an androgen-induced anti-parallel AR dimer.
)is a ligand-activated
transcription factor that requires high affinity androgen binding to
initiate a series of molecular events leading to specific gene
activation required for male sex development. In its unliganded state,
AR resides in the cytoplasm(1) , where it rapidly degrades (2) and is regulated by a cytoplasmic dnaJ
homologue(3) . High affinity androgen binding slows AR
degradation in a concentration-dependent manner, accounting at least in
part for the physiological differences between the biologically active
androgens(4) . Androgen binding activates a bipartite nuclear
targeting signal (5) and triggers receptor dimerization and
acquisition of DNA binding that involves distal regions of the
AR(6) . Once activated, AR binds androgen response elements
that resemble the simple consensus glucocorticoid response element (7) or more distinct, specific complex response
elements(8, 9, 10) . Little is known,
however, about transcription factors that interact with AR during gene
activation or the role of AR phosphorylation(11) . That AR is
crucial for specific gene regulation required for male sex development
is demonstrated by an abundance of AR gene mutations that result in
different degrees of impaired male sex development characteristic of
the androgen insensitivity
syndrome(12, 13, 14, 15) . -terminal and
steroid-binding domains and four-leaf clover shaped dimers. It was not
established, however, whether dimer orientation was parallel or
anti-parallel(29) . Additional protein-protein interactions and
alterations in DNA structure are indicated by increased gene activation
following cooperative dimer binding to tandem hormone response
elements(30) . ER dimerization involves hydrophobic
interactions between the steroid-binding domains (31) and, for
thyroid hormone receptor 
, regions outside the steroid-binding
domain(32) . In addition, a leucine zipper-like structure in
the thyroid receptor ligand-binding domain mediates heterodimerization
with the retinoic acid receptor(33) .- and
carboxyl-terminal domains that raises the possibility of an
anti-parallel oriented AR dimer.
Materials
The following reagents were
purchased. Monkey kidney COS-1 and Chinese hamster ovary (CHO) cells
were from the American Type Culture Collection (Rockville, MD);
minimum essential medium and prestained protein molecular weight
standards were from Life Technologies, Inc.; Dulbecco's modified
essential medium with high glucose with or without phenol red was from
JRH Biosciences; fetal calf serum was from Hyclone Laboratories (Logan,
UT), and bovine calf serum was from Irvine Scientific (Santa Ana, CA);
AR PG-21 rabbit polyclonal AR anti-peptide antiserum was from Affinity
Bioreagents, Inc. (Neshanic Station, NJ);
[
H]methyltrienolone
[17-methyl-H]R1881, 80 Ci/mmol was from
DuPont NEN; hydroxyflutamide was provided by R. O. Neri (Schering
Corporation, Bloomfield, NJ); deep vent polymerase, T4 DNA ligase, and
restriction enzymes were from New England Biolabs (Beverley, MA);
Sequenase was from U. S. Biochemical Corp.; diethylaminoethyl dextran
was from Pharmacia Biotech Inc.; D-luciferin was from
Analytical Luminescence; cell lysis buffer was from Ligand
Pharmaceuticals (San Diego, CA); phenylmethylsulfonyl fluoride and
general chemicals were from Sigma; ECL Western blotting detection kit
was from Amersham Corp.; and Immobilon-P was from Millipore (Bedford,
MA).Expression Plasmids
Eukaryotic expression
vectors pGALO containing the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein (amino acid residues
1-147) and pNLVP coding for the transcriptional activation domain
of the herpes simplex virus VP16 protein (residues 411-456) were
previously described (36) and kindly provided by Gordon
Tomaselli, Johns Hopkins University. Expression vectors contain the
SV40 promoter, nuclear targeting signals, and a 3` multiple cloning
site. Control plasmid pGAL-Fos contains the Fos leucine zipper region
(amino acid residues 137-216) and pVP-Jun, the Jun leucine zipper
region (residues 250-334) as described previously (36) and kindly provided by Gordon Tomaselli and Chi V. Dang,
Johns Hopkins University. Full-length human AR was cloned in frame in
both vectors using polymerase chain reaction (PCR) mutagenesis to place
an NdeI site at the initiation codon. A triple ligation
reaction was performed with the NdeI/AflII
NH-terminal AR PCR fragment, the AflII/XbaI carboxyl-terminal AR fragment from
pCMVhAR(1) , and the NdeI/XbaI fragments of
the pGALO or pNLVP expression vectors. pGAL-A1 (A1 comprising human AR
amino acids 1-503) and pVP-A1 were constructed by deleting
sequence coding for carboxyl-terminal residues 504-919 using KpnI/XbaI followed by ligation of the filled ends.
NH-terminal deletion and insertion mutants
pCMVhAR14-150, pCMVhAR142-337,
pCMVhAR339-499, and pCMVhARGln66 previously described (4) were used to clone the corresponding deletions into the
pGAL-A1 and pVP-A1 vectors or into the full-length pGAL-hAR and
pNLVP-hAR vectors using unique sites in the AR coding sequence to
exchange the deletion fragment or, in the case of the 14-150
deletion, using PCR to include the NdeI site at the initiation
codon. pGALD-H (human AR amino acids 624-919; 624-627 from
exon C, 628-919 exons D-H) was constructed using PCR to
create an NdeI site at methionine 624 and was cloned into the NdeI/XbaI sites in the polylinker of pGALO.
pGAL-LNCaPD-H was constructed in the same manner using pCMV-LNCaP AR (37) as template for PCR. Plasmids were amplified using
DH5 E. coli cells and DNA isolated by banding in CsCl.
Fusion genes created using PCR were sequenced using Sequenase to verify
the absence of random mutations.Cell Culture and Transient
Transfections
CHO cells were maintained in minimum
essential medium containing 10% fetal calf serum, 20 mM Hepes,
pH 7.2, and penicillin and streptomycin. CHO cells at 50% confluence
were transiently transfected with expression and reporter plasmids
using DEAE-dextran as described previously(38) . Unless
otherwise stated for the protein-protein interaction assay 1 µg
each of the GAL4 and VP16 fusion protein plasmids and 5 µg of
reporter plasmid G5E1bLuc (provided by Gordon Tomaselli) were
cotransfected into CHO cells (0.4 
10
cells/60-mm
dish). The reporter vector had five tandem copies of the GAL4-binding
sites, the E1bTATA promoter as described previously(39) , and
the luciferase coding sequence. Immediately after transfection, cells
were placed in 0.2% serum-containing medium and 24 h later into
serum-free, phenol red-free medium in the presence or the absence of
steroid. Medium with or without hormone was replaced 3 h prior to
harvest. Plates were washed with phosphate-buffered saline, harvested
in 0.5 ml of cell lysis buffer, and frozen at -80 °C until
assayed for luciferase activity. Lysates were supplemented with 8
mM MgCl, 1 mM dithiothreitol, and 0.4
mM phenylmethylsulfonyl fluoride. Relative light units were
determined using a monolight 2010 Analytical Luminescence Laboratory
luminometer after combining 0.4 ml of reaction buffer (15 mM MgCl, 5 mM ATP, 0.5 mg/ml bovine serum
albumin, and 15 mM glycylglycine, pH 7.8), 0.1 ml of cell
lysate, and 0.1 ml of 1 mMD-luciferin injected
automatically. All experiments were performed in duplicate and repeated
at least three times.Immunoblots and Binding Assays
Expression
of GAL4- and VP16-AR fusion proteins was assessed on immunoblots and by
whole cell steroid binding assays. Monkey kidney COS-1 cells were grown
in Dulbecco's modified essential medium supplemented with 4.5
g/liter glucose and L-glutamine, 10% fetal calf serum, 20
mM Hepes, pH 7.2, and antibiotics. For immunoblot analysis,
COS cells plated in 60-mm dishes (0.45 10 cells/dish) were transiently transfected with 5 µg of
wild-type or mutant AR chimeric expression plasmids using
DEAE-dextran(38) . Cells were maintained in Dulbecco's
modified essential medium with 4.5 g/liter glucose and L-glutamine with 10% bovine calf serum and, for cells
transfected with plasmids expressing the AR steroid-binding domain,
supplemented with 0.1 µM dihydrotestosterone. 48 h after
transfection, cells were washed in phosphate-buffered saline and
harvested in 0.1 ml of 2% SDS, 10% glycerol, and 10 mM Tris,
pH 6.8. 
-Mercaptoethanol (4%) and bromphenol blue (1%) were added,
and the samples were boiled for 5 min. 20-µl aliquots were analyzed
in SDS containing 8% acrylamide gels as described previously using AR
anti-peptide rabbit antiserum AR32 (40) or AR PG-21 (41) and the ECL immunoblotting detection kit.
cells/well) or 35-mm (0.25 10 cells/plate for
Scatchard plot analysis) tissue culture plates were transiently
transfected using DEAE-dextran with 1 µg of GAL4 or VP16 fusion
plasmids containing wild-type or LNCaP AR steroid-binding domain. Cells
were maintained for 48 h in Dulbecco's modified essential medium
with 4.5 g/liter glucose and L-glutamine media containing 10%
bovine calf serum and labeled for 2 h at 37 °C with 5 nM [H]R1881 in serum-free, phenol red-free
medium in duplicate. For Scatchard analysis, cells were incubated with
0.05-4 nM [H]R1881 in the presence
and the absence of a 100-fold excess of unlabeled R1881 for 2 h at 37
°C. Aliquots of free [H]R1881 were taken, and
the cells were washed in phosphate-buffered saline and collected in SDS
sample buffer for scintillation counting. Nonspecific binding was
determined by parallel incubations in the presence of a 100-fold excess
unlabeled R1881. Labeling medium was removed, and cells were washed
twice with phosphate-buffered saline and harvested in 0.2 ml of 2% SDS,
10% glycerol, and 10 mM Tris, pH 6.8. Radioactivity was
determined by scintillation counting.
Expression of GAL4-AR and VP16-AR Fusion
Proteins
Intermolecular interactions within the AR dimer
were investigated using a protein-protein interaction assay in
mammalian cells. Fusion protein expression vectors contained partial AR
sequence cloned in frame and carboxyl-terminal to the S. cerevisiae GAL4 DNA-binding domain or the herpes simplex virus VP16 protein
transactivation domain (shown schematically in Fig. 1).
Activation of the GAL4-luciferase reporter vector results when regions
of AR interact stably after expression of the chimeras in CHO cells.
GAL-Fos and VP-Jun vectors expressing Fos and Jun leucine zipper fusion
proteins (36) were used as a positive control.
-terminal to AR steroid-binding
domain residues 624-919. Epitopes for AR antibodies were not
present in this region, so these fusion proteins were quantitated by
ligand binding after expression in CHO and COS cells. GALD-H and VPD-H
displayed high affinity (K
= 0.11 ±
0.02 nM) saturable binding of the synthetic androgen
[H]R1881. Equilibrium binding affinity was
indistinguishable from full-length wild-type AR; however, these
fragments would be expected to have increased ligand dissociation rates
based on results with AR deletion mutants lacking the
NH-terminal domain(4) . Expression levels were
similar for the two constructs and were approximately 10-fold greater
in COS cells than in CHO cells (data not shown).-terminal to AR
NH-terminal residues 1-503 (A1), and express at
similar levels as 90-95-kDa proteins on immunoblots using an AR
anti-peptide antibody, shown for VP-A1 in Fig. 2(lane
1). Deletions within the NH-terminal region resulted
in correspondingly smaller proteins expressed either in CHO and COS
cells (Fig. 2, lanes 2-4), whereas expansion of
the glutamine repeat from 21 to 66 residues resulted in a slightly
larger peptide (Fig. 2B, lane 5). All were
smaller than the 120-kDa full-length, wild-type AR (Fig. 2, lane 6).
-terminal peptides. Molecular mass standards were
analyzed in parallel for gel calibration. Shown are VP-A1 (lane
1), VP-A114-150 (lane 2),
VP-A1142-337 (lane 3), VP-A1339-499 (lane 4), VP-A1Gln66 (B, lane 5), pCMVhAR wild-type
human AR expression vector (lane 6), and the parent pCMV5
vector lacking AR sequence (lane
7).
Androgen-dependent Interaction between the AR
Steroid-binding and NH
In the absence of androgen, luciferase activity was
negligible when cotransfecting GALD-H with VP-A1 (Fig. 3).
Treatment with 1 nM DHT-induced luciferase activity greater
than that observed with the Fos/Jun leucine zipper chimeras relative to
background activity (Fig. 3). Similar results were obtained
using COS cells for transfection (data not shown); however, CHO cells
were used in subsequent experiments due to lower background activity.
No significant transactivation was observed in CHO cells in the
presence of androgen when either plasmid was transfected with the other
parent plasmid lacking AR sequence (Fig. 3). The results
indicate an androgen-dependent stable interaction between the AR
steroid-binding domain and the NH-terminal
Domains-terminal region.
10 CHO cells)
were cotransfected into CHO cells together with 5 µg of G5E1bLuc
reporter vector. Luciferase activity is expressed as optical units,
where fold induction reflects the ratio of activity determined in the
presence and the absence of 1 nM DHT or the ratio of activity
of the interacting vectors over the GAL chimera alone. GAL-Fos and
VP-Jun containing the leucine zipper regions as described under
``Experimental Procedures'' served as a positive
control.
-/carboxyl-terminal interaction using this construct.
Cotransfecting GALD-H and VPD-H both containing the AR steroid-binding
domain failed to activate the reporter vector in the presence or
absence of androgen (Fig. 3). Transfecting GAL-A1 with VP-A1
increased activation 4-5-fold over the activity of GAL-A1 alone (Fig. 3), indicating a ligand-independent interaction between
the AR NH-terminal domains. The high transcriptional
activity induced by GAL-A1 with VP-A1 was likely due in part to the
presence of three transactivation domains, two from the AR
NH-terminal domains and one from VP-16. The lower fold
induction by the NH-/NH-terminal interaction
may therefore reflect a weaker interaction than that observed for the
androgen-induced NH-/carboxyl-terminal interaction
(4-5- versus 59-fold; Fig. 3).Steroid Specificity and Concentration
Dependence
Steroid specificity of the GALD-H and VP-A1
interaction was investigated by determining transcriptional activation
in the presence of androgens, the antiandrogens hydroxyflutamide and
cyproterone acetate, and other steroids. The strongest transcriptional
activation was observed at 1 nM DHT and between 1 (Fig. 4) and 10 nM R1881 or testosterone.
Hydroxyflutamide up to 1 µM failed to activate luciferase
activity (Fig. 4).
Ala mutation in the steroid-binding domain.
Fold induction, shown above the error bars, was determined
from the ratio of activity in the presence and absence of ligand. A
representative of three experiments is
shown.
-/carboxyl-terminal interaction in the presence of
androgen. Increasing concentrations of hydroxyflutamide between
0.2-1 µM inhibited androgen-induced gene activation (Fig. 4). Estradiol, progesterone, and cyproterone acetate
failed to induce luciferase activity and at 0.5 µM inhibited transcriptional activation induced by 1 nM DHT (Fig. 5). The results indicate that the
NH-/carboxyl-terminal interaction induced by androgens is
blocked by moderate affinity ligands such as hydroxyflutamide,
progesterone, and estradiol, paralleling the activation and inhibition
properties of these ligands with wild-type, full-length AR.
-estradiol (E),
progesterone (P), and cyproterone acetate (CA) in the
absence or the presence of 1 nM DHT as indicated. Shown are
optical units and fold induction relative to the activity determined in
the absence of ligand. Shown is a representative of three independent
experiments.
Activity of the LNCaP AR Mutant
It was
shown previously that the mutant AR in the androgen-dependent human
prostate cancer cell line, LNCaP, contains a single base mutation in
the steroid-binding domain that changes threonine 877 to alanine that
results in increased affinity for hydroxyflutamide with concomitantly
increased agonist activity(43, 44, 45) . We
inserted the LNCaP Thr-877 Ala mutation into GALD-H and tested
for the NH
-terminal/steroid-binding domain interaction.
Whole cell binding assays using [H]R1881 showed
similar binding affinities for GALD-H and GAL-LNCaPD-H (data not
shown). In the presence of 1 nM DHT, greater than 100-fold
induction of luciferase activity indicated an
NH-/carboxyl-terminal interaction similar to wild-type AR (Fig. 4). At 1 µM, hydroxyflutamide-induced
luciferase activity almost 10-fold and was a less active inhibitor of
androgen-induced complex formation, with luciferase activity remaining
approximately 10-fold (Fig. 4). This result is in agreement with
the known agonist activity of hydroxyflutamide acquired by the LNCaP
mutation(43, 44, 45) . Estradiol and
progesterone similarly induced luciferase activity by GAL-LNCaPD-H and
VP-A1, whereas no detectable activity was observed with these steroids
with the wild-type GALD-H and VP-A1 chimeras (Fig. 5).
Cyproterone acetate did not induce luciferase activity of the wild-type
or LNCaP mutant chimeras. All three ligands, estradiol, progesterone,
and cyproterone acetate, at 0.5 µM were less active
inhibitors of DHT-induced luciferase activity of GAL-LNCaPD-H and VP-A1
relative to wild-type GALD-H and VP-A1 chimeras (Fig. 5). Thus,
although the magnitude of the interaction was 5-10-fold less than
that observed with DHT, the NH-/carboxyl-terminal
interaction induced by hydroxyflutamide, estradiol, and progesterone
with the threonine 877 to alanine mutation correlated with the agonist
activities of these ligands with the full-length LNCaP mutant AR.NH
To characterize the dimerization region
within the AR NH-terminal Deletions Define the
Dimerization Domain-terminal domain, PCR mutagenesis was used
to create deletions within VP-A1 (shown schematically in Fig. 6and Fig. 7) for cotransfection with GALD-H,
GAL-LNCaPD-H, and GAL-A1. Expression levels of the deletion mutants
were similar as determined by immunoblotting (see Fig. 2).
Deletion of the AR transactivation region (VP-A1142-337)
resulted in 14-fold stimulation of luciferase activity (Fig. 6),
representing a significant decrease relative to the wild-type sequence.
Because the AR transactivation domain likely contributes to luciferase
induction, it is possible that the decrease reflects deletion of the AR
activating region rather than a decrease in dimerization. Deletion
immediately NH-terminal to the DNA-binding domain
(VP-A1339-499) abolished luciferase activity (Fig. 6)
even after a 5-fold increase in plasmid concentration (data not shown).
Deletion near the NH terminus (VP-A114-150) also
inhibited transcriptional activation, with 2-5-fold induction of
luciferase activity using a 5-fold higher plasmid DNA concentration (Fig. 6). Expansion of the NH-terminal polymorphic
glutamine repeat from 21 (residues 58-78 in wild-type AR) to 66
glutamine residues (VP-A1Gln66) had little effect on transcriptional
activation of luciferase (Fig. 6). Expansion of the glutamine
repeat is associated with spinal/bulbar muscular atrophy
(Kennedy's disease)(46) , and thus, the amplified repeat
does not interfere with this aspect of AR dimerization, in agreement
with its wild-type level of activation. ()Results using the
NH-terminal deletion and insertion mutants with the
GAL-LNCaPD-H mutant were essentially identical to those with wild-type
GALD-H fusion protein (data not shown).
-terminal
deletions on VP-A1 interaction with GALD-H. Several mutants with
portions of the AR NH-terminal domain deleted, including
VP-A114-150 (5 µg), VP-A1142-337 (1 µg),
VP-A1339-499 (1 µg), VP-A1Gln66 (1 µg), or VP-A1 (1
µg), were cotransfected with GALD-H (1 µg). The expanded
glutamine repeat replaces 21 Gln residues with 66 Gln residues
identified in a patient with spinal/bulbar muscular
atrophy(52) . Relative luciferase activities are shown for
GALD-H cotransfected in CHO cells with wild-type and deletion mutants
of VP-A1 in the absence and presence of 1 nM DHT. Amino acid
residues deleted from AR are indicated by . Shown are optical
units and fold induction relative to activity in the absence of DHT.
GAL-Fos cotransfected with VP-Jun was a positive control and the parent
vector VP16 lacking AR sequence cotransfected with GALD-H served as
negative controls. The data shown are representative of three
independent experiments.
-terminal
deletions on the VP-A1/GAL-A1 interaction. The VP-A1 mutant vector DNAs
described in legend to Fig. 6were cotransfected with GAL-A1,
and luciferase activity was determined as described in the legend to Fig. 6. The data shown are representative of three
experiments.
-/NH-terminal interaction was investigated
using the VP-A1 deletion mutants described above. Inhibition was
observed after deleting transactivation domain residues 142-337
and to a lower extent by deletion of residues 339-499 or
14-150 (Fig. 7). Expanding the glutamine repeat to 66
residues as described above slightly enhanced the interaction. Thus,
different regions appear to be involved in the
NH-/NH-terminal interaction than in
NH-terminal interaction with the androgen-bound
steroid-binding domain.Smaller Domains within the Interacting
Regions
Insertion of shorter regions of AR coding sequence
into GAL4 and VP16, such as NH-terminal fragments
1-150 or 339-503 linked to VP16, failed to activate the
reporter plasmid when cotransfected with GALD-H and analyzed in the
presence of androgen (data not shown). Similarly, subdividing the
steroid-binding domain into regions encoded by exons D-E
(residues 624-780) and F-H (residues 774-919)
resulted in no reporter vector activation. The results suggest that the
sites of interaction localize to larger domains that may involve
concerted actions within the NH-terminal and entire
steroid-binding domains, with the latter required for androgen binding.
-terminal and
steroid-binding domains in androgen-induced AR dimer formation. Domain
interactions were analyzed by reporter gene activation using fusion
proteins that linked the NH- and carboxyl-terminal regions
of human AR to the GAL4 DNA-binding or VP16 transactivation domains.
The results support previous evidence that in vivo dimerization of human AR is mediated through direct intermolecular
interactions between the androgen-bound steroid-binding domain and the
NH-terminal region. The dependence on androgen binding and
inhibition by an antiandrogen and other steroids parallels properties
of native AR and raises the possibility of an androgen-activated,
anti-parallel AR dimer.-/carboxyl-terminal interaction
occurs intramolecularly, previous studies using baculovirus-expressed
AR fragments support a ligand-induced intermolecular interaction.
Androgen-dependent dimerization was observed between
NH-terminal plus DNA-binding domain and DNA plus
steroid-binding domain fragments(6) . However, as the
DNA-binding domain is implicated in receptor dimerization through the
so-called D box region (47, 48, 49) with the
other monomer(50, 51) , the DNA-binding domain could
have accounted for the observed AR dimerization. In the present study,
the AR DNA-binding domain, which itself dimerizes, ()was
excluded from the chimeric proteins, indicating an additional
dimerization interface between the NH-terminal and
androgen-bound steroid-binding domain of AR. Further support for AR
NH- and carboxyl-terminal interactions comes from kinetic
studies where the dissociation rate of bound androgen slows about
5-fold by the presence of the NH-terminal domain despite no
change in equilibrium dissociation constant(4) .
Crystallographic data of glucocorticoid receptor/DNA interactions (50) and the asymmetric dimer proposed for the vitamin D
receptor (53) suggest a ligand-activated anti-parallel dimer
may be the active conformation for other members of the steroid
receptor family.-terminal
domain required most for carboxyl-terminal interaction were immediately
NH-terminal to the DNA-binding domain and near the NH terminus. Lack of a direct role of the more centrally positioned
transactivation domain might allow this region to remain accessible for
transcription factor interaction. In our unpublished studies and the
work of others(54) , deletion of the transactivation domain
creates a strong dominant negative AR inhibitor, suggesting that loss
of the transactivation domain does not interfere with receptor
dimerization. It is interesting, therefore that this region or the
region NH-terminal to the DNA-binding domain (residues
142-337 and 339-499, respectively) may be involved in an
interaction between the NH-terminal domains and may reflect
an association that occurs in the unliganded receptor that could
contribute to suppression of activation in the absence of ligand. -/carboxyl-terminal interaction
appears to be similar to that observed for Fos-Jun leucine zipper
binding. When the Fos/Jun leucine zipper regions were fused to
progesterone receptor, agonist-induced progesterone receptor
dimerization persisted through the receptor dimerization domain,
suggesting that ligand-induced receptor dimerization was of equal or
greater affinity than the Fos/Jun leucine zipper
interaction(55) . Leucine zipper motifs are often involved in
transcription factor dimerization resulting in efficient DNA
binding(56) . A heptad repeat of hydrophobic amino acid
residues in the steroid-binding domain of mouse ER resembles a leucine
zipper, is conserved among the family of steroid receptors, and is
implicated in dimerization and high affinity estrogen
binding(20) . Like ER(57) , other steroid receptors
appear to have two dimerization interfaces: a constitutive region in
the DNA-binding domain and a stronger, hormone-dependent region in the
hormone-binding domain that may be involved in stable dimer formation
required for high affinity DNA binding. The carboxyl-terminal end of
the thyroid hormone receptor was also implicated in receptor
dimerization(58, 59) .-terminal domains, i.e. 559
amino acid residues in AR versus 185 in ER. It is noteworthy,
therefore, that a transcriptionally inactive AR deletion mutant
AR507-919, lacking all but 52 NH-terminal amino acid
residues(4) , dimerizes and binds DNA independent of ligand
binding (6) as observed with full-length ER (20, 21, 22, 23) but not full-length
AR(6) . The androgen-independent dimerization of this AR
deletion mutant suggests two forms of DNA-binding homodimers: one for
the AR deletion mutant AR507-919 and perhaps ligand free ER and
another for androgen-bound full-length AR and perhaps ligand-bound ER.
A parallel dimer capable of binding DNA may form constitutively through
interactions between the DNA-binding domains if no extended
NH-terminal region interferes, in the case of ER and the AR
deletion mutant. However, the active configuration requiring ligand
binding for full-length AR might be anti-parallel and depends on the
presence of the NH-terminal domain. This hypothesis is
supported by studies using glucocorticoid receptor deletion mutants,
where deletion of the NH-terminal domain changed the
contact points within the dimer in cross-linking studies and reduced
the specificity of DNA binding(60) . Androgen-induced
conformational effects on full-length AR that might establish the
anti-parallel dimer may be required for DNA binding that results in
transcriptional activation.
),,-trifluoro-2-methyl-4`-nitro-m-lactotoluidide.))
We are grateful for the technical assistance of Jon A.
Kemppainen and Michelle Cobb and thank Frank S. French for reading the
manuscript, and Gordon Tomaselli and Chi V. Dang for plasmids.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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J. Lim, F. J. Ghadessy, A. A. R. Abdullah, L. Pinsky, M. Trifiro, and E. L. Yong Human Androgen Receptor Mutation Disrupts Ternary Interactions between Ligand, Receptor Domains, and the Coactivator TIF2 (Transcription Intermediary Factor 2) Mol. Endocrinol., August 1, 2000; 14(8): 1187 - 1197. [Abstract] [Full Text]
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H. Fuse, H. Kitagawa, and S. Kato Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1) Mol. Endocrinol., June 1, 2000; 14(6): 889 - 899. [Abstract] [Full Text]
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S.-M. Huang and M. R. Stallcup Mouse Zac1, a Transcriptional Coactivator and Repressor for Nuclear Receptors Mol. Cell. Biol., March 1, 2000; 20(5): 1855 - 1867. [Abstract] [Full Text]
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T. J. Schrader and G. M. Cooke Examination of Selected Food Additives and Organochlorine Food Contaminants for Androgenic Activity in Vitro Toxicol. Sci., February 1, 2000; 53(2): 278 - 288. [Abstract] [Full Text] [PDF]
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R. A. Irvine, H. Ma, M. C. Yu, R. K. Ross, M. R. Stallcup, and G. A. Coetzee Inhibition of p160-mediated coactivation with increasing androgen receptor polyglutamine length Hum. Mol. Genet., January 22, 2000; 9(2): 267 - 274. [Abstract] [Full Text] [PDF]
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J.-a. Tan, S. H. Hall, K. G. Hamil, G. Grossman, P. Petrusz, J. Liao, K. Shuai, and F. S. French Protein Inhibitor of Activated STAT-1 (Signal Transducer and Activator of Transcription-1) Is a Nuclear Receptor Coregulator Expressed in Human Testis Mol. Endocrinol., January 1, 2000; 14(1): 14 - 26. [Abstract] [Full Text]
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B. He, J. A. Kemppainen, J. J. Voegel, H. Gronemeyer, and E. M. Wilson Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain J. Biol. Chem., December 24, 1999; 274(52): 37219 - 37225. [Abstract] [Full Text] [PDF]
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C. L. Bevan, S. Hoare, F. Claessens, D. M. Heery, and M. G. Parker The AF1 and AF2 Domains of the Androgen Receptor Interact with Distinct Regions of SRC1 Mol. Cell. Biol., December 1, 1999; 19(12): 8383 - 8392. [Abstract] [Full Text] [PDF]
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W. Huang, Y. Shostak, P. Tarr, C. Sawyers, and M. Carey Cooperative Assembly of Androgen Receptor into a Nucleoprotein Complex That Regulates the Prostate-specific Antigen Enhancer J. Biol. Chem., September 3, 1999; 274(36): 25756 - 25768. [Abstract] [Full Text] [PDF]
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P. Alen, F. Claessens, G. Verhoeven, W. Rombauts, and B. Peeters The Androgen Receptor Amino-Terminal Domain Plays a Key Role in p160 Coactivator-Stimulated Gene Transcription Mol. Cell. Biol., September 1, 1999; 19(9): 6085 - 6097. [Abstract] [Full Text] [PDF]
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H. Ma, H. Hong, S.-M. Huang, R. A. Irvine, P. Webb, P. J. Kushner, G. A. Coetzee, and M. R. Stallcup Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins Mol. Cell. Biol., September 1, 1999; 19(9): 6164 - 6173. [Abstract] [Full Text] [PDF]
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M. J. Tetel, P. H. Giangrande, S. A. Leonhardt, D. P. McDonnell, and D. P. Edwards Hormone-Dependent Interaction between the Amino- and Carboxyl-Terminal Domains of Progesterone Receptor in Vitro and in Vivo Mol. Endocrinol., June 1, 1999; 13(6): 910 - 924. [Abstract] [Full Text]
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X. Sui, K. S. Bramlett, M. C. Jorge, D. A. Swanson, A. C. von Eschenbach, and G. Jenster Specific Androgen Receptor Activation by an Artificial Coactivator J. Biol. Chem., April 2, 1999; 274(14): 9449 - 9454. [Abstract] [Full Text] [PDF]
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J. A. Kemppainen, E. Langley, C.-i. Wong, K. Bobseine, W. R. Kelce, and E. M. Wilson Distinguishing Androgen Receptor Agonists and Antagonists: Distinct Mechanisms of Activation by Medroxyprogesterone Acetate and Dihydrotestosterone Mol. Endocrinol., March 1, 1999; 13(3): 440 - 454. [Abstract] [Full Text]
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S. A. Leonhardt, M. Altmann, and D. P. Edwards Agonist and Antagonists Induce Homodimerization and Mixed Ligand Heterodimerization of Human Progesterone Receptors in Vivo by a Mammalian Two-Hybrid Assay Mol. Endocrinol., December 1, 1998; 12(12): 1914 - 1930. [Abstract] [Full Text]
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A. Scheller, E. Hughes, K. L. Golden, and D. M. Robins Multiple Receptor Domains Interact to Permit, or Restrict, Androgen-specific Gene Activation J. Biol. Chem., September 11, 1998; 273(37): 24216 - 24222. [Abstract] [Full Text] [PDF]
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C. A. Berrevoets, P. Doesburg, K. Steketee, J. Trapman, and A. O. Brinkmann Functional Interactions of the AF-2 Activation Domain Core Region of the Human Androgen Receptor with the Amino-Terminal Domain and with the Transcriptional Coactivator TIF2 (Transcriptional Intermediary Factor 2) Mol. Endocrinol., August 1, 1998; 12(8): 1172 - 1183. [Abstract] [Full Text]
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E. Langley, J. A. Kemppainen, and E. M. Wilson Intermolecular NH2-/Carboxyl-terminal Interactions in Androgen Receptor Dimerization Revealed by Mutations That Cause Androgen Insensitivity J. Biol. Chem., January 2, 1998; 273(1): 92 - 101. [Abstract] [Full Text] [PDF]
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T. Ikonen, J. J. Palvimo, and O. A. Janne Interaction between the Amino- and Carboxyl-terminal Regions of the Rat Androgen Receptor Modulates Transcriptional Activity and Is Influenced by Nuclear Receptor Coactivators J. Biol. Chem., November 21, 1997; 272(47): 29821 - 29828. [Abstract] [Full Text] [PDF]
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L. L. Heckert, E. M. Wilson, and J. H. Nilson Transcriptional Repression of the {alpha}-Subunit Gene by Androgen Receptor Occurs Independently of DNA Binding but Requires the DNA-Binding and Ligand-Binding Domains of the Receptor Mol. Endocrinol., September 1, 1997; 11(10): 1497 - 1506. [Abstract] [Full Text]
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M. J. Tetel, S. Jung, P. Carbajo, T. Ladtkow, D. F. Skafar, and D. P. Edwards Hinge and Amino-Terminal Sequences Contribute to Solution Dimerization of Human Progesterone Receptor Mol. Endocrinol., July 1, 1997; 11(8): 1114 - 1128. [Abstract] [Full Text]
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U. Karvonen, P. J. Kallio, O. A. Janne, and J. J. Palvimo Interaction of Androgen Receptors with Androgen Response Element in Intact Cells. ROLES OF AMINO- AND CARBOXYL-TERMINAL REGIONS AND THE LIGAND J. Biol. Chem., June 20, 1997; 272(25): 15973 - 15979. [Abstract] [Full Text] [PDF]
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Z. Zhou, J. L. Corden, and T. R. Brown Identification and Characterization of a Novel Androgen Response Element Composed of a Direct Repeat J. Biol. Chem., March 28, 1997; 272(13): 8227 - 8235. [Abstract] [Full Text] [PDF]
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N. L. Chamberlain, D. C. Whitacre, and R. L. Miesfeld Delineation of Two Distinct Type 1Activation Functions in the Androgen Receptor Amino-terminal Domain J. Biol. Chem., October 25, 1996; 271(43): 26772 - 26778. [Abstract] [Full Text] [PDF]
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A. Bubulya, S. C. Wise, X.-Q. Shen, L. A. Burmeister, and L. Shemshedini c-Jun Can Mediate Androgen Receptor-induced Transactivation J. Biol. Chem., October 4, 1996; 271(40): 24583 - 24589. [Abstract] [Full Text] [PDF]
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M. Xu, P. K. Chakraborti, M. J. Garabedian, K. R. Yamamoto, and S. S. Simons Jr. Modular Structure of Glucocorticoid Receptor Domains Is Not Equivalent to Functional Independence. STABILITY AND ACTIVITY OF THE STEROID BINDING DOMAIN ARE CONTROLLED BY SEQUENCES IN SEPARATE DOMAINS J. Biol. Chem., August 30, 1996; 271(35): 21430 - 21438. [Abstract] [Full Text] [PDF]
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J. P. Aumais, H. S. Lee, C. DeGannes, J. Horsford, and J. H. White Function of Directly Repeated Half-sites as Response Elements for Steroid Hormone Receptors J. Biol. Chem., May 24, 1996; 271(21): 12568 - 12577. [Abstract] [Full Text] [PDF]
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B. He, J. A. Kemppainen, and E. M. Wilson FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor J. Biol. Chem., July 21, 2000; 275(30): 22986 - 22994. [Abstract] [Full Text] [PDF]
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J. Rao, P. Lee, S. Benzeno, C. Cardozo, J. Albertus, D. M. Robins, and A. J. Caplan Functional Interaction of Human Cdc37 with the Androgen Receptor but Not with the Glucocorticoid Receptor J. Biol. Chem., February 16, 2001; 276(8): 5814 - 5820. [Abstract] [Full Text] [PDF]
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K. Takahashi, T. Taira, T. Niki, C. Seino, S. M. M. Iguchi-Ariga, and H. Ariga DJ-1 Positively Regulates the Androgen Receptor by Impairing the Binding of PIASxalpha to the Receptor J. Biol. Chem., September 28, 2001; 276(40): 37556 - 37563. [Abstract] [Full Text] [PDF]
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T. Slagsvold, I. Kraus, K. Fronsdal, and F. Saatcioglu DNA Binding-independent Transcriptional Activation by the Androgen Receptor through Triggering of Coactivators J. Biol. Chem., August 10, 2001; 276(33): 31030 - 31036. [Abstract] [Full Text] [PDF]
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X. Wang, S. Yeh, G. Wu, C.-L. Hsu, L. Wang, T. Chiang, Y. Yang, Y. Guo, and C. Chang Identification and Characterization of a Novel Androgen Receptor Coregulator ARA267-alpha in Prostate Cancer Cells J. Biol. Chem., October 26, 2001; 276(44): 40417 - 40423. [Abstract] [Full Text] [PDF]
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A. Bubulya, S.-Y. Chen, C. J. Fisher, Z. Zheng, X.-Q. Shen, and L. Shemshedini c-Jun Potentiates the Functional Interaction between the Amino and Carboxyl Termini of the Androgen Receptor J. Biol. Chem., November 21, 2001; 276(48): 44704 - 44711. [Abstract] [Full Text] [PDF]
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