Functional Activities of the A and B Forms of the Human Androgen Receptor in Response to Androgen Receptor Agonists and Antagonists
Tianshu Gao and
Michael J. McPhaul
Department of Internal Medicine, The University of Texas
Southwestern Medical Center, Dallas, Texas 75235
 |
ABSTRACT
|
---|
The androgen receptor (AR) is present in
many cells in two forms. The B form migrates with an apparent mass of
110 kDa and constitutes more than 80% of the immunoreactive receptor
in most cell types. The A form of the AR migrates with an apparent mass
of 87 kDa, appears to derive from internal translation initiation at
methionine-188 in the AR open-reading frame, and usually constitutes
20% or less of the immunoreactive AR present. Previous experiments
designed to examine the functional capacity of the A and B forms of the
AR have been hampered by marked differences in the expression levels of
the two isoforms, as the nucleotide sequence surrounding the codon
encoding methionine-188 causes it to be used inefficiently as a
translation initiation site. To circumvent this, we altered the
nucleotide sequence surrounding methionine-188 to render it more
similar to that surrounding the codon encoding methionine-1.
Transfection of a cDNA containing these changes resulted in similar
levels of expression of A and B forms of the AR as assessed by
immunoblot assays using antibodies directed at an epitope preserved in
both. Functional activities of these cDNAs were assessed using
cotransfection assays that employed two model androgen-responsive genes
(MMTV-luciferase and PRE2-tk-luciferase) in
response to mibolerone, a potent androgen agonist, in three different
cell lines. These studies demonstrated subtle differences in the
activities of the A and B isoforms, which depended on the promoter and
cell context. Additional studies failed to reveal any major differences
in the responses of the AR-A and AR-B isoforms to a variety of androgen
agonists and antagonists, suggesting that the previously reported
functional defect of the AR-A is due principally to its level of
expression. When assays of AR function are performed under conditions
in which levels of expression of the two isoforms are equivalent, the
AR-A and AR-B possess similar functional activities.
 |
INTRODUCTION
|
---|
The androgen receptor (AR) is a member of the steroid
hormone-thyroid hormone-retinoic acid family of nuclear receptors (1, 2) and mediates the responses of tissues to the male hormones,
testosterone and 5
-dihydrotestosterone. The cDNA nucleotide sequence
predicts a protein approximately 917 amino acids long comprised of a
highly conserved DNA-binding domain, a carboxy-terminal hormone-binding
domain, and a large amino terminus (3, 4, 5, 6). The form of the AR predicted
by the translation of the complete open reading frame (termed AR-B)
migrates with an apparent molecular mass of 110 kDa on
SDS-polyacrylamide gels.
During our studies of affected members of a family with complete
testicular feminization, we detected a novel shortened form of the
human AR (7). Subsequent experiments revealed that this AR isoform
[termed AR-A because of its similarity in structure to the A form of
the progesterone receptor (PR)] differed from the structure predicted
for the B form of the AR in that it lacked an intact amino terminus,
yet contained intact DNA- and hormone- binding domains. Further
analysis revealed that this form of the AR is expressed in a variety of
normal tissues, albeit at low levels, and is derived from initiation of
translation at methionine-188 instead of methionine-1 of the AR open
reading frame (8, 9).
The detection of two distinct forms of the AR raised a number of
issues. First, since the original family in which the AR-A form of the
receptor was detected exhibited a phenotype of complete testicular
feminization, it was not clear whether this defect of androgen action
was caused by the reduced levels of receptor expressed or to the
impaired function of this form of the AR (7). Second, as noted above,
the structures of the A and B forms of the AR bear a striking
resemblance to the more fully characterized A and B forms of the PR. A
number of studies have suggested that the A and B forms of the PR
exhibit distinct differences of function, with respect to the target
genes that they regulate, the ligands to which they respond, and the
way in which they interact with target DNA sequences (10, 11, 12, 13, 14, 15). The
similarities evident between the isoforms of the AR and PR suggested
that specific roles might exist for the AR-A and AR-B isoforms in the
regulation of androgen-responsive genes as well.
 |
RESULTS
|
---|
Alteration of Nucleotide Sequence Surrounding Methionine-188
Permits the Expression of the AR-A and AR-B Isoforms at Equivalent
Levels in the Absence of Hormone in Transfection Assay
Previous studies suggested that the defect of AR function in
patient 776 was caused, at least in part, by the reduced levels of AR-A
expressed in fibroblasts established from the affected subject (7).
This low level of expression is likely caused by the nucleotide
sequence environment surrounding methionine-188, which is a poor fit to
Kozak consensus sequence rules for translation initiation and predicts
poor translation efficiency (16). To circumvent this limitation,
sequences 5' to methionine-188 were replaced by those that are normally
5' to methionine-1 by site-directed mutagenesis (Fig. 1
). The effect of this change was
assessed by measuring the level of expression in transfected cells
using antibodies that recognize both receptor isoforms (amino acid
residues 200220 in AR-B). As shown in Fig. 2
, transfection of the resulting
expression plasmid (PS367) into COS cells resulted in the synthesis of
levels of immunoreactive AR equivalent to those observed after
transfection with a cDNA encoding the full-length AR (CMV 3.1).

View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Schematic Structures of the AR-A and AR-B Isoforms
The AR-A and AR-B isoforms differ in the length of their amino termini.
The cDNA used in the initial studies of the functional activity of the
AR-A isoform (7) contained a premature termination codon at amino acid
residue 60, and the surrounding nucleotide sequence predicted a poor
efficiency of translation initiation at methionine-188. To permit the
expression of the isoforms in a fashion where equivalent amounts of
each are expressed, the cDNA segment 5' to methionine-188 in plasmid
776 (CMV776) was mutated to correspond to that 5' to methionine-1 in
CMV3.1 (Ref. 6). The resulting plasmid (in which the codon encoding
methionine-188 is located immediately 3' to the segment normally 5' to
the ATG encoding methionine-1) was designated PS367. The black
boxes shown in the plasmids CMV3.1 and CMV776 indicate the
positions of the glutamine repeats. Underlined sequences
shown in PS367 indicate a BglII restriction endonuclease
cleavage site by which the segment amplified by PCR was ligated into
CMV5 vector.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Figure 2. Alteration of the Segment 5' to Methionine-188
Results in High Level Expression of the AR-A Isoform
Monolayers of COS cells were transfected in parallel with expression
plasmids encoding either the hAR (CMV3.1; AR 1917), CMV776, or PS367.
Forty hours after transfection, the cells were harvested, and extracts
were prepared and analyzed by Western analysis. The antibody used
(U407) recognizes an epitope (amino acids 200220) that is common to
both the AR-A and AR-B isoforms. The numbers in
parentheses indicate the quantity of protein loaded in each
lane.
|
|
AR-A and AR-B Demonstrate Differences of Function When Assayed in
the CV-1 Cell Line Using Mouse Mammary Tumor Virus (MMTV) and
PRE2-tk Promoter Fusions
Having demonstrated that the levels of
immunoreactive AR expressed were similar when equivalent levels of cDNA
were transfected, we then performed experiments to compare their
functional activities. To do so, we transfected increasing amounts of
the human (h) AR-A and hAR-B expression vectors and constant amounts of
the MMTV-luciferase reporter gene into CV1 cell monolayers and
incubated the transfected cultures with 2 nM
dihydrotestosterone (DHT) in each experiment. The results (shown in
Fig. 3A
) revealed substantial differences
in the dose-response curves of the two isoforms. After transfection
with low concentrations of cDNAs encoding either isoform, increasing
activation of the reporter gene was observed. Although in both
instances the maximum stimulation of the reporter gene was achieved
between 0.1 µg and 0.4 µg of AR cDNA, at the region of peak
activity AR-A exhibits approximately half the level of reporter gene
activity measured for cells transfected with the AR-B cDNA (Fig. 3A
).
This result suggests that the amino-terminal segment (amino acid
residues 1188) that is not present in the AR-A isoform is important
for the formation of receptor complexes capable of stimulating maximal
activation of the MMTV promoter in CV1 cells.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 3. Dose Response of AR-A and AR-B Function Analyzed in
CV1 Cells Using the MMTV Luciferase and PRE2-tk Luciferase
Reporter Gene
Monolayers of CV1 cells were transfected with different quantities of
cDNA encoding AR-A (PS367) and AR-B (CMV3.1) and the
androgen-responsive reporter gene MMTV luciferase (A) and
PRE2-tk luciferase (B). Transfection efficacy was assessed
by measurement of the ß-galactosidase activity encoded by the control
plasmid, CMV-ß-galactosidase, which was included in each
transfection. Each point on this graph is derived from
one of six separate cell samples (triplicate points each from cells
stimulated in parallel with no hormone or 2 nM mibolerone).
This experiment is representative of three independent experiments.
|
|
Different results were obtained when a different promoter,
PRE2-tk, was used to assay the functions of the AR isoforms
in the same cells (CV1 cells). When assayed using this reporter gene,
AR-A functioned approximately twice as well as AR-B (Fig. 3B
). This
result suggests that the two AR isoforms interact with components of
the transcription apparatus in a fashion that is promoter-specific.
Experiments conducted transfecting cDNAs encoding both isoforms
demonstrated no evidence that the AR-A isoform acted in a dominant
negative fashion (data not shown).
Cell- and Promoter-Specific Behavior of AR Isoforms in Stimulating
the Activity of Androgen-Responsive Reporter Genes
To determine how the A and B isoforms would function in different
cell types to activate these model androgen-responsive reporter genes,
we transfected 200 ng of AR-A and AR-B cDNA in parallel with either the
MMTV or PRE2-tk reporter plasmids into the CV1, DU145, and
PPC1 cell lines and assayed reporter gene activity after incubation
with 2 nM DHT in each experiment. This protocol was chosen
as this amount of AR cDNA induced the activity of the
androgen-responsive genes by both isoforms to maximal levels (Fig. 3
).
When CV1 cells were transfected with cDNAs encoding the AR
isoforms and the MMTV promoter reporter plasmids, AR-A exhibited
approximately half of the level of activity observed in transfections
using the AR-B cDNA. These results correspond to those obtained in the
dose response curves presented in Fig. 3A
. Although the measurements of
AR function in the different cell lines cannot be compared directly, it
is clear that similar patterns in the relative activities of the A and
B isoforms are seen when AR function is measured in the DU145 and PPC1
cell lines using the MMTV reporter, as when performed in the CV1 cell
line. In each instance, the B form of the AR is approximately twice as
active in stimulating reporter gene activity as the A form assayed in
parallel (Fig. 4A
).

View larger version (34K):
[in this window]
[in a new window]
|
Figure 4. The Effects of Cell Types and Reporter Gene
Promoters on the Activities of AR-A and AR-B
Cells were transfected with a constant amount of expression plasmid
(200 ng) encoding the AR-A or AR-B isoforms, reporter plasmid [MMTV
luciferase (A) or PRE2-tk luciferase (B), 10 µg], and
transfection control plasmid CMV-ß-galactosidase (1 µg). After
stimulation with no hormone or 2 nM mibolerone for 48
h, luciferase and ß-galactosidase activities were measured. Each
value is derived from six separate points (three transfections treated
with no hormone and three treated with 2 nM mibolerone).
Owing to the different cell lines and reporter genes used, only the
values for the AR-A and AR-B isoforms in a given cell line and promoter
can be directly compared.
|
|
The results obtained when the capacities of these AR isoforms to
activate the PRE2-tk promoter fusion were assayed in the
same panel of cell lines were less constant. When assayed in the CV1
cell line, AR-A isoform was found to be more potent than the AR-B
isoform (Fig. 4B
, results consistent with those depicted in Fig. 3B
).
When assayed in the DU145 cell line, the two isoforms exhibited similar
capacities to activate the reporter gene. In the PPC1 cell line, the
level of reporter gene activation achieved by AR-A was lower than that
of AR-B. These differences suggest that the AR-A and AR-B
hormone-receptor complexes interact with the PRE2-tk
promoter in a fashion that varies among cell types. Despite these
differences, each reporter gene used displayed similar levels of
activation in response to saturating concentrations of DHT and
testosterone (data not shown).
AR-A and AR-B Cannot Be Distinguished by Antiandrogens in
Cotransfection Assay
The A and B forms of the PR have functions that can be
distinguished pharmacologically. To examine whether this was true for
the A and B AR isoforms, we examined the response of these isoforms to
different AR agonists and antagonists. These assays were performed by
transfecting the AR-A and AR-B cDNAs in parallel into CV1 cells in
combination with the MMTV-luciferase reporter gene and treating the
transfected cells with varying concentrations of antiandrogens alone or
in combination with saturating concentrations of 5
-DHT. The results
that we obtained are shown in Fig. 5
and
are intriguing in several respects.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 5. Effects of Antiandrogens on AR-A and AR-B Function
CV1 cells were transiently transfected by the addition of a calcium
phosphate precipitate containing the cDNAs (200 ng) encoding either
AR-A (PS367) or AR-B (CMV3.1), the reporter plasmid MMTV-luciferase (10
µg), and a control plasmid, CMV-ß-galactosidase (1 µg), and
incubated with the indicated combinations of ligands for 48 h.
Panels A, B, and C display the activities observed in experiments using
hydroxyflutamide and flutamide, while panel D indicates the results of
an experiment using bicalutamide (Casodex).
|
|
As expected, hydroxyflutamide functioned as a potent antagonist to the
activation of the AR isoforms by DHT (Fig. 5A
). In these experiments,
the inclusion of 0.5 µM hydroxyflutamide inhibited the
reporter gene activity induced by saturating concentrations of 5
-DHT
by more than 90%. Additional inhibition of AR function was not evident
when the concentration of hydroxyflutamide was increased further.
Instead, as the concentration of hydroxyflutamide was increased,
increasing levels of reporter gene activation were observed. When these
experiments were performed using hydroxyflutamide alone, it was
possible to demonstrate that the agonism observed at the higher
hydroxyflutamide concentrations was a function of the properties of
hydroxyflutamide itself.
When this same type of experiment was repeated using flutamide, a
different result was observed. Flutamide showed less potent capacity to
antagonize DHT action than hydroxyflutamide, requiring concentrations
of 2 µM to achieve a 90% inhibition of DHT-stimulated AR
function. Of interest, as the concentrations of flutamide were
increased (from 0.5 to 2 µM), only a progressive
inhibition of AR function was observed. In keeping with this
observation, flutamide was devoid of any intrinsic agonism in
functional assays when added alone to the transfected cells (Fig. 5B
).
Experiments in which side-by-side comparisons were performed confirmed
this finding (Fig. 5C
).
The findings presented above are not confined to hydroxyflutamide, and
other high-affinity AR antagonistseven those that have been suggested
to be pure antiandrogensexhibit a mixture of agonist and antagonist
properties. A representative experiment is shown in Fig. 5D
for Casodex
(bicalutamide). At low concentrations, Casodex exhibits a potent
capacity to antagonize the activation of the AR-B by 2 nM
5
-DHT. As was observed with hydroxyflutamide, however, as the
concentration of Casodex is increased, increasing agonism becomes
evident. As shown in Table 1
, the AR
antagonists demonstrate similar behaviors when assayed in the PPC-1 and
DU145 cell lines.
View this table:
[in this window]
[in a new window]
|
Table 1. AR Antagonists Display Similar Activities in the
PPC-1, DU145, and CV1 Cell Lines When Assayed Using the MMTV Luciferase
Reporter Gene
|
|
In the experiments depicted in Fig. 5
, A, B, and D, the activity of
AR-A isoform was measured in parallel with those of the AR-B isoform.
In each instance, the patterns of response observed for the AR-B and
AR-A isoforms were similar.
Activation of the AR Isoforms Is Not Always Accompanied by
Increased Levels of Immunoreactive AR
Androgen causes the levels of AR to increase when assayed by
ligand binding (17) or by immunoblot assays (18), and this increase
appears to result from a change in the half-life of the receptor
protein. To determine whether the agonism observed for the AR
antagonists (such as hydroxyflutamide and Casodex) also caused changes
in the levels of AR, we performed immunoblot assays of cells
transfected with cDNAs encoding the different isoforms after treatment
with either no hormone or with the different antagonists. The results
of these experiments are shown in Fig. 6
and are interesting in two respects. The first is that the level of AR
does not change in cells transfected with AR-B and treated with AR
antagonists, even under circumstances in which substantial agonism is
observed (e.g. at 5 µM hydroxyflutamide).
These experiments also show, unexpectedly, that the increase in the
level of AR-B that is observed after treatment with androgen agonists,
such as mibolerone, is not observed in cells transfected with the AR-A
isoform. This finding suggests that the amino- terminal segment of AR-B
is critical for the increase in receptor levels after treatment with
ligand.

View larger version (56K):
[in this window]
[in a new window]
|
Figure 6. Increase in the Levels of Immunoreactive AR Do Not
Parallel the Agonistic Effects Observed in Response to Selected
Antiandrogens
Monolayers of CV1 cells were transfected in parallel with expression
plasmids encoding either the hAR-B (CMV3.1 AR 1917) and AR-A (PS367
AR 188917). Twenty four hours after transfection, cells were treated
with DHT and different antiandrogens at different concentrations in MEM
containing 5% charcoal- treated serum. Forty hours later, the cells
were harvested, and extracts were prepared and analyzed by Western
analysis. The antibody used (U407) recognizes an epitope (amino acids
200220) that is common to both the AR-A and AR-B isoforms. The
quantity of protein loaded in each lane is the same ( 100 µg).
|
|
 |
DISCUSSION
|
---|
Different receptor forms derived from a single structural gene
have been described for a number of different members of the
steroid-thyroid-retinoic acid family of nuclear receptors (1, 19, 20, 21).
Of these, the two different forms of the hAR bear the most striking
similarity to the A and B forms of the PR. As in the case of the PR,
the expression of two different forms of the hAR has been detected in a
number of different tissues. Unlike the PR-A isoform, the hAR-A has
been detected at relatively low and constant levels and no cell type or
tissues have been identified to date in which only the AR-A is
expressed as the predominant isoform (8, 9). Despite this, the similar
structures of the AR and PR isoforms suggested that the AR-A isoform
might display distinctive properties in the regulation of
androgen-responsive genes.
In our prior studies, a patient with complete testicular feminization
expressed only a shortened form of AR (AR-A), which appeared to be
derived from initiation of translation at methionine-188 (7). The low
and relatively constant level of AR-A that was detected in the
fibroblasts of this patient was also found to be present in the
fibroblasts of normal subjects and in most target tissues (8). The
unfavorable context surrounding the AUG triplet encoding methionine-188
appeared to account for the low level of expression in vivo
and in transfection studies and complicated studies to analyze the
functional capacity of the two isoforms. For this reason, we
constructed an AR-A expression vector (PS367) in which the sequence
upstream of methionine-1 was introduced 5' to methionine-188. As the
result of these changes, the expression levels between AR-A and AR-B
were similar and permitted a more thorough analysis of their functional
activities.
When assayed in CV1 cells, AR-A and AR-B showed different dose-response
curves in transfection assays using an MMTV-luciferase reporter gene
fusion to measure receptor function. The activities of both isoforms
were maximal at low concentrations of transfected cDNA, and decreasing
levels of reporter gene activity were observed as increasing amount of
cDNA are added. Of note, at the level where maximal activation was
achieved, the AR-B isoform was approximately twice as active as the
AR-A isoform. When such assays are repeated using a reporter gene
controlled by the PRE2-tk promoter in CV1 cells, the level
of induction by AR-B was half that stimulated by AR-A. This finding
suggests that the amino-terminal segment of the AR (amino acid residues
1187) interacts differently with the proteins that regulate
transcription from these two promoters. These inferences are further
supported by the results of assays using these same reporter genes in
different cell lines. In these experiments, while the activity of the
AR-B was higher using the MMTV promoter in all of the cell lines, the
PRE2-tk promoter gene fusion was activated to varying
levels by the AR-A in the DU145, PPC1, and CV1 cell lines. The
demonstration that the amino terminus confers upon the AR-B isoform
activities not exhibited by the AR-A isoform is consistent with studies
that have localized a distinct transactivation domain to the
corresponding segment of the human PR (22).
In some instances, isoforms of nuclear receptors have demonstrated
differential responses to pharmacological manipulations (10, 11, 12). For
this reason, we explored the effects of a variety of AR antagonists on
AR function. In these experiments, using a reporter gene controlled by
the MMTV promoter in CV1 cells, a variety of antiandrogens failed to
display any major qualitative differences in the activities of AR
isoforms. In each instance the results of assays using AR-A were
similar to those obtained using AR-B.
While the results of these functional studies did not reveal unique
activities of the AR isoforms, the properties exhibited by the
different antagonists in these experiments were intriguing. First, it
is evident that of the compounds tested, only flutamide failed to
exhibit agonism in our assays. While low concentrations of each of the
higher affinity antagonists (Casodex, hydroxyflutamide) effectively
blocked the stimulation of the AR by saturating concentrations of
agonist (DHT), increasing agonism was evident as the concentrations of
these AR antagonists were further increased. Of note, our observations
differ somewhat from the findings of other groups who have examined the
activities of hydroxyflutamide and Casodex in reporter gene assays. In
two studies, these compounds exhibited little agonistic activity and
appeared to function as pure antagonists (23, 24). By contrast,
Kemppainen and Wilson (25) noted the agonism inherent in the behaviors
of hydroxyflutamide and Casodex. The results reported in the present
study are in best agreement with these latter investigations. The
reasons for the discordances are not clear, but may be explained in
part by the reporter genes used, by the concentrations of antagonist
assayed, or by the specific cell types employed.
The mechanisms by which ligands for nuclear receptors exhibit
both agonism and antagonism are not clear. Molecules exhibiting such a
mixture of activities have been identified that act to antagonize the
actions of ligands for many members of the nuclear receptor family,
including androgens, estrogens, and progestins (12, 24, 25, 26, 27, 28). Detailed
studies of the activities of such compounds have suggested models in
which the receptor assumes distinct conformations that permit the
liganded receptor to interact with the transcription apparatus in a
productive or nonproductive fashion (29, 30). The degree of agonism
observed has been found to vary substantially, depending on cell type
and promoter context. In the case of the ER, the agonistic properties
of some antiestrogens have been correlated with the activity of a
specific transactivation domain (TAF-1) located within the amino
terminus of the receptor (27, 28, 31). Recent work suggests that the
levels and capacity of the liganded receptor to recruit specific
coactivators and repressors to the transcription complex are important
determinants of the degree of agonism or antagonism that is observed
(32, 33).
While such models provide a framework with which to explain the
tissue- and cell type-specific behaviors of some antagonists, they do
not provide a ready explanation for one aspect of the behaviors of the
AR antagonists studied in the current work. Namely, these models do not
offer a rationale for the emergence of agonistic behavior of some AR
antagonists at concentrations well above that required to inhibit the
activation of the AR by saturating concentrations of DHT. Such
observations could be reconciled by a contrasting view of the actions
of steroid hormone receptor antagonists, such as that afforded by
Jensen and co-workers (34, 35). In this view, a two-site model has been
proposed to assist in accounting for the mixed agonist-antagonist
properties displayed by some receptor antagonists. In such a model, in
addition to competing for the cognate hormone-binding site,
antihormones react with a second domain in the receptor, which is not
recognized by cognate ligand and which plays a role in antagonist
action (34, 35). Such a model would consider the difference between
antihormones of different classes to result from their different
relative affinities for the two binding regions. While such models have
not been experimentally tested extensively, differences in the number
of binding sites have been detected using [3H]tamoxifen
and estradiol as ligands in binding assays. In a similar vein, the
demonstration that a mutant PR deleted for the receptor carboxy
terminus is able to bind and respond to RU486 but not to progesterone
is also consistent with the existence of separable sites for the
binding of progesterone antagonists and agonists within the PR
hormone-binding domain (36). Finally, with respect to the mechanisms by
which AR antagonists function, the existence of additional binding
sites would offer a ready explanation for the appearance of agonism at
concentrations of antagonist well above that needed to interrupt the
action of AR agonists.
Flutamide is rapidly and extensively metabolized in
vivo to hydroxyflutamide by hydroxylation. While both molecules
have been shown to block the effects of androgens in target tissues
(37, 38, 39, 40), flutamide has been estimated to do so with a 25-fold lower
affinity relative to hydroxyflutamide for AR in vitro (41).
For this reason, subsequent attempts to develop higher affinity AR
antagonists have used hydroxyflutamide as a starting point. Our results
suggest that antagonists with such structures (particularly with larger
side chain substituents) may possess, by their very nature, a certain
degree of inherent agonism that is unmasked at higher concentrations.
It is conceivable that increased formation or accumulation of
metabolites (such as hydroxyflutamide) within cells might contribute to
the apparent resistance of prostate cancers that occurs in patients
treated with such compounds. Such a mechanism would offer an
alternative explanation for the flutamide withdrawal syndrome,
particularly in those cases in which an AR mutation is not
identified.
Finally, the addition of ligand to cultured cells has been shown to
result in an increase in the level of AR, measured either using
ligand-binding assays (17) or Western blotting (18). Previous studies
have demonstrated that this effect is posttranslational and requires
both an intact ligand-binding domain and the amino-terminal segment of
the receptor molecule. The studies of Zhou et al. concluded
that an interaction of the amino and carboxy termini of the protein
were required to observe a ligand-induced stabilization of the receptor
(42). These inferences were further supported by the results of
immunoblot assays that measured the levels of expression of the intact
AR and a number of mutant ARs containing deletions of varying sizes
within the amino terminus (43). The present work suggests that amino
acids within the most amino-terminal segments of the receptor
participate directly in these interactions.
 |
MATERIALS AND METHODS
|
---|
Chemicals
Restriction enzyme were purchased from New England Biolabs, Inc.
(Beverly, MA). PCR reagents were obtained from Perkin Elmer
(Branchburg, NJ). 125 I-labeled goat F-(AB) antirabbit IgG
was purchased from DuPont NEN research products (Boston, MA). Samples
of the antiandrogens flutamide (batch no. IRQ-BTA-7-D-76D) and
hydroxyflutamide (batch no. 2649279) were generously provided by R.
Neri (Schering Co., Bloomfield, NJ). Sample of the Casodex (ICI 176334
batch no PP-0195) was provided by G. Kolvenbag (Zeneca Pharmaceuticals,
Macclesfield, Cheshire, U.K.). The solutions used were freshly prepared
before use. 5
-Dihydrotestosterone was purchased from Steraloids,
Inc. (Wilton, NH).
Site-Directed Mutagenesis
The segment 5' to methionine-188 in the original plasmid
encoding the A-form of the AR [CMV776 (Ref.7)] was modified by
replacing it with the sequence that is 5' to methionine-1 in the AR
expression plasmid CMV3.1. This mutagenesis was accomplished by using
two oligonucleotides (AR-As:
5'-ACACAGATCTAGGTGGAAGATTCAGCCAAGCTCAAGGATGCAACTCCTTCAGCAACAGCAGCAGGAA-3'
and AR-Aas: 5'-GGCTGAGGGTGACCCAGAACCGGGT-3') as
primers and the plasmid CMV3.1 (6) as template in a PCR reaction. The
resulting PCR fragment, encoding the methionine-188 as the initiator
methionine [i.e. deleting nucleotides 163723 of the AR
cDNA sequence (6)], was digested with the restriction enzymes
NcoI and BglII and ligated into the 0.6-kb
fragment purified after digestion of a sample of the CMV 3.1 hAR
expression plasmid digested with the same restriction endonucleases.
Vector fragments derived from the expression vector (CMV3.1) cleaved
with the same enzymes. As shown in Fig. 1
, the resulting plasmid
(designated PS367) encodes a protein that is identical to hAR-B, except
it lacked the segment encoding amino acids 1187 of the hAR open
reading frame. In this environment, the initiator methionine of the
hAR-B (the codon encoding methionine-188 in CMV 3.1) is predicted to be
a much better fit to the Kozak consensus sequence for translation
initiation (16).
Cell Culture and Transient Transfection Assays
Stock cultures of the CV1 (monkey kidney fibroblast-like cell
line) and DU145 cell lines (a metastatic human prostate carcinoma
cell line) were obtained from the American Type Culture Collection and
were maintained in MEM (GIBCO/BRL, Gaithersburg, MD) containing 10%
(vol/vol) FCS and 1% penicillin and streptomycin. PPC1 cell line
(considered to be a variant of PC-3 cell line) was obtained from Arthur
Brothman (Salt Lake City, UT) and maintained in RPMI 1640
containing 10% (vol/vol) FCS and 1% penicillin and
streptomycin.
Transfection assays were performed as described (43). The day before
transfection, cells were trypsinized and plated at a density of 2
x 105 cells per well in six-well plates (each well 35
mM diameter) for reporter gene assays of AR function and a
density of 1 x 106 cells per 10-cm dish for
immunoblot analyses. Each six-well plate or 10-cm dish was transiently
transfected by the addition of calcium phosphate precipitate containing
the indicated concentrations of hAR isoform expression plasmid, the
androgen-responsive reporter plasmid (10 µg), and 1 µg of a control
plasmid (CMV-ß-galactosidase) in 12 ml (for six-well plates) or 10 ml
(for each 10-cm dish) of culture medium for 24 h. After these
incubations, the medium was removed and replaced with fresh MEM
containing 5% charcoal-stripped serum alone or containing the various
ligands. Forty-eight hours later, the cell cultures were harvested and
assayed for luciferase activity and ß-galactosidase activity or
harvested for immunoblot assays. In each experiment, measurements of
the function of individual ARs were assessed in at least three separate
transfections (wells) for each in the absence or presence of hormone.
The results of these individual measurements were averaged and compared
with the results obtained using the AR-B included in each experiment.
In the text and legends, the functional assay results are presented
either as stimulated luciferase values or as fold induction. This
latter value is calculated by dividing the stimulated luciferase values
by the basal luciferase values.
Immunoblots
Immunoblots were prepared as previously described. After
transfer, the filters were incubated with affinity-purified antibody
(anti-Internal A antibody) from rabbit U407 (8) that recognizes amino
acids 200220 of the hAR protein, which is preserved in both the AR-A
and AR-B isoforms.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael J. McPhaul, M.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.
This work was supported by NIH Grants DK-03892 and 47657, a grant from
the Robert A. Welch foundation (I-1090), and a grant from the Perot
Family Foundation.
Received for publication July 14, 1997.
Revision received January 2, 1998.
Accepted for publication January 29, 1998.
 |
REFERENCES
|
---|
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS,
Wilson EM 1988 Cloning of human androgen receptor complementary DNA and
localization to the X chromosome. Science 240:327330[Medline]
-
Chang C, Kokontis J, Liao S 1988 Molecular cloning of human
and rat complementary DNA encoding androgen receptors. Science 240:324326[Medline]
-
Trapman JP, Klaassen, Kuiper GGJM, van der Korput JAGM, Faber
PW, van Rooij HCJ, Geurts A, van Kessel, Voorhorst M, Mulder E,
Brinkmann AO 1988 Cloning, structure and expression of a cDNA encoding
the human androgen receptor. Biochem Biophys Res Commun 153:241248[Medline]
-
Tilley WD, Marcelli M, Wilson JD, McPhaul MJ 1989 Characterization and expression of a DNA encoding the human androgen
receptor. Proc Natl Acad Sci USA 86:327331[Abstract]
-
Zoppi S, Wilson CM, Harbison MD, Griffin JE, Wilson JD,
McPhaul MJ, Marcelli M 1993 Complete testicular feminization caused by
an amino-terminal truncation of the androgen receptor with downstream
initiation. J Clin Invest 91:11051112[Medline]
-
Wilson CM, McPhaul MJ 1994 A and B forms of the androgen
receptor are present in human genital skin fibroblasts. Proc Natl Acad
Sci USA 91:12341238[Abstract]
-
Wilson CM, McPhaul MJ 1996 A and B forms of the androgen
receptor are expressed in a variety of human tissues. Mol Cell
Endocrinol 120:5157[CrossRef][Medline]
-
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, OMalley BW,
McDonnell DP 1993 Human progesterone receptor A form is a cell- and
promoter-specific repressor of human progesterone receptor B function.
Mol Endocrinol 7:12441255[Abstract]
-
McDonnell D, Shahbaz MM, Vegeto E, Goldman ME 1994 The human
progesterone receptor A-form functions as a transcriptional modulator
of mineralocorticoid receptor transcriptional activity. J Steroid
Biochem Mol Biol 48:425432[CrossRef][Medline]
-
Meyer ME, Pornon A, Ji J, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonist and antagonist activities of RU 486 on the functions of
the human progesterone receptor. EMBO J 9:39233932[Abstract]
-
Tora L, Gronemeyer H, Turcotte B, Gaub MP, Chambon P 1988 The
N-terminal region of the chicken progesterone receptor specifies target
gene activation. Nature 333:185188[CrossRef][Medline]
-
Meyer ME, Quirin-Stricker C, Lerouge T, Bocquel MT, Gronemeyer
H 1992 A limiting factor mediates the differential activation of
promoters by the human progesterone receptor isoforms. J Biol Chem 267:1088210887[Abstract/Free Full Text]
-
Prendergast P, Pan Z, Edwards DP 1996 Progesterone
receptor-induced bending of its target DNA: distinct effects of the A
and B receptor forms. Mol Endocrinol 10:393407[Abstract]
-
Kozak M 1989 The scanning model for translation: an update.
J Cell Biol 108:229241[Abstract]
-
Kaufman M, Pinsky L, Feder-Hollander R 1981 Defective
up-regulation of the androgen receptor in human androgen insensitivity.
Nature 293:735737[Medline]
-
Krongrad A, Wilson CM, Wilson JD, Allman DR, McPhaul M 1991 Androgen increases androgen receptor protein while decreasing receptor
mRNA in LNCaP cells. Mol Cell Endocrinol 76:7988[CrossRef][Medline]
-
Carson-Jurica MA, Schrader WT, OMalley BW 1990 Steroid
receptor family: structure and functions. Endocr Rev 11:201220[Abstract]
-
Wahli W, Martinez E 1991 Superfamily of steroid nuclear
receptors: positive and negative regulators of gene expression. FASEB J 5:22432249[Abstract/Free Full Text]
-
Horwitz KB 1992 The molecular biology of RU486. Is there a
role for antiprogestins in the treatment of breast cancer? Endocr Rev 13:146163[Medline]
-
Sartorius CA Melville MY Hovland AR Tung L Takimoto GS Horwitz
KB 1994 A third transactivation function (AF3) of human progesterone
receptors located in the unique N-terminal segment of the B-isoform.
Mol Endocrinol 8:13471360[Abstract]
-
Fuhrmann U, Bengtson C, Repenthin G, Schillinger E 1992 Stable
transfection of androgen receptor and MMTV-CAT into mammalian cells:
inhibition of CAT expression by anti-androgens. J Steroid Biochem Mol
Biol 42:787793[CrossRef][Medline]
-
Kuil CW, Berrevoets CA, Mulder E 1995 Ligand-induced
conformational alterations of the androgen receptor analyzed by limited
trypsinization. J Biol Chem 270:2756927576[Abstract/Free Full Text]
-
Kemppainen JA Wilson EM 1996 Agonist and antagonist activities
of hydroxyflutamide and Casodex relate to androgen receptor
stabilization. Urology 48:157163[CrossRef][Medline]
-
Shull JD, Beams FE, Baldwin TM, Gilchrist CA, Hrbek MJ 1992 The estrogenic and anti-estrogenic properties of tamoxifen in
GH4 C1 pituitary tumor cells are gene specific.
Mol Endocrinol 6:529535[Abstract]
-
McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals
three distinct classes of antiestrogens. Mol Endocrinol 9:659669[Abstract]
-
Berry M, Metzger D, Chambon P 1990 Role of the two activating
domains of the oestrogen receptor in the cell-type and promoter-context
dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.
EMBO J 9:28112818[Abstract]
-
Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ,
OMalley BW 1992 Hormone and antihormone induce distinct
conformational change which are central to steroid receptor activation.
J Biol Chem 267:1951319520[Abstract/Free Full Text]
-
Schulman IG, Juguilon H, Evans RM 1996 Activation and
repression by nuclear hormone receptors: hormone modulates an
equilibrium between active and repressive states. Mol Cell Biol 16:38073813[Abstract]
-
Tzukerman M, Esty A, Santiso-Mere D, Danielian P, Parker MG,
Stein RB Pike JW, McDonnell DP 1994 Human estrogen receptor
transactivational capacity is determined by both cellular and promoter
context and mediated by two functionally distinct intramolecular
regions. Mol Endocrinol 8:2130[Abstract]
-
Smith CL, Nawaz Z, OMalley BW 1997 Coactivator and
corepressor regulation of the agonist/antagonist activity of the mixed
antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657666[Abstract/Free Full Text]
-
Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz
KB 1997 The partial agonist activity of antagonist-occupied steroid
receptors is controlled by a novel hinge domain-binding coactivator
L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693705[Abstract/Free Full Text]
-
Martin PM, Berthois Y, Jensen EV 1988 Binding of antiestrogens
exposes an occult antigenic determinant in the human estrogen receptor.
Proc Natl Acad Sci USA 85:25332537[Abstract]
-
Hedden A, Müller V, Jensen EV 1995 A new interpretation
of antiestrogen action. Ann NY Acad Sci 761:109120[Abstract]
-
Vegeto E, Allan GF, Schrader WT, Tsai MJ, McDonnell DP,
OMalley BW 1992 The mechanism of RU486 antagonism is dependent on the
conformation of the carboxy-terminal tail of the human progesterone
receptor. Cell 69:703713[Medline]
-
Neri R, Florance K, Koziol P, Van Cleave S 1972 A biological
profile of a nonsteroidal antiandrogen Sch 13521
(4'-nitro-trifluor-methy-lisobutyranillide). Endocrinology 91:427437[Medline]
-
Clark CR, Nowell NW 1980 The effect of the non-steroidal
antiandrogen flutamide on neural receptor binding of testosterone and
intermale aggressive behaviour of mice. Psychoneuroendocrinology 5:3945[CrossRef][Medline]
-
Peets AE, Henson MF, Neri R 1974 On the mechanism of the
anti-androgenic action of flutamide
(Trifluoro-2-methyl-4'-nitro-m-propionotoluidide) in the rat.
Endocrinology 94:532540[Medline]
-
Katchen B, Buxbaum S 1975 Disposition of a new, non-steroid,
antiandrogen (flutamide), in men following a single oral 200 mg dose.
J Clin Endocrinol Metab 41:373379[Abstract]
-
Simard J, Luthy I, Guay J, Belanger A, Labrie F 1986 Characteristics of interaction of the antiandrogen flutamide with the
androgen receptor in various target tissues. Mol Cell Endocrinol 44:261270[CrossRef][Medline]
-
Zhou ZX, Lane MV, Kemppainen JA, French FS Wilson EM 1995 Specificity of ligand-dependent androgen receptor stabilization:
receptor domain interactions influence ligand dissociation and receptor
stability. Mol Endocrinol 9:208218[Abstract]
-
Gao TS, Marcelli M, McPhaul MJ 1996 Transcription
transactivation and transient expression of the human androgen
receptor. J Steroid Biochem Mol Biol 59:920[CrossRef][Medline]