Structural alignment of the human androgen
receptor dimer was investigated by introducing steroid binding domain
mutations that cause partial or complete androgen insensitivity into
fusion proteins containing the full-length androgen receptor or the
steroid binding domain. Most of the mutants had unchanged apparent
equilibrium androgen binding affinity and increased dissociation rates
of [3H]methyltrienolone and required increased
dihydrotestosterone concentrations for transcriptional activation. In a
2-hybrid protein interaction assay in mammalian cells, the steroid
binding domain interacts with an NH2-terminal-DNA binding
domain fragment and with the full-length androgen receptor at
physiological androgen concentrations in a dose-dependent
manner. However, mutations at Val-889 and Arg-752 disrupt the
NH2-/carboxyl-terminal interaction when introduced into the
steroid binding domain fragment but not when present in the full-length
androgen receptor. The N-C bimolecular interaction reduces the
dissociation rate of bound androgen and slows the degradation rate of
the carboxyl-terminal steroid binding domain fragment. The results
suggest that steroid binding domain residues Val-889 and Arg-752 are
critical to the NH2-/carboxyl-terminal interaction and that
an intermolecular N-C interaction occurs during receptor dimerization
that results in an antiparallel arrangement of androgen receptor
monomers.
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INTRODUCTION |
Steroid hormone receptor dimerization required for optimal DNA
binding (1-5) is mediated by the second zinc finger region in the DNA
binding domain (6) and by a carboxyl-terminal region in the steroid
binding domain (7-10). Some steroid receptors do not require steroid
binding for dimerization (11-13) or dimerize independent of DNA
binding (5, 14-16). For the androgen receptor (AR),1
binding to androgen response element DNA
requires androgen-dependent AR dimerization (17). Recent
evidence using a 2-hybrid protein interaction assay in mammalian cells
indicates that AR dimerization involves an
androgen-dependent interaction between the
NH2-terminal and carboxyl-terminal steroid binding domains
(N-C interaction) that is of sufficiently high affinity to occur
bimolecularly, independent of the AR DNA binding domain (18). Similar
N-C interactions in the estrogen receptor (ER) led to a parallel
dimerization model (19). An unresolved issue in the steroid receptor
field, therefore, is whether receptor dimerization has a parallel or
antiparallel orientation (20). In support of the parallel model,
evidence was reported for ER dimerization through direct hydrophobic
interactions between steroid binding domains (21-23). An Src homology
2 domain-type interaction with phosphorylated tyrosine 537 in the ER
steroid binding domain may be involved in ER dimerization although
dimer orientation was not addressed (24). Another report suggested dimerization occurs in a head-to-toe, antiparallel arrangement (25). In
support of the antiparallel model, the glucocorticoid receptor
NH2-terminal domain was implicated in dimerization (26). For the thyroid hormone and vitamin D receptors, the DNA binding domain
has rotational flexibility relative to the dimerization interface in
the steroid binding domain, allowing a 180° change in conformation
(27). The vitamin D receptor may form a symmetrical, head-to-tail,
antiparallel dimer through the DNA binding domain but is influenced by
the steroid binding domain dimerization interface (28).
Several lines of evidence argue against a parallel dimer for
androgen-activated AR. First, no interaction is observed between the AR
steroid binding domains in the 2-hybrid protein interaction assay in
the presence or absence of androgen (18). Second, the N-C interaction
is of sufficiently high affinity to promote a bimolecular interaction.
Third, the steroid specificity of N-C-mediated dimerization parallels
that of full-length AR. Fourth, antiparallel interactions between N-C
regions of AR NH2- and carboxyl-terminal fragments promote
high affinity DNA binding. In the present report, evidence for an
intermolecular N-C interaction in an antiparallel AR dimer was obtained
by the introduction of single base mutations into the AR steroid
binding domain that cause partial or complete androgen insensitivity
without altering the apparent equilibrium androgen binding constant and
by examining the influence of the NH2-terminal domain on
androgen dissociation rates and protein turnover.
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EXPERIMENTAL PROCEDURES |
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 (29)
and kindly provided by Gordon Tomaselli, Johns Hopkins University.
These vectors were used to construct the full-length AR vector
pNLVP-hAR (designated here as pVPAR) and the carboxyl terminal AR
fragment vector pGALD-H (containing AR amino acid residues 624-919)
(18). pVPAR1-660 contains the VP16 transactivation domain 5
of human
AR residues 1-660, including the NH2-terminal, DNA
binding, and part of the hinge regions, and was constructed from the
full-length vector by deleting the carboxyl terminal portion using
TthIII/XbaI followed by ligation of the filled
ends. Androgen insensitivity mutations V889M and V866M were introduced
into pGALD-H and pVPAR by polymerase chain reaction (PCR) amplification
of pCMVhAR-V889M (30, 31) and pCMVhAR-V866M (32). Amplified fragments
were cloned into pGALO at NdeI/XbaI, in the case
of the D-H fragment, or into VPAR using CspXI/XbaI.
Bi-directional PCR mutagenesis was used to construct pGALD-H-R752Q,
which was used to construct pCMVhAR-R752Q at the TthIII/XbaI
restriction sites and pVPAR-R752Q at BstEII/XbaI. pCMVhAR-Y763C was constructed by PCR mutagenesis changing TAC codon 763 to TGC. PCR fragments were digested with
HincII/XbaI, and a triple ligation reaction was
performed with the NdeI/HincII fragment of pVPAR
or pGALD-H. pCMVhAR-Y763C was constructed in the
HindIII/XbaI fragment from pGALD-H-Y763C. The
sequence of all PCR-amplified regions was verified.
Cell Culture, DNA Transfections, Binding Assays, and
Immunoblots--
Chinese hamster ovary (CHO) cells were maintained
in alpha minimum essential medium containing 10% bovine calf serum, 20 mM Hepes, pH 7.2, penicillin, and streptomycin, plated at
4.5 × 105 cells/6-cm dish, and transfected with 1-2
µg of expression vector DNA and 5 µg of G5E1bLuc reporter vector
using DEAE-dextran as described (18). After addition of
increasing concentrations of DHT and incubation for 24 h, cells
were harvested and assayed for luciferase activity as described
(18).
Apparent equilibrium binding affinity was determined in whole cell
binding assays as described (33). Monkey kidney COS-1 cells were plated
at 3.5 × 105 cells/well of 6-well culture plates and
transfected using DEAE-dextran with 3 µg of DNA for AR fragments
containing the steroid binding domain and 1-2 µg for full-length AR.
After 48 h, cells were incubated with 0.25-5 nM
[3H]R1881 for 2 h at 37 °C. Dissociation rate
constants were determined by labeling with 5 nM
[3H]R1881 for 2 h at 37 °C followed by the
addition of a 10,000 molar excess of unlabeled R1881. Cells were washed
twice with phosphate-buffered saline and harvested in 0.5 ml of 2%
SDS, 10% glycerol, and 10 mM Tris, pH 6.8. Specific
binding was determined from the difference in radioactivity in the
presence and absence of a 100-fold molar excess unlabeled R1881 as
described previously (31).
Protein expression levels were compared by immunoblot analysis of
wild-type and mutant full-length AR and partial AR fusion proteins
expressed in COS-1 cells and analyzed in 9 or 12% acrylamide SDS gels
using AR52 antipeptide antibody (34) or the GAL4 DNA binding domain
antibody (Santa Cruz Biotechnology). Secondary antibody was anti-rabbit
IgG (Promega).
Degradation rates were determined in COS-1 cells (1 × 106/10-cm dish) transiently transfected using DEAE-dextran
with 4 µg of AR507-919 plus either 8 µg of parent expression
plasmid pCMV5 or 8 µg of AR1-660. Cells were maintained in 10%
serum and, after 48 h, were incubated in serum-free,
methionine-free media as described previously (31) except with 100 µCi
[35S]L-methionine/L-cysteine
PRO-MIX in vitro cell labeling mix (1000 Ci/mmol) (Amersham
Life Science, Inc.). After 30 min of labeling, cells were washed,
placed in serum-free media with or without 100 nM DHT at
37 °C, and harvested at time intervals up to 7 h. Cells were
harvested in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1%
SDS, 0.15 M NaCl, 5 mM EDTA, and 50 mM Tris, pH 7.4), and cell extracts were incubated
with AR52 anti-peptide antibody and Pansorbin and processed for
polyacrylamide gel analysis as described previously (31).
 |
RESULTS |
Steroid Binding Domain Amino Acid Residues Involved in the N-C
Interaction and Polarity of AR Dimerization--
The polarity of AR
dimerization was investigated by introducing naturally occurring
mutations that cause partial or severe forms of androgen insensitivity.
The mutations were constructed by mutagenesis of GALD-H, which contains
human AR steroid binding domain residues 624-919 (coded by exons D-H)
fused to the carboxyl-terminal end of GAL4 DNA binding domain residues
1-147, or into VPAR, which contains full-length human AR residues
1-919 fused to the carboxyl-terminal end of VP16 transactivation
domain residues 411-456. AR mutations that result in partial or
complete androgen insensitivity either do not significantly alter
(V889M, R752Q, Y763C, Y763H) or decrease (V866M) the apparent
equilibrium binding affinity for the synthetic androgen,
[3H]R1881 (Table I).
Equilibrium androgen binding affinities of the full-length AR mutants
shift toward higher affinities when the NH2-terminal region
is deleted in AR507-919 and in the GALD-H fusion proteins (Table I).
However all of the full-length androgen insensitivity mutants have
increased rates of [3H]R1881 dissociation (Table
II) relative to wild-type AR, suggesting correspondingly increased association rates in mutants with equilibrium binding constants similar to that of wild-type AR. Deletion of the
NH2-terminal domain results in increased dissociation rates of [3H]R1881 (Table II) for all mutants as previously
reported for wild-type AR (18). Expression levels of the wild-type and
mutants are similar for the partial AR fusion proteins in GALD-H (Fig. 1A), for full-length ARs
expressed as fusion proteins with the VP16 transactivation domain (Fig.
1B), or full-length AR (Fig. 1C) as determined
from immunoblots of extracts from transiently transfected COS-1
cells. However, protein expression from pCMV5 is at least 2-fold higher
than from pVP16. When introduced into the mammalian expression vector
pCMVhAR and assayed with the MMTV-luciferase reporter vector, most of
the androgen insensitivity mutants require DHT concentrations above the
physiological range to promote full transcriptional activation (Fig.
2).
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Table I
Equilibrium dissociation constant, Kd (nM)
Apparent equilibrium binding constants and standard deviations were
determined in COS cells using increasing [3H]R1881 as
described under "Experimental Procedures."
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Table II
Dissociation half-time of [3H]R1881 (min)
Dissociation half-times were determined at 37 °C as described under
"Experimental Procedures" with averages and standard deviation from
at least three independent experiments.
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Fig. 1.
Immunoblots of partial and full-length AR
with wild-type or androgen insensitivity mutation sequence expressed
alone or as fusion proteins. COS-1 cells were transfected using
DEAE-dextran as described under "Experimental Procedures" with 10 µg of DNA/1.2 × 106 cells/10-cm dish. Forty-eight h
after transfection and in the absence of androgen, COS cells were
harvested in SDS containing buffer and analyzed in 12% (A)
or 9% (B, C) acrylamide gels. A, GALD-H expression vectors contain the S. cerevisiae GAL4 DNA
binding domain residues 1-147 (GAL DBD) and AR steroid
binding domain residues 624-919 (coded by exons D-H) with the
indicated androgen insensitivity mutations. Expressed GALD-H fusion
proteins (50 kDa apparent Mr; 49 kDa calculated;
50 µl) were detected using a rabbit anti-GAL DNA binding domain
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The GAL4 DNA
binding domain (19 kDa apparent Mr; 16.3 kDa
calculated) is evident in lane 7 from the expressed parent
vector, pGALO, that lacks AR sequence. B, VPAR expression vectors contain the herpes simplex virus VP16 transactivation domain
residues 411-456 and full-length AR residues 1-919 with the indicated
mutations. VPAR fusion proteins (120 and 126 kDa apparent
Mr; 112 kDa calculated; 50 µl) were detected
using antipeptide AR52 IgG previously described (34). Expression of the
parent vector pVP16 without AR sequence is shown in lane 7.
C, pCMVhAR expression vectors contain full-length AR
residues 1-919 with the indicated mutations. Full-length AR (113 kDa
apparent Mr; 102 kDa calculated) was detected
using AR52 IgG. Expression of the parent vector pCMV5 lacking AR
sequence is shown in lane 7. Shown are extracts (40 µl)
from cells expressing parent vectors with wild-type AR sequence
(lane 1, WT) or with the following androgen insensitivity AR
mutations: V889M (lane 2, VM889), V866M (lane 3, VM866), R752Q (lane 4, RQ752), Y763C (lane 5, YC763), Y763H (lane 6, YH763), and the parent vectors
lacking AR sequence (lane 7).
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Fig. 2.
Androgen-induced transcriptional activation
of the MMTV promoter-luciferase reporter vector by full-length
wild-type AR and mutant ARs that cause partial or complete androgen
insensitivity. Monkey kidney CV1 cells (3.5 × 105/6-cm dish) were transiently transfected with 100 ng of
pCMVhAR with wild-type sequence (AR) or with the indicated human
androgen insensitivity mutations and the testicular feminized
androgen-resistant rat mutation (R734Q) in pCMVrAR and 5 µg of
MMTV-luciferase reporter vector using calcium phosphate precipitation
as described previously (47). Cells were incubated for 30 h with
increasing concentrations of DHT from 0.001 to 100 nM as
indicated, and optical light units were determined using an automatic
luminometer. Shown is a representative experiment of at least three
independent determinations.
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GALD-H interacts with VPAR1-660 (AR NH2-terminal, and DNA
binding domain residues 1-660 fused carboxyl terminal to the VP16 transactivation domain) in the presence of DHT in a
concentration-dependent manner. Luciferase activity of
3-16 fold becomes detectable at 0.01 nM DHT, with full
activation between 22-31 fold at 1-10 nM DHT (Figs.
3A, 4A,
5A, and 6A, left). VP-full-length AR (VPAR) interacts with GALD-H in a similar concentration dependent manner with
DHT, with up to an 11-fold increase in luciferase activity at 0.01 nM DHT and full activation of 12-34 fold at 1-5
nM DHT (Figs. 3B, 4B, 5B,
and 6B, left). These results indicate that the
N-C interaction occurs as a bimolecular reaction when one monomer is
either full-length AR or the NH2-terminal and DNA binding regions and the other monomer contains only the AR steroid
binding domain. The similar dose-dependent N-C interaction
between the steroid binding domain and either full-length AR or the
NH2-terminal fragment supports the antiparallel dimer
model. Self dimerization of VPAR apparently does not interfere with its
interaction with GALD-H.

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Fig. 3.
2-hybrid protein interaction assay between AR
NH2- and carboxyl-terminal domains. The 2-hybrid assay
was performed in CHO cells as described previously (18). A,
VPAR1-660, coding for AR NH2-terminal residues 1-660 and
the VP16 activation domain, was cotransfected with GALD-H, coding for
wild-type AR steroid binding domain residues 624-919 (left)
or with GALD-H-V889M (right) (codon 889 GTG ATG).
B, VPAR, coding for wild-type full-length AR residues 1-919
(left) or VPAR-V889M (middle) expressed as fusion proteins with the VP16 activation domain were transfected with GALD-H
with wild-type sequence, or VPAR was transfected with GALD-H-V889M (right). DNA (1 µg of each expression plasmid) was
transfected with 5 µg of G5E1bLuc reporter plasmid using DEAE-dextran
and incubated for 30 h with 0.01 nM to 1 µM DHT as indicated. Luciferase activity is shown as
optical light units and is representative of at least three independent
experiments. Schematic diagrams of the constructs are shown where the
shaded area represents the AR DNA binding domain, and × represents the V889M mutation. Fold induction relative to luciferase
activity in the absence of DHT is indicated above the
bars.
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V889M--
V889M results from a GTG
ATG mutation in exon H
that causes grade 6 androgen insensitivity (female external phenotype
with pubic hair in a 46XY genetic male) (30, 35) without altering equilibrium androgen binding (30) but increasing the dissociation rate
of [3H]R1881 (31) from the full-length mutant AR (Table
II). The dissociation rate of [3H]R1881 increases about
4-fold further with deletion of the NH2-terminal region in
AR507-919 (DNA and steroid binding domains) (Table II), suggesting
that, at the high androgen concentration (50 µM unlabeled R1881) used in the dissociation experiment, the
NH2-terminal region slows androgen dissociation from V889M
to an extent similar to wild-type AR. The 5-10-fold faster androgen
dissociation rate from AR507-919-V889M and GALD-H-V889M relative to
these wild-type AR fragments suggests, in addition, that increased
androgen dissociation is an inherent defect of the V889M mutation in
the steroid binding domain (Table II).
The interaction between GALD-H-V889M and VPAR1-660 occurs only at DHT
concentrations of 5 nM or greater, about 500 times the concentration required for the wild-type GALD-H and VPAR1-660 interaction, and increases to only 12-16-fold, up to 1 µM DHT (Fig. 3A, right). On the
other hand, reactivity of VPAR-V889M with GALD-H was similar to
wild-type VPAR (Fig. 3B, middle), and little
interaction is observed between VPAR and GALD-H-V889M except at high
DHT concentrations (Fig. 3B, right). The results
support an intermolecular N-C interaction where GALD-H interacts with the NH2-terminal region of VPAR-V889M, which is unaffected
by the V889M steroid binding mutation in VPAR-V889M. The similar dose
response in interaction between GALD-H with VPAR or VPAR-V889M suggests
that low physiological DHT concentrations are sufficient to promote
this bimolecular reaction and may reflect the retained high androgen
binding affinity of V889M (Table I).
R752Q--
Androgen insensitivity in the testicular feminized
(Tfm) rat results from a single base mutation CGG
CAG in exon E of
the AR steroid binding domain changing Arg 734 to Gln (33). This mutation lowers androgen binding capacity with only slightly lower apparent equilibrium binding affinity (Table I) (33). The Tfm rat AR
mutation occurs in human AR (Arg 752 to Gln, CGA
CAA) in two
unrelated families with complete androgen insensitivity; however, in
these cases, androgen binding activity was not reported (36, 37). The
R752Q human AR mutant and the R734Q Tfm rat mutant (33) require at
least 10,000-fold higher DHT concentrations than wild-type AR for
transcriptional activation of a mouse mammary tumor virus reporter
vector (Fig. 2). Like V889M, the apparent binding affinity of R752Q for
[3H]R1881 is similar to that of wild-type AR (Table I),
and dissociation of [3H]R1881 is at least 11 times faster
in full-length AR and increases or is similar in the truncated
fragments containing the DNA and steroid binding domains (AR507-919)
or the steroid binding domain fusion protein in GALD-H (Table II).
These results suggest that the R752Q binding defect is inherent to the
steroid binding domain. This previously unrecognized rapid androgen
dissociation kinetics from human AR R752Q and Tfm rat AR R734Q,
combined with the low binding capacity, likely account for the
inability of other laboratories (36, 37) to detect androgen
binding using conventional radiolabeling binding assays of endogenous
AR in fibroblasts from affected individuals with the R752Q mutation and
for the 10% binding levels relative to wild-type siblings resulting
from the synonymous R734Q mutation in the Tfm rat (33).
In the 2-hybrid protein interaction assay, R752Q disrupts the N-C
interaction between VPAR1-660 and GALD-H-R752Q (Fig.
4A, right) to an
extent similar to that observed for V889M (Fig. 3A), suggesting that Val-889 and Arg-752 are both critical residues in the
steroid binding domain for the N-C interaction. Also like V889M, the
interaction between VPAR-R752Q and GALD-H is similar to that of
wild-type VPAR (Fig. 4B, left and
middle), and GALD-H-R752Q fails to interact with VPAR at
physiological androgen concentrations (Fig. 4B,
right), supporting the antiparallel N-C dimer orientation. In both cases, retention of high affinity binding with rapid binding and dissociation kinetics suggests that perturbations around the androgen binding pocket are critical to the N-C interaction.

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Fig. 4.
2-hybrid protein interaction assay of
R752Q. The 2-hybrid assay was performed as described (18). The
R734Q AR mutation of the testicular feminized rat (33) was introduced
into pCMVhAR at codon 752 CGA CAA of human AR. A,
VPAR1-660 coding for AR NH2-terminal residues 1-660 fused
to the VP16 transactivation domain was transfected with GALD-H
(left) or GALD-H-R752Q (right). B,
VPAR (left) or VPAR-R752Q (middle) was
transfected with GALD-H, or VPAR was transfected with GALD-H-R752Q
(right). DNA (1 µg of each plasmid) was cotransfected with
5 µg of G5E1bLuc reporter plasmid using DEAE-dextran and incubated
for 30 h with 0.01 nM to 1 µM DHT as
indicated. Luciferase activity shown as optical light units is
representative of at least three independent experiments. Schematic
diagrams of the constructs are shown where the shaded area
represents the AR DNA binding domain, and × represents the
R752Q mutation. Fold induction relative to luciferase activity without
DHT is indicated above the bars.
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Y763C--
Tyr at 763 in exon E changes to Cys (TAC
TGC) in a
family with partial androgen insensitivity (grade 2/3, male phenotype with hypospadias and virilization at puberty) (38) and to His (TAC
CAC) in an unrelated family with complete androgen insensitivity (35).
The Y763C mutation (reported previously as Y761C) occurred together
with shortening of the NH2-terminal Gln repeat from an average length of 22 residues to 12 residues (38). Y763C is reported to
increase the androgen dissociation rate 2- to 3-fold with a slight
increase in apparent equilibrium binding affinity (Kd) (38). When recreated in full-length AR, we
observe a similar increase in the dissociation rate of
[3H]R1881 from Y763C with the apparent equilibrium
binding affinity indistinguishable from wild-type AR (Tables I and II).
The 2-hybrid protein interaction assay results indicate only a slight
decrease in transcriptional activity (Fig.
5), suggesting that Tyr-763 is not
critical for the N-C interaction. This conclusion is supported by
2-hybrid results with Y763H (data not shown). Y763H had similar equilibrium binding but 4-fold faster dissociation kinetics of [3H]R1881 relative to wild-type AR (Tables I and II) and
causes more severe androgen insensitivity.

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Fig. 5.
2-hybrid protein interaction assay of Y763C.
A, VPAR1-660 cotransfected with GALD-H (left) or
GALD-H-Y763C (TAC TGC) (right). B, VPAR
(left) or VPAR-Y763C (middle) was cotransfected with GALD-H, or VPAR was cotransfected with GALD-H-Y763C
(right). DNA (1 µg) was cotransfected with 5 µg of
G5E1bLuc reporter plasmid using DEAE-dextran and incubated for 30 h with DHT as indicated. Luciferase activity shown as optical light
units is representative of three independent experiments. Schematic
diagrams of the constructs are shown where the shaded area
represents the AR DNA binding domain, and × represents the Y763C
mutation. Fold induction relative to luciferase activity without DHT is
indicated above the bars.
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V866M--
Because the V889M and R752Q mutations shorten the
dissociation half-time of bound androgen 7-12-fold and Y763C and Y763H
only 2-4 fold, and whereas only the former two were implicated in the N-C interaction, we investigated androgen insensitivity mutant V866M to
determine whether increased androgen dissociation kinetics is
associated with disruption of the N-C interaction. This GTG
ATG
mutation in exon G of the steroid binding domain causes complete
androgen insensitivity with a reported 4-fold reduction in androgen
binding affinity (32). We also observe a 4-5-fold reduction in
apparent equilibrium binding affinity of [3H]R1881 and a
3-4-fold increase in the dissociation rate of [3H]R1881
(Tables I and II). GALD-H-V866M reacted with VPAR1-660 in a
dose-dependent manner with the dose response shifted to
about 5-fold higher DHT concentrations (Fig.
6A), a shift that can be accounted for by the decreased apparent equilibrium androgen binding affinity (Table I). Decreased sensitivity to DHT is also observed in
the interaction between GALD-H with VPAR-V866M and between GALD-H-V866M
and VPAR (Fig. 6B). The parallel shift in dose response by
the V866M mutant proteins with the reduced binding affinity suggests
that increased androgen dissociation kinetics greater than 4-fold is
associated with disruption of the N-C interaction and that Val 866 is
not a critical residue for the N-C interaction.

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Fig. 6.
2-hybrid protein interaction assay of
V866M. The V866M mutation GTG ATG was introduced into VPAR and
GALD-H as described under "Experimental Procedures."
A,VPAR1-660 was transfected with GALD-H (left)
or GALD-H-V866M (right). B, VPAR
(left) or VPAR-V866M (middle) was transfected
with GALD-H, or VPAR was transfected with GALD-H-V866M
(right). DNA (1 µg each) was cotransfected with 5 µg of
G5E1bLuc reporter plasmid using DEAE-dextran and incubated for 30 h with DHT as indicated. Luciferase activity shown as optical light
units is representative of at least three independent experiments. Schematic diagrams are shown, where the shaded area
represents the AR DNA binding domain, and × represents the
V866M mutation. Fold induction relative to luciferase activity without
DHT is indicated above the bars.
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Effect of the N-C Interaction on Androgen Dissociation--
It was
shown previously that deletion of AR NH2-terminal residues
1-507 causes a 3-fold increase in the dissociation rate of
[3H]R1881 from the steroid binding domain with the
apparent equilibrium binding affinity unchanged (31), indicating that
the association rate increases in parallel, and the
NH2-terminal region slows androgen association and
dissociation kinetics. It was not clear, however, whether slow androgen
dissociation necessary for AR stabilization and transcriptional
activity (31, 39) is caused by an intramolecular N-C interaction in the
AR monomer or as part of the intermolecular N-C interaction in an
antiparallel dimer. Coexpression of the DNA and steroid binding domain
fragment AR507-919 with a 3-fold molar excess of transfected AR
NH2-terminal fragment AR1-660 DNA slowed the dissociation
rate of [3H]R1881 from AR507-919 about 2-fold (Fig.
7A). The dissociation rate of
[3H]R1881 from AR507-919 with the V889M mutation also
decreases by coexpression of AR1-660 in the presence of a high
concentration of unlabeled androgen in the dissociation experiments (50 µM DHT). The dissociation rate of V889M remained,
however, about 10-fold faster than wild-type AR (Fig. 7). In this
bimolecular dimerization reaction, therefore, androgen dissociation
rates slow toward that observed for wild-type AR, suggesting that the
N-C intermolecular interaction associated with dimerization reduces the
kinetics of androgen binding and dissociation in the steroid binding
domain pocket.

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Fig. 7.
Effect of the bimolecular N-C dimerization on
[3H]R1881 dissociation. AR507-919 (2 µg) (44)
containing the coding region for the AR DNA and steroid binding domains
with wild-type (A) or V889M (B) sequence in pCMV5
was transiently transfected in COS-1 cells alone (0) or with a 3-fold
molar excess (6 µg) pCMV5 either lacking AR sequence ( ) or with
AR1-660 in pCMV5 ( ). AR1-660 contains the AR
NH2-terminal and DNA binding domain residues 1-660 (44).
Full-length pCMVhAR (AR1-919) was included as a control ( ). COS
cells (3.5 × 105/6 cm dish) transfected using
DEAE-dextran were labeled with 5 nM [3H]R1881
for 2 h at 37 °C. Dissociation was initiated by the addition of
10,000-fold molar excess of unlabeled R1881. Cells were washed twice
with phosphate-buffered saline and harvested in SDS sample buffer, and
radioactivity was determined by scintillation counting. Nonspecific
binding was determined by the addition of 100-fold excess unlabeled
R1881 to parallel samples at the initiation of the binding
reaction.
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Effect of the N-C Interaction on AR Degradation--
To
investigate whether the antiparallel N-C interaction also mimics the
decrease in AR turnover observed with the transcriptionally active
full-length AR, the carboxyl-terminal AR507-919 vector was coexpressed
with a 2-fold molar excess of the NH2-terminal AR1-660
expression vector or with the parent vector as a control. Degradation
half-time of the [35S]methionine/cysteine-labeled
AR507-919 was 2 h at 37 °C in the presence or absence of 100 nM DHT, or in the presence of the NH2-terminal fragment in the absence of DHT (Fig. 8).
The degradation half-time slowed to 4 h, however, in the presence
of the NH2-terminal fragment and 100 nM DHT
(Fig. 8). The results suggest that the antiparallel structural
orientation in the bimolecular N-C interaction of the AR fragments
recapitulates the reduced androgen dissociation and receptor turnover
that occur in the transcriptionally active full-length AR.

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Fig. 8.
Influence of the AR
NH2-terminal and DNA binding domain fragment on degradation
of the steroid binding domain fragment. Transient transfections in
COS-1 cells were performed using DEAE-dextran and 4 µg of AR507-919
plus 8 µg of the parent vector pCMV5 (p5) or 8 µg AR1-660 coding
for the NH2-terminal and DNA binding regions. After 48 h, cells were incubated for 30 min with 100 µCi of
35S-labeled cysteine-methionine as described under
"Experimental Procedures" followed by a chase period. AR fragments
were harvested at increasing time intervals, immunoprecipitated using
AR52 anti-peptide antibody, and analyzed on a 9% acrylamide gel
containing SDS (A and B). C, shown are
degradation rate data for AR 507-919 + pCMV5 (p5) in the absence of
androgen ( ), AR507-919 + p5 + 100 nM DHT ( ),
AR507-919 + AR1-660 in the absence of androgen ( ), and AR507-919 + AR1-660 + 100 nM DHT ( ). Intensities of the
AR507-919 bands shown in A and B were determined
on an LKB laser densitometer and are shown on a semi-log scale.
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DISCUSSION |
The N-C interaction observed for AR (18) and ER (19) has raised at
least two dimerization models for steroid receptors. The parallel dimer
model proposed for ER (19) implicates an intramolecular N-C
interaction within the ER monomer that potentiates ER dimerization
through conformational changes and subsequent intermolecular
interactions between the steroid binding domains. The antiparallel
dimer model proposed for AR implicates an intermolecular N-C
interaction between monomers (18) in androgen-dependent AR
dimerization and specific DNA binding (17). Natural mutations in the AR
steroid binding domain that cause partial or complete androgen
insensitivity were introduced into AR fusion proteins to identify
steroid binding domain residues critical for the N-C interaction and to
investigate further the molecular alignment of the AR dimer. Because
naturally occurring mutations in the steroid binding domain often
decrease the apparent equilibrium androgen binding affinity, it can be
difficult to assess their role in AR dimerization and DNA binding. More
than 70 missense mutations that cause partial or complete androgen
insensitivity are located throughout the AR steroid binding domain
(35). The few that do not change the apparent androgen binding affinity yet are associated with partial or complete androgen insensitivity are
particularly helpful in revealing additional mechanisms involving the
steroid binding domain.
A series of AR gene mutations that cause androgen insensitivity without
altering the apparent equilibrium androgen binding affinity were
introduced into full-length AR expressed as a fusion protein with the
VP16 transactivation domain or into the AR steroid binding domain
expressed as a fusion protein with the GAL4 DNA binding domain to
investigate dimerization polarity. Two residues, Val-889 and Arg-752,
were identified as steroid binding domain residues critical for the N-C
interaction. Both V889M and R752Q mutations disrupt the N-C
interaction, suggesting that loss of function by these mutations in
individuals with androgen insensitivity relates to ineffective N-C
interaction at physiological androgen concentrations. AR with either
mutation is transcriptionally active but requires androgen
concentrations above the physiological range to overcome the
defect.
Steroid binding domain mutation V889M causes grade 6 (35) androgen
insensitivity (30). Its unaltered apparent equilibrium binding constant
with 8-fold increased dissociation and presumably increased association
rates suggest that Val-889 is not directly part of the steroid binding
pocket but forms a structural barrier to androgen binding and
dissociation. Val-889 is situated 30 residues from the AR carboxyl
terminus in a region with sequence similarity to the region between
helices 11 and 12 near the steroid binding pocket of the thyroid
hormone
receptor based on crystal structure analysis (40). Homo-
and heterodimerization interfaces of the thyroid hormone
receptor
(41) include helical regions 10 and 11 (40) corresponding to human AR
residues 855-883 preceding the V889M mutation. The proximity of
Val-889 to the steroid binding pocket suggests this region undergoes
conformational changes with androgen binding. From the crystal
structure of the human RXR-
ligand binding domain, it was proposed
that the region of helix 11 changes dramatically upon ligand binding
(42). A region between residues 389-429 in the predicted helices 9 and
10 has a role in establishing the specificity of RXR homodimerization
and heterodimerization with RAR and thyroid hormone receptor and
subsequent DNA binding (13). Ligand binding to thyroid hormone receptor
and RAR exposes a region different from the ninth heptad repeat to
promote interactions with RXR (43).
Val-889 precedes by four amino acids the AF2 activation core region
predicted for AR based on sequence similarity among the family of
steroid hormone receptors (44). Unlike other steroid receptors,
however, a transcriptional activation function in the AR steroid
binding domain has remained elusive (18, 45, 46). It is conceivable
that this region contributes to transcriptional activation through its
interaction with the NH2-terminal region. Val-889 follows
by two amino acids a region in the steroid binding domain implicated in
ER dimerization and high affinity DNA binding (9). A preponderance of
indirect evidence implicates, therefore, the region of Val-889 not only
in major conformational changes upon androgen binding that could alter
the availability of the steroid binding pocket but in association with
AR dimerization and transcriptional activation. We postulate that one
of the underlying mechanisms in these processes involves the
intermolecular N-C interaction that occurs during androgen-induced AR
dimerization.
Mutations at both residues implicated in the N/C interaction, Val-889
and Arg-752, cause an 8-12-fold increase in androgen dissociation
rate, whereas dissociation rates of the other mutations were only
3-4-fold increased. This result suggests that very rapid androgen
dissociation either promotes or results from disruption of the N-C
interaction. The further increase in dissociation rate with deletion of
the NH2-terminal domain suggests stabilization of bound
androgen by the NH2-terminal domain. In the case of V889M and R752Q, some degree of stabilization of bound androgen in the full-length mutants likely resulted from the high, pharmacological androgen levels used in the dissociation experiments, concentrations at
which these mutants are transcriptionally active. The
NH2-terminal region not only stabilizes androgen in the
steroid binding pocket but also slows the turnover rate of the steroid
binding domain in the bimolecular N-C interaction with wild-type AR
fragments, both of which parallel characteristics of the full-length
transcriptionally active AR. Whether the rapid androgen dissociation
kinetics of V889M and R752Q is an inherent property of the steroid
binding domain or results from the disrupted N-C interaction is
unclear. Both Val-889 and Arg-752 are not likely directly part of the
androgen binding pocket but could influence dissociation and
association rates without significantly changing the apparent
equilibrium binding constant while also having a role in the N-C
interaction. Based on sequence similarity with the thyroid hormone
receptor, the crystal structure places Val-889 on the same surface as
Arg-752 across the steroid binding pocket adjacent to a mixed
sheet region (40). Val-866, on the other hand, not implicated in the N-C
interaction, is on a different surface of the steroid binding domain.
None of the AR mutations introduced into the full-length AR-VP16 fusion
protein significantly alter the N-C interaction with the wild-type GAL4
DNA binding domain-AR steroid binding domain fusion protein. However,
some of the same mutations introduced into the GALD-H steroid binding
domain fragment eliminate the interaction with full-length AR at
physiological androgen concentrations. The failure of full-length VPAR
to interact with V889M-GALD-H or R752Q-GALD-H except at high androgen
concentration, whereas GALD-H interacts with the mutant VPARs,
indicates that the AR N-C interaction is intermolecular and that any
resulting conformation changes do not impose a C-C interaction between
the steroid binding domains. The lack of C-C interactions between human
AR monomers agrees with previous results in the 2-hybrid protein
interaction assay (18).
We are grateful to K. Michelle Cobb and
De-Ying Zang for technical assistance; Frank S. French for helpful
discussions; and Gordon Tomaselli, Chi V. Dang, and Dominique Ashen
from the Johns Hopkins University School of Medicine, for
providing the mammalian 2-hybrid expression parent plasmids and
reporter vectors.