A C619Y Mutation in the Human Androgen Receptor Causes Inactivation and Mislocalization of the Receptor with Concomitant Sequestration of SRC-1 (Steroid Receptor Coactivator 1)
Lynne V. Nazareth,
David L. Stenoien,
William E. Bingman, III,
Alaina J. James,
Carol Wu,
Yixian Zhang,
Dean P. Edwards,
Michael Mancini,
Marco Marcelli,
Dolores J. Lamb and
Nancy L. Weigel
Department of Molecular and Cellular Biology (L.V.N., D.L.S.,
W.E.B., A.J.J., C.W., Y.Z., M.M., M.M., D.J.L., N.L.W.), Scott
Department of Urology (D.J.L.), and Department of Medicine
(M.M.) Baylor College of Medicine Houston, Texas 77030
Department of Pathology (D.P.E.) University of Colorado
Health Science Center Denver, Colorado 80262
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ABSTRACT
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Androgen ablation therapy is a primary treatment
for advanced prostate cancer, but tumors become refractive to therapy.
Consequently, the role of the androgen receptors (ARs) and of mutations
in the AR in prostate cancer has been a subject of much concern. In the
course of analyzing tumors for mutations, we identified a somatic
mutation that substitutes tyrosine for a cysteine at amino acid 619
(C619Y), which is near the cysteines that coordinate zinc in the DNA
binding domain in the AR. The mutation was re-created in a wild-type
expression vector and functional analyses carried out using
transfection assays with androgen-responsive reporters. The mutant is
transcriptionally inactive and unable to bind DNA. In response to
ligand treatment, AR619Y localizes abnormally in numerous, well
circumscribed predominantly nuclear aggregates in the nucleus and
cytoplasm. Interestingly, these aggregates also contain the bulk of the
coexpressed steroid receptor coactivator SRC-1, suggesting, in analogy
to AR in spinal bulbar muscular atrophy, that this mutant may
alter cellular physiology through sequestration of critical proteins.
Although many inactivating mutations have been identified in androgen
insensitivity syndrome patients, to our knowledge, this is the first
characterization of an inactivating mutation identified in human
prostate cancer.
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INTRODUCTION
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Because of the importance of androgens in prostate growth and
development and the initial success of androgen ablation in treatment
of advanced prostate cancer (1, 2), much attention has been devoted to
the role of androgen receptors (ARs) (3, 4) in prostate cancer. In
normal prostate, androgens induce production of growth factors in the
stromal cells that cause growth of the epithelial cells (5). The ARs in
the epithelial cells induce the expression of various proteins
characteristic of the differentiated state including prostate-specific
antigen (6). Tumors develop from the epithelial cells and ultimately
become independent of the prostate stromal cells. After an initial
response to androgen ablation, the tumors in the prostate and their
metastases eventually become unresponsive to endocrine therapy (7).
Whether AR continues to play a role in androgen-independent disease is
an open question. In many cases, it is likely that the cells have
developed independent means of growth. AR usually continues to be
expressed after androgen ablation therapy (4, 8) and the gene is
sometimes amplified (9). Moreover, under some circumstances, activation
of cell-signaling pathways induces AR activity in the absence of
hormone (10, 11). Because the AR is encoded on the X chromosome and
therefore is present as a single copy in males, attention has also been
focused on whether the AR is mutated in prostate cancer tumors and the
potential consequences of these mutations.
There have been several investigations of AR gene alterations/mutations
in prostate cancer (3, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). It appears that the incidence of
mutations increases dramatically in patients with more advanced disease
(3, 12, 16, 17, 18) as there are fewer reports of mutations in early stage
disease (3, 12, 17). AR mutants that respond to the AR antagonist,
flutamide, as an agonist have been detected in patients who have failed
flutamide therapy (20), suggesting a role for these mutations in the
response of the tumors. In the course of searching for AR mutations in
prostate cancer samples, we identified a mutation at cysteine 619, a
non-zinc-coordinating cysteine immediately carboxyl terminal of the
second zinc finger that is conserved in all steroid receptors. The
consequences of amino acid substitutions at this position in the
glucocorticoid receptor vary from no effect to partial inactivation,
depending upon the substitution and the species of glucocorticoid
receptor (25, 26). We find that this mutation in the AR not only
inactivates the receptor, but also causes hormone-dependent aggregation
of the AR. Protein aggregates have been detected in a number of
diseases caused by expansion of CAG repeats, resulting in extended
polyglutamine tracts (27, 28, 29, 30, 31). Included among these is spinal bulbar
muscular dystrophy (SBMA), which is caused by an expansion of the
polyglutamine tract in the amino terminus of the AR (30). We and others
(32, 33) have found that this expansion induces hormone-dependent
aggregates, although the morphology differs from those of the C619Y
mutant. Our finding that the C619Y mutant binds and causes
colocalization of SRC-1 (steroid receptor coactivator 1), suggests that
transcriptionally inactive AR may nonetheless alter cellular
physiology.
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RESULTS
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The AR gene has been cloned and mapped to the X chromosome
(34, 35, 36, 37, 38, 39). It is comprised of eight exons generally containing between
910 and 919 amino acids, depending on the variable length of the
polyglutamine repeat in the amino terminus of the receptor. In the
course of screening primary prostate cancer tumors and associated lymph
node metastases for AR mutations using single-strand conformational
polymorphism, we found a substitution of a tyrosine for the
conserved cysteine carboxyl terminal of the second zinc finger (M.
Marcelli, M. Ittmann, S. Mariani, R. Sutherland, R. Nigam, L. Murthy,
Y. Zhou, D. DiConcini, E. Puxeddu, A. Esen, J. Eastham, N. L. Weigel,
and D. J. Lamb, submitted). The location of the mutation in the AR DNA
binding domain is depicted in Fig. 1A
.
Because a length of 919 amino acids is used for Web-based
collections of AR mutations (http://www.mcgill.ca/androgendb), we have
termed this mutation C619Y to conform to this nomenclature.

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Figure 1. Functional Analysis of the Mutant AR
The location of the mutation in the AR DNA binding domain is indicated
by the arrow in panel A. CV-1 cells (panel B) and PC-3
cells (panel C) were transfected with 0.5 µg of the
GRE2E1bCAT reporter alone (first set of
bars) or in conjunction with 62.5 ng (CV1 cells) or 5 ng (PC-3
cells) of the WT or mutant AR (C619Y) indicated by the second
and third sets of bars, respectively. The cells were treated
with ethanol vehicle, 10 nM, or 0.1 nM R1881.
Error bars are as indicated (SD of the mean for
triplicates).
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Mutation of Cysteine 619 to Tyrosine Results in a Loss of
Transcriptional Activity
To determine whether this mutant is transcriptionally active, the
androgen-inducible GRE2E1bCAT reporter was transfected into
CV1 cells (Fig. 1B
) or PC-3 prostate cancer cells (panel C) in the
absence or presence of the wild type (WT) or mutant (C619Y) AR
expression vectors. Cells were treated with vehicle (ethanol), or the
synthetic androgen, R1881 (10 nM or 0.1 nM). As
expected, the reporter alone is not activated by R1881 in either cell
line (shown in the first set of bars). The WT receptor is strongly
activated by either concentration of R1881 (the second set of bars).
However, the C619Y receptor is not activated at either level of
androgen (the third group). In data not shown, the C619Y receptor,
cotransfected with an estrogen-responsive ERE2E1bCAT
reporter, was also found to be inactive, indicating that the mutation
did not alter the specificity of interaction with this DNA response
element.
C619Y Does Not Undergo the Hormone-Dependent Change in Mobility on
SDS-PAGE Analysis That Is Characteristic of WT AR
To be certain that mutation did not alter receptor expression,
high-salt extracts were prepared from COS-1 cells transfected with WT
or C619Y receptors and analyzed by SDS-PAGE followed by immunoblotting.
One of the characteristics of the WT AR is hormone-dependent
phosphorylation resulting in reduced mobility on SDS gels. The AR
is detected as a doublet of 112110 kDa corresponding to different
phosphorylation isoforms (40, 41), as indicated by the arrow
(Fig. 2
). Lanes 14 represent WT
receptor and lanes 58 represent C619Y receptor (duplicates). Lanes 1
and 2 and lanes 5 and 6 were from control (ethanol-treated cells)
extracts, and lanes 3 and 4 and lanes 7 and 8 were from cells treated
with 10 nM R1881. Expression levels for the receptors are
similar. The WT receptor shows an increase in the 112-kDa form in the
presence of R1881 (the upper band becomes more pronounced compared with
control) as previously reported (41). In contrast, the C619Y receptor
does not show a corresponding increase in the 112-kDa form on steroid
treatment, suggesting a failure to undergo a hormone-dependent
posttranslational modification.

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Figure 2. Expression of WT and C619Y AR
An immunoblot of COS-1 cells transfected with WT or C619Y receptors and
treated with ethanol vehicle or 10 nM R1881 is shown.
Thirty micrograms of total protein were electrophoresed (in duplicate)
on a 6.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and
probed with monoclonal antibody AR441 to the AR. Lanes 14 correspond
to WT AR and lanes 58 to C619Y AR-transfected extracts. Lanes 1, 2,
5, and 6 correspond to vehicle treatment, and lanes 3, 4, 7, and 8
represent R1881-treated extracts. The AR protein is visualized as a
doublet of 112110 kDa as shown by the arrow.
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The Mutant Receptor Has A Slightly Lower Steroid Binding Affinity
Than WT Receptor
Since the mutation is carboxyl terminal of the second zinc finger
of the DNA binding domain, and we did not detect a hormone-dependent
change in mobility on SDS-PAGE gels, we next tested the hormone binding
affinity of the receptors. COS-1 cells were mock transfected
(GRE2E1bCAT DNA) or transfected with WT or C619Y expression
vectors, and a whole-cell binding assay was performed using
[3H]R1881. Scatchard analysis (Fig. 3A
) showed that the dissociation
constants (Kds) for the WT and C619Y receptors are 0.84
nM and 1.90 nM, respectively. This difference
of a factor of 2 has been detected consistently in five separate
experiments. Because transactivation was tested at concentrations of
hormone that would essentially saturate the hormone binding site (10
nM), this difference is unlikely to explain the lack of
transcriptional activity.

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Figure 3. Effect of the C619Y Mutation on Steroid Binding
A, Hormone binding affinity. COS-1 cells were mock transfected or
transfected with 10 ng of the WT and C619Y AR expression vectors and
treated with doses of [3H]R1881 ranging from 0.1
nM to 4 nM for 24 h. Cells were washed
with PBS and bound receptor was extracted with 100% ethanol.
Nonspecific counts were subtracted from total counts using the
mock-transfected values to yield specific counts. A graph of bound/free
vs. bound (nM) was plotted. The WT receptor
is indicated by the solid circles, whereas the C619Y
receptor is shown by the open circles. B, R1881
dissociation rate of the mutant AR is not altered. COS-1 cells were
transfected with WT and C619Y receptors and were incubated with
[3H]R1881 or radioinert R1881 for varying lengths of time
as described in Materials and Methods. A graph of
exponential counts bound vs. time (hours) was plotted to
compare the R1881 dissociation rates for the two receptors. The WT
receptor is depicted by the solid circles and the C619Y
receptor by the open circles.
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Dissociation Rates of R1881 for the C619Y and WT Receptors Are
Similar
Zhou et al. (42) have described AR mutations with
near-normal hormone binding affinity, but dramatically increased
dissociation rates that reduce activity of the mutant. We next measured
the dissociation rate of R1881 from C619Y. COS-1 cells were transfected
with WT or C619Y expression vectors, and a dissociation rate assay was
performed using [3H]R1881. From the plot of exponential
counts bound over time (Fig. 3B
), it can be seen that the two receptors
have nearly parallel slopes, implying that their off rates are similar.
In fact, when a plot of percent counts bound over time was done, the
t1/2 values for the WT and C619Y receptors were very
similar (217 and 204 min, respectively; data not shown).
In Vitro Analysis of DNA Binding
To test for DNA binding, extracts from R1881-treated COS cells
containing WT AR, C619Y, or no receptor were incubated with labeled ARE
(androgen response element) +/- AR441 antibody and analyzed by
electrophoretic mobility shift assay (EMSA) as described in
Materials and Methods (Fig. 4A
). Although there are a number of DNA
complexes formed by COS cell extracts alone (lane 5), there is an
additional complex in cells transfected with AR (lane 1) that is
supershifted by addition of AR441 (lane 2). In contrast, AR619Y fails
to form a DNA complex either in the absence (lane 3) or presence (lane
4) of AR441. That this was not due to differences in expression levels
is shown in the Western blot in Fig. 4B
.

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Figure 4. Electrophoretic Mobility Shift Assay
COS cell extracts containing WT receptor, C619Y, or no receptor were
incubated with radiolabeled ARE -/+ monoclonal antibody AR441 and
analyzed by EMSA as described in Materials and methods.
Lane 1, WT receptor; lane 2, WT + AR441; lane 3, C619Y; lane 4, C619Y +
AR441; lane 5, mock; lane 6, probe alone. The position of the AR-ARE
complex is as indicated by the arrow on the gel.
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The Mutant Receptor Does Not Bind to AREs in Whole Cells
Because EMSA conditions may be more stringent than in
vivo DNA binding, a promoter interference assay was used as
described previously to directly assess DNA binding in vivo
(43, 44). The promoter interference reporter plasmid contains three
consensus progesterone response element (PRE)/AREs positioned
between the TATA box and the transcription start site of the
chloramphenicol acetyltransferase (CAT) gene, all driven by the
constitutive cytomegalovirus (CMV) enhancer/promoter. In theory,
binding of AR to the AREs will sterically hinder assembly of the
transcription complex and reduce CAT gene expression. As shown in Fig. 5
, as increasing amounts of AR WT
expression vector are transfected into cells, the transcription of the
CMV-PRE3-CAT reporter is inhibited in a dose-dependent
manner. However, the C619Y receptor does not significantly inhibit
expression of the reporter gene, indicating that it does not bind to
the AREs in vivo.

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Figure 5. Promoter Interference Assay
COS-1 cells were transfected with increasing amounts of WT or C619Y
receptor expression vectors (ranging from 0 ng to 2 ng) and with the
0.1 µg of CMV-PRE3-CAT reporter. The cells were treated
with 0.1 nM R1881 and harvested to assay for potential
inhibition of CMV-driven CAT activity by the receptors binding to the
AREs in the reporter construct. The WT receptor is represented by the
solid circles and the C619Y receptor by the open
circles.
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Subcellular Localization of the AR and the Steroid Receptor
Coactivator SRC-1
To determine whether the subcellular localization of AR was
altered in the C619Y mutant, we transfected HeLa cells and looked at
receptor distribution using the AR441 antibody. As shown in Fig. 6
, in the absence of hormone, both WT and
C619Y receptors exhibited a diffuse and predominantly cytoplasmic
distribution [panels A (WT) and D (C619Y)]. The addition of 5
nM R1881 for 16 h resulted in the nuclear localization
of both receptors (panels B and E). The WT AR localizes in a
hyperspeckled distribution in the nucleoplasm (panel B). Remarkably,
after 16 h of hormone treatment, the C619Y receptor is found
localized to ligand-dependent aggregates in the nucleus and sometimes
in the cytoplasm (panel E). Nuclear aggregates are uniform in shape and
can range from barely detectable to 12 µm in diameter. That this
pattern is not a fixation artifact is shown in panels C and F in which
GFPAR [green fluorescent protein (GFP)-linked AR] displays normal
nuclear distribution whereas the GFPAR619Y mutant forms aggregates. The
differences in appearance of these aggregates in panels E
vs. F is at least partially due to the inability of the
antibody to penetrate the aggregates, resulting in nonuniform staining,
whereas all ARs in panel F contain GFP. Although aggregate formation
has been reported previously for artificially generated AR mutations in
the zinc-coordinating cysteines (45) and for mutants with deletions
that eliminate one or more of these cysteines, not all DNA binding
mutants form aggregates (D. L. Stenoien and M. Mancini,
unpublished results). To our knowledge, this is the first observation
of hormone-dependent aggregate formation by an AR containing a
single amino acid substitution. This suggests that substitution of the
cysteine by the tyrosine affects the protein folding of the AR
molecule, such that it forms aggregates with other AR molecules and
possibly with other factors.

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Figure 6. The C619Y Mutation Alters the Hormone-Dependent
Subnuclear Localization of the AR
Immunofluorescence was performed on HeLa cells transfected with WT or
C619Y receptors. Panels A (WT) and D (619Y) show untreated cells
immunostained with anti-AR and a FITC-conjugated secondary antibody.
Both WT and C619Y have a diffuse and predominantly cytoplasmic
distribution in the absence of hormone. Panels B (WT) and E (C619Y)
show cells treated 16 h with 5 nM R1881. WT receptor
translocates to the nucleus and adopts a hyperspeckled distribution in
response to hormone. C619Y also translocates to the nucleus in response
to hormone but forms intranuclear aggregates of varying size that are
substantially different from foci containing WT receptor. Nuclear
aggregates containing C619Y range in size and can be up to 2 µm in
diameter. C619Y aggregates are found in almost all cells with
detectable levels of immunofluorescence, suggesting that aggregates do
not result from overexpression. In some cells cytoplasmic C619Y
aggregates can be seen. To demonstrate that C619Y aggregates are not a
staining or fixation artifact, GFP fusions of WT and C619Y were
transfected into HeLa cells and microscopy was performed on live,
unfixed cells. Panels C (GFP-WT) and F (GFP-C619Y) show live cells
after hormone treatment. Both GFP-WT and GFP-C619Y localize similarly
to their immunostained, untagged counterparts. Bar
= 10 µm.
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Because the C619Y retains hormone binding activity, we next asked
whether the mutant would cause SRC-1 to associate with the aggregates
in vivo. To test this, cotransfection experiments were
performed with a green fluorescent SRC-1 (GFP-SRC-1) construct (32).
This construct behaves similarly as WT SRC-1 in terms of its
coactivation and localization properties (D. L. Stenoien, S.
Onate, and M. Mancini, unpublished observations). When
cotransfected with C619Y in the absence of hormone, C619Y is
predominantly cytoplasmic whereas GFP-SRC-1 exhibits diffuse nuclear
staining (Fig. 7D
) indistinguishable from
its distribution in the absence of AR (Fig. 7C
). In the presence of
R1881, GFP-SRC-1 has the same hyperspeckled distribution as AR and
overlaps considerably with AR staining (Fig. 7A
). In parallel
experiments, cotransfection with the C619Y construct in the presence of
R1881 leads to GFP-SRC-1 localizing with C619Y aggregates (Fig. 7B
).
After cell division, these aggregates can also be seen in the cytoplasm
(data not shown). Thus, this transcriptionally inactive form of AR
appears to retain the ability to associate with and, in this case,
sequester SRC-1.

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Figure 7. GFP-SRC-1 Colocalizes with Both WT and C619Y
Receptors
HeLa cells were transfected with WT+GFP-SRC-1 (A), C619Y+GFP-SRC-1 (B
and D), or GFP-SRC-1 alone (C). Cells were treated 16 h with 5
nM R1881 (A and B), and immunofluorescence was performed on
fixed cells using anti-AR antibody and Texas Red-conjugated secondary
antibody. In cells treated with hormone (A and B), there is substantial
overlap of both WT and C619Y signal (top row) with
GFP-SRC-1 fluorescence (middle row); merged images
(bottom row) demonstrate the high degree of overlap. In
the absence of cotransfected receptor (C), GFP-SRC-1 has a nuclear
distribution. To demonstrate that C619Y and GFP-SRC-1 colocalization is
dependent upon hormone, immunofluorescence was performed on cells in
the absence of hormone (D). No overlap of staining is observed with the
predominantly cytoplasmic C619Y and the nuclear GFP-SRC-1.
Bars = 10 µm.
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DISCUSSION
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The mutation described in this paper, in contrast to most of the
previously described mutations in prostate cancer, is a somatic
mutation causing loss of transcriptional function (an inactivating
mutation). Germline inactivating mutations are often found in androgen
insensitivity syndrome (AIS), and Takahashi et al. (46)
reported the presence of inactivating mutations (frame-shift, nonsense,
and missense) in latent carcinomas in the Japanese but not American
populations. In contrast, no inactivating mutations were identified in
clinical carcinoma cases from either country. Fenton et al.
(20) have identified several AR mutations in patients that failed
flutamide treatment which allow the AR to utilize flutamide as an
agonist. In contrast, the C619Y mutation was identified in a patient
who was initially classified as having organ-confined cancer until
lymph node metastases were identified during surgery. This
naturally occurring mutation involved substitution of a highly
conserved cysteine in the DNA binding domain, which obliterates AR
function. Although this residue is not one of the cysteines involved in
binding zinc, it is conserved across members of the steroid receptor
superfamily. Replacement of the corresponding cysteine with a glycine
residue in the human glucocorticoid receptor results in a receptor that
is unable to bind DNA and unable to stimulate transcription of an
inducible MMTVCAT reporter, but represses the
-glycoprotein hormone
promoter up to 70% of WT function (26). In contrast, replacement of
the same residue by alanine or serine in the rat glucocorticoid
receptor did not compromise receptor function (25). Hence, it was not
immediately clear what effect the tyrosine substitution at this
position in AR would have on receptor function. The AR mutant is
transcriptionally inactive and does not bind DNA in vitro or
in vivo. The hormone binding affinity is slightly lower, but
this difference is insufficient to account for loss of activity.
The immunolabeling studies performed shed some light on how this
mutation may alter AR function. The C619Y receptor, like the WT
receptor, translocates from the cytoplasm to the nucleus during 16
h of androgen treatment, indicating that the mutation does not
completely block the hormone-induced localization of AR. However, once
inside the nucleus, it adopts an altered distribution and forms
discrete foci that develop into large clusters of mutant receptor.
These aggregates are observed in more than 90% of the cells expressing
detectable levels of AR. In the absence of hormone, the apparently
normal cytoplasmic appearance of the mutant suggests that the heat
shock proteins bind to the mutant and either correct or mask the
misfolded area. Hormone treatment results in dissociation from heat
shock proteins, thus allowing potentially abnormal interactions of the
AR containing the misfolded region with other AR molecules and possibly
with other proteins. Whether the change in folding induced by the
mutation produces a hydrophobic surface that induces aberrant
interaction with additional AR molecules leading to aggregration or
whether the misfolded protein is recognized by the chaperone machinery
inducing aggregration remains to be determined. Abnormal protein
aggregation through expanded polyglutamine tracts has been reported in
a number of diseases (27, 28, 29, 30). Among these is SBMA or Kennedys
disease, in which AR aggregates have been detected in the cytoplasm
and/or nucleus of affected motor neurons (31), upon immunocytochemical
staining (30). The AR aggregates in the patients and in transfected
cells differ from the C619Y aggregates both in their size (much larger)
and cellular localization (30, 32). Interestingly, many SBMA patients
also exhibit symptoms of AIS as they get older, suggesting that the
activity of the receptors with expanded polyglutamine repeats is
compromised. SBMA is not due to a simple lack of sufficient AR as
demonstrated by the fact that most AIS patients do not have SBMA. Hence
the presence of the abnormal protein must have a deleterious effect on
cell physiology.
The C619Y mutant also fails to undergo the characteristic
hormone-dependent increase in phosphorylation detected by altered
mobility on SDS-PAGE analysis of the WT AR. This implies that the
phosphorylation either requires specific subcellular localization to a
region in the nuclear matrix containing the appropriate kinases
(47, 48, 49) or DNA binding. Alternatively, misfolding within the
aggregates (or elsewhere) blocks access of the kinase to the
phosphorylation sites. Nevertheless, the C619Y receptor colocalizes
with the coexpressed SRC-1 in the aggregates, demonstrating that
interactions between steroid receptors and coactivators may occur in
the absence of DNA binding and transcription. This interaction
presumably occurs through interactions of SRC-1 with the
hormone-binding domain as in the WT, since, as judged by hormone
binding affinity, the mutation has little effect on the hormone-binding
domain. The ability of C619Y to sequester SRC-1, and potentially other
factors that interact with AR, suggests that mutants that are
transcriptionally inactive may still have profound effects on cell
function through reduction in available levels of other factors.
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MATERIALS AND METHODS
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Materials
All cell culture media and reagents were purchased from
Life Technologies, Inc. (Gaithersburg, MD). R1881
(radiolabeled and radio inert forms) was obtained from NEN
Life Science Products (Boston, MA), [
-32P]dGTP
was from ICN Pharmaceuticals (Costa Mesa, CA) and
poly-L-lysine was from Sigma. All other
chemicals were reagent grade.
Case Report
The patient was a 62-yr-old white male found to have metastatic
prostate cancer (stage D1, Gleason grade 3+3) during exploratory
surgery. Metastatic deposits were found in the right iliac and
hypogastric lymph nodes. The mutation was identified in this tissue (M.
Marcelli, M. Ittmann, S. Mariani, R. Sutherland, R. Nigam, L. Murthy,
Y. Zhou, D. DiConcini, E. Puxeddu, A. Esen, J. Eastham, N. L. Weigel,
and D. J. Lamb, in preparation).
Transfection
Monkey kidney CV-1 and COS-1 cells and human prostate PC-3 cells
were transfected by the nonrecombinant adenoviral-mediated DNA transfer
technique (11, 50). Plasmid DNA for transfection (62.5 ng of AR
expression vector for CV1 cells, 5 ng for PC-3 cells, and 10 ng for
steroid binding assays along with 0.5 µg of the androgen-inducible
GRE2E1bCAT reporter) (51) were incubated with the coupled
virus (at a multiplicity of infection of 750:1 for CV1 cells, and 500:1
for PC-3 and COS-1 cells) for 30 min. For EMSAs, 100 ng AR expression
vector and 0.5 µg CMV-ß-galactosidase carrier DNA were used.
Subsequently, additional poly-L-lysine (1.3 µg/µg of
DNA) was added to shrink the DNA onto the viral surface. The virus-DNA
complex was added to the cells and allowed to infect the cells for
2 h in serum-free medium, after which time the medium was
supplemented with charcoal-stripped serum to a final concentration of
5%. Each experiment was performed a minimum of three times.
Cell Treatment
Twenty-four hours after transfection, the transfected CV-1,
COS-1, and PC-3 cells were treated with 10 nM or 0.1
nM R1881 (methyltrienolone from NEN Life
Science Products or 0.2% ethanol vehicle (control) or as
described in the individual procedures.
Chloramphenicol Acetyltransferase (CAT) Assay
After 24 h of treatment, the cells were harvested by
scraping in TEN buffer (40 mM Tris, 1 mM EDTA,
150 mM NaCl, pH 8.0) and pelleted by centrifugation. Cells
were resuspended in 0.25 M Tris, pH 7.5, and lysed by
freeze thawing. Protein concentrations were determined by a microtiter
plate assay (Bio-Rad Laboratories, Inc. Hercules, CA) that
is a modification of the Bradford assay (52). The CAT activity was
determined by incubation of 2.520 µg of total protein with
[3H] chloramphenicol (20 µCi/µmol)(NEN
Life Science Products and butyryl-CoA as previously described
(53). Acylated chloramphenicol was extracted using a 2:1 mixture of
tetramethyl pentadecane:xylene and counted in a scintillation
counter.
AR Antibody Generation and Characterization
The peptide CSTEDTAEYSPFKGGYTK, corresponding to amino acids
301317 of the human AR, with an added cysteine residue at the amino
terminus for cross-linking, was synthesized and coupled to Keyhole
Limpet Hemocyanin by Macromolecular Resources, Colorado State
University (Ft. Collins, CO). Immunizations, cell fusions, and
construction of hybridomas were done as previously described (54). The
antibody was purified from a cell culture supernatant by 50% ammonium
sulfate precipitation followed by purification on a protein G Sepharose
column. The antibody specifically detects AR by Western blot analysis,
immunoprecipitation, and immunocytochemistry (D. P. Edwards and N.
Weigel, unpublished observations).
Western Blot Analysis
Cells were harvested in 0.4 M NaCl homogenization
buffer (50 mM potassium phosphate, pH 7.4, 50
mM sodium fluoride, 1 mM EDTA, 1 mM
EGTA, and 12 mM
-monothioglycerol) with protease
inhibitors (leupeptin, antipain, aprotinin, benzamidine HCl,
chymostatin, pepstatin, phenylmethylsulfonyl fluoride). Thirty
micrograms of protein were electrophoresed on a 6.5% SDS-PAGE gel,
transferred to nitrocellulose using a liquid transfer apparatus
(Bio-Rad Laboratories, Inc.), and the filter was processed
as follows. The membrane was blocked with a solution of Tris-buffered
saline (TBS, 10 mM Tris, 150 mM NaCl, pH 7.5
solution) containing 1% milk for 10 min, incubated with the anti-AR
antibody described above (at a concentration of 0.05 µg/ml) for
1 h, washed with TBS, incubated with a secondary rabbit antimouse
antibody (2:10,000 dilution of a 1 mg/ml stock) for 1 h, washed
with TBS, followed by TBS + 0.1% Tween. The membrane was then
incubated with antirabbit horseradish peroxidase (1:10,000 dilution)
for 1 h and washed with TBS + 0.3% Tween solution, followed by
TBS + 0.1% Tween. The signal was detected using the ECL kit from
Amersham Pharmacia Biotech (Arlington Heights, IL).
Scatchard Analysis
A whole-cell binding assay was performed in transfected COS-1
cells as described in Ref. 55 . Cells were transfected with WT, C619Y,
or GRE2E1bCAT DNA as a control. After 24 h, the cells
were treated with [3H]R1881 (specific activity = 86
Ci/mmol) at doses ranging from 0.1 to 4 nM for 24 h.
Cells were washed five times with ice-cold PBS and
[3H]R1881 extracted with ethanol. Samples were counted in
scintillation cocktail in a LS6500 counter (Beckman Coulter,
Inc., Fullerton, CA). Nonspecific counts (from
mock-transfected cells) were subtracted from total counts to yield
specific counts bound. The Kd values for the two receptors
were determined by Scatchard analysis.
R1881 Dissociation Rate Analysis
The off-rate for steroid dissociation from the receptor was
calculated as described by Zhou et al. (42). Briefly, COS-1
cells in six-well plates were transfected with 10 ng of AR WT or C619Y
receptors. The next day, the medium was replaced with serum-free
medium. Five nanomolar [3H]R1881 was added to all of the
wells, and then cells were incubated in the absence (total) or presence
of a 100-fold molar excess of radioinert R1881 (nonspecific) for 2
h at 37 C. Cells were washed twice with ice-cold PBS, the medium was
replaced, and once again the cells were incubated in the absence or
presence of a 1000-fold molar excess radioinert R1881 at 37 C. At the
designated time points (0, 1, 2, 3, 4, and 5 h), cells were washed
twice with PBS, and the hormone was extracted in ethanol and counted in
scintillation cocktail. Specific binding was obtained by subtracting
nonspecific counts from the total counts. A plot of bound counts
vs. time was used to determine whether the two receptors
have similar t1/2 values (time required for dissociation of
half of the counts bound to the receptor). The t1/2 for
each receptor was calculated from a plot of percentage counts bound
vs. time.
Electrophoretic Mobility Shift Assay
The ARE oligo DNA used for band shift analysis is a single
steroid response element from the tyrosine amino transferase gene as
described by Tsai et al. (56). The probe was labeled with
-[32P]dGTP (3000 Ci/mmol) using DNA polymerase (Klenow
fragment) and purified using a Nensorb column (NEN Life
Science Products). COS-1 cells that were transfected with WT AR
or C619Y DNA and treated with 10 nM R1881 were freeze
thawed and harvested in 0.4 M NaCl TEDG (10 mM
Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol,
and 10% glycerol), and cell extracts were prepared as before.
Additional R1881 was added to the cell extract to a final concentration
of 10 nM. A reaction mix was prepared containing 4 µl
labeled ARE (
300,000 cpm), 14 µl 10% Ficoll, 15 µl 1
mg/ml nuclease-free BSA, 15 µl 0.1 M
dithiothreitol, 45 µl 35 µg/ml pBR322 cut with HinF, and 15
µl of TEDG as described previously (57). Incubations included two
µg of cell extracts, 5 µl of reaction mix +/- 1 µl AR441 anti-AR
antibody in a final volume of 17 µl. Samples were incubated on ice
for 40 min and run on a 5% gel in TAE (40 mM Tris acetate,
1 mM EDTA, pH 8.3). The gel was prerun at 200 V for 30 min
in the cold room and, after loading, was run at 200 V until the
bromophenol blue dye moved to one inch from the bottom of the gel.
Labeled complexes were detected by autoradiography after the gel was
dried.
Promoter Interference Assay
A promoter interference reporter plasmid
(CMV-PRE3-CAT) described previously (43) was used to
determine the ability of the AR to bind to DNA in whole cells. The
plasmid has the constitutive CMV promoter followed by three synthetic
PRE/ARE oligonucleotides inserted into the unique SacI site
of CMV-TATA-CAT (kindly provided by Dr. Benita Katzenellenbogen)
located between the TATA box and the start of transcription of the CAT
gene. COS-1 cells were cotransfected as before with 02 ng of AR WT or
C619Y expression vectors and 0.1 µg of CMV-PRE3-CAT
plasmid and treated with 0.1 nM R1881 for 24 h before
harvesting. Two micrograms of total protein were assayed for CAT
activity.
Immunolabeling and Image Acquisition
Hela cells were grown in OptiMEM medium (Life
Technologies, Inc.) supplemented with 4% FBS. For the
transfections, cells were plated onto poly-D-lysine coated
coverslips in 35-mm wells at a density of 1 x 105
cells per well. The GFP-WTAR and GFP-SRC-1 plasmids were made as
described previously (32). The GFP-C619Y plasmid was generated using
the same protocol as for GFP-WT. The C619Y sequence was cut out of the
original vector using an internal XbaI and a
3'-BglII site. This segment was subcloned into a
pEGFP-C1-AR(aa1108) vector using the XbaI and
BamHI sites to generate the GFP-C619Y plasmid. Cells were
transfected with 0.10.5 µg of DNA (AR expression vectors and/or the
GFP-SRC-1 construct) using the calcium phosphate mammalian cell
transfection kit (5 Prime-> 3 Prime, Inc, Boulder, CO). Cells were
shocked with 10% dimethylsulfoxide for 3 min and then
transferred to DMEM with 5% stripped FBS. After 4 h, the cells
were treated with ethanol or 5 nM R1881 for 16 h.
Cells were then fixed with a 4% paraformaldehyde solution in PEM
buffer (80 mM
piperazine-N,N,'-bis(2-ethanesulfonic
acid), 1 mM EDTA, 1 mM
MgCl2, pH 7.4), extracted with 0.5% Triton X-100 in PEM,
and blocked with 5% milk in TBST (0.1 M Tris, 0.15
M NaCl, 0.1% Tween-20, pH 7.4). Cells were incubated with
the monoclonal antibody to the AR (1 µg/ml in TBST with 5% milk) for
2 h, followed by incubation with fluorescein- or Texas
Red-conjugated goat antimouse secondary antibody (Southern
Biotechnology Associates, Birmingham, AL) diluted 1:600 in TBST
with 5% milk for 1 h. A Z-series (0.1 µm steps) of optical
sections was digitally imaged on a Delta Vision Deconvolution
Microscopy System (Applied Precision, Inc., Issaquah, WA) and
deconvolved using a constrained iterative algorithm to generate
high-resolution images. All image files were digitally processed for
presentation in Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).
Shown in each image is a single Z-section from typical cells. For live
microscopy, HeLa cells were plated onto 40-mm coverslips in 6-cm dishes
and transfected with 1 µg of either GFP-WTAR or GFP-C619Y. After
hormone treatment, cells were transferred to a live cell chamber
(Bioptechs, Inc., Butler, PA) and maintained in DMEM with 5% stripped
FBS and 5 nM R1881 at 37 C. Image acquisition and
processing were performed as above.
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Dr. John Cidlowski (NIEHS, Research
Triangle Park, NC) for providing us with the
GRE2E1bCAT reporter, as well as Kurt Christensen and Teri
Ladtkow, University of Colorado Cancer Center Tissue Culture/Monoclonal
Antibody Core Facility (Boulder, CO), for assistance with production of
the AR antibody. Imaging was performed in the Department of Cell
Biologys Integrated Microscopy Core; we thank Frank Herbert for
technical support.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Nancy L. Weigel, Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030.
This work was supported in part by NIH Training Grant HD-07165
(L.V.N.), a Cancer Research Foundation of America fellowship to L.V.N.,
a Merck United Negro College Fund fellowship (A.J.J.), NIH
Training Grant T32 DK-07696 (A.J.J.), and NIH Grant CA-68615 as well as
a generous gift from the Association for the Cure of Cancer of the
Prostate to D.J.L., M.M., and N.L.W.
Received for publication September 18, 1998.
Revision received August 10, 1999.
Accepted for publication August 16, 1999.
 |
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