Mutations at the Boundary of the Hinge and Ligand Binding Domain of the Androgen Receptor Confer Increased Transactivation Function
Grant Buchanan,
Miao Yang,
Jonathan M. Harris,
Hyun S. Nahm,
Guangzhou Han,
Nicole Moore,
Jacqueline M. Bentel,
Robert J. Matusik,
David J. Horsfall,
Villis R. Marshall,
Norman M. Greenberg and
Wayne D. Tilley
Flinders Cancer Centre (G.B., M.Y., N.M., J.B., D.J.H., V.R.M.,
W.D.T.) Flinders University and Flinders Medical Centre
Adelaide SA 5042, Australia Institute for Molecular
Biosciences (J.M.H.) University of Queensland Brisbane Qld
4072, Australia Department of Cell Biology and Department of
Medicine (H.S.N., G.H., N.M.G.) aylor College of Medicine
Houston, Texas 77030 Department of Cell Biology (R.J.M.)
Urological Surgery and the Vanderbilt Cancer Center Nashville,
Tennessee 37232
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ABSTRACT
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The androgen receptor (AR), a member of the
steroid receptor superfamily of nuclear transcription factors, mediates
androgen signaling in diverse target tissues. Here we report AR gene
mutations identified in human prostate cancer and the autochthonous
transgenic adenocarcinoma of the mouse prostate model that colocate to
residues 668QPIF671 at
the boundary of the hinge and ligand-binding domain, resulting in
receptors that exhibit 2- to 4-fold increased activity compared with
wild-type AR in response to dihydrotestosterone, estradiol,
progesterone, adrenal androgens, and the AR antagonist,
hydroxyflutamide, without an apparent effect on receptor levels, ligand
binding kinetics, or DNA binding. The expression of these or similar
variants could explain the emergence of hormone refractory disease in a
subset of patients. Homology modeling indicates that amino acid
residues 668QPIF671
form a ridge bordering a potential protein-protein interaction
surface. The naturally occurring AR gene mutations reported in this
study result in decreased hydrophobicity of this surface, suggesting
that altered receptor-protein interaction mediates the precocious
activity of the AR variants.
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INTRODUCTION
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The androgen receptor (AR) is a ligand-activated nuclear
transcription factor that mediates the cellular effects of androgens
including differentiation, homeostasis, morphogenesis, and growth (1).
The AR consists of four broadly defined functional domains: a large
amino-terminal domain containing a strong constitutive transactivation
function (AF-1); a DNA binding domain; a hinge region; and a
carboxy-terminal ligand binding domain (LBD) that contains a highly
conserved ligand-dependent transactivation function (AF-2) (2). The
activity of both AF-1 and AF-2 is mediated by interaction with
accessory proteins termed cofactors, which function to either
up-regulate (coactivators) or down-regulate (corepressors) AR activity
(3, 4).
In the inherited form of androgen insensitivity, germline mutations in
the AR gene result in loss of receptor function and cause abnormal male
sexual development (5). In contrast, somatic missense mutations in the
AR gene that have been detected in primary and metastatic forms of
prostate cancer at frequencies of up to 44% and 50%, respectively
(6, 7, 8, 9), as well as in prostate cancer cell lines and xenografts (10, 11), consistently exhibit a gain of function. The majority of these
mutations colocate to two discrete regions in the LBD of the AR
[i.e. the signature sequence which corresponds to helix 3
(codons 669728) and a region containing AF-2 (codons 872908)] that
play key roles in regulation of receptor function, and result in
receptor variants with reduced specificity for ligand binding and/or
activation (7, 9, 11, 12, 13, 14, 15). While mutations identified in the signature
sequence and the AF-2 region of the AR gene potentially explain the
failure of androgen ablation therapies in a subset of patients with
metastatic disease, the genetic and pathological heterogeneity of
clinical prostate cancer has hindered a comprehensive analysis of the
incidence and nature of AR gene mutations in vivo.
In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model,
generated with a probasin-directed SV40 T/t antigen in C57Bl/6 inbred
mice, expression of the transgene is initially regulated by androgens
and restricted to the prostate epithelial cells of the dorsolateral and
ventral lobes (16, 17). Spontaneous prostate tumors that histologically
resemble the human disease arise with a short latency period and
exhibit progression from prostatic intraepithelial neoplasia to severe
hyperplasia and adenocarcinoma. We present here the first report of a
spontaneous AR gene mutation in an autochthonous animal model of
prostate cancer (i.e. TRAMP) and the colocation of this
mutation with somatic AR gene mutations identified in human prostate
tumors to amino acids
668QPIF671 at the boundary
of the hinge and LBD. This colocation to
668QPIF671 suggests that
mutations in this poorly characterized region of the AR, like those in
the signature sequence and AF-2, might have functional consequences
relevant to tumor progression. In this study, we demonstrate that the
naturally occurring mutations identified in prostate cancer in the
668QPIF671 region result in
AR variants with a 2- to 4-fold greater transactivation capacity in
response to dihydrotestosterone (DHT) and other nonclassical ligands
compared with wtAR. Moreover, homology modeling indicates that the
668QPIF671 residues define
a motif that forms a major structural component of a protein-protein
interaction surface that is disrupted by the mutations reported in this
study. Collectively, these findings implicate
668QPIF671 as an important
motif for AR function and suggest that mutations in this motif may
contribute to progression of prostate cancer in a subset of
patients.
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RESULTS AND DISCUSSION
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PCR single-stranded conformational polymorphism analysis of the
entire coding region of the AR in four tumors derived from TRAMP mice
(29 weeks of age) identified, in one tumor, a region of exon 4 with
altered mobility compared with the wild-type DNA sequence. DNA
sequencing of independently amplified fragments of exon 4 of the AR
gene in this tumor demonstrated that the mobility shift resulted from a
T-A base transition leading to substitution of phenylalanine for
isoleucine at codon 653. Phe653 is conserved in the human AR
(i.e. Phe671) and is located at the boundary of the hinge
region and LBD (Fig. 1
). This represents
the first report of a naturally occurring AR gene mutation in an
autochthonous animal model of prostate cancer. Moreover, Phe-Ile671
colocates with a subset of AR gene mutations (i.e. A-G,
Gln-Arg668; and T-C, Ile-Thr670) identified in human prostate tumors
(8, 9), the latter variant being identified in tumors derived from two
individuals (Fig. 1
). The colocation of these mutations to
668QPIF671 residues
suggested that they, like the mutations colocating to the signature
sequence and AF-2, might also have functional consequences relevant to
tumor progression.

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Figure 1. Schematic Showing the DNA Binding Domain (DBD),
Hinge Region (H), and LBD of the AR
The T-A base transition identified in the AR gene in a TRAMP tumor
results in an amino acid substitution (Phe-Ile653), which is equivalent
to Phe-Ile671 in the human AR. Phe-Ile671 colocates to a small peptide
668QPIF671 at the boundary of the hinge and LBD
with two AR gene mutations (i.e. A-G, Gln-Arg668; and
T-C, Ile-Thr670) identified in human prostate tumors. Additional
regions of the AR where AR gene mutations identified in human prostate
cancer colocate (i.e. the signature sequence/helix-3,
and AF-2) are indicated (stippled boxes). Together,
mutations in these three regions account for 73% of the amino acid
substitutions identified in the hinge and LBD of the AR in clinically
important prostate cancer, and collectively span only 8% of the
receptor coding sequence.
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In vitro analysis using the androgen-responsive probasin
reporter gene in PC-3 cells demonstrated that the level of
transactivation activity of the transfected AR variants was greater
than wtAR for each concentration of AR plasmid (Fig. 2A
) and all concentrations of DHT
(0.0110 nM) examined (Fig. 2B
). Furthermore,
the increased transactivation capacity of the
668QPIF671 receptor
variants in the presence of 1 nM DHT was
independent of promoter and cellular context, in that a similar
increase in receptor activity was also observed using the AR-responsive
mouse mammary tumor virus promoter in both PC-3 and CV-1 cells (data
not shown).

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Figure 2. Transactivation Capacity of wtAR and
668QPIF671 AR Variants in Transiently
Transfected PC-3 Cells
Data are expressed as a percentage of the luciferase activity induced
by wtAR in the presence of 1 nM DHT and represents the mean
(±SEM) of three to seven independent experiments (BE),
or mean of a representative experiment performed in triplicate (A). A,
Optimization of transactivation analysis. Maximal transactivation
response was obtained with transfection of 10 ng of either wtAR or
variant AR expression vector. BD, Transactivation of wtAR and
668QPIF671 AR variants in
the presence of (B) increasing concentrations (010 nM)
DHT; (C) nonandrogenic ligands 17ß-estradiol
(17ß-E2, 1 nM), progesterone (PROG,
1 nM) and hydroxyflutamide (OHF, 1000 nM); (D)
adrenal androgens androstenedione (1 nM) and
dehydroepiandrosterone (DHEA, 1 nM). E,
Comparison of transactivation activities of wtAR, the naturally
occurring 668QPIF671 AR
variants identified in prostate tumors (Arg668, Thr670, Ile671), and a
receptor variant, Pro-Arg669, generated by in vitro
mutagenesis, induced by 1 nM DHT.
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Consistent with a precocious receptor phenotype, the
668QPIF671 variants
displayed up to 10-fold greater transactivation of the
androgen-responsive probasin reporter gene induced by 17ß-estradiol,
progesterone, and the adrenal androgens, androstenedione and
dehydroepiandrosterone, compared with wtAR (Fig. 2
, C and D). Moreover,
agonist activity of the AR antagonist, hydroxyflutamide, was observed
for each receptor variant (Fig. 2C
). Taken together, our findings
suggest that the 668QPIF671
or other AR variants with a similar gain of function phenotype could
provide a significant growth advantage in the presence of normal
physiological levels of DHT before treatment, and have sufficient
activity in the presence of 1) alternative ligands (e.g.
estrogens, progestins and antiandrogens such as hydroxyflutamide) or 2)
low levels of DHT after androgen ablation therapy, to promote the cell
survival functions of the AR in cells expressing these variants. In
contrast to these naturally occurring AR variants, in vitro
mutagenesis of the only amino acid (Pro669) in this region conserved
among the closely related receptors for progestins, glucocorticoids,
and mineralocorticoids (18) resulted in a receptor that exhibited
reduced capacity to activate transcription of the probasin reporter
gene compared with wtAR (Fig. 2E
), presumably due to decreased receptor
stability (see below). An Arg669 AR variant is unlikely to be detected
in vivo as it would not be expected to provide a growth
advantage to prostate tumor cells.
Previous studies have shown that mutations in the AR gene in both the
androgen insensitivity syndrome and human prostate cancer can alter AR
mRNA or protein levels and/or the kinetics of ligand binding and
dissociation (10, 19). Immunoblot analysis demonstrated that AR
variants identified in this study exhibited similar steady-state AR
protein levels compared with wtAR in transfected PC-3 cells (Fig. 3
, AD). Consistent with the immunoblot
analysis, similar AR steady state RNA levels of the
668QPIF671 variants and
wtAR were observed by RNAse protection assay (RPA) in transfected PC-3
cells (Fig. 3
, E and F). In addition, there was no appreciable change
in the affinity of the
668QPIF671 AR variants for
DHT (Table 1
) or the synthetic
nonmetabolizable androgen, methyltrienolone (R1881; data not shown)
when compared with wtAR. Furthermore, the naturally occurring
668QPIF671 AR variants
exhibited no change in either receptor stability or rate of
dissociation of DHT compared with wtAR (Fig. 4
, A and B). The observation that the
amino acid substitutions in
668QPIF671 residues do not
alter receptor-ligand binding kinetics suggests that these residues do
not contribute to the formation of the ligand-binding pocket, which is
consistent with observations from the recently determined AR crystal
structure (14). In contrast, the reduced transactivation capacity of
the in vitro derived Arg669 receptor variant appears, in
part, to be due to reduced stability (Fig. 4A
).

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Figure 3. Relative Levels of wtAR and AR Variant Protein and
mRNA Determined by Immunoblot and RNAse Protection Analysis A, AR and cytokeratin 8 (CK) immunoblot using 20 µg of total cellular
protein derived from PC-3 cells transfected with either wtAR or an
668QPIF671 AR variant. B, Relative protein
levels of wtAR and 668QPIF671 AR variants
determined by densitometric analysis of immunoblots in panel A,
normalized for CK internal control and expressed relative to wtAR. Data
represents the mean (±SEM) of three individual
transfections. C, Immunoblot analysis of AR and CK using increasing
amounts (550 µg) of total cellular protein derived from the
AR-positive human prostate cancer cell line, LNCaP. D, Densitometric
quantitation of AR and CK protein levels in panel C above,
demonstrating the linear detection of both proteins over the range of
550 µg total cellular protein. Data shown are a representative
standard curve. E, RPA of AR and cyclophilin (CY) using total RNA
extracted from PC-3 cells transiently transfected with wtAR or
668QPIF671 AR expression vectors. F, Relative
wtAR and 668QPIF671 AR variant RNA levels
determined by densitometric analysis of RPA in panel E, normalized for
CY internal control and expressed relative to wtAR. Data are
representative of three independent transfections.
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Figure 4. Stability (A) and Dissociation Rate (B) of AR-DHT
Complexes in COS-1 Cells Transiently Transfected with wtAR or
668QPIF671 AR Variants
Dissociation rate was also determined for an AR containing an amino
acid substitution at position 778 (Met-Thr778). In contrast to
668QPIF671 AR variants, the Thr778 variant
exhibited rapid dissociation compared with wtAR, consistent with
previous reports for AIS mutations at this residue (15 ). Results
represent the mean of two independent determinations.
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AR electrophoretic mobility shift assays using different amounts of
nuclear protein extracts from transiently transfected PC-3 cells
demonstrated that the
668QPIF671 AR variants bind
to a similar extent as wtAR to a specific, high-affinity androgen
response element derived from the probasin gene [i.e.
PB-ARE-2 (20); Fig. 5A
]. The specificity
of the AR/PB-ARE-2 interaction was demonstrated by the addition of an
AR antibody (21) that resulted in a supershift of the AR/PB-ARE-2
complex (data not shown). Formation of the AR/PB-ARE-2 complex was
specifically inhibited by 100-fold excess of unlabeled PB-ARE-2, but
not by a similar 100-fold excess of a nonspecific competitor (Fig. 5A
).
AR/PB-ARE-2 complex formation by wtAR and AR-Ile671 was equally
competed by 10-, 50-, or 100-fold excess unlabeled PB-ARE-2 (data not
shown). Increasing the salt concentration did not discriminate between
binding of wtAR and the
668QPIF671 AR variants to
PB-ARE-2. The conclusions from mobility shift assays were confirmed by
densitometric analysis (data not shown).

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Figure 5. Relative DNA Binding Ability of wtAR and
668QPIF671 AR Variants
Gel shift analysis of AR DNA binding ability in PC-3 cells transiently
transfected with wtAR, the naturally occurring
668QPIF671 AR variants, or an in
vitro derived mutation (Pro-Arg669). Nuclear extract (5 or 10
µg) from the transfected PC-3 cells and from nontransfected LNCaP
control cells were incubated with equal amounts of
32P-labeled double-stranded PB-ARE2 oligonucleotide probe
in the presence or absence of 100-fold excess unlabeled probe or
100-fold excess of labeled or unlabeled nonspecific (N/S) probe, as
described in Materials and Methods.
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The transactivation capacity of steroid receptors is dependent upon
ligand-induced release of corepressors and the active recruitment of
coactivators (22, 23). The best characterized of these are the p160
coactivators glucocorticoid receptor-interacting protein 1 (GRIP1),
steroid receptor coactivator 1 (SRC1), and amplified in breast cancer-1
(AIB1) (24, 25, 26), which contain conserved LXXLL motifs (L for
leucine and X for any amino acid) termed LX domains (LXDs), that confer
high-affinity binding to a specific hydrophobic surface containing AF-2
in the LBD of steroid receptors in a ligand-dependent manner (27, 28). Structural changes in regions of steroid receptors responsible for
cofactor binding have been shown to impact on receptor activity. For
example, mutations in AF-2 in the AR and the estrogen receptor have
been shown to either abolish receptor activity or result in receptors
that can be activated by antagonists, without affecting the ability of
the receptor to bind ligand (7, 10, 11, 23, 29). In addition, amino
acid substitutions in the LBD of the AR have been shown to enhance
receptor interaction with the coactivator RFG/ELE1 6-fold compared with
the wtAR-RFG/ELE1 interaction (30). This evidence suggests that one
mechanism whereby an AR variant could exhibit a gain of function is
altered interaction with receptor cofactors. However, transactivation
analysis demonstrated that the increased activity of the
668QPIF671 AR variants is
not due to an altered response to the well-characterized p160
coactivator GRIP1 (Fig. 6
), consistent
with a requirement for regions of the AR distinct from the
668QPIF671 residues for
p160 binding. While GRIP1 remains an effective coactivator of the
naturally occurring
668QPIF671 AR variants, the
fold coactivation was marginally reduced compared with wtAR, possibly
reflecting either a reduced requirement for p160 coactivators, or an
altered interaction with other cofactors in PC-3 cells.

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Figure 6. Effect of GRIP1 on the Transactivation Capacity of
wtAR and the 668QPIF671 AR Variants in
Transiently Transfected PC-3 Cells
Transactivation was assessed as in Fig. 2 using a 20:1 ratio of
transfected GRIP1:AR expression vectors, and in the presence of 1
nM DHT. Data are expressed as mean
(±SEM) of four independent determinations.
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To examine whether mutations in
668QPIF671 residues
resulted in structural alterations in the receptor that could
potentially influence ligand binding and/or cofactor interactions, we
applied a homology modeling protocol to generate a model of the AR-LBD
(14). This approach was recently used to produce a model of the AR-LBD
that accurately predicted the AR crystal structure (14). Our analyses
indicate that residues
668QPIF671 do not
contribute directly to ligand binding or to binding of the p160
coactivators (Fig. 7A
;
Refs. 14, 31, 32). Moreover, from this modeling we have identified
that AR residues 668QPIF671
and the carboxy-terminal residues of helix 9 form complete opposing
ridges that frame a small highly hydrophobic cleft (Fig. 7
, B and Ca).
A salt bridge between residues Glu835 and Arg838 forms the hydrophobic
floor of this pocket (Fig. 7C
) which, together with the ridges, is
conserved in the closely related progesterone receptor (PR), but not
other nuclear receptors (data not shown). Crystal structure analysis of
other unliganded nuclear receptors reveals minimal repositioning of
helix 9 after ligand binding (33), strongly suggesting that the cleft
bordered by the 668QPIF671
ridge is a ligand-independent structure. Significantly, each of the
naturally occurring AR gene mutations identified in prostate tumors
reduced the hydrophobicity of the cleft while having no discernable
effect on residues required for ligand or p160 binding (Fig. 7C
, b,c,e,f). A more pronounced reduction in hydrophobicity was predicted
for the in vitro derived mutation in the conserved proline
residue (Pro-Arg669) which, in addition to perturbation of the
668QPIF671 ridge, appears
to decrease the structural stability of the receptor due to a loss of
backbone cyclization (Fig. 7C
d), possibly accounting for the reduced
receptor stability and transactivation capacity observed with this
mutant (Figs. 2E
and 4A
). Ligand-independent formation of the cleft
framed by 668QPIF671
suggests that mutations that disrupt this motif would alter AR activity
independent of the nature of the bound ligand, supporting the
transactivation data (Fig. 2
). The hydrophobic cleft bordered by the
668QPIF671 motif exhibits a
number of features in common with the region of the AR formed by
helices 3, 4, and 12 that binds the LXDs of p160 coactivators (Fig. 7A
). This suggests that it may accommodate related motifs such as the
two LXD-like peptides in the amino-terminal transactivation domain
(NTD) of the AR, which were recently shown to mediate the N-C
interaction between the AR NTD and the LBD resulting in stabilization
of the receptor-ligand complex (34). In that report, the FXXLF peptide
was shown to interact with high affinity to the hydrophobic p160
binding site after ligand binding. While the interaction surface for
the second motif (i.e. WXXLF) is not known, it is possible
that it could bind to the cleft bordered by
668QPIF671 residues.
However, we did not observe altered dissociation of the ligand-receptor
complex during characterization of the
668QPIF671 variants (Fig. 4B
), as would be predicted if the WXXLF motif bound to this region
(34).

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Figure 7. Homology Modeling of the AR LBD
A, MOLSCRIPT/Raster3D ribbon diagram (60 61 ) of the AR LBD bound to
testosterone showing -helix (H112) and ß-sheet (S14)
structures. Ligand (green) is shown docked in the ligand
binding pocket; the NR box LXXLL peptide from the p160 coactivator
GRIP1 (purple) docked in the hydrophobic coactivator
binding cleft formed by helices 3, 4, and 12; and the
668QPIF671 region (red).
Leucines in the GRIP1 motif and the side chains of the
668QPIF671 residues are depicted in
stick form. There are no direct interactions between
668QPIF671 residues and those in either the
ligand binding pocket or the hydrophobic coactivator binding cleft. B,
Electrostatic potential surface of the AR LBD. Regions of the surface
with negative electrostatic potential are shown in red,
and positive electrostatic potential in blue;
hydrophobic surfaces appear as white regions. The GRIP1
peptide and leucine side chains of the LXXLL motif are shown in
purple. Side chains from the
668QPIF671 residues form a short ridge
delineating a small hydrophobic cleft (indicated by an
arrow) bordering a pronounced hydrophobic area. C,
Disruption of a small hydrophobic cleft by mutations in
668QPIF671 residues. a,
Semitransparent surface diagram of the AR
668QPIF671 region showing a
small pocket with a hydrophobic floor bordered on one side by a ridge
consisting of 668QPIF671
residues and on the other by a ridge formed by the carboxy terminus of
helix 9. 668QPIF671 residue
side chains are depicted as sticks and colored according to
the standard CPK scheme. A hydrophobic floor is
formed between these ridges by a salt bridge between Glu835 and Arg838
in helix 9. bf, Electrostatic potential surface diagrams showing that
each of the naturally occurring mutations (c, e, f) in the
668QPIF671 region alter the
hydrophobicity of the
668QPIF671 ridge around the
cleft, and that this is more pronounced for the in vitro
derived Pro-Arg669 substitution (d). Surface coloring is as for panel
B.
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The region of the AR containing the
668QPIF671 residues has
previously been shown to contain a repressor function that impairs
ligand-dependent activity directed by AF-2 (35, 36). In addition, a
four-amino acid deletion in the hinge region has been shown to relieve
the dependence of the AR on the hinge-binding coactivator, Ubc9, for
maximal activity (37), presumably by disrupting the binding of a
corepressor normally displaced by Ubc9. Therefore, one possible
explanation for increased activity of the
668QPIF671 AR variants is a
reduced interaction with a nuclear corepressor. The best characterized
nuclear receptor corepressors, N-CoR (nuclear receptor corepressor) and
SMRT (38, 39), contain extended nuclear receptor interaction motifs
(LXXI/HIXXXI/L) termed the CoRNR box (40, 41, 42). CoRNR boxes appear to
mediate N-CoR and SMRT binding to the hydrophobic cleft of nuclear
receptors normally occupied by the LXDs of coactivators on the
addition of ligand. Moreover, ligand-induced repositioning of AF-2
results in dissociation of N-CoR and SMRT, consistent with their
apparent inability to interact with nuclear receptors in the presence
of ligand (42, 43, 44). Collectively, these data suggest N-CoR or SMRT are
not likely to interact with the cleft bordering the
668QPIF671 motif, and
therefore unlikely to mediate the enhanced ligand-dependent activity of
the 668QPIF671 AR variants.
The observations presented in this study suggest that perturbation of
the interaction between the AR and an undetermined receptor cofactor
mediates the precocious activity of the naturally occurring
668QPIF671 AR variants.
Identification of the critical protein factor(s) interacting with the
hydrophobic cleft adjacent to the
668QPIF671 motif may
provide new insights into AR signaling.
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MATERIALS AND METHODS
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Cell Culture
COS-1, CV-1, and PC-3 cells were obtained from the
American Type Culture Collection (ATCC;
Manassas, VA) and maintained in RPMI 1640 medium (Life Technologies, Inc., Melbourne, Australia) supplemented with 5%
FBS and penicillin (100 U/ml) and streptomycin (100 U/ml).
Single-Stranded Conformational Polymorphism Analysis
Total RNA was isolated from four TRAMP mice at
24 weeks
of age as previously described (45). RT-PCR single-stranded
conformational polymorphism analysis was performed on the entire open
reading frame of the AR gene and analyzed on a nondenaturing acrylamide
gel (6% polyacrylamide, 5% glycerol gel; either 10 C or 28 C for
16 h) according to previously published methods (46, 47) using 10
sets of overlapping primers spanning the full length of the mouse AR
cDNA.
DNA Sequencing
Sequencing was performed using a SEQUENASE 7-deaza-dGTP DNA
sequencing kit (Amersham Pharmacia Biotech,
Buckinghamshire, UK) and a 373A automated DNA sequencer (PE Applied Biosystems, Foster City, CA).
Construction of AR Variant Expression Plasmids
The required base substitutions were introduced into an
expression vector containing the complete human AR gene coding sequence
under the control of the cytomegalovirus promoter (48) by PCR-based
megaprimer in vitro mutagenesis (49) using pCMV-AR as the
template. Megaprimers were created using a common downstream primer
(5'-CCTCTAGAGTCGACCTGCAGG-3'), which spans an
XbaI restriction site unique to pCMV-AR
(underlined), and one of the following upstream primers:
AR-Arg668, 5'-GCTATGAATGTCGGCCCATCTT-3';
AR-Arg669, 5'-GAATGTCAGCGCATCTTTCTG-3';
AR-Thr670, 5'-GTCAGCCCACCTTTCTGAATGTC-3';
AR-Ile671 5'-CAGCCCATCATTCTGAATGTC-3'.
Megaprimers were purified and used in a second PCR with a common
upstream primer (5'-TGGAGATGAAGCTTCTGGGTGT-3') spanning a
HindIII site (underlined) unique to pCMV-AR.
Products were digested with HindIII and XbaI and
cloned into the pCMV-AR digested with the same enzymes using normal
techniques. The presence of the required base substitution
(underlined in the primers above) and the integrity of each
AR variant plasmid construct were confirmed by DNA sequencing of both
strands of the recreated expression plasmid including the entire region
amplified in the PCR-based mutagenesis procedure.
In Vitro Transactivation Assay
The AR-negative human prostate cancer cell line, PC-3 (20,000
cells per well in 96-well plates) was cotransfected with wtAR or AR
variant expression vectors (040 ng), an androgen-responsive minimal
probasin luciferase reporter construct (100 ng) (50), and a control
Renilla luciferase plasmid (pRL-tk, Promega Corp., Sydney,
Australia; 1 ng) using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturers
protocol. Cofactor analysis was performed in an analogous manner using
2.5 ng AR expression vector and cotransfecting 50 ng of GRIP1 (51).
Five hours after transfection, the reaction mixture was replaced with
DMEM (Life Technologies, Inc.) containing 5%
charcoal-stripped FBS and supplemented with 0 or 0.0110
nM steroids. After 36 h, cells were lysed using
Passive Lysis Buffer (Promega Corp.) according to the
manufacturers specifications and assayed immediately for reporter and
control gene activities with the Dual-Luciferase Reporter Gene Assay
(Promega Corp.) using a plate reading luminometer (Packard
Top Count, Canberra Packard, Mount Waverley, Australia). The luciferase
activity induced by 1 nM DHT on wtAR was determined in each
experiment and assigned as 100%. All other data are expressed as a
percentage of this value.
Dissociation Rate, Stability, and Affinity (kDa) of DHT-AR
Complexes
Dissociation of
[1,2,4,5,6,7-3H]-5
-dihydrotestosterone
([3H]-DHT; Amersham Pharmacia Biotech) from wtAR and
668QPIF671 AR variants was
examined in monolayer cultures of COS-1 cells
(106 cells per 10-cm culture dish) as previously
described (52). Cells were transiently transfected with 20 µg of wtAR
or 668QPIF671 AR variant
expression vectors using diethylaminoethyl (DEAE) Dextran (0.5 mg/ml;
Sigma, Castle Hill, Australia; 30 min at 37 C), and
subsequently cultured in DMEM (Life Technologies, Inc.)
containing 5% charcoal-stripped FBS for 48 h. Parallel cultures
were preincubated in DMEM (Life Technologies, Inc.)
containing 5% charcoal-stripped FBS and 3 nM
[3H]-DHT in the presence or absence of a
200-fold excess of unlabeled DHT for 1 h at 37 C. Media were
replaced with DMEM (Life Technologies, Inc.) containing
5% charcoal-stripped FBS and 0.5 µmol/liter unlabeled DHT.
Cyclohexamide (500 µmol/liter) was included to minimize synthesis of
new AR. Assays for AR stability and affinity for DHT were performed as
previously described (19, 48). COS-1 cells transfected as above with
wtAR or 668QPIF671 AR
variants were harvested into 1.2 ml ice-cold cytosol buffer [10
mM Tris·HCl, 1.0 mM EDTA, 10% (wt/vol)
glycerol, 10 mM sodium molybdate, 0.2 mM
dithiothreitol, 1.0 mM phenylmethylsulfonylfluoride; pH
7.2]. Soluble cytosol fractions were prepared by centrifugation at
100,000 x g for 30 min. For analysis of receptor
stability, cytosol fractions were prelabeled with 3
nM [3H]-DHT for 4 h
at 4 C in the presence and absence of a 200-fold excess of unlabeled
DHT and incubated at 37 C for 0, 0.5, 1, and 3 h. Unbound steroid
was removed by addition of dextran-coated charcoal, and the amount of
[3H]-DHT specifically bound was measured by
scintillation counting. The affinity of AR variants for DHT was
determined in cytosol fractions incubated with 0.13.0
nM [3H]-DHT for 16 h
at 4 C. Specific binding was determined as above, and the binding data
were analyzed by Scatchard plot analysis and linear regression.
Immunoblot Analysis
Lysates of PC-3 cells transfected with wtAR or
668QPIF671 AR variant
expression vectors or untransfected LNCaP cells were prepared as for
transfected COS-1 cells above, and the protein concentration was
determined using the Bradford protein assay kit (Bio-Rad Laboratories, Inc. Regents Park, NSW, Australia). Total cellular
protein (20 µg) was electrophoresed on a 6% SDS-polyacrylamide gel,
transferred to Hybond-C membrane (Amersham Pharmacia Biotech), and immunostained using an affinity-purified rabbit
polyclonal antibody (U402; 1:200 dilution) specific for the N-terminal
21 amino acids of the AR (21) and a mouse monoclonal cytokeratin 8
antibody (Sigma; 1:1,000 dilution). Immunoreactivity was
detected using horseradish peroxidase-conjugated sheep antirabbit IgG
(Silenus; 1:2,000) and horseradish peroxidase-conjugated goat antimouse
IgG (DAKO Corp., Carpinteria, CA; 1:4,000), respectively,
and visualized using ECL Western blotting reagents (Amersham Pharmacia Biotech). Relative AR and cytokeratin 8 protein levels
were determined from immunoblots using an Imaging Densitometer
(Bio-Rad Laboratories, Inc.).
RPA
PC-3 cells (5 x 106 cells per 10-cm
dish) were transiently transfected with wtAR or
668QPIF671 AR variant
expression vectors (2 µg) using DEAE-Dextran as described above and
incubated for 24 h in the presence or absence of DHT. Total RNA
was isolated from cells using guanidinium isothiocyanate as previously
described (45). [32P]-UTP-labeled cRNA probes
specific for AR nucleotides 1,6821,918 (48) and cyclophilin
nucleotides 38 to 140 (53) were synthesized using a T7 RNA polymerase
labeling kit (Ambion, Inc., Austin, TX) and hybridized
with 10 µg total RNA. The protection assay was performed using the
Ambion, Inc. RNAse Protection Assay Kit according to the
manufacturers protocol. Products were separated on a 6% denaturing
polyacrylamide gel and visualized using a PhosphorImager
(Molecular Dynamics, Inc. Sunnyvale, CA). AR and
cyclophilin- protected fragments were quantified using Image Quant
software.
Electrophoretic Mobility Shift Assays (EMSAs)
Nuclear extracts of untransfected LNCaP and PC-3 cells and of
PC-3 cells transfected with wtAR or 668QPIF671
AR variant expression vectors were prepared as previously described
(54). EMSAs were performed using labeled specific double-stranded
oligonucleotide probe [PB-ARE-2;
5'-AGCTTAATAGGTTCTTGGAGTACTTTACGTCGA-3'; (20); 0.2 ng] incubated with
510 µg crude nuclear extracts in 20 µl binding buffer (10
mM HEPES, pH 7.9, 50 mM KCl, 1 mM
MgCl2 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol) for 15 min at
room temperature (55). For competition studies, nuclear extracts were
preincubated with 10- to 100-fold excess of unlabeled PB-ARE-2 or a
nonspecific double-stranded oligonucleotide [C/EBP;
5'-GATCCGACCTTACCACTTTCACAATCTGCCAG-3'; (56)]. For EMSAs with
increasing salt concentrations, the binding buffer was supplemented
with 0450 mM KCl. Unbound probe was separated from
protein-DNA complexes by electrophoresis on a 4% nondenaturing
polyacrylamide gel for 3 h at 4 C. Products were visualized using
a PhosphorImager, and shifted DNA-protein complexes were quantified
using Image Quant software.
Homology Modeling
A model of the human AR-LBD was constructed based on the crystal
structure coordinates of the human PR LBD in a complex with
progesterone (18), as described elsewhere (14). Testosterone and the
GRIP1 peptide were docked into the AR model by superimposing the AR-LBD
model onto crystal structures of the PR-LBD/progesterone (18) and
estrogen receptor-LBD/estrogen/GRIP1 peptide (32) complexes. An
energy-minimized structure for testosterone was constructed using
ISIS/Draw and Sculpt 3 (57) and aligned with progesterone by a Maximal
Common Substructure Search (58). Electrostatic and van der Waals
interactions of testosterone and the GRIP1 peptide with the AR-LBD were
minimized locally using Monte Carlo Minimization (59); Van der Waals
interactions were modeled using a modified Lennard-Jones function
between atoms within 6 Å of each other, and electrostatic interactions
were modeled with a distance-dependent dielectric between atoms within
10 Å. Molecular surfaces were calculated using a 1.4-Å probe, and
transparencies were rendered with the Ray Tracing program
POV-RAY 3 (http://ww.povray.org).
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Petra Neufing for critical review of this
manuscript and Ms. Marie Pickering for technical assistance. Also Dr.
Mike R. Stallcup (Department of Pathology, University of Southern
California, Los Angeles, CA) for providing the GRIP1 expression
vector.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Wayne D. Tilley, Flinders Cancer Centre, Flinders Medical Centre, Adelaide SA 5042, Australia. E-mail:
Wayne.Tilley{at}flinders.edu.au
G.B. and M.Y. contributed equally to this work.
This work was supported by grants from the Anti-Cancer Foundation of
South Australia, the Prostate Cancer Foundation of Australia, and the
National Health and Medical Research Council of Australia (W.D.T.,
D.J.H., V.R.M.), the T. J. Martell Foundation (R.J.M.), the
National Cancer Institute Grant CA-74737 (N.M.G.), Prostate Cancer
Specialist Program of Research Excellence Grant CA-58204 (N.M.G.), and
CaP CURE (N.M.G.).
Received for publication December 21, 1999.
Revision received September 1, 2000.
Accepted for publication September 25, 2000.
 |
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