(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- 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.
The androgen receptor (AR) ()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) .
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-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) .
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- and
carboxyl-terminal domains that raises the possibility of an
anti-parallel oriented AR dimer.
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
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.
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-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).
GAL-A1 and VP-A1
comprise the GAL4 DNA-binding and VP16 transactivation domains,
respectively, fused NH-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).
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-terminal peptides. Molecular mass standards were
analyzed in parallel for gel calibration. Shown are VP-A1 (lane
1), VP-A1
14-150 (lane 2),
VP-A1
142-337 (lane 3), VP-A1
339-499 (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).
Figure 3:
Luciferase activity induced by the
chimeric vectors. Combinations of the indicated parent and chimeric
expression vectors (1 µg DNA/0.45 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.
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-/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).
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 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.
Because hydroxyflutamide is a potent
antiandrogen, we investigated whether it would disrupt the
NH-/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.
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-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.
Figure 6:
Effect of AR NH-terminal
deletions on VP-A1 interaction with GALD-H. Several mutants with
portions of the AR NH
-terminal domain deleted, including
VP-A1
14-150 (5 µg), VP-A1
142-337 (1 µg),
VP-A1
339-499 (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.
Figure 7:
Effect of AR NH-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.
The
NH-/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.
The objective of the present study was to establish whether a
direct interaction occurs between the NH-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.
Although the present data do not rule out
that the ligand-induced NH-/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.
The two regions of the AR NH-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.
The affinity of the NH-/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) .
Human AR and ER differ in
the length of their NH-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.