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Department of Cellular and Structural Biology (R.K.T., Y.L., S.C.A., C.S.S., B.C., A.K.R.) The University of Texas Health Science Center at San Antonio and Audie L. Murphy Memorial Veterans Affairs Hospital (B.C.) San Antonio, Texas 78284
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-dihydrotestosterone
(DHT). All AR ligands with transactivation potential, including the
mixed agonist/antagonist cyproterone acetate, caused translocation of
the GFP-AR into a subnuclear compartment indicated by its punctate
intranuclear distribution. However, translocation caused by casodex, a
pure antagonist, resulted in a homogeneous nuclear distribution.
Subsequent exposure of the casodex-treated cell to DHT rapidly (15–30
min) altered the homogeneous to punctate distribution of the already
translocated nuclear AR. When transported into the nucleus either by
casodex or by DHT, GFP-AR was resistant to 2 M
NaCl extraction, indicating that the homogeneously distributed AR is
also associated with the nuclear matrix. Taken together, these results
demonstrate that AR requires ligand activation for its nuclear
translocation where occupancy by only agonists and partial agonists can
direct it to a potentially functional subnuclear location and that one
receptor molecule can undertake multiple rounds of hormonal signaling;
this indicates that ligand dissociation/inactivation rather than
receptor degradation may play a critical role in terminating hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-dihydrotestosterone
(DHT), cells transfected with the chimeric (GFP-AR) expression vector
showed about one third transactivation function as compared with the
nonchimeric AR (relative CAT activities of 183 ± 19 for GFP-AR
vs. 588 ± 11 for AR). The partial loss of
transactivational activity of the GFP-AR may be due to the GFP
interference with the amino-terminal TAF-1 function of the AR protein.
However, transactivation function of both of these proteins was almost
totally dependent on the presence of the androgen (relative CAT
activity without DHT of less than 3).
We then examined the localization of the GFP-AR in three different cell
lines, i.e. prostate-derived PC3, uterus-derived HeLa, and
kidney-derived COS1 cells. In the absence of hormone, GFP-AR was found
to be mostly cytoplasmic in all three cell types (Fig. 1
, A–C). However, addition of DHT to the
culture medium resulted in a time-dependent translocation of the GFP-AR
into the nuclear compartment. Nuclear migration of the receptor was
rapid and clearly evident within 15 min after hormone treatment, and
the receptor became primarily or exclusively nuclear within 60 min. A
similar DHT-mediated nuclear translocation was also observed by
immunostaining of both the endogenous AR in AR-positive LNCaP cells and
COS1 cells transfected with the pCMV-AR expression vector (Fig. 2, A and B).
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show the kinetics of hormone-dependent
nuclear translocation of the GFP-AR and immunodetected wild-type AR in
transfected COS1 cells. In both cases, a linear increase in nuclear
import after DHT treatment was observed, and the import was complete
between 60 to 90 min.
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show a reversal of cytoplasmic to nuclear migration of the GFP-AR after
hormone withdrawal in the presence of 50 µg/ml cycloheximide. At
12 h after hormone withdrawal, no significant difference in the
intensity of fluorescence between nuclear and cytoplasmic compartments
can be distinguished. Since export of the receptor from the nuclear to
the cytoplasmic compartment only became evident between 6 to 12 h
after hormone withdrawal, it was of interest to compare the rate of
ligand dissociation with nuclear export. Data presented in Fig. 4B show
about 50% ligand dissociation within the same time frame when nuclear
export became clearly evident. A discordant relationship between ligand
dissociation and nuclear export of GR has been reported earlier (21).
We speculate that a dynamic process involving capture of the
dissociated ligand from one receptor molecule by another unoccupied
receptor provides a threshold effect of nuclear retention after hormone
withdrawal. The fact that the concentration of cycloheximide used in
these experiments (50 µg/ml) caused a total inhibition of
immunodetectable GFP-AR synthesis is demonstrated by results presented
in Fig. 4C. Even at 10 µg/ml, no immunodetectable GFP-AR can be seen
within 15 h of culture period while untreated cells produced a
high level of this protein. The GFP-AR exported from the nucleus
after 12 h of hormone withdrawal could again be transported into
the nuclear compartment after reexposure to its hormonal ligand (Fig. 5A). Quantitative analysis of this
reutilization process for up to four cycles of import and three cycles
of export by sequential hormone treatment and withdrawal (Fig. 5B)
showed a gradual decline of the recycling competency of the receptor
protein. We interpret this observation to indicate a time-dependent
structural damage (degradation) of the receptor protein. However, a
decreased level of importins or other proteins in nucleocytoplasmic
transport after inhibition of protein synthesis by cycloheximide may
also be a contributing factor. During the first two rounds of
recycling, the receptor was almost equally translocation competent.
Upon subsequent hormone treatment the same batch of cells at their
third and fourth import cycles showed a gradual decline of
ligand-dependent nuclear import. Thus, the same receptor may be able to
mediate multiple rounds of hormonal signaling, which suggests that
inactivation and/or dissociation of hormonal steroids, rather than
nuclear degradation of the receptor protein after its initial import,
may play a dominant role in the termination mechanism of hormone
action.
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B (NFB), has been described (30). Using p65-GFP as
our positive control, we examined the effect of LMB on the nuclear
export of the GFP-AR after androgen withdrawal. Although LMB failed to
prevent the export of GFP-AR from the nuclear to the cytoplasmic
compartment, the conditioned medium derived from the GFP-AR transfected
cells containing LMB was able to prevent the nuclear export of p65-GFP
with consequent accumulation of this protein in the nucleus (Fig. 6). The latter observation shows that LMB
added to the culture medium of cells transfected with GFP-AR is still
biologically active and that the nuclear export of the
ligand-dissociated AR may involve an as-yet-uncharacterized
exportin-independent transport mechanism.
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show
that both 17ß-estradiol and progesterone, especially at a higher
concentration (10-7 and
10-6 M), were effective in
translocating the GFP-AR into the nucleus. However, the gluococorticoid
dexamethasone and EGF were totally ineffective. Two antiandrogens,
cyproterone acetate (a mixed agonist/antagonist) (37) and casodex (a
pure antagonist) (38), also showed a relatively limited ability to
translocate GFP-AR into the nucleus. When these hormones and
antihormones were tested for their relative transactivation function
for the AR-dependent promoterreporter construct derived from the
rat probasin gene (i.e. ARR3-TK-Luc) (39), it showed that in
addition to DHT, 17ß-estradiol, progesterone, and cyproterone acetate
were transactivation competent (Fig. 7B). However, the relative
abilities of these steroids for nuclear import and transactivation
function were different. Next to DHT, estradiol showed the best
translocation function, while cyproterone acetate was more competent in
transactivation than estradiol. Among the translocation-competent
ligands that we have tested, casodex, a pure antagonist, was found to
be the least effective. Again, some cells were more sensitive to
casodex-mediated translocation than others and after a prolonged
exposure (>20 h) to 10-6
M casodex, about 70% of the cells showed
primarily nuclear fluorescence (data not shown).
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). Since proteasome-dependent nuclear
degradation of the estrogen receptor has been observed (40), we wanted
to rule out the possibility of proteasomic inclusion of GFP-AR as the
cause of the punctate distribution. Treatment of GFP-AR transfected
cells with the proteasome inhibitor MG132 (3 µM) before
DHT treatment did not alter the intranuclear punctate distribution of
the GFP-AR (data not presented). This observation indicates that the
formation of the punctate foci is an intermediate step in the signaling
rather than degradation process. That ligand-mediated nuclear
translocation and subnuclear compartmentalization represent two
distinct steps in the process of hormonal signaling was further
indicated by a rapid conversion of the nucleoplasmic GFP-AR of the
casodex-treated cell by subsequent treatment with DHT (Fig. 9A). In this case punctate appearance was
evident within 15 min, and formation of the punctate foci was complete
within 1 h after DHT treatment. Salt extraction of nuclear
proteins followed by immunoblot analysis showed that most of the GFP-AR
within the nuclei of either casodex-treated or DHT-treated cells is
present within the salt (2 M NaCl)-resistant nuclear matrix
fraction. Thus, the punctate foci may represent a distinct step in
steroid receptor function after its binding to the nuclear
matrix.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In all of the cell lines that we have examined, the unliganded GFP-AR
is primarily localized in the cytoplasmic compartment, and upon
androgen exposure, rapidly migrates into the nucleus. Translocation of
AR can be detected within a few minutes after hormone treatment and is
almost complete within 60 min. Translocation kinetics of the GFP-AR are
very similar to the pattern observed in immunostained COS1 cells
containing unmodified AR. We have also observed that both the
unliganded GFP-ER and GFP-peroxisome proliferator-activated receptor
(PPAR) are primarily nuclear in COS1 cells (data not shown). From
all of these results and the existing literature it is reasonable to
conclude that members of the steroid hormone receptor family can be
classified into two distinct groups, one that requires ligand binding
for nuclear translocation [e.g. glucocorticoid receptor
(GR) and AR] and another that is nuclear, even without ligand binding
[e.g. estrogen receptor (ER), progesterone receptor (PR),
and PPAR]. Structural features that differentiate these two groups
are presently unknown. Cytoplasmic proteins that are larger than 60 kDa
are transported into the nuclear compartment through a
chaperone-mediated transport process (44). In the case of unliganded GR
and AR, the NLS, which interacts with chaperone proteins (importins),
is likely to be masked by receptor-associated heat shock proteins and
immunophilins. This group of receptors may require ligand-mediated
conformational change to unmask the NLS site for its appropriate
interaction with importins, whereas in the case of ER and PPAR, the
NLS may be easily accessible to importins, even in the absence of their
respective ligands.
Our observation of the nucleocytoplasmic recycling of the GFP-AR in living cells has an important bearing on the termination mechanism of the signaling process. We have observed multiple cycles of the DHT-dependent import-export process of GFP-AR after inhibition of new protein synthesis in the presence of cycloheximide. These results suggest that termination of nuclear signaling may depend on dissociation of the hormonal ligands due to ligand withdrawal or ligand inactivation. However, the receptor protein, after its dissociation from the ligand and chromatin, may be reutilized for another round of the signaling process when the ligand is again made available. A recent report has suggested an alternative mechanism for the termination of hormonal signal by nuclear degradation of the specific receptor protein through ubiquitination followed by proteasomal degradation (40). However, the quantitative contribution of such a process of receptor degradation in the termination of hormonal signaling has not been established. Our results indicate that, upon ligand withdrawal, most of the GFP-AR is exported back into the cytoplasmic compartment and is still competent to undergo the ligand-dependent translocation process. Multiple rounds of hormonal signaling by recycled receptors and cross-reactive ligands under certain clinical conditions, such as recurrent forms of AR-positive prostate cancer, may contribute to the etiology of the disease. In a large number of these cases there are abnormally high levels of AR, due to either AR gene amplification or possibly to AR up-regulation after prolonged periods of androgen deprivation and antiandrogen therapy (45, 46). These recurrent cancer cells expressing high levels of AR may experience adequate androgenic response caused by multiple rounds of receptor recycling through intermittent exposure of the AR to cross-reactive partial agonists of nongonadal origin.
The role of exportin/CRM1 chaperone proteins in the nuclear-to-cytoplasmic export of a number of proteins has been well established (27, 47, 48, 49). This mechanism is exquisitely sensitive to inhibition by LMB. LMB sensitivity to nuclear export of PR and GR by the immunostaining method has been examined earlier. Nuclear export of PR is insensitive to LMB (50), and contradictory results have been reported for GR (51, 52). Our results are consistent with the conclusion that the nuclear export of AR is mediated through an LBM-insensitive exportin-independent process, the nature of which has yet to be established. We feel that the results presented in this report concerning the LMB insensitivity of the export process are especially convincing, because 1) our observations were made in the living cell, without any additional intervention; and 2) inhibition of the nuclear export of GFP-p65 by the LMB-containing conditioned medium derived from the GFP-AR-transfected cells.
We have observed a general correlation between the dose-dependent transactivational function of AR by two other natural steroid hormones (i.e. 17ß-estradiol and progesterone, that are capable of cross-reacting with AR), and their effectiveness for translocating the receptor into the nucleus. Both dexamethasone and EGF were unable either to cause translocation of GFP-AR into the nucleus or to transactivate the ARR3-thymidine kinase (TK)-Luc in the cell transfection assay. A number of studies have suggested the potential role of EGF in enhancing steroid receptor function (32, 33, 34, 35, 36). The results presented in this article indicate that such a modulating influence of EGF may not be due to a direct influence on either the nuclear translocation or transactivation function of the unliganded AR and may be dependent on initial androgenic activation (32).
It is also of interest to note that both a mixed agonist/antagonist (cyproterone acetate) and a pure antagonist (casodex) were moderately effective in mediating the nuclear translocation of GFP-AR as compared with DHT. However, the patterns of nuclear distribution of the translocated receptor in these two cases are distinctly different. Similar to other agonists, the cyproterone acetate treatment led to a punctate nuclear distribution of GFP-AR, and, in the case of casodex, the fluorescence was homogeneously distributed within the nuclear compartment. However, upon subsequent exposure to DHT, the receptor moved to a distinct subnuclear compartment. A similar difference in the distribution pattern of GR and ER after agonist and antagonist treatment has also been reported (41, 53, 54). Similar to ER (53), our results show that both homogeneous and punctate forms of GFP-AR are associated with the nuclear matrix. Thus, formation of the punctate foci appears to be a distinct step in the mechanism of steroid hormone action, and this step lies beyond the initial matrix association of the receptor protein. It is of interest to note that only ligands with agonist or partial agonist activity can carry the process up to this step and beyond to initiate transcriptional activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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was
constructed similarly by inserting the human ER cDNA into the
BamHI site of the pEGFP-C2 vector. For generating the
chimeric GFP construct of the p65 subunit of NFB, the human p65
cDNA fragment was excised from the pCMV-p65 expression vector (provided
by Dr. John Cidlowski, NIEHS, Research Triangle Park, NC) and
inserted into the BamHI site of the GFP expression plasmid,
pEGFP-N3. Nucleotide sequences of all DNA constructs were
authenticated by manual sequencing. Construction of the expression
vectors pCMV-AR and ARR3-TK-Luc has been described previously (39). The
promoter-reporter construct pMMTV-CAT was a gift from Dr. Stephen
Harris (The University of Texas Health Science Center, San Antonio,
TX).
Cell Culture, Transfection, Cell Fractionation, and Western Blot
Analysis
PC3, HeLa, LNCaP, and COS1 cells were obtained from
American Type Culture Collection (Manassas, VA) and
cultured in serum-containing media as recommended by the supplier.
Cells were plated in six-well culture flasks at 150 x
103 cells per well in the growth medium (MEM
supplemented with 5% charcoal-stripped FBS), grown overnight, and
cotransfected with either pARR3-TK-Luc or pMMTV-CAT promoter-reporter
(1 µg) along with either wild-type steroid receptor-expression
plasmid (pCMV-rAR) or the chimeric GFP plasmids (pCMV-GFP-rAR,
pCMV-GFP-ER), using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) reagent, 6 µl/well, or
LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD),
8 µl/ml.
Media were changed at 24 h after transfection and were replaced with fresh media with or without the hormonal ligands. Cells were incubated for an additional 24 h before harvesting and extraction of proteins. Cell extracts were assayed for protein concentration (Bradford protocol), luciferase activity (assay kit, Promega Corp., Madison, WI), and CAT by enzyme-linked immunoassay (CAT ELISA kit, Roche Molecular Biochemicals). For CAT activity results were expressed as optical density (x 103) per µg protein.
For experiments involving imaging of GFP fluorescence, after transfection, cells were allowed to express the appropriate chimeric protein for 30 h before any hormonal treatment. For import/export experiments after hormone treatment and withdrawal, hormone-treated cells were rinsed at 0, 30, 60, and 120 min (twice at each time point with 3 ml of the culture medium containing the charcoal-stripped serum) before the next round of the translocation process was initiated. To prevent de novo protein synthesis, the replacement medium was supplemented with cycloheximide (50 µg/ml). Cycloheximide-mediated inhibition of GFP-AR synthesis in transfected cells was monitored by Western blot analysis with monoclonal antibody to GFP (primary antibody) and horseradish peroxidase-conjugated antimouse IgG. Peroxidase signal was visualized with ECL plus reagent according to the manufacturer’s recommendation (Amersham Pharmacia Biotech, Arlington, IL). Leptomycin B (a gift from Dr. M. Yoshida, University of Tokyo, Tokyo, Japan) was used at a final concentration of 15 ng/ml.
Isolation of cell nuclei and nuclear fractionation were performed according to the modified procedure described by Htun et al. (53). Briefly, freshly harvested cells were suspended in TNM buffer (10 mM Tris, pH 8.0, 300 mM sucrose, 100 mM NaCl, 2 mM MgCl2, 1% dithioglycol, and 1 mM phenylmethylsulfonyl fluoride) and after homogenization cells were treated with 0.5% Triton X-100. Isolated nuclei were washed in TNM buffer and resuspended in DIG buffer (TNM buffer with 50 mM NaCl and 3 mM MgCl2) and digested with 168 µ/ml DNase I. Digested nuclei were first fractionated with 0.25 M ammonium sulfate (fraction 1), and the pellet was subjected to two sequential extractions with 2 M NaCl (combined supernatants were used as fraction 2), and the remaining pellet containing the nuclear matrix was used as the fraction 3. After freeze-drying, all three fractions were dissolved in 100 µl Laemli’s SDS buffer for Western blot analysis according to the procedure described above.
Fluorescence Imaging
Fluorescence imaging of live cells was performed through a E400
Eclipse epifluorescence microscope and water immersion objectives
(Nikon, Melville, NY) connected to a video monitor through
a cooled charge-coupled device camera (Princeton Instruments, Princeton, NJ; Roper Scientific Inc., Trenton, NJ).
The microscope contains a temperaturecontrolled stage, a stepper
motor for optical sectioning, and an automatic filter wheel for
appropriate filter selection. Optical sectioning was performed at
1-µm steps and images were reconstructed through Metamorph
software (Universal Imaging Corp., West Chester, PA).
For immunodetection, cells were cultured in two chambered glass slides and fixed with 100% methanol at -20 C for 10 min. Fixed cells were rinsed twice with PBS (10 min each wash) and air dried. Before antibody treatment, slides were incubated in a humid chamber for 30 min, followed by overnight incubation (at 4 C) with polyclonal anti-AR antibody (affinity-purified IgG) produced in the rabbit. The antibody was generated by conjugating the first 20 N-terminal amino acids of AR to keyhole hemocyanin. After removal of the anti-AR antibody by rinsing three times with PBS, cells were treated for 1 h with sheep antirabbit IgG antibody conjugated with the fluorescent dye CY3. This step was followed by rinsing and mounting with coverslips and observation under the fluorescence microscope.
Dissociation Rate of AR
Binding of 3H-DHT and its rate of
dissociation were assayed according to Zhou et al. (55).
Briefly, COS1 cells transfected with GFP-AR expression plasmid for
48 h were subsequently placed in a serum-free medium containing 5
nM 3H-DHT in the presence
or absence of a 100-fold molar excess of unlabeled DHT and incubated
for 2 additional hours. Cells were then washed twice in PBS and
incubated further in the serum-free medium containing 50
µM unlabeled DHT for various time periods. Cell
samples collected at different time intervals were washed twice with
PBS, dissolved in Tris-SDS-glycerol buffer, and counted for
radioactivity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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B.C. is a career scientist with the Department of Veterans Affairs. S.C.A. was partially supported by a fellowship from the Korean Science and Engineering Foundation. This work was supported by NIH Grants DK-14744 and AG-10486.
Received for publication December 31, 1999. Revision received April 4, 2000. Accepted for publication April 24, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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,
steroid receptor coactivator-1. Mol Endocrinol 14:518–534This article has been cited by other articles:
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 H.-J. Kim, Y. I. Park, and M.-S. Dong Effects of 2,4-D and DCP on the DHT-Induced Androgenic Action in Human Prostate Cancer Cells Toxicol. Sci., November 1, 2005; 88(1): 52 - 59. [Abstract] [Full Text] [PDF]
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 R. Ilagan, L. J. Zhang, J. Pottratz, K. Le, S. Salas, M. Iyer, L. Wu, S. S. Gambhir, and M. Carey Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model Mol. Cancer Ther., November 1, 2005; 4(11): 1662 - 1669. [Abstract] [Full Text] [PDF]
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P. Farla, R. Hersmus, J. Trapman, and A. B. Houtsmuller Antiandrogens prevent stable DNA-binding of the androgen receptor J. Cell Sci., September 15, 2005; 118(18): 4187 - 4198. [Abstract] [Full Text] [PDF]
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I. G. Mills, L. Gaughan, C. Robson, T. Ross, S. McCracken, J. Kelly, and D. E. Neal Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors J. Cell Biol., July 18, 2005; 170(2): 191 - 200. [Abstract] [Full Text] [PDF]
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M. J. M. Schaaf, L. J. Lewis-Tuffin, and J. A. Cidlowski Ligand-Selective Targeting of the Glucocorticoid Receptor to Nuclear Subdomains Is Associated with Decreased Receptor Mobility Mol. Endocrinol., June 1, 2005; 19(6): 1501 - 1515. [Abstract] [Full Text] [PDF]
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J. N. Feige, L. Gelman, C. Tudor, Y. Engelborghs, W. Wahli, and B. Desvergne Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand J. Biol. Chem., May 6, 2005; 280(18): 17880 - 17890. [Abstract] [Full Text] [PDF]
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R. F. Walther, E. Atlas, A. Carrigan, Y. Rouleau, A. Edgecombe, L. Visentin, C. Lamprecht, G. C. Addicks, R. J. G. Hache, and Y. A. Lefebvre A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus J. Biol. Chem., April 29, 2005; 280(17): 17549 - 17561. [Abstract] [Full Text] [PDF]
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 L. Gaughan, I. R. Logan, D. E. Neal, and C. N. Robson Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation Nucleic Acids Res., January 7, 2005; 33(1): 13 - 26. [Abstract] [Full Text] [PDF]
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X.-F. Lin, B.-X. Zhao, H.-Z. Chen, X.-F. Ye, C.-Y. Yang, H.-Y. Zhou, M.-Q. Zhang, S.-C. Lin, and Q. Wu RXR{alpha} acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells J. Cell Sci., November 1, 2004; 117(23): 5609 - 5621. [Abstract] [Full Text] [PDF]
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 H. C. Whitaker, S. Hanrahan, N. Totty, S. C. Gamble, J. Waxman, A. C. B. Cato, H. C. Hurst, and C. L. Bevan Androgen Receptor Is Targeted to Distinct Subcellular Compartments in Response to Different Therapeutic Antiandrogens Clin. Cancer Res., November 1, 2004; 10(21): 7392 - 7401. [Abstract] [Full Text] [PDF]
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H.-K. Lin, Y.-C. Hu, D. K. Lee, and C. Chang Regulation of Androgen Receptor Signaling by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) Tumor Suppressor through Distinct Mechanisms in Prostate Cancer Cells Mol. Endocrinol., October 1, 2004; 18(10): 2409 - 2423. [Abstract] [Full Text] [PDF]
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S. Dong, D. L. Stenoien, J. Qiu, M. A. Mancini, and D. J. Tweardy Reduced Intranuclear Mobility of APL Fusion Proteins Accompanies Their Mislocalization and Results in Sequestration and Decreased Mobility of Retinoid X Receptor {alpha} Mol. Cell. Biol., May 15, 2004; 24(10): 4465 - 4475. [Abstract] [Full Text] [PDF]
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B. E. Black, M. J. Vitto, D. Gioeli, A. Spencer, N. Afshar, M. R. Conaway, M. J. Weber, and B. M. Paschal Transient, Ligand-Dependent Arrest of the Androgen Receptor in Subnuclear Foci Alters Phosphorylation and Coactivator Interactions Mol. Endocrinol., April 1, 2004; 18(4): 834 - 850. [Abstract] [Full Text] [PDF]
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 R. L. Arnett-Mansfield, A. deFazio, P. A. Mote, and C. L. Clarke Subnuclear Distribution of Progesterone Receptors A and B in Normal and Malignant Endometrium J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1429 - 1442. [Abstract] [Full Text] [PDF]
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I. Ochiai, K.-i. Matsuda, M. Nishi, H. Ozawa, and M. Kawata Imaging Analysis of Subcellular Correlation of Androgen Receptor and Estrogen Receptor {alpha} in Single Living Cells Using Green Fluorescent Protein Color Variants Mol. Endocrinol., January 1, 2004; 18(1): 26 - 42. [Abstract] [Full Text] [PDF]
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 W. J. Lee, R. W. Thompson, J. M. McClung, and J. A. Carson Regulation of androgen receptor expression at the onset of functional overload in rat plantaris muscle Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2003; 285(5): R1076 - R1085. [Abstract] [Full Text] [PDF]
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A. J. Saporita, Q. Zhang, N. Navai, Z. Dincer, J. Hahn, X. Cai, and Z. Wang Identification and Characterization of a Ligand-regulated Nuclear Export Signal in Androgen Receptor J. Biol. Chem., October 24, 2003; 278(43): 41998 - 42005. [Abstract] [Full Text] [PDF]
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D. K. Lee and C. Chang Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4043 - 4054. [Abstract] [Full Text] [PDF]
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L. Zhang, M. Johnson, K. H. Le, M. Sato, R. Ilagan, M. Iyer, S. S. Gambhir, L. Wu, and M. Carey Interrogating Androgen Receptor Function in Recurrent Prostate Cancer Cancer Res., August 1, 2003; 63(15): 4552 - 4560. [Abstract] [Full Text] [PDF]
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 L. L. DonCarlos, D. Garcia-Ovejero, S. Sarkey, L. M. Garcia-Segura, and I. Azcoitia Androgen Receptor Immunoreactivity in Forebrain Axons and Dendrites in the Rat Endocrinology, August 1, 2003; 144(8): 3632 - 3638. [Abstract] [Full Text] [PDF]
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H. T. Le, C. M. Schaldach, G. L. Firestone, and L. F. Bjeldanes Plant-derived 3,3'-Diindolylmethane Is a Strong Androgen Antagonist in Human Prostate Cancer Cells J. Biol. Chem., May 30, 2003; 278(23): 21136 - 21145. [Abstract] [Full Text] [PDF]
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M. J. M. Schaaf and J. A. Cidlowski Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity Mol. Cell. Biol., March 15, 2003; 23(6): 1922 - 1934. [Abstract] [Full Text] [PDF]
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 L. Jia, J. Kim, H. Shen, P. E. Clark, W. D. Tilley, and G. A. Coetzee Androgen Receptor Activity at the Prostate Specific Antigen Locus: Steroidal and Non-Steroidal Mechanisms Mol. Cancer Res., March 1, 2003; 1(5): 385 - 392. [Abstract] [Full Text] [PDF]
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G. Liao, L.-Y. Chen, A. Zhang, A. Godavarthy, F. Xia, J. C. Ghosh, H. Li, and J. D. Chen Regulation of Androgen Receptor Activity by the Nuclear Receptor Corepressor SMRT J. Biol. Chem., February 7, 2003; 278(7): 5052 - 5061. [Abstract] [Full Text] [PDF]
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Y. Okada, Y. Fujii, J. P. Moore Jr., and S. J. Winters Androgen Receptors in Gonadotrophs in Pituitary Cultures from Adult Male Monkeys and Rats Endocrinology, January 1, 2003; 144(1): 267 - 273. [Abstract] [Full Text] [PDF]
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O. J. Rivera, C. S. Song, V. E. Centonze, J. D. Lechleiter, B. Chatterjee, and A. K. Roy Role of the Promyelocytic Leukemia Body in the Dynamic Interaction between the Androgen Receptor and Steroid Receptor Coactivator-1 in Living Cells Mol. Endocrinol., January 1, 2003; 17(1): 128 - 140. [Abstract] [Full Text] [PDF]
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K.-i. Matsuda, I. Ochiai, M. Nishi, and M. Kawata Colocalization and Ligand-Dependent Discrete Distribution of the Estrogen Receptor (ER){alpha} and ER{beta} Mol. Endocrinol., October 1, 2002; 16(10): 2215 - 2230. [Abstract] [Full Text] [PDF]
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D. Gioeli, S. B. Ficarro, J. J. Kwiek, D. Aaronson, M. Hancock, A. D. Catling, F. M. White, R. E. Christian, R. E. Settlage, J. Shabanowitz, et al. Androgen Receptor Phosphorylation. REGULATION AND IDENTIFICATION OF THE PHOSPHORYLATION SITES J. Biol. Chem., August 2, 2002; 277(32): 29304 - 29314. [Abstract] [Full Text] [PDF]
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L. Gaughan, I. R. Logan, S. Cook, D. E. Neal, and C. N. Robson Tip60 and Histone Deacetylase 1 Regulate Androgen Receptor Activity through Changes to the Acetylation Status of the Receptor J. Biol. Chem., July 12, 2002; 277(29): 25904 - 25913. [Abstract] [Full Text] [PDF]
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D. B. DeFranco Navigating Steroid Hormone Receptors through the Nuclear Compartment Mol. Endocrinol., July 1, 2002; 16(7): 1449 - 1455. [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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D. J. Mulholland, H. Cheng, K. Reid, P. S. Rennie, and C. C. Nelson The Androgen Receptor Can Promote beta -Catenin Nuclear Translocation Independently of Adenomatous Polyposis Coli J. Biol. Chem., May 10, 2002; 277(20): 17933 - 17943. [Abstract] [Full Text] [PDF]
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M. Saitoh, R. Takayanagi, K. Goto, A. Fukamizu, A. Tomura, T. Yanase, and H. Nawata The Presence of Both the Amino- and Carboxyl-Terminal Domains in the AR Is Essential for the Completion of a Transcriptionally Active Form with Coactivators and Intranuclear Compartmentalization Common to the Steroid Hormone Receptors: A Three-Dimensional Imaging Study Mol. Endocrinol., April 1, 2002; 16(4): 694 - 706. [Abstract] [Full Text] [PDF]
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T. E. Akiyama, C. T. Baumann, S. Sakai, G. L. Hager, and F. J. Gonzalez Selective Intranuclear Redistribution of PPAR Isoforms by RXR{alpha} Mol. Endocrinol., April 1, 2002; 16(4): 707 - 721. [Abstract] [Full Text] [PDF]
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E. Holter, N. Kotaja, S. Makela, L. Strauss, S. Kietz, O. A. Janne, J.-A. Gustafsson, J. J. Palvimo, and E. Treuter Inhibition of Androgen Receptor (AR) Function by the Reproductive Orphan Nuclear Receptor DAX-1 Mol. Endocrinol., March 1, 2002; 16(3): 515 - 528. [Abstract] [Full Text] [PDF]
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M. Nishi, H. Ogawa, T. Ito, K.-I. Matsuda, and M. Kawata Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants Mol. Endocrinol., July 1, 2001; 15(7): 1077 - 1092. [Abstract] [Full Text] [PDF]
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C. F. Bunn, J. A. Neidig, K. E. Freidinger, T. A. Stankiewicz, B. S. Weaver, J. McGrew, and L. A. Allison Nucleocytoplasmic Shuttling of the Thyroid Hormone Receptor {{alpha}} Mol. Endocrinol., April 1, 2001; 15(4): 512 - 533. [Abstract] [Full Text]
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Y. Wan, K. K. Coxe, V. G. Thackray, P. R. Housley, and S. K. Nordeen Separable Features of the Ligand-Binding Domain Determine the Differential Subcellular Localization and Ligand-Binding Specificity of Glucocorticoid Receptor and Progesterone Receptor Mol. Endocrinol., January 1, 2001; 15(1): 17 - 31. [Abstract] [Full Text]
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A. Tomura, K. Goto, H. Morinaga, M. Nomura, T. Okabe, T. Yanase, R. Takayanagi, and H. Nawata The Subnuclear Three-dimensional Image Analysis of Androgen Receptor Fused to Green Fluorescence Protein J. Biol. Chem., July 20, 2001; 276(30): 28395 - 28401. [Abstract] [Full Text] [PDF]
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DHT). Monoclonal mouse
antibody to GFP was used to detect the GFP-AR. Three lanes for each of
the treatment condition represent as follows: F1, supernatant after
extraction with 0.25 M ammonium sulfate; F2,
supernatant after extraction with 2 M NaCl; F3, pellet
containing nuclear matrix.