From the Division of Urologic Surgery, Department of
Surgery, ¶ Renal Division, Department of Medicine, Brigham and
Women's Hospital and Harvard Medical School,
Boston, Massachusetts 02115
Received for publication, July 24, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Androgen receptor (AR) belongs to the steroid
hormone nuclear receptor superfamily. It functions as an
androgen-dependent transcriptional factor that regulates
genes for cell proliferation and differentiation. Caveolin is a
principal component of caveolae membranes serving as a scaffold protein
of many signal transduction pathways. Recent results correlate
caveolin-1 expression with androgen sensitivity in murine prostate
cancer. Furthermore, immunohistochemical staining of patient specimens
suggests that caveolin expression may be an independent predictor of
progression of prostate cancer. In this study, we investigate the
potential interactions between AR signaling and caveolin-1 and
demonstrate that overexpression of caveolin-1 potentiates
ligand-dependent AR activation. Conversely, down-regulation
of caveolin-1 expression by a caveolin-1 antisense expression construct
can down-regulate ligand-dependent AR activation. Association between these two molecules is also demonstrated by co-localization of AR with caveolin-rich, low-density membrane fractions isolated by an equilibrium sucrose gradient centrifugation method. Co-immunoprecipitation and glutathione
S-transferase fusion protein pull-down experiments
demonstrate that interaction between AR and caveolin-1 is an
androgen-dependent process, offering further evidence for a
physiological role of this interaction. Using a mammalian two-hybrid
assay system, we determine that the NH2 terminus region of
caveolin-1 is responsible for the interaction with both the
NH2-terminal domain and the ligand-binding domain of
AR.
Androgen receptor (AR)1
belongs to the steroid hormone nuclear receptor superfamily. It
functions as a ligand-dependent transcriptional factor that
regulates genes for cell proliferation and differentiation. Similar to
glucocorticoid and mineralocorticoid steroid receptors, AR remains in
the cytoplasm until it is activated by ligand binding (1, 2). In a
classic activation paradigm, AR is dissociated from the heat-shock
protein chaperone complex upon ligand binding and dimerized AR
translocates into the nucleus where transcriptional activation is
initiated by binding to the cognate regulatory sequence on target
genes. There is evidence that signal cross-talk between AR activation
and growth factors such as epidermal growth factor, keratinocyte
growth factor, and insulin-like growth factor-I (3-5) mediates signal
transduction. Other nongrowth factor-related signal pathways reported
to modulate AR include protein kinase A, which mediates
ligand-independent AR activation (6, 7), and protein kinase C, which
negatively regulates AR-dependent transcription (8). It is
unclear how signaling by these nonsteroid growth factors or kinase
pathways overlap with the AR activation pathway. Either they directly
affect the assembly or transport of the transformed AR or they affect
AR transcriptional activity by interacting with overlapping general
transcriptional regulators.
Recent studies by Thompson and colleagues (9) suggest that expression
of caveolin-1 may regulate androgen responsiveness in prostate cancer.
They found, in samples from patients with prostate cancer, a positive
correlation between expression of caveolin-1 and progression of the
cancer (9). Using tumor cells derived from the mouse prostate
reconstitution model (10, 11), these authors link the expression of
caveolin-1 to androgen sensitivity in hormone-resistant metastatic
prostate cancer (12). Moreover, immunohistochemical staining of
caveolin-1 in tumor samples from patients who had undergone radical
prostatectomy suggested that caveolin-1 immunostaining is an
independent predictor of disease progression (13). Among 187 specimens
from lymph node-negative cancers, 47 were found with caveolin-1
immunoreactivity, which correlates with a shorter interval to
postsurgical recurrence.
Caveolin-1, a 21-24-kDa integral membrane protein, is a major
component of the caveolae membrane structures, with a flask-shaped invagination that are enriched with cholesterol and glycosphingolipid as well as with lipid-modified signaling proteins. Caveolin-1 has been
implicated as a principal structural scaffold for the oligomerization
and organization of cytoplasmic signal complexes (14-16). Interaction
with and modulation by caveolin-1 has been shown in many signal
transduction pathways, including those regulated by receptor or soluble
tyrosine kinases. Caveolin-1 has been shown to regulate the activity of
phosphatidylinositol 3-kinase associated with receptor tyrosine kinase
(17) and to associate with and regulate endothelial nitric-oxide
synthase (18, 19), epidermal growth factor receptor (20), and insulin
receptor (21). Targeted down-regulation of caveolin-1 expression in 3T3
cells results in hyperactivation of mitogen-activated p42/44 protein
kinases as well as loss of anchorage-dependent cell growth
(22, 23). However, the physiological consequences of caveolin-1
overexpression remain controversial. Interaction of caveolin-1 with
many of the signal transduction components is thought to have important
consequences for cellular transformation. In lung and breast cancer
cells, overexpression of caveolin-1 results in reduced transformation phenotypes (24, 25), suggesting a tumor suppressor role of caveolin-1.
A reciprocal relationship between Her2/Neu tyrosine kinase activity and
caveolin-1 expression has been documented in mammary adenocarcinoma.
Ectopic overexpression of caveolin-1 inhibits Her2/Neu activity
in vivo, a further suggestion of a tumor suppressor role of
caveolin-1 (26). In cells derived from an mouse prostate reconstitution
model, however, overexpression of caveolin-1 promotes resistance to
apoptosis induced by androgen withdrawal, suggesting a promoter role of
caveolin-1 in prostate tumor progression (12). Steroid hormone estrogen
receptors have been shown to be potentiated by caveolin-1 in their
transcriptional activities (27). Furthermore, nongenomic estradiol
stimulation of nitric oxide release has been shown to be mediated by
estrogen receptors localized in caveolae (28).
In light of these findings, we investigate the potential interactions
between AR signaling and caveolin-1. We demonstrate that overexpression
of caveolin-1 potentiates ligand-dependent AR activation
and, conversely, that down-regulation of caveolin-1 expression by a
caveolin-1 antisense expression construct can down-regulate
ligand-dependent AR activation. We also demonstrate an
association between these two molecules by finding co-localization of
AR with caveolin-rich, low-density membrane fractions isolated by an
equilibrium sucrose gradient centrifugation method.
Co-immunoprecipitation and GST fusion protein pull-down experiments
demonstrated that interaction between AR and caveolin-1 is an
androgen-dependent process, offering further evidence for a
physiological role of this interaction. Using a mammalian two-hybrid
assay system, we determine that the caveolin-1 NH2 terminus
region is responsible for the interaction with both the
NH2-terminal domain and the ligand-binding domain (LBD) of
AR.
Materials--
Dihydrotesteosterone (DHT) was purchased from
Sigma, polyclonal antibody to caveolin-1 from Transduction Laboratories
(Lexington, KY), monoclonal antibody against AR from Santa Cruz
Biotechnology (Santa Cruz, CA), and monoclonal antibody to
hemagglutinin antigen (HA) from Berkeley Antibody (Berkeley, CA). A kit
for selection of transfected cells, Capture-Tec System, was purchased
from Invitrogen (Carlsbad, CA) and charcoal-dextran treated fetal
bovine serum from HyClone (Denver, CO).
Expression Vector Constructs--
Human AR pSV-hAR (29)
was cloned into pCDNA3.0 (Invitrogen) downstream of a CMV immediate
early promoter. Sense and antisense caveolin-1 constructs, a gift from
H. Chapman (Harvard Medical School), were cloned into the pCEP4 and
pMEP4 vectors, respectively (Invitrogen). An androgen-responsive
luciferase reporter construct driven by a minimal promoter was
constructed by inserting four synthetic tandem repeats of the
androgen-responsive element (ARE) primers
(5'-TGTACAGGATGTTCTGAATTCCATGTACAGGATGTTCT-3' and
5'-AGAACATCCTGTACATGAATTCAAGAACATCCTGTACA-3') in front of an E1b
minimal TATA box sequence, followed by a firefly luciferase gene. A
renilla luciferase reporter gene driven by a CMV promoter was used as a
transient transfection internal control. Mammalian two-hybrid
expression vectors expressing fusion protein of VP16-AR (full-length),
AR-N-(1-500), DBD-(501-660), and LBD-(661-919) were
constructed by cloning the corresponding fragments in-frame with
partial herpes transactivating protein (VP16, residues 411-456) as a
fusion gene into the pACT vector (Promega, Madison, WI). Caveolin-1
full-length and truncated mutants (Cav-(1-60), Cav-(58-178), Cav-(60-100), and Cav-(135-178)) were cloned in-frame fused with GAL4-DBD (residues 1-147) in a pBIND vector (Promega). Luciferase reporters pTet-off (encoding a tetracycline repressor protein driven by
CMV promoter) and pTRE-luc (luciferase reporter driven by seven tandem
repeats of tetracyclin-responsive element-fused minimal promoter)
(CLONTECH, Palo Alto, CA), pGAL-VP16 (CMV-driven expression vector of Gal4 DNA-binding domain fused with VP16
transactivating domain) (O. Gjoerup, Dana-Farber Cancer Institute), and
pG5-Luc (luciferase driven by five tandem-repeats of Gal4 binding
sequence-fused minimal promoter) (Promega) were used as nonandrogen
responsive reporter control vector for monitoring general
transcriptional activities.
Cell Culture and Transient Transfection--
PC3 and LNCap cells
were kept in RPMI 1640 supplemented with antibiotics (penicillin and
streptomycin) and 10% fetal bovine serum. HEK293 cells were grown in
Dulbecco's modified Eagle's medium (high glucose) supplemented with
antibiotics and 10% fetal bovine serum. HEK293 cells overexpressing
caveolin (293-Cav) or harboring an antisense caveolin driven by a
metallothionine promoter (293-AS), gift of Dr. Chapman (30), were kept
in the same medium as 293 cells supplemented with hygromycin. For
androgen stimulation experiments, cells were grown in the same medium
supplemented with 10% charcoal stripped fetal bovine serum (HyClone).
Cells were transfected by electroporation with a total of 10 µg of
plasmid DNA using a Bio-Rad Gene Pulser (Bio-Rad), and a luciferase
assay was performed 48 h after transfection.
Establishing Stable LNCap Cells Expressing Caveolin-1--
The
pCDNA3.0 vector (Invitrogen) was used to subclone the full-length
human caveolin-1 cDNA downstream of the CMV promoter. LNCap cells
were transfected with pCDNA-Cav (LNCap-Cav), and an empty vector
was used as a noncaveolin expressing LNCap parental control. Clones
were selected in medium containing 300 µg/ml G418.
Preparation of Caveolae-enriched Membrane by Subcellular
Fractionation--
Low-density, caveolae-rich membrane fractions were
isolated as described previously (31). Four 100-mm dishes of
caveolin-1-expressing LNCap cells (LNCap-Cav) were grown to confluence
(a total 2 × 107 cells), scraped into 1.5 ml of 0.5 M sodium carbonate buffer (pH 11.0), homogenized with a
hand-held Polytron on ice (three 3-s bursts at medium speed), and
sonicated (three 1-min bursts at 90% output with a Branson 450 sonicator). The resulting cell lysates were cleared by centrifugation
on a bench-top centrifuge at 600 × g for 5 min.
Supernatants were mixed with equal amounts of 90% sucrose in
Mes-buffered saline (MBS; 50 mM Mes, pH 6.8, 150 mM NaCl) to make a concentration of 45% sucrose. Lysates
containing the 45% sucrose were transferred to a centrifuge tube and
overlaid with 4 ml each of 35 and 5% sucrose in MBS containing 0.25 M carbonate. The gradient was centrifuged at 39,000 rpm
(200,000 × g) for 21 h with a Beckman SW41Ti
rotor. The resulting gradient fractions were analyzed by collecting 12 1-ml fractions from the bottom of the gradient. The fractions were
subjected to Western blot analysis with specific antibodies against
either AR or caveolin-1.
Mammalian Two-hybrid Assay--
A mammalian two-hybrid assay was
performed with the vector system, pBIND and pACT, commercially
available from Promega. Interaction between AR and caveolin-1 was
determined with pACT-AR and its truncated mutants derived from
full-length AR with pBIND-caveolin constructs. HeLa cells were
transfected with the above expression constructs along with a
luciferase reporter construct driven by a minimal promoter fused with
GAL4-binding element. Activity was determined by a dual luciferase
assay (Promega) in the presence and absence of 1 nM DHT.
Fold induction relative to basal activity of cells transfected with
straight pACT and pBIND control vectors was calculated and normalized
to the renilla luciferase internal transfection control.
Immunoprecipitation--
A standard protocol was used for
immunoprecipitation of AR and caveolin-1. In brief, cells were lysed in
immunoprecipitation RIPA buffer containing 50 mM Tris (pH
7.4), 135 mM NaCl, 1% (v/v) Triton X-100, and 60 mM octylglucoside and supplemented with protease inhibitors
(2 mM phenylmethylsulfonyl fluoride, 5 mM
diisopropyl fluorophosphate, 5 µg/ml pepstatin, and 1 mM
EDTA). Lysates were cleared by centrifugation at 12,000 × g for 30 min at 4 °C. Supernatants were incubated with
individual antibodies (1 µg) and protein A-Sepharose beads (20 µl
of packed beads) at 4 °C for 1 h. At the end of incubation, beads were washed 5 times with lysis buffer. The resulting
immunoprecipitated immunocomplexes were solubilized in 40 µl of
Laemmli sample buffer, resolved by SDS-PAGE, and transferred to a
nitrocellulose membrane. The protein complex was detected by Western
blot analysis and developed by ECL (Amersham Pharmacia Biotech,
Piscataway, NJ).
GST Fusion Protein Pull-down Experiment--
GST-caveolin fusion
protein was created by fusing full-length caveolin-1 in-frame to the 3'
end of glutathione transferase using a PGEX-2T vector (Amersham
Pharmacia Biotech) under the regulation of a tac promoter. In
vitro translated AR labeled with [35S]methionine was
obtained with the TNT system for coupled transcription and translation
in vitro (Promega). GST-Cav fusion proteins were immobilized
on glutathione beads and resuspended in binding buffer (100 mM NaCl, 1 mM EDTA, 0.05% Nonidet P-40, 0.2%
bovine serum albumin, 20 mM Tris, pH 8.0). GST-Cav packed
beads (20 µl) were incubated with in vitro translated AR
labeled with [35S]methionine in a total of 40 µl of
binding buffer and incubated at 4 °C in the presence or absence of
10 nM DHT for 4 h. After incubation, the beads were
washed 5 times with 1.5 ml of washing buffer (100 mM NaCl,
1 mM EDTA, 0.5% Nonidet P-40, 20 mM Tris, pH
8.0). The resulting GST·Cav complexes were resolved by
SDS-PAGE and visualized by autoradiography.
Enrichment of Positive Transfectants using Hapten-coated Magnetic
Colloidal Beads--
To enrich positive transfectants in a transient
transfection experiment, we employed the commercially available kit
Capture-Tec (Invitrogen). The procedure for isolation of positive
transfectants was performed following the manufacturer's instructions.
In brief, PC3 cells for AR reporter assay experiments were
co-transfected with pHook-1 plasmid encoding a membrane-anchored
single-chain antibody (sFv) directed against the hapten
4-ethoxymethylene-2-phenyl-2-oxazolin-5-one. 16 h after
transfection, cells were harvested with phosphate-buffered saline/EDTA
and resuspended in phosphate-buffered saline; 10 µl of hapten-coated
colloidal beads was added to 2 × 106 transfected
cells and incubated at 37 °C with constant mixing for 30 min.
Transfected cells expressing sFv were selected by immobilizing cells on
a magnetic stand in an Eppendorf tube, while cells not expressing sFv
were washed away. The resulting cells were subjected to biochemical
analysis or replating for the AR reporter gene assay. To determine the
recovery efficiency for positive transfectants, we used PC3 cells that
were co-transfected with pHook-1 and green fluorescence protein and
then counterstained with Hochst dye in a parallel experiment to monitor
recovery rate by counting green fluorescent cells under a fluorescence
microscope. We were constantly able to enrich cells from 90% to 95%
with positive green fluorescence protein expression as compared with
<10% positive transfectants in a preselected population.
Gel Electrophoresis and Immunoblotting--
Proteins were
separated by SDS-PAGE with a standard reducing condition protocol.
After electrophoresis, proteins were electroblotted to a nitrocellulose
membrane. The protein bands were visualized by Ponceau S staining.
Blots were blocked by 5% nonfat dry milk, 0.05% Tween 20 in
Tris-buffered saline (10 mM Tris, pH 8.0, 135 mM NaCl). Immunoblotting was performed with designated
antibodies and visualized with an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech) following the manufacturer's protocol.
Overexpression of Caveolin-1 Enhances AR-mediated Transcriptional
Activity and Increases the Sensitivity of AR to
Ligand-dependent Activation--
High levels of caveolin-1
expression have been observed to correlate with the sensitivity of
murine metastatic prostate cancer cells to androgen withdrawal (12), a
finding that suggests caveolin plays a role in AR signaling. To test
this hypothesis, we used a cell culture model to determine whether
changes in the levels of caveolin-1 expression alter
ligand-dependent AR activation. The HEK293 cells expressed
detectably lower levels of caveolin-1 compared with the PC3 cells,
allowing for greater manipulation of caveolin-1 expression by
transfection (data not shown). We used HEK293 lines stably transfected
with either a wild-type caveolin-1 construct (293-Cav) or an antisense
caveolin-1 construct (293-AS) driven by either CMV or an inducible
metallothionine promoter, respectively. As shown in Fig.
1A, 293-Cav constitutively
expresses levels of caveolin-1 ~10 times higher than those expressed
by the parental vector control HEK293 line, as determined by video densitometry. Expression of caveolin-1 in the 293-AS cells was further
reduced to 25% of the level of parental 293 control group when an
antisense construct driven by a metallothionine promoter was induced by
inclusion of 1 µM cadmium in the culture medium.
Responses of AR signaling in cells expressing different levels of
caveolin-1 expression were determined by transient co-transfection of
pCDNA-hAR and p(ARE)4-Luc into the above mentioned cell
lines, followed by exposure to DHT (1 nM). As shown in Fig.
1B, in the absence of DHT stimulation, overexpression of
caveolin-1 resulted in a 4-fold elevation of basal ARE-luciferase
reporter activity in 293-Cav cells as compared with that of parental
293 vector control cells. These results suggest that caveolin-1
overexpression sensitizes cells to AR mediated signaling which induces
a moderate ligand-independent activation of AR. This observation is
consistent with the previous finding that caveolin-1 overexpression
promotes estrogen receptor
We hypothesize that cultured cell lines overexpressing caveolin-1
sensitize AR (making it "hyperactive") by lowering the critical concentration of androgen required for AR activation. Using the same
transient transfection assay, we tested this hypothesis by establishing
an androgen dose-response curve for ligand-dependent AR
activation in HEK293 cells expressing various levels of caveolin-1. As
shown in Fig. 2, the IC50 for
the ligand-dependent activation of vector control parental
293 cells was approximated at 0.5 nM DHT. In contrast, the
293-Cav cells required only 0.02 nM DHT to achieve the same
fold stimulation (determined by interpolation), 25 times lower than
that required by the parental 293 cells. The left-shift of the
dose-response curve indicated an increase in fold stimulation of AR in
response to DHT in 293-Cav cells compared with parental 293 cells and
293-AS cells. This experiment demonstrated that caveolin-1
overexpression in 293-Cav cells dramatically increased androgen
receptor-mediated transcriptional activation as 293-CAV cells required
a much lower concentration of androgen to achieve the same androgenic
responses as that achieved by the 293 parental vector control
cells.
To further confirm these results, we tested the prostate cancer PC3
cell line. Since PC3 expresses very high levels of caveolin-1, efforts
to increase levels of caveolin expression appreciably by heterologous
overexpression proved futile (data not shown). We used a transient
system with an antisense caveolin-1 construct to down-regulate the
expression. By co-transfecting a membrane-anchored mouse sFv receptor
(see "Experimental Procedures") with antisense caveolin-1 into PC3
cells, we were able to enrich the positive transfectants harboring the
caveolin antisense expression vector in a transient transfection
experiment with the aid of colloidal magnets coated with sFv-binding
hapten. As shown in Fig. 3A,
an antisense vector dose-dependent down-regulation of
caveolin-1 expression in PC3 cells was observed, whereas the levels of
AR protein remained equal in various groups. This system was used in
parallel to test our hypothesis concerning caveolin-1 modulation of AR
transcription. As shown in Fig. 3B, down-regulation of
caveolin-1 expression in PC3 cells by antisense caveolin-1 reduced the
androgen sensitivity of AR. This reduction in AR transactivation
activity, as shown in Fig. 3A, was not due to reduced levels
of AR expression. Down-regulation of caveolin-1 expression by antisense
did not affect AR-unresponsive general transcriptional activity of the pGAL-VP16/pG5-Luc reporter system (Fig. 3C). These results
strongly suggest that cross-talk occurs between the AR activation
pathway and the caveolin-1-associated signal complex and further
indicate that the level of caveolin expression correlates positively
with AR-mediated transcriptional activity.
Cosedimentation of AR with Caveolin-1-enriched Caveolae Membrane
Domain Fractions--
The results of AR activity modulated by
caveolin-1 expression suggest potential physical interactions between
these two molecules. To evaluate the association of AR with
caveolin-rich caveolae membrane complex, we performed equilibrium
sucrose density gradient centrifugation (31) to determine whether these
two molecules co-localize in the caveolin-enriched membrane fractions.
Equilibrium sucrose density gradient centrifugation is widely used for
isolation of caveolae-enriched membrane fractions because these
membrane subdomains contain high levels of cholesterol and
sphingolipids with characteristic low buoyant density (16). Since LNCap
cells express AR but do not express detectable amounts of caveolin-1 (9, 32),2 we established a
stable LNCap cell line expressing caveolin-1 constitutively
(LNCap-Cav). LNCap-Cav cells were stimulated with vehicle or 1 nM DHT for 10 and 60 min. Caveolin-containing low-density membrane fractions were collected by equilibrium sucrose gradient as
described under "Experimental Procedures." As shown in Fig. 4, only a small amount of AR was
associated with the caveolin-rich density membrane fractions in
nonandrogen-stimulated LNCap-Cav cells (upper panel,
control), as detected by Western blot. Association of AR with the
low-density, caveolin-rich membrane fractions increased with androgen
stimulation (middle panel, 10 min). By 60 min, this association became undetectable (lower panel) as most of the
AR became nucleus bound. Distribution of caveolin-1 in the caveolae membrane fractions remained constant throughout the time course. These
results indicate that AR redistributes to the caveolin-rich membrane
fractions in response to androgen stimulation and that such
redistribution is a dynamic transient process since AR is no longer
detected in the caveolin-rich membrane fractions once it becomes
nucleus bound.
Co-immunoprecipitation of AR with Caveolin-1 in Response to
Androgen Stimulation--
The association of AR with caveolin-1-rich,
low-density membrane fractions provides further evidence that this
receptor is somehow interacting with caveolin-1 in response to androgen
stimulation. However, the co-localized subcellular distribution does
not indicate a direct interaction between these two molecules. Two
approaches, in vivo co-immunoprecipitation and in
vitro GST fusion protein pull-down, were undertaken to further
address this issue. Co-immunoprecipitation studies in intact cells were
performed with LNCap cells stably expressing caveolin-1 (LNCap-Cav).
These cells were maintained in hormone-free medium for 72 h prior
to the experiment. They were stimulated with 1 nM DHT or
vehicle for 10 min at 37 °C. Cell lysates were collected for
immunoprecipitation with antibodies against caveolin-1 (rabbit
polyclonal), against AR (mouse monoclonal), or with mouse or rabbit IgG
and then Western blotted. The membranes were probed reciprocally with
antibodies against either AR or caveolin-1 and developed with
horseradish peroxidase-coupled specific second antibodies in an
enhanced ECL system. As shown in Fig. 5
(upper panel), association of AR with caveolin-1 was
detected in anti-caveolin-1 immunoprecipitates. Consistent with the
sucrose gradient AR partition assay described above, AR associated with caveolin-1 was detected at a low level in the nonandrogen-treated group, and the association increased after cells were treated with 1 nM DHT for 10 min. Reciprocally (Fig. 5, lower
panel), caveolin-1 also was detected in the anti-AR
immunoprecipitation complex. In the control IgG groups, neither AR nor
caveolin-1 was detected by Western blot analysis.
Direct Interaction between AR and Caveolin-1 as Determined by
GST-caveolin Fusion Protein Pull-down Experiment--
The
co-immunoprecipitation of AR and caveolin-1 was further substantiated
by a GST fusion protein pull-down experiment. Bacteria-expressed GST-caveolin fusion protein was immobilized on glutathione-Sepharose beads, which were incubated with in vitro translated AR
labeled with [35S]methionine in the presence or absence
of androgen. As shown in Fig. 6, in
vitro association of AR with immobilized caveolin is also an
androgen-dependent event. Together, these results demonstrate that
association between AR and caveolin-1 is a ligand-dependent process.
Determination of Interaction between AR Submolecular Domain and
Caveolin-1 by Mammalian Two-hybrid Assay--
The results described
above indicate a direct interaction between AR and caveolin-1 during AR
ligand-dependent activation. To further characterize this
interaction at the submolecular level, we performed a mammalian
two-hybrid assay. This approach was previously described in mapping the
interaction between caveolin-1 and endothelial nitric-oxide synthase
(33). As depicted in Fig. 7A,
we divided AR into several submolecular domains:
NH2-terminal (AR-N, residues 1-500),
NH2-terminal with DNA-binding domain (AR-N/DBD, residues 1-660), DNA-binding domain alone (DBD, residues 500-660), and ligand-binding domain alone (LBD, residues 660-919). The full-length AR and submolecular domains were cloned in-frame with a herpes VP16
transactivation protein epitope tagged with HA using a pACT vector (see "Experimental Procedures"). Full-length caveolin-1 was
cloned in-frame with Gal4 DNA-binding protein tagged with HA in a pBIND
vector. Expression of the fusion proteins was confirmed by Western blot
(Fig. 7B) using 12CA5 monoclonal antibody against HA tag.
Consistent with the biochemical results, the two-hybrid results show
that interaction between full-length AR and caveolin-1 is
androgen-dependent, i.e. 46.6 ± 6.5-fold
of induction (p < 0.001; two-tailed Student's
t test for samples with unequal variances) as compared with
vector control (Fig. 7C). Inclusion of bicalutamide (Casodex), an anti-androgen, also promoted the interaction between AR
and caveolin (64.5 ± 6.5-fold of induction, p < 0.005), similar to an agonist effect, a finding consistent with the
ligand dependence of this interaction observed above. The higher
luciferase induction in the Casodex group may reflect a higher
concentration of Casodex used for the treatment. Both AR-N and LBD
domains showed an interaction with caveolin-1, with induction of
73.6 ± 10.4-fold and 14.2 ± 0.7-fold, respectively
(p < 0.005), whereas AR-DBD showed no detectable interaction with caveolin-1. Constructs with overlapping DBD domains, AR-N/DB, exhibited the same levels of interaction as their
nonoverlapping counterpart AR-N, an indication that DBD does not
participate in the interaction. The interaction between caveolin-1 and
LBD, although weaker, appeared to be androgen-dependent. It
is interesting that the anti-androgen bicalutamide (Casodex) did not
promote caveolin/LBD interaction. The interaction between the
NH2-terminal of AR (AR-N or AR-N/DBD) and caveolin-1 was
not affected by treatment with the antagonist.
Mapping the Submolecular Regions of Caveolin-1 Supports the
Interaction with AR--
To determine the submolecular region required
for the interaction of caveolin-1 with AR, we cloned caveolin-1
truncated mutants, Cav-(1-60), Cav-(58-100), and Cav-(135-178) (Fig.
8A), which encompass both the
amino and carboxyl termini of cytoplasmic domains of caveolin-1
in-frame with the GAL4DBD in a pBIND vector with an HA tag. The
expression of the corresponding constructs was confirmed by a Western
blot analysis with use of anti-HA 12CA5 monoclonal antibody (Fig.
8B). A mammalian two-hybrid assay was carried out to detect
the interactions between these various fragments with various AR
domains, amino terminus (AR-N), DBD, or carboxyl domains (LBD), by
transient co-transfection. As shown in Fig. 8C, a strong interaction was detected between caveolin NH2-terminal
amino acid residues 1-60 and the AR-N (25.8 ± 2.5-fold of
induction; p < 0.005), while a weaker interaction was
detected between the LDB domain of AR and the same caveolin-1 fragment
(6.0 ± 0.3-fold of induction; p < 0.05). Since
no functional domain was previously designated in the
NH2-terminal region of caveolin-1 (14), our result defines
for the first time a potentially new interacting domain for caveolin-1.
It was previously reported that deletion of the
NH2-terminal corresponding region in caveolin-3, a
muscle-specific isoform of caveolin-1, results in no functional defect
in the mediation of Ha-Ras signals (34). In this context, our
results suggest that this domain may function in special signal events. Cav-(58-101) and Cav-(135-178) exhibit no detectable interactions with any of the submolecular domains of AR since only basal luciferase activities were detected in these groups.
Caveolin-1 Mutants with Deleted AR-binding Domain Do Not Interact
with AR--
To validate the physiologic relevance of the caveolin
AR-binding domain identified from the two-hybrid assay, we used two of
our deletion mutants of caveolin-1, Cav-(1-60) (the AR-binding domain), and Cav-(58-178) (deletion of AR-binding domain), to test
their ability to interact with AR in vivo in
co-immunoprecipitation and AR response transcriptional reporter assays.
AR- and HA-tagged full-length caveolin (Cav-FL), Cav-(1-60), or
Cav-(58-178) were co-transfected into 293 cells. HA-tagged full-length
caveolin and deletion mutants were immunoprecipitated by anti-HA (clone 12CA5). The resulting immunoprecipitates were probed with polyclonal antibody to AR in a Western blot analysis. As expected, AR could be
co-immunoprecipitated by HA-tagged Cav-(1-60), although at a lower
level than the full-length caveolin (Fig.
9A). Consequently, the
AR-binding domain deletion mutant Cav-(58-178) exhibits no physical
interaction with AR, as evidenced by the absence of AR in the
immunoprecipitation complex. Next, using a transient co-transfection reporter assay, we characterized these deletion mutants of caveolin for
their ability to modulate AR transactivation activity. As shown in Fig.
9B, co-transfection of caveolin wild type up-regulates AR
transactivation activity as compared with activity in the vector control (1.73 ± 0.13-fold; p < 0.005).
Co-transfection of the newly identified AR-binding domain deletion
mutant Cav-(58-178) had no modulatory effect on AR transactivation.
However, co-transfection of the caveolin AR-binding domain
(Cav-(1-60)) with AR down-regulated AR transcriptional activity to
40% of the vector control. Since 293 cells expressed moderate levels
of caveolin-1, down-regulation of AR transactivation by overexpression
of the cytosolic soluble Cav-(1-60) fragment may have resulted from
the interference of the interaction between AR and endogenous
caveolin-1. The expression of AR in different transfection groups
remained at similar levels as determined by Western blot (Fig.
9C).
In the current study, we demonstrate the interaction between AR
and caveolin-1 and show that overexpression of caveolin-1 potentiates
AR-mediated transcription. Our results support the notion that
overexpression of caveolin-1 sensitizes AR signaling by lowering the
critical concentration of androgen required for AR activation. It is
therefore plausible that, in some prostate cancers, overexpression of
caveolin-1 is one way by which the tumors become unresponsive to
androgen deprivation and survive in the milieu characterized by a
castration-like androgen concentration. The correlation of the level of
caveolin-1 expression with disease progression demonstrates the
changing physiology of cancer cells during cancer progression. The
"neo-expression" of caveolin-1 in cancerous prostate cells appears
to be a gain-of-function step during tumor progression.
Immunohistochemical analysis of a prostate specimen (9, 13)
demonstrated that normal human prostate and BPH epithelial cells do not
express detectable levels of caveolin-1. Furthermore, detection of
caveolin-1 expression in an organ-confined prostate cancer specimen
predicts a shorter time to disease progression (13). These observations
suggest that, in normal prostate epithelium, AR activation pathways
interact with signal components associated with a caveolae functional
equivalent, the cholesterol-rich raft microdomain, for signal
transduction (35).
Caveolin-1 is a versatile protein that has a very complex
functional domain organization. Several recent studies have elegantly defined domains involved in targeting of Golgi (residues 60-80 and
135-178), homologous oligomerization (residues 61-101), heterologous oligomerization (residues 168-178), scaffolding domain (residues 80-101), and transmembrane domain (residues 102-134) (36-38).
Caveolin has been well recognized as a primary scaffolding protein in
the membrane invagination caveolae. It has also been implicated in membrane trafficking of nonclathrin-dependent endocytosis
and intracellular cholesterol transport. As a signal complex scaffold protein, caveolin-1 has been postulated to organize and modulate the
signal outputs. The conventional "caveolae signaling hypothesis" proposed by Lisanti and colleagues (22, 26) suggests that caveolar
localization of various signaling molecules provides a
compartmental basis for their regulation and serves as a convergence point for cross-talk between different signaling pathways. Interaction between AR and caveolin may represent one such regulatory mechanism. Our data on the transient interaction between AR and caveolin upon
ligand stimulation suggest a functional interplay between the
caveolin-scaffolding signal complex and liganded AR. As demonstrated in
the present study, AR does not associate with caveolin until it is
activated by ligand binding. Interaction between AR and the
"preassembled scaffold signal complex" may function by providing an
organizational mechanism for molecular interaction and by maximizing the signal output. This hypothesis is consistent with our finding of a
positive correlation between the level of caveolin expression and AR
transactivation activity.
The association of various signal molecules with caveolin is mediated
by a conserved caveolin scaffolding domain located in the
membrane-proximal region as determined by domain mapping (39, 40). This
domain recognizes a well defined caveolin-binding motif
( Alternatively, multiple lines of evidence demonstrate that caveolin-1
is also involved in the intracellular membrane trafficking. Caveolin
has been shown to directly bind to cholesterol (46, 47). Treating cells
with cholesterol oxidase causes a "retrograde" movement of caveolin
from membrane caveolae to endoplasmic reticulum. The returning of
caveolin-1 from endoplasmic reticulum to membrane caveolae has been
determined to be microtubule-dependent (48). Furthermore,
it has been shown that, while transporting cholesterol intracellularly,
caveolin forms a complex with chaperone consisting of hsp56,
cyclophylin 40, and cyclophilin A (49). The same subset of chaperones
is also involved in AR transformation and nuclear translocation (50,
51), suggesting a potential overlap in the intracellular trafficking
machinery. It is estimated that up to 10 to 15% of caveolin resides in
the cytosol rather than being membrane-bound in 3T3 cells (49). It
raises the possibility that the interaction between AR and caveolin may
be targeted at AR nuclear translocation as part of the steroid-receptor
transformation process. Moreover, a recent study by Ozanne et
al. (52) shows that filamin, an actin cross-linking protein, is an
AR-interacting protein. The authors show that
ligand-dependent AR nuclear translocation is facilitated by
filamin interaction since AR remains in the cytoplasm even after
prolonged incubation with androgen in filamin-deficient M2 cells. They
propose a potential role of filamin in the organization of an active
chaperone complex for AR nuclear translocation. Filamin has previously
been identified as interacting with caveolin through the caveolin
NH2-terminal half (residues 1-101) (53). Inter-relations between these observations and our results further underscore the
possibility that the functional role of the AR-caveolin interaction may
be targeted at AR nuclear translocation. Indeed, in the study by
Schlegel et al. (27), overexpression of caveolin appears to
potentiate estrogen receptor Our finding that the NH2-terminal 60 amino acids of
caveolin interact with AR is somewhat unexpected since the
NH2 terminus of caveolin (residues 1-60) has not
previously been designated to be a functional domain. Schlegel et
al. (27) has previously demonstrated that Cav-(1-60) expresses as
a soluble polypeptide in cytoplasm. The ability of this peptide
fragment to down-regulate AR transactivation in 293 cells, which
express moderate level of caveolin-1, may be a result of competitive
interference of AR and endogenous caveolin interaction. This notion is
further mirrored by the ineffectiveness of Cav-(58-178) to modulate AR transcriptional activity in the transient transfection reporter assay.
Unlike that of NH2-terminal of AR with caveolin,
interaction between LBD and caveolin depends on androgen but not on
Casodex (Fig. 8C), suggesting that this interaction is
conformation-dependent. A potential caveolin binding motif
composite ( In summary, we have demonstrated the potential cross-talk between the
caveolin-1-associated signal pathway and AR-mediated transcriptional
activity in a cell culture model. Several lines of evidence, biological
as well as biochemical, support the notion of an
androgen-dependent physiological interaction between these two pathways. Overall, our results favor the notion that a transient and dynamic association between AR and caveolin-1 may play a role in
promoting AR ligand-dependent transcriptional activation.
Although the functional significance of this interaction remains to be determined, it suggests that caveolin-1 plays a role as a convergent point for AR cross-talk with other cellular signal transduction pathways. These findings pave the way to further define the underlying signal cross-talk in AR-mediated transactivation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Caveolin-1 overexpression potentiates
ligand-dependent AR transcriptional activity.
A, levels of expression of caveolin-1 protein were
determined by Western blots in the 293 parental vector control, 293 cells overexpressing caveolin-1 (293-Cav), or 293 cells stably
expressing antisense caveolin-1 (293-AS) under a cadmium
(Cd2+)-inducible metallothionine promoter. In the
lower panel, the levels of transient expression of AR from
each transfection group were equal, as determined by anti-AR Western
blot analysis. B, androgen-dependent AR
transcriptional activities in 293 cells expressing different levels of
caveolin-1 were determined by transient transfection with 0.5 µg of
pCDNA-AR and 9.5 µg of p(ARE)4-Luc. DHT (1 nM), vehicle or bicalutamide Casodex (100 nM)
was added to the culture 24 h after cells were transfected with AR
and luciferase reporter genes. The luciferase reporter assay was
performed 24 h later. The luciferase activity was normalized by
the internal control and the non-DHT-treated control groups. All
experiments were repeated three times, with consistent results.
Relative light unit (RLU) data represent the mean.
C, non-AR-responsive control promoter/reporter systems,
pGAL-VP16/pG5-Luc and ptetR-VP16/pTRE-Luc, are included to show that
general transcription was not affected by changes in the levels of
caveolin expression. The same 293 cell groups were transiently
transfected with 0.5 µg of pGAL-VP16 expression vector with 9.5 µg
of pG5-Luc reporter; or 0.5 µg of ptetR-VP16 expression vector with
9.5 µg of pTRE-Luc reporter. The luciferase reporter assay was
carried out 48 h after transfection. Data represent the mean ± S.D. (n = 3).
ligand-independent signaling (27). In
the DHT-stimulated groups (293 versus 293-Cav),
overexpressing caveolin-1 dramatically up-regulated the expression of
ARE-luciferase reporter which may be the result of cell sensitization.
Down-regulation of caveolin-1 expression by a caveolin antisense
construct limited the induction of ligand-dependent AR
transcription activation to one-half of level of the control parental
cells. Thus, caveolin-1 overexpression is sufficient to induce a
moderate ligand-independent activation of AR, and caveolin-1
potentiates AR transcriptional activity in the presence of ligand. On
the other hand, the anti-androgen bicalutamide (Casodex) completely
blocked the AR-mediated transactivation of luciferase gene expression
regardless of caveolin-1 overexpression. Overexpression of caveolin-1
does not alter AR ligand specificity since estrogen does not stimulate
the AR response (data not shown). We determined that this modulated AR
transactivation response was not the result of different levels of AR
expression in various transfection groups. As demonstrated in Fig.
1A (lower panel), the expression level of AR is
maintained at similar levels in each group. Two AR unresponsive
negative control promoter/reporter systems, pGAL-VP16/pG5-luc and
ptetR-VP16/pTRE-luc, were tested to demonstrate that changes in the
level of caveolin-1 expression do not affect the general transcription
activities (Fig. 1C).
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Fig. 2.
Caveolin-1 overexpression increases the
sensitivity of AR to ligand-dependent transactivation.
This figure shows the dose-dependent (DHT: 0.01-10
nM) AR transcriptional response of 293 cells expressing
various levels of caveolin-1. The IC50 for the
ligand-dependent activation of 293 is approximated at 0.5 nM. To achieve the same fold stimulation, 293-Cav cells
required 0.02 nM DHT, as determined by interpolation, which
is 25-fold lower than that required by the parental 293 cells. The
left-shift of the dose-response curve indicates an increase in the fold
stimulation of AR in response to DHT in 293-Cav cells compared with
parental 293 cells and 293-AS cells. Data represent the mean ± S.D. (n = 3)
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Fig. 3.
Antisense caveolin-1 down-regulates AR
transactivation in PC3 cells. A, antisense caveolin-1
reduces caveolin-1 protein expression in PC3 cells as determined by
Western blot analysis. Various quantities (0, 1.0, 5.0, and 10.0 µg)
of antisense caveolin-1 expression vector (pCEP-Cav-AS) were
transiently transfected into PC3 cells together with 1 µg of pHook-1
expression vector for enrichment of the transfected cells (see
"Experimental Procedures"). Cell lysates were collected 48 h
afterwards. Caveolin expression was determined by immunoblot. Level of
AR in the co-transfectants remained the same in the different
transfection groups, as shown in the lower panel.
B, transient AR transactivation experiments were performed
in parallel to establish a dose-response curve for AR transactivation
by co-transfecting pCDNA-AR and p(ARE)4-Luc. Data
represent the mean ± S.D. of firefly luciferase activity
normalized to renilla luciferase activity by a dual luciferase assay
(n = 3). C, non-AR-responsive control
promoter/reporter system, pGAL-VP16 and pG5-Luc, are included to show
that general transcription was not affected by changing levels of
caveolin expression. The luciferase reporter assay was carried out
48 h post-transfection. Data represent the mean ± S.D.
(n = 3).
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Fig. 4.
Co-sedimentation of AR with caveolin-rich
caveolae membrane domain fractions. LNCap cells stably expressing
caveolin-1 (LNCap-Cav) were treated without (vehicle) or with DHT (1 nM) for 10 and 60 min. Cells were lysed in alkaline lysis
buffer as described under "Experimental Procedures." The resulting
cell lysates were subjected to equilibrium sucrose density gradient
centrifugation. Twelve 1-ml fractions were collected from the gradients
and resolved by SDS-PAGE and then by transblotting to a nitrocellulose
membrane. Immunoblot analysis was performed with anti-Cav and anti-AR,
respectively. As shown, the association of AR with the caveolin-rich
caveolae membrane fraction in response to DHT treatment was transiently
increased, whereas the distribution of caveolin-1 remained
unchanged.
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Fig. 5.
Co-immunoprecipitation of AR and caveolin-1
from intact cells. Cell lysates from LNCap-Cav cells stimulated
for 10 min with 1 nM DHT or vehicle were subjected to
immunoprecipitation by antibodies against caveolin-1 (polyclonal,
anti-CAV) or AR (monoclonal, anti-AR). Immunoprecipitates were resolved
by SDS-PAGE and transblotted to nitrocellulose membranes. Membranes
were subjected to immunoblot analysis, reciprocally probed with anti-AR
or anti-CAV, respectively, and then developed with enhanced ECL. Rabbit
IgG and mouse IgG were used, respectively, as the control IgG in these
experiments. As indicated in the blot, an increased association of AR
and caveolin-1 was detected in response to DHT treatment.
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Fig. 6.
In vitro GST fusion protein
binding studies of AR interactions with caveolin-1.
Androgen-dependent interaction of AR and caveolin-1 was
tested by incubating in vitro translated
[35S]methionine AR with glutathione-agarose-immobilized
GST-caveolin fusion protein or with GST alone in the presence or
absence of 10 nM DHT. The incubation and washing were
performed as described under "Experimental Procedures." The input
lane represents 5% of the [35S]methionine AR used per
reaction.
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Fig. 7.
Mapping of AR submolecular domain responsible
for caveolin-1 interaction by mammalian two-hybrid assay.
A, full-length AR and truncated mutants of AR encompassing
various functional domains were cloned in-frame downstream of VP16
transactivation domain with a HA tag using a pACT vector. Caveolin-1
was cloned in-frame downstream of GAL4 DBD with a HA tag using a pBIND
vector. B, immunoblot analysis of correspondent fusion
proteins transiently expressed in HeLa cells, Gal4-caveolin
(pBIND-Cav), VP-AR full-length (pACT-AR-FL), and truncated domains
(AR-N, AR-N/DBD, LBD, DBD), by the anti-HA antibody 12CA5.
C, a mammalian two-hybrid assay was performed by
co-transfecting HeLa cells with 5 µg of pACT-AR or AR truncated
mutants, as indicated, with 5 µg pBIND-caveolin and 5 µg of pG5-Luc
vector. Cells were treated with vehicle control, DHT (10 nM), or bicalutamide Casodex (5.0 µM) for
20 h before the dual luciferase assay. A renilla luciferase
reporter activity was used as a transfection internal control. The
luciferase activity was normalized by the internal control and the
vector basal control groups. All experiments were repeated three times,
with consistent results. Data represent the mean ± S.D.
(n = 3).
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Fig. 8.
Mapping of caveolin-1 submolecular domain
responsible for AR interaction by mammalian two-hybrid assay.
A, three truncated fragments of caveolin-1 were cloned
in-frame with a GAL-4 DNA-binding domain using a pBIND vector with an
HA tag toward NH2 terminus. These domains encompass
cytoplasmic portions of the caveolin-1. B, Western blot
analysis of the expressed GAL-caveolin truncated fragment fusion
protein with anti-HA (clone 12CA5). C, a mammalian
two-hybrid assay was performed by co-transfecting HeLa cells with VP
fusion of various AR domains (using a pACT vector), GAL4-caveolin
truncated domain fusion (using a pBIND vector), and pG5-Luc vector.
Cells were treated with vehicle control or DHT (10 nM) for
20 h before the dual luciferase assay. A renilla luciferase
reporter was used as a transfection internal control. The luciferase
activity was normalized by the internal control and the vector basal
control groups. All experiments were repeated three times, with
consistent results. Data represent the mean ± S.D.
(n = 3).
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Fig. 9.
Caveolin-1 AR-binding domain deletion mutant
does not potentiate AR transactivation activity. A,
co-immunoprecipitation of AR with caveolin and Cav-(1-60) but not with
Cav-(58-178). 293 cells were co-transfected with 5 µg of
pCDNA-AR and 5 µg of pCDNA harboring HA-tagged caveolin-1,
truncated fragments, or an empty pCDNA3 vector. 24 h later,
cell lysates from each group were subjected to immunoprecipitation by
antibodies against HA (clone 12CA5). Immunoprecipitates were resolved
by SDS-PAGE and transblotted to nitrocellulose membranes. Membranes
were then subjected to immunoblot analysis probed with anti-AR and
developed with enhanced ECL. B,
androgen-dependent AR transcriptional activity in 293 cells
transiently expressing caveolin-1 deletion mutants. 293 cells were
co-transfected with 0.5 µg of pCDNA-AR and 4.5 µg of
pCDNACav(FL), pCDNA-CAV-(1-60), or pCDNA-CAV-(58-178)
along with 5 µg of p(ARE)4-Luc. DHT (1 nM)
was added to the culture 24 h after transfection. The luciferase
assay was performed 48 h post-transfection. The luciferase
activity was normalized by the internal control and the non-DHT-treated
control groups. All experiments were repeated three times with
consistent results. Data represent the mean ± S.D.
(n = 3). C, the expression level of AR
remained the same in each transfection group, as determined by anti-AR
Western blot analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
X
XXXX
or
XXXX
XX
, where
is a hydrophobic
residue and X is any amino acid residue). Many signal
components, including small GTPase, protein kinase-
, protein
kinase-
, phospholipase C
, Src family kinases, mitogen-activated
protein kinase, and receptor tyrosine kinases, have been proven
to interact with caveolin-1 via this domain (14). Therefore, it is
conceivable that AR is brought to the proximity of a preformed
activation complex for further biochemical interaction via caveolin-1
association. Because AR is a phosphoprotein with dynamic regulation of
its phosphorylation status (41, 42), many signal pathways regulated
through the caveolin complex are also implicated in AR transactivation
process, including mitogen-activated protein kinase (43) and epidermal growth factor receptor (44). It is reasonable to predict, although it
remains to be proven, that AR-caveolin interaction may facilitate at
least part of the AR phosphorylation and/or dephosphorylation process.
Consequently, a recent report by Migliaccio et al. (45) demonstrates an agonist-dependent interaction and
activation of tyrosine kinase Src by the androgen receptor and the
estrogen receptor in prostate cancer cells. Although the occurrence of this interaction has not yet been clearly defined, it will be interesting to determine whether AR is brought to the proximity of Src
by association with caveolin-1 upon ligand-dependent
activation. Moreover, these interactions may be part of a general
mechanism of steroid receptor regulation since caveolin has been shown
to potentiate the transactivation process of estrogen receptor
(27). Furthermore, caveolin-1 also co-localizes with estrogen receptor
in an estradiol-dependent manner in a
co-immunoprecipitation study. In agreement with our data,
4-hydroxytamoxifen, an antiestrogen, remains effective in
inhibiting estrogen receptor
transactivation in the presence of
caveolin-1 overexpression (27).
nuclear translocation in a
ligand-independent manner.
X
XXXX
+
XXXX
XX
=
X
XXXX
XXXX
XX
)
from residue
739YSWMGLMVFAMGWRSF754
of AR is spotted in helix 5 of the ligand-binding domain (54, 55). This
composite binding motif is similar to a previously identified composite
binding motif in the platelet-derived growth factor receptor and the
endothelin receptor (20). In two-hybrid studies, we were unable to
demonstrate the interaction between LBD and submolecular domains of
caveolin. We did, however, detect a moderate interaction between LBD
and the full-length caveolin in an agonist-dependent
fashion. It remains to be determined whether this putative motif
actually participates in the interaction between AR and caveolin. It
may suggest that a ligand-induced conformational change is involved in
the interaction. A weak, ligand-dependent interaction, less
than 1% of AR input, was detected in a GST pull-down experiment. On
the one hand, this weak interaction is consistent with the nature of a
transient association of these two molecules. On the other hand, it
suggests that other auxiliary interactions or molecules, which may not
be present in the in vitro translation lysate, are required
for the interaction.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Harold Chapman for providing the plasmids and cell lines, Dr. Yontong Zhao for technical help with the cloning work, and Dr. Dean Hartley for critical reading of this manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institute of Health Grant R29GM54713.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Urology Research (LMRC-BLI143), Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-6430; Fax: 617-264-6338; E-mail: mlu@rics.bwh.harvard.edu.
Present address: Division of Genetics, Southern Illinois
University School of Medicine.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M006598200
2 M. L. Lu and X. Zhang, unpublished observations
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ABBREVIATIONS |
---|
The abbreviations used are: AR, androgen receptor; GST, glutathione transferase; DHT, dihydrotesteosterone; ARE, androgen-responsive element; DBD, DNA-binding domain; LBD, ligand-binding domain; HA, hemagglutinin antigen; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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