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Laboratories for Reproductive Biology and the Departments of
Pediatrics (J.A.K., E.L., C-i.W., E.M.W.), and Biochemistry and
Biophysics (E.L., E.M.W.) University of North Carolina Chapel
Hill North Carolina 27599
Endocrinology Branch (K.B.,
W.R.K.) Reproductive Toxicology Division National Health and
Environmental Effects Research Laboratory United States
Environmental Protection Agency Research Triangle Park, North
Carolina 27711
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Previous studies from this laboratory identified an AR NH2-terminal and carboxyl-terminal (N/C) interaction that requires high-affinity androgen binding (4). The androgen-induced N/C interaction is inhibited by the androgen antagonist hydroxyflutamide. These results raised the possibility that an N/C interaction is required for AR agonist activity and that its interruption is a prerequisite for antagonist activity. Similar studies with the estrogen receptor revealed a ligand-dependent N/C interaction that predicted parallel dimerization (5). Recent studies on AR suggest that its N/C interaction is intermolecular and results in the formation of an antiparallel homodimer (6). A feature common to both models is the requirement for high-affinity agonist binding to promote the N/C interaction. In the present report we tested the requirement for the AR N/C interaction in relation to AR dimerization, DNA binding, and transcriptional activity in transient and stable cotransfection assays to distinguish the activities of several natural and pharmaceutical agonists and antagonists. The results suggest that at higher concentrations, certain weak AR agonists such as medroxyprogesterone acetate (MPA) activate AR through a mechanism that does not involve the N/C interaction, although potent agonists capable of AR activation at low ligand concentrations induce the N/C interaction. Furthermore, inhibition of the N/C interaction does not necessarily reflect the activity of an antagonist.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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are indicated as apparent
equilibrium binding constant (Kd) determined by Scatchard
analysis as previously reported, inhibition constant Ki of
endogenous AR in rat prostate extracts, or the concentration
required for 50% inhibition of [3H]methyltrienolone
(R1881) binding to recombinant AR in transfected COS cells (Table 1).
Relative competitive binding affinities for [3H]R1881 in
transfected COS cells were mibolerone 
R1881 >
RU56187 > dihydrotestosterone (DHT) MPA
progesterone > estradiol (E2) > cyproterone
acetate > testosterone oxandrolone
fluoxymesterone > hydroxyflutamide. Chemical structures of
several of the ligands are shown in Fig. 1. Binding of DHT and testosterone was
weaker in the COS cell assay when compared with apparent equilibrium
binding affinities in tissue cytosols (Table 1) likely resulting from
partial metabolism during the 2-h 37 C incubation. Analysis of COS
cells transfected with the parent plasmid lacking the AR sequence
showed binding only of [3H]progesterone. Lack of binding
of [3H]R1881 and [3H]mibolerone in the
absence of AR expression suggested that this endogenous binding
activity was not due to the progesterone receptor (7, 8, 9, 10). Inhibition
constants for the anabolic steroids oxandrolone (Ki 62
nM) and fluoxymesterone (Ki 44 nM)
were less than that of hydroxyflutamide (Ki 175
nM), but about 100 times greater than the equilibrium
binding constant (Kd) for high affinity agonists such as
DHT. Competitive binding by MPA and RU56187 were similar to DHT,
although the reported Kd values for MPA were slightly
greater than that for DHT (Table 1).
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mibolerone
> testosterone R1881 > oxandrolone >
fluoxymesterone (Fig. 2). No N/C
interaction was detected with MPA, RU56187, E2,
progesterone, hydroxyflutamide, or cyproterone acetate up to
concentrations of 1 µM. Induction of the N/C interaction
did not correlate with relative binding affinities (Fig. 2 and Table 1
). The relatively high-affinity ligands, MPA and RU56187, failed to
promote the N/C interaction, whereas the lower affinity anabolic
steroids, oxandrolone and fluoxymesterone, induced the N/C interaction.
The inability of MPA to induce the N/C interaction was not limited to
CHO cells, as it was also ineffective in monkey kidney CV1 or COS cells
where 5-fold induction of the N/C interaction was observed with 1
nM DHT (data not shown).
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and Table 1). RU56187 was a slightly
less potent inhibitor of the N/C interaction than MPA, exhibited a high
AR equilibrium binding affinity, and is reported to have antagonist
activity in vivo (14, 15). Ligands with less inhibitory
activity than MPA or RU56187 at concentrations between 50 and 500
nM were hydroxyflutamide, cyproterone acetate,
E2, and progesterone (Fig. 3 and Table 1). The anabolic
steroids, oxandrolone and fluoxymesterone, and the potent androgen
agonists, DHT, mibolerone, testosterone, and R1881, showed little or no
inhibition of the DHT-induced N/C interaction.
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Transcriptional Activation
Agonist and antagonist activities were determined in CV1
cells transiently transfected with a mouse mammary tumor virus
(MMTV)-luciferase reporter and full-length human AR expression vectors.
Ligands with more than 10-fold agonist activity at 0.001 nM
were DHT, mibolerone, and R1881 (Fig. 4
and Table 1). Similar induction was achieved by 0.01 nM
testosterone, 0.1 nM MPA, and 1 nM oxandrolone
or fluoxymesterone. Cyproterone acetate, progesterone, E2,
and RU56187 induced luciferase activity at concentrations between 10
and 100 nM, but transcriptional activity remained low at
100 nM hydroxyflutamide, the latter requiring
concentrations of 1–10 µM for agonist activity in this
assay (16). Agonist potency, therefore, tended to parallel the
ligand-induced N/C interaction. Lack of an N/C interaction induced by
MPA is associated with 100-fold higher MPA concentrations necessary for
transcriptional activity compared with DHT.
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Antagonist activity of the ligands was tested in CV1 cells by
coincubation with 0.1 nM DHT. Hydroxyflutamide was the most
effective antagonist with about 50% inhibition at 100 nM
(Fig. 5). Cyproterone acetate was
slightly less effective, and RU56187 had some inhibitory activity but
decreased in effectiveness at higher concentrations. Antagonist
activity was also observed with increasing concentrations of
progesterone and E2, but none was observed with MPA (Fig. 5) or with the high-affinity agonists or the anabolic steroids (results
not shown). Thus, except for MPA, at least partial inhibition of
DHT-induced transcriptional activity correlated with inhibition of the
DHT-induced N/C interaction.
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The weak in vivo AR agonist activity reported for MPA (11, 12, 13, 19) is reflected in MMTV-luciferase assays by the requirement for higher MPA concentrations relative to DHT for reporter gene activation. Nevertheless, the agonist activity of MPA was surprising considering that MPA inhibits the N/C interaction better than most antagonists. We therefore tested a CHO cell line in which the MMTV-luciferase reporter and human AR expression vectors were stably integrated in the genome using pcDNA3.1/Zeo vector with the zeocin gene and the human AR-coding sequence (K. Bobseine and W. R. Kelce, unpublished data). Cell lines such as this were used previously to distinguish agonist activities not detected by transient transfection (24). DHT at 0.1 nM stimulated luciferase activity 2-fold while MPA required a 10-fold higher concentration for similar induction (data not shown). A greater overall response to MPA (10-fold) compared with DHT (6-fold) likely resulted from MPA activation of endogenous glucocorticoid receptor since coincubation with 500 nM hydroxyflutamide inhibited MPA-activated gene transcription about 50% and DHT activity by 95%, but had no effect on induction by dexamethasone (data not shown). The AR-mediated MPA response was therefore similar to that of DHT, but required higher steroid concentrations as observed in the transient assays. The chromatin arrangement of the reporter gene seemed to have little effect on the relative concentration-dependent gene activation by DHT and MPA.
Dimerization and DNA Binding
Because binding of baculovirus expressed full-length AR to
androgen response element DNA requires exposure of Sf9 cells to
androgen and is inhibited by coincubation with the antagonist
hydroxyflutamide (25), we tested the activity of these ligands to
promote AR DNA binding in vitro. DNA binding of full-length
AR was observed with 50 nM DHT, mibolerone, MPA,
oxandrolone, fluoxymesterone (Fig. 6A),
or 50 nM R1881 or testosterone (Fig. 6B). Concentrations of
DHT or R1881 less than 50 nM reduced AR DNA binding
probably due to insufficient saturation of baculovirus-expressed AR
(data not shown). AR DNA binding was observed at 1 µM
RU56187 or hydroxyflutamide but was barely detectable with 1
µM progesterone, E2, or cyproterone acetate
(Fig. 6).
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, lanes C) each homodimerize and bind
DNA independently of hormone, agonists are required for dimerization
and DNA binding of the N/C complex and an antagonist such as
hydroxyflutamide inhibits this DHT-induced DNA binding (25, 26). Since
both fragments contain the DNA-binding domain, dimerization could be
mediated by the DNA-binding domain and/or by the N/C interaction.
Results of this assay (25) and others (22) nevertheless predicted the
AR N/C interaction, which was later confirmed in the two-hybrid
interaction assay (4).
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, C+N)
but required 0.5–1 µM oxandrolone or fluoxymesterone for
similar activity. MPA-induced dimerization and DNA binding of the two
AR fragments were somewhat less efficient than DHT but similar to the
anabolic steroids (Fig. 7, A and B). Only slightly weaker DNA binding
of the N/C hybrids was detected using cyproterone acetate and
E2, whereas 1 µM RU56187 or 1
µM progesterone was required for DNA binding, and
essentially no DNA binding was detected with 1 µM
hydroxyflutamide (Fig. 7 and Table 2).
Binding of the homodimer C fragment alone was most effective with
mibolerone, testosterone, and R1881; it was not detected with 50
nM DHT, required 500 nM MPA, and was weak to
undetectable with the anabolic steroids and other ligands (Fig. 7, lanes C). Similar high-level AR expression was observed by immunoblot
analysis of full-length AR or the AR507–919 fragment after the
different hormone treatments and expression levels were independent of
the extent of AR dimerization and DNA binding (data not shown).
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). Yet none of these ligands,
except for MPA, promote DNA binding of full-length AR and none induce
the N/C two-hybrid interaction (Fig. 7A). The effectiveness of these
ligands to induce dimerization and DNA binding of the AR
NH2- and carboxyl-terminal fragments in mobility shift
assays is confounded by the presence of the dimerization region in the
DNA-binding domain. We therefore attempted to address the contribution
of the AR DNA-binding domain in the two-hybrid assay by testing the
interaction of a fusion protein comprised of the GAL4 DNA-binding
domain and AR DNA and ligand-binding domains (GAL-AR507–919) with
VPAR1–660 or VPAR. However, lack of an interaction in this and
additional experiments using GAL-AR (GAL4 DNA-binding
domain-full-length AR fusion protein) suggests that the presence of two
DNA-binding domains in a fusion protein (i.e. from GAL4 and
AR) interferes with hybrid formation.
AR Stabilization
One property that has distinguished androgen agonists from
antagonists is their ability to stabilize AR against degradation (27).
It was therefore of interest to determine the concentration dependence
of AR stabilization by MPA, RU56187, and the anabolic steroids.
Transfected COS cells were incubated with
[35S]methionine/cysteine and increasing
concentrations of ligands, followed by chase periods with unlabeled
methionine for 2–7 h as previously described (27). The results
shown for several ligands in Fig. 8 and
summarized in Table 2 indicate that more than 100 nM MPA
was required to increase the AR degradation half-time to almost 5
h at 37 C (Fig. 8C). A similar degree of AR stabilization was achieved
by 1 nM DHT, mibolerone, or R1881, 5 nM
testosterone (Table 2), or 10 nM fluoxymesterone or
oxandrolone (Fig. 8, B and D). Cyproterone acetate and progesterone
stabilized AR only at 1 µM (Table 2), and almost no AR
stabilization was observed with 1 µM RU56187 (Fig. 8A),
hydroxyflutamide, or E2 (Table 2). The results tend to
parallel the MMTV-luciferase and DNA-binding activities in that ligands
that efficiently stabilize AR are more effective agonists. It is
noteworthy that the lower affinity anabolic steroids, oxandrolone and
fluoxymesterone, promote the N/C interaction and stabilize AR at
concentrations of 5–10 nM, concentrations only slightly
higher than those required for the high-affinity agonists, DHT,
mibolerone, R1881, and testosterone. However, higher concentrations of
the anabolic steroids were required for DNA binding of the AR
fragments, perhaps reflecting the lower AR binding affinity for these
ligands.
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) compared with t1/2 of
2.5–3.5 h for [3H]R1881 (Fig. 9), [3H]DHT,
and [3H]mibolerone (Table 2). A rapid dissociation rate
for [3H]estradiol (t1/2 0.67 h) (Table 2) was also observed. The results raise the possibility that rapid
ligand dissociation is associated with the lack of an N/C interaction
and with in vivo antagonist activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The apparent discrepancy in ligand potency between maximal induction in the N/C assay using the GAL4-AR carboxyl-terminal and the VP16-AR NH2-fragment fusion proteins (0.1–1 nM) vs. agonist potency of full-length AR in the luciferase assay (0.001–0.01 nM) probably reflects deletion of the NH2-terminal domain. We demonstrated previously that although the AR ligand-binding domain retains high-affinity binding after deletion of the NH2-terminal region, the ligand dissociation rate increases 5- to 7-fold, and androgen no longer stabilizes this truncated receptor as it does full-length AR (22). Thus, higher ligand concentrations are likely required for the N/C interaction between AR fragments than between monomers of full-length AR.
Equilibrium dissociation constants (MPA, 1.7–2.9 nM; DHT,
0.9–2.6 nM) and saturation binding capacities (MPA,
107–249 fmol/mg protein; DHT, 42–257 fmol/mg protein) for MPA and DHT
binding to AR were similar when measured in rat pituitary and
hypothalamic extracts (28). However, direct measurement of in
vivo bioactivity classifies MPA as a weak androgen. MPA increases
the synthesis of ß-glucuronidase in mouse kidney but only at 100-fold
higher doses relative to testosterone (11, 12). In the androgen-
insensitive Tfm mouse, ß-glucuronidase activity did not increase,
indicating that gene activation in response to MPA is AR mediated (12).
MPA doses up to 1000 times higher than testosterone were required to
increase ventral prostate weight in castrated rats (13). High-dose (0.9
mg/day) MPA was less effective than low-dose DHT (0.2 mg/day) in
stimulating the synthesis of rat prostatic binding protein mRNA, and
the effects of MPA were inhibited by flutamide (19), again indicating
that its in vivo activity is AR mediated. MPA, rather than a
metabolite, was shown to bind AR, and its low in vivo
androgenic activity correlated with reduced nuclear uptake (29, 30).
MPA was reported to dissociate rapidly from AR (13, 31), although these
studies did not account for possible degradation of the MPA-AR complex.
X-ray crystal analysis indicates that MPA has an inverted 1ß,2
half-chair conformation of the A-ring resulting from steric strain by
the 6-methyl group that restricts side chain flexibility (32). This
predicted rigid structure of MPA is in contrast to the flat, flexible
structure of methyltrienolone (R1881), which can undergo large shape
changes (33). It is conceivable that ligand flexibility facilitates the
conformational changes required for the AR N/C interaction.
Acetate derivatives of steroids often have slower metabolic breakdown rates, making them candidates for use in hormone therapy (34). MPA, available for clinical use as Provera or Depo-Provera, has been used in the treatment of sexual precocity; its progestin and weak androgen effects inhibit pituitary gonadotropin secretion and lower gonadal steroid production (35). Stimulation of the growth of pubic hair in female patients without a significant slowdown in skeletal maturation (35) suggested weak androgenic activity of large doses of MPA (200–300 mg every 7–10 days). 21-Hydroxylated metabolites of MPA bind the glucocorticoid receptor and suppress pituitary secretion of ACTH. Because of its progestational effect, MPA was formerly used to prevent spontaneous abortion. However, prenatal exposure to MPA was reported to cause mild clitoral hypertrophy and posterior labial fusion in the female and hypospadias in the male (36, 37, 38, 39), contraindicating its use during pregnancy. The virilizing effect of MPA in the female fetus can be explained by its androgenic activity. Antiandrogen effects of MPA in the male fetus could result from competition for DHT binding to AR and subsequent insufficient agonist activity. Anogenital distance, a measure of antiandrogen activity in rodents (1), was lengthened in females and shortened in males exposed to MPA during fetal development. More recently, MPA was approved for use in the United States as an injectable contraceptive based on its effectiveness in suppressing gonadotropin secretion, inhibiting follicular maturation and preventing ovulation. Doses of 150 mg im every 6 weeks to 3 months lack androgen effects in the adult female.
MPA has also been used in breast cancer therapy (40, 41). In an MFM-223 mammary cancer cell line that has high levels of AR, but low levels of estrogen, progesterone, and glucocorticoid receptors, cell proliferation was inhibited by 1 nM DHT or 10 nM MPA (42), indicating the AR agonist effect of MPA inhibits breast cancer cell growth. Response rates of breast cancer patients to MPA therapy correlated with higher AR levels (43). The antiproliferative activity of DHT and MPA on breast cancer cells was attributed to increased 17ß-hydroxysteroid dehydrogenase activity, which promotes increased oxidation of estradiol to the weak estrogen, estrone (44).
Agonists vs. antagonist activity is influenced by
metabolism, binding affinity, association and dissociation rates, and
ligand-induced receptor conformation, stabilization, dimerization, DNA
binding, and interactions with associating proteins. Clearly,
equilibrium binding affinity is of limited usefulness in predicting
in vivo bioactivity unless combined with measurements of
ligand dissociation rates. AR binding affinity of RU56187 is similar to
that for DHT, yet RU56187 is an antagonist in vivo (14, 15).
The anabolic steroids, oxandrolone and fluoxymesterone, have high
inhibition constants for binding, yet induce the N/C interaction and
stabilize AR at relatively low ligand concentrations and are AR
agonists in vivo. Oxandrolone induces male- specific liver
P450 enzymes (45). Oxandrolone and fluoxymesterone are structurally
related 17-alkylated synthetic anabolic steroids used clinically to
promote weight gain, stimulate growth of the bone matrix, and improve
libido and sexual performance (46). In low doses oxandrolone (47, 48)
or fluoxymesterone (49) accelerate linear growth in children with
constitutional growth delay and Turner’s syndrome (50, 51).
Evidence from crystal structure analysis of the retinoic acid
receptor-
(52), thyroid hormone receptor (53), and estrogen receptor
(54) indicates that hormone binding causes helix 12 [helix 11 in
retinoid X receptor- (55)] at the carboxyl terminus to undergo a
conformational change closing down over the ligand-binding pocket. For
the estrogen receptor, binding of the antagonist raloxifene prevents
alignment of helix 12 over the binding pocket (54). MPA binding to AR
may distort the position of helix 12 causing an increased rate of
ligand dissociation and interference with the N/C interaction. Proper
closure of helix 12 might be expected to slow ligand dissociation from
the pocket and form a new interface for the N/C interaction. Alignment
of helix 12 by MPA binding may differ from that induced by potent
agonists or antagonists, a distortion that may account for the high MPA
concentrations required to stabilize AR.
Part of the discrepancy between ligand binding affinity and agonist and antagonist activities relates to differences in ligand binding kinetics. Association and dissociation rate kinetics can be fast or slow for high-affinity ligands. Slow dissociation of the most potent AR agonists, DHT, mibolerone, and R1881, is associated with AR stabilization at low ligand concentration. Fast dissociating ligands such as RU56187 fail to stabilize AR and have agonist activity in transcriptional activation assays but are antagonists in vivo. Mutations in the AR hormone-binding domain at valine 889 and arginine 752 cause severe androgen insensitivity, increase the rate of dissociation of bound androgen without altering high-affinity equilibrium binding (22), disrupt the N/C interaction (6), and cause loss of AR stabilization by low ligand concentrations (22). These mutations likely increase the rate of ligand dissociation and AR degradation by preventing helix 12 from closing the binding pocket and interfering with the N/C interaction. Rapid ligand dissociation could reduce in vivo agonist activity and enhance dose-dependent antagonist activity as suggested previously for some antiestrogens and antiandrogens (13).
The most reliable in vitro indicators of in vivo
AR antagonist activity therefore appear to be failure of a ligand to
stabilize AR against degradation at steroid concentrations of 500
nM or more and an inability to induce AR DNA binding. DNA
binding itself, however, appears to be a poor indicator of agonist
potency. In vivo agonist activity is best reflected by a
slow dissociation rate of bound ligand, AR stabilization at low ligand
concentrations (
10 nM), and induction of the N/C
interaction. Concentrations at which a ligand activates AR in
MMTV-luciferase assays can indicate agonist potency. MPA is an agonist
in transient transcription assays but requires 100-fold higher
concentrations than DHT. A similar shift in in vitro
sensitivity to DHT results from certain AR missense mutations that
cause partial or complete androgen insensitivity (56), indicating the
critical importance of AR activation by low ligand concentrations.
However, even though acetate derivatives of steroids have increased
metabolic half-lives, it cannot be ruled out that the in
vivo pharmacology of MPA limits its bioavailability to the AR.
A model for androgen-induced AR dimerization suggests an antiparallel orientation of monomers interacting through the DNA-binding domain and a ligand-dependent N/C interaction (4). Similar studies with the estrogen receptor (5) and AR fragments expressed in yeast (57) predict a parallel interaction model, and studies on solution dimerization of the human progesterone receptor favor a parallel model (58). More recent studies on AR made use of androgen insensitivity mutations in the steroid-binding domain that do not interfere with high-affinity equilibrium binding of androgen but increased the dissociation rate of bound androgen and disrupted the N/C interaction. Placement of the mutations in different AR fragments allowed assessment of directional dimerization in association with AR transcriptional activation, and the results were consistent with an antiparallel activated AR dimer model (6). Lack of an interaction between the ligand-binding domains bound to MPA or DHT argue against a parallel dimer model for AR with either ligand. Taken together the results suggest that the N/C interaction is required for potent in vivo agonists to be effective at low concentrations, but is not required for AR DNA binding in vitro or weak in vivo agonist activity at higher ligand concentrations. Formation of the N/C interaction likely contributes to in vivo potency by stabilizing AR at low ligand concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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N/C Luciferase Assay
Recombinant fusion proteins included GALD-H which contained the
Saccharomyces cerevisiae GAL4 DNA-binding domain amino acid
residues 1–147 linked in frame with human AR steroid-binding domain
amino acid residues 624–919. VPAR1–660 contained the herpes simplex
virus VP16 transactivation domain amino acid residues 411–456 linked
in frame with AR NH2-terminal and DNA-binding domain amino
acid residues 1–660 (4). CHO cells (0.4 x 106 cells
per 6-cm dish) were transfected using DEAE-dextran and 1 µg GALD-H, 1
µg VPAR1–660, and 5 µg G5E1b-luciferase per plate, the latter
containing five GAL-4 DNA-binding sites (60). DNA was added to 0.42 ml
H20 plus 0.5 ml 2xTBS (0.14 M NaCl, 3
mM KCl, 1 mM CaCl2, 0.5
mM MgCl2, 0.9 mM
NaH2PO4, and 25 mM Tris-HCl, pH
7.4), and then 0.11 ml DEAE-dextran (0.5%) was added, after which the
mixture was added to the aspirated plates and incubated for 1 h at
37 C. Plates were aspirated and 4 ml -MEM containing 10% calf
serum, penicillin/streptomycin, and 20 mM HEPES, pH 7.2,
were added and incubated at 37 C for 3 h followed by a 4-min 15%
glycerol shock in -MEM. Cells were washed twice with 4 ml TBS, and 4
ml 0.2% calf serum-- MEM media were added. The medium was changed
24 and 48 h later to serum-free medium and ligands were added.
Cells were washed 4 h after the last addition with 4 ml PBS and
harvested in 0.5 ml lysis buffer (Ligand Pharmaceuticals Inc.,
San Diego, CA). Luciferase light units were measured on a Monolight
2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA).
To test for inhibitory activity, cells were incubated with 1
nM DHT in the presence of increasing concentrations of
ligands. Decreases due to toxicity of up to 1 µM ligand
were monitored using an AR fusion plasmid GAL-A1 (4) coding for a
constitutively active fusion protein containing AR
NH2-terminal residues 1–503 linked to the GAL-4
DNA-binding domain. Decreases in constitutive transcriptional activity
after exposure were minimal.
MMTV-Luciferase Assay
Monkey kidney CV1 cells (0.4 x 106 cells per
6-cm dish) were transfected 24 h after plating using calcium
phosphate with 100 ng human AR expression vector pCMVhAR and 5 µg
MMTV-luciferase reporter vector per plate. DNA is added to 0.28
M NaCl, 1.5 mM Na2HPO4,
0.05 M HEPES, pH 7.11 (0.14 ml/plate; 0.28
M NaCl, 1.5 mM Na2HPO4,
50 mM HEPES, pH 7.12); 0.25 M CaCl2
(0.14 ml/plate) was added dropwise with vortexing and incubated 5 min,
followed by the addition of DMEM-H media containing 10% calf serum,
penicillin, streptomycin, and 20 mM HEPES, pH 7.2 (0.8
ml/plate) to minimize particle size, and then incubated for 15 min. The
mixture was added to the aspirated plates followed by the addition of 3
ml DMEM-H containing 10% calf serum and incubated for 4 h at 37
C. Cells are washed twice with TBS, and 4 ml phenol red-free medium
containing 0.2% calf serum was added. Ligands were added and cells
were harvested and assayed as described above for the N/C luciferase
assay.
DNA Mobility Shift Assay
Spodoptera frugiperda (Sf9) cells plated at 3.5
x 106 cells per 6-cm dish or 1 x 107
cells per 10-cm dish were infected for 45 h at 27 C at
multiplicity of infection of 1–5 with AR recombinant
baculovirus in Autographa californica nuclear polyhedrosis
virus (AcMNPV) coding for full-length human AR, AR1–660
coding for the NH2-terminal, DNA-binding, and hinge regions
(amino acid residues 1–660), and AR507–919 coding for the DNA- and
steroid-binding domains (amino acid residues 507–919) (25). The
indicated concentrations of ligands were added 24 h and again
4 h before cell harvest. Cells were washed once in PBS at 4 C,
pelleted, and resuspended in 0.15 ml/6-cm dish or 0.4 ml/10-cm dish in
high-salt extraction buffer containing 0.5 M NaCl, 1
mM EDTA, 1 mM dithiothreitol, 10% glycerol, 10
mM Tris, pH 7.4 with protease inhibitors, 40
µM leupeptin, 5 µM aprotinin, 10
µM pepstatin A, 2 mM Pefabloc, 5
mM benzamidine, and 10 mM
-amino-n-caproic acid. Cells were frozen and thawed three
times, incubated on ice for 40 min, and microfuged for 15 min.
Supernatants were dialyzed against the above buffer except containing
25 mM KCl and 0.5 mM phenylmethylsulfonyl
fluoride as the only protease inhibitor. The reaction mixture contained
approximately 20 µg total cell protein of either full-length AR,
AR1–660 (N), or AR507–919 (C), or when combined, 10 µg total
protein each for extracts of N and C. The reaction mix also contained 4
µg of poly dI-dC, 80 µg BSA, and DNA-binding buffer (25
mM KCl, 10% glycerol, 0.2 mM EDTA, 1
mM dithiothreitol, 10 mM Tris-HCl, pH 7.5) to a
final volume of 20 µl. 32P-labeled oligonucleotides
(0.2–0.3 ng, 20,000–25,000 cpm) were added and incubated for 1 h
on ice. Annealing oligos
5'-CGACCAGAGTACGTGATGTTCTCAGG-3' with
AccI-5' compatible end and
5'-GATCCCTGAGAACATCACGTACTCTGGT-3' with 3'
BamHI compatible end were 32P-labeled using the
Klenow fragment of DNA polymerase. The androgen response element
(underlined) derives from the 0.5-kb first intron fragment
of the rat C3 prostatein gene (61). Before electrophoresis, 2 µl
0.2% bromophenol blue were added, and the 5% nondenaturing acrylamide
gel was preelectrophoresed at 100 V for 30 min at 4 C. Samples are
electrophoresed at 150 V for 4 h at 4 C. Gels are dried under
vacuum at 80 C for 1 h and exposed to Biomax MR x-ray film
(Eastman Kodak, Rochester, NY) at -80 C.
AR Stabilization
Full-length AR was expressed from pCMVhAR (8 µg) in COS cells
(1.2 x 106 cells/10-cm dish) transfected using
DEAE-dextran. After 48 h, cells were incubated in methionine-free
medium for 20 min followed by the addition of methionine-free medium
containing 100 µCi [35S]L-methionine/cysteine (PRO-MIX,
Amersham, >1000 Ci/mmol) in vitro labeling mix. Cells were
incubated for increasing times in the presence of the indicated
concentrations of ligands, washed twice with PBS, and harvested in RIPA
buffer and immunoprecipitated using AR52 antipeptide AR antibody as
previously described (22).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Grants HD-16910 and IU54-HD-35041 from the National Institute of Child Health and Human Development Center for Population Research, and by ES-08265 from the National Institute of Environmental Health Sciences.
1 Present address: Departamento de Biotecnología, Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma
de México, México, D. F. México. 
2 Present address: Monsanto Company, 645 South Newstead Avenue, St.
Louis, Missouri 63110.
Received for publication May 20, 1998. Revision received November 4, 1998. Accepted for publication December 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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  S. Lenie and J. Smitz Functional AR Signaling Is Evident in an In Vitro Mouse Follicle Culture Bioassay That Encompasses Most Stages of Folliculogenesis Biol Reprod, April 1, 2009; 80(4): 685 - 695. [Abstract] [Full Text] [PDF]
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 Q. Ma, W. Fu, P. Li, S. V. Nicosia, G. Jenster, X. Zhang, and W. Bai FoxO1 Mediates PTEN Suppression of Androgen Receptor N- and C-Terminal Interactions and Coactivator Recruitment Mol. Endocrinol., February 1, 2009; 23(2): 213 - 225. [Abstract] [Full Text] [PDF]
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M. M. Centenera, J. M. Harris, W. D. Tilley, and L. M. Butler Minireview: The Contribution of Different Androgen Receptor Domains to Receptor Dimerization and Signaling Mol. Endocrinol., November 1, 2008; 22(11): 2373 - 2382. [Abstract] [Full Text] [PDF]
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 R. Gogoi, M. Kudla, O. Gil, and D. Fishman The Activity of Medroxyprogesterone Acetate, an Androgenic Ligand, in Ovarian Cancer Cell Invasion Reproductive Sciences, October 1, 2008; 15(8): 846 - 852. [Abstract] [PDF]
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S. D. Conzen Minireview: Nuclear Receptors and Breast Cancer Mol. Endocrinol., October 1, 2008; 22(10): 2215 - 2228. [Abstract] [Full Text] [PDF]
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 S. Bai and E. M. Wilson Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor Mol. Cell. Biol., March 15, 2008; 28(6): 1947 - 1963. [Abstract] [Full Text] [PDF]
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 M. C. Hodgson, I. Astapova, A. N. Hollenberg, and S. P. Balk Activity of Androgen Receptor Antagonist Bicalutamide in Prostate Cancer Cells Is Independent of NCoR and SMRT Corepressors Cancer Res., September 1, 2007; 67(17): 8388 - 8395. [Abstract] [Full Text] [PDF]
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 E. B. Askew, R. T. Gampe Jr., T. B. Stanley, J. L. Faggart, and E. M. Wilson Modulation of Androgen Receptor Activation Function 2 by Testosterone and Dihydrotestosterone J. Biol. Chem., August 31, 2007; 282(35): 25801 - 25816. [Abstract] [Full Text] [PDF]
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 S. N. Birrell, L. M. Butler, J. M. Harris, G. Buchanan, and W. D. Tilley Disruption of androgen receptor signaling by synthetic progestins may increase risk of developing breast cancer FASEB J, August 1, 2007; 21(10): 2285 - 2293. [Abstract] [Full Text] [PDF]
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 S. Lu, A. Wang, S. Lu, and Z. Dong A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells Mol. Cancer Ther., July 1, 2007; 6(7): 2057 - 2064. [Abstract] [Full Text] [PDF]
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 J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies Endocr. Rev., October 1, 2006; 27(6): 575 - 605. [Abstract] [Full Text] [PDF]
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D. Kazmin, T. Prytkova, C. E. Cook, R. Wolfinger, T.-M. Chu, D. Beratan, J. D. Norris, C.-y. Chang, and D. P. McDonnell Linking Ligand-Induced Alterations in Androgen Receptor Structure to Differential Gene Expression: A First Step in the Rational Design of Selective Androgen Receptor Modulators Mol. Endocrinol., June 1, 2006; 20(6): 1201 - 1217. [Abstract] [Full Text] [PDF]
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B. He, R. T. Gampe Jr., A. T. Hnat, J. L. Faggart, J. T. Minges, F. S. French, and E. M. Wilson Probing the Functional Link between Androgen Receptor Coactivator and Ligand-binding Sites in Prostate Cancer and Androgen Insensitivity J. Biol. Chem., March 10, 2006; 281(10): 6648 - 6663. [Abstract] [Full Text] [PDF]
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 W. Arlt Androgen therapy in women Eur. J. Endocrinol., January 1, 2006; 154(1): 1 - 11. [Abstract] [Full Text] [PDF]
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G. Buchanan, S. N. Birrell, A. A. Peters, T. Bianco-Miotto, K. Ramsay, E. J. Cops, M. Yang, J. M. Harris, H. A. Simila, N. L. Moore, et al. Decreased Androgen Receptor Levels and Receptor Function in Breast Cancer Contribute to the Failure of Response to Medroxyprogesterone Acetate Cancer Res., September 15, 2005; 65(18): 8487 - 8496. [Abstract] [Full Text] [PDF]
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 F. Schaufele, X. Carbonell, M. Guerbadot, S. Borngraeber, M. S. Chapman, A. A. K. Ma, J. N. Miner, and M. I. Diamond The structural basis of androgen receptor activation: Intramolecular and intermolecular amino-carboxy interactions PNAS, July 12, 2005; 102(28): 9802 - 9807. [Abstract] [Full Text] [PDF]
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S. Bai, B. He, and E. M. Wilson Melanoma Antigen Gene Protein MAGE-11 Regulates Androgen Receptor Function by Modulating the Interdomain Interaction Mol. Cell. Biol., February 15, 2005; 25(4): 1238 - 1257. [Abstract] [Full Text] [PDF]
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C.-L. Hsu, Y.-L. Chen, H.-J. Ting, W.-J. Lin, Z. Yang, Y. Zhang, L. Wang, C.-T. Wu, H.-C. Chang, S. Yeh, et al. Androgen Receptor (AR) NH2- and COOH-Terminal Interactions Result in the Differential Influences on the AR-Mediated Transactivation and Cell Growth Mol. Endocrinol., February 1, 2005; 19(2): 350 - 361. [Abstract] [Full Text] [PDF]
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 T. R. Brown Nonsteroidal Selective Androgen Receptors Modulators (SARMs): Designer Androgens with Flexible Structures Provide Clinical Promise Endocrinology, December 1, 2004; 145(12): 5417 - 5419. [Full Text] [PDF]
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D. Masiello, S.-Y. Chen, Y. Xu, M. C. Verhoeven, E. Choi, A. N. Hollenberg, and S. P. Balk Recruitment of {beta}-Catenin by Wild-Type or Mutant Androgen Receptors Correlates with Ligand-Stimulated Growth of Prostate Cancer Cells Mol. Endocrinol., October 1, 2004; 18(10): 2388 - 2401. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, O. U. Bolden-Tiller, I. Huhtaniemi, D. J. Handelsman, and M. L. Meistrich Effects of Medroxyprogesterone and Estradiol on the Recovery of Spermatogenesis in Irradiated Rats Endocrinology, October 1, 2004; 145(10): 4461 - 4469. [Abstract] [Full Text] [PDF]
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 G. Buchanan, M. Yang, A. Cheong, J. M. Harris, R. A. Irvine, P. F. Lambert, N. L. Moore, M. Raynor, P. J. Neufing, G. A. Coetzee, et al. Structural and functional consequences of glutamine tract variation in the androgen receptor Hum. Mol. Genet., August 15, 2004; 13(16): 1677 - 1692. [Abstract] [Full Text] [PDF]
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S. A. Hild, B. J. Attardi, and J. R. Reel The Ability of a Gonadotropin-Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone Biol Reprod, July 1, 2004; 71(1): 348 - 358. [Abstract] [Full Text] [PDF]
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 K. Pazol, M. E. Wilson, and K. Wallen Medroxyprogesterone Acetate Antagonizes the Effects of Estrogen Treatment on Social and Sexual Behavior in Female Macaques J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2998 - 3006. [Abstract] [Full Text] [PDF]
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Q. Wang, T. S. Udayakumar, T. S. Vasaitis, A. M. Brodie, and J. D. Fondell Mechanistic Relationship between Androgen Receptor Polyglutamine Tract Truncation and Androgen-dependent Transcriptional Hyperactivity in Prostate Cancer Cells J. Biol. Chem., April 23, 2004; 279(17): 17319 - 17328. [Abstract] [Full Text] [PDF]
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G. Sathya, C.-y. Chang, D. Kazmin, C. E. Cook, and D. P. McDonnell Pharmacological Uncoupling of Androgen Receptor-mediated Prostate Cancer Cell Proliferation and Prostate-specific Antigen Secretion Cancer Res., November 15, 2003; 63(22): 8029 - 8036. [Abstract] [Full Text] [PDF]
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A. L. Amir, M. Barua, N. C. McKnight, S. Cheng, X. Yuan, and S. P. Balk A Direct {beta}-Catenin-independent Interaction between Androgen Receptor and T Cell Factor 4 J. Biol. Chem., August 15, 2003; 278(33): 30828 - 30834. [Abstract] [Full Text] [PDF]
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I. U. Agoulnik, W. C. Krause, W. E. Bingman III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel Repressors of Androgen and Progesterone Receptor Action J. Biol. Chem., August 15, 2003; 278(33): 31136 - 31148. [Abstract] [Full Text] [PDF]
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 B. Terouanne, P. Nirde, F. Rabenoelina, W. Bourguet, C. Sultan, and G. Auzou Mutation of the Androgen Receptor at Amino Acid 708 (Glyright-arrowAla) Abolishes Partial Agonist Activity of Steroidal Antiandrogens Mol. Pharmacol., April 1, 2003; 63(4): 791 - 798. [Abstract] [Full Text] [PDF]
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B. He and E. M. Wilson Electrostatic Modulation in Steroid Receptor Recruitment of LXXLL and FXXLF Motifs Mol. Cell. Biol., March 15, 2003; 23(6): 2135 - 2150. [Abstract] [Full Text] [PDF]
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D. Yin, Y. He, M. A. Perera, S. S. Hong, C. Marhefka, N. Stourman, L. Kirkovsky, D. D. Miller, and J. T. Dalton Key Structural Features of Nonsteroidal Ligands for Binding and Activation of the Androgen Receptor Mol. Pharmacol., January 1, 2003; 63(1): 211 - 223. [Abstract] [Full Text] [PDF]
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 S. Y. Euling, C. Gennings, E. M. Wilson, J. A. Kemppainen, W. R. Kelce, and C. A. Kimmel Response-Surface Modeling of the Effect of 5{alpha}-Dihydrotestosterone and Androgen Receptor Levels on the Response to the Androgen Antagonist Vinclozolin Toxicol. Sci., October 1, 2002; 69(2): 332 - 343. [Abstract] [Full Text] [PDF]
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D. Masiello, S. Cheng, G. J. Bubley, M. L. Lu, and S. P. Balk Bicalutamide Functions as an Androgen Receptor Antagonist by Assembly of a Transcriptionally Inactive Receptor J. Biol. Chem., July 12, 2002; 277(29): 26321 - 26326. [Abstract] [Full Text] [PDF]
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J. E. Pawlowski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, and M. E. Wierman Liganded Androgen Receptor Interaction with beta -Catenin. NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS J. Biol. Chem., May 31, 2002; 277(23): 20702 - 20710. [Abstract] [Full Text] [PDF]
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V. S. Wilson, K. Bobseine, C. R. Lambright, and L. E. Gray Jr. A Novel Cell Line, MDA-kb2, That Stably Expresses an Androgen- and Glucocorticoid-Responsive Reporter for the Detection of Hormone Receptor Agonists and Antagonists Toxicol. Sci., March 1, 2002; 66(1): 69 - 81. [Abstract] [Full Text] [PDF]
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P. C. Hartig, K. L. Bobseine, B. H. Britt, M. C. Cardon, C. R. Lambright, V. S. Wilson, and L. E. Gray Jr. Development of Two Androgen Receptor Assays Using Adenoviral Transduction of MMTV-Luc Reporter and/or hAR for Endocrine Screening Toxicol. Sci., March 1, 2002; 66(1): 82 - 90. [Abstract] [Full Text] [PDF]
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 C. M. Kuhn Anabolic Steroids Recent Prog. Horm. Res., January 1, 2002; 57(1): 411 - 434. [Abstract] [Full Text] [PDF]
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B. He, N. T. Bowen, J. T. Minges, and E. M. Wilson Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 Coactivator Recruitment by Activation Function 2 J. Biol. Chem., November 2, 2001; 276(45): 42293 - 42301. [Abstract] [Full Text] [PDF]
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D. A. Nazarenko, S. D. Dertinger, and T. A. Gasiewicz In Vivo Antagonism of AhR-Mediated Gene Induction by 3'-Methoxy-4'-nitroflavone in TCDD-Responsive lacZ Mice Toxicol. Sci., June 1, 2001; 61(2): 256 - 264. [Abstract] [Full Text] [PDF]
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T. Raivio, J. J. Palvimo, L. Dunkel, S. Wickman, and O. A. Jänne Novel Assay for Determination of Androgen Bioactivity in Human Serum J. Clin. Endocrinol. Metab., March 1, 2001; 86(4): 1539 - 1544. [Abstract] [Full Text]
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J. Kwong, J. W. Xuan, P. S. F. Chan, S.-M. Ho, and F. L. Chan A Comparative Study of Hormonal Regulation of Three Secretory Proteins (Prostatic Secretory Protein-PSP94, Probasin, and Seminal Vesicle Secretion II) in Rat Lateral Prostate Endocrinology, December 1, 2000; 141(12): 4543 - 4551. [Abstract] [Full Text] [PDF]
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D. Deplewski and R. L. Rosenfield Role of Hormones in Pilosebaceous Unit Development Endocr. Rev., August 1, 2000; 21(4): 363 - 392. [Abstract] [Full Text] [PDF]
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J.-a. Tan, S. H. Hall, K. G. Hamil, G. Grossman, P. Petrusz, J. Liao, K. Shuai, and F. S. French Protein Inhibitor of Activated STAT-1 (Signal Transducer and Activator of Transcription-1) Is a Nuclear Receptor Coregulator Expressed in Human Testis Mol. Endocrinol., January 1, 2000; 14(1): 14 - 26. [Abstract] [Full Text]
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B. He, J. A. Kemppainen, J. J. Voegel, H. Gronemeyer, and E. M. Wilson Activation Function 2 in the Human Androgen Receptor Ligand Binding Domain Mediates Interdomain Communication with the NH2-terminal Domain J. Biol. Chem., December 24, 1999; 274(52): 37219 - 37225. [Abstract] [Full Text] [PDF]
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N. Poujol, J.-M. Wurtz, B. Tahiri, S. Lumbroso, J.-C. Nicolas, D. Moras, and C. Sultan Specific Recognition of Androgens by Their Nuclear Receptor. A STRUCTURE-FUNCTION STUDY J. Biol. Chem., July 28, 2000; 275(31): 24022 - 24031. [Abstract] [Full Text] [PDF]
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B. He, J. A. Kemppainen, and E. M. Wilson FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor J. Biol. Chem., July 21, 2000; 275(30): 22986 - 22994. [Abstract] [Full Text] [PDF]
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