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Laboratory of Receptor Biology and Gene Expression National Cancer Institute Bethesda, Maryland 20892-5055
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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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). PRA and PRB exhibit cell- and
promoter-specific differences in transcriptional activity. In cases
where it is inactive, PRA may act as a strong trans-dominant
repressor of PRB-mediated transcription (1). Recent studies have
suggested a possible clinical significance of the ratios of A:B forms
in breast cancer. Specifically, a very low level of PRB (and hence a
high PRA:PRB ratio) was found in a significant proportion of PR-
positive breast tumors (2). The presence of PR in breast tumors has
been linked to a better prognosis due to improved responsiveness to
endocrine therapy. In light of the fact that the A form may act as a
strong trans-dominant repressor of PRB-mediated
transcription, an altered A:B ratio may contribute to the poor response
of some patients to hormone therapy.
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To date, no detailed studies on the possible differences in localization of the A and B forms of the PR have been performed, due to the lack of anti-PRA antibody used for immunolocalization studies. The real-time localization of functional steroid receptors is now possible. Tagging receptors with the green fluorescent protein (GFP) allows for direct visualization of these receptors in living cells (3). GFP is a 238-amino acid naturally fluorescent chromophore that was first isolated from the jellyfish Aequorea victoria. It is the brightest naturally occurring fluorescent chromophore reported and has been shown to be a useful tag with which to study interactions between receptors and chromosomal DNA during transcription (3).
We report here the development and characterization of GFP chimeras for both forms of the PR. The GFP-PRA and GFP-PRB fusion proteins are efficiently expressed, and their transactivation properties and ligand response are not significantly altered by the presence of the GFP tag. Using these chimeras, we have investigated the localization of PRA and PRB for the first time in living cells. We find that the two forms of the receptors have distinct subcellular distributions in the absence of ligand. The A form of the receptor is located primarily in the nucleus of untreated cells, whereas a significant fraction of the B form is present in the cytoplasm. Both forms give complete nuclear translocation when activated with ligand. These findings are unexpected, since the two forms of the PR have identical sequences in the regions of the molecule that contain the nuclear translocation signals and heat shock protein interaction domains. We conclude that nuclear/cytoplasmic shuttling for the PR is significantly impacted by the presence of the N-terminal extension present in the B form of the receptor. This differential in cellular distribution may in turn contribute to altered biological activities for the two forms of the receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) were constructed using
pCI-nGFP-C656G (3) and phPRB (1, 6). Activation studies with
agonist/antagonist were then performed using pGFP-PRA, phPRA, pGFP-PRB,
and phPRB vectors.
Upon induction with agonist, PRA and PRB are known to activate the MMTV
long terminal repeat (LTR) promoter. The plasmid pLTRluc, containing
the luciferase gene under MMTV control, was used as a reporter gene in
these experiments. The MMTV LTR contains multiple PR-binding sites, as
well as sites for other transcription factors that are recruited to the
promoter by receptor activation. PRA has been shown previously to have
a modest effect on MMTV transcription, whereas PRB is a potent
activator of this promoter. Typical hormone-dependent activation
(induction) of the MMTV promoter is 1- to 15-fold (over noninduced,
basal levels of luciferase activity) by PRA, and 16- to 80-fold by PRB,
depending on the cell line used (1, 7). As shown in Fig. 2, like PRB alone, GFP-PRB also activates
pLTRluc in an agonist-dependent manner in 1471.1 cells (using R5020, a
synthetic progestin). At maximal dose, a 30- to 40-fold induction is
seen for both PRB and GFP-PRB. Neither PRA nor GFP-PRA activates
pLTRluc to the same extent as the B form. In these studies, 0.5 µg
GFP-PR or PR plasmids was transfected; transfection of greater than 5
µg of GFP-PR plasmid leads to squelching of activity (data not
shown). Clearly, despite the presence of the GFP group, the
transcriptional activation potential of GFP-PR chimeras is still
maintained.
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, panels A and B, induced, with panels C
and D, uninduced).
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, which shows that the
progesterone-dependent induction of pLTRluc by GFP-PRB and PRB is
antagonized by RU486. Studies with the A form of the receptor are not
shown here because the A form is transcriptionally inactive in this
system. With 10 nM agonist (R5020), fold induction is
18-fold for PRB and 24-fold for GFP-PRB, for this experiment.
Antagonist alone (100 nM RU486) has no effect on induction
for either PRB or GFP-PRB. However, when agonist (10 nM)
and antagonist (100 nM) are added simultaneously,
agonist-dependent induction is reduced to baseline. These results show
that the GFP-PR chimeras are fully functional, responding to antagonist
in the same manner as untagged PR.
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indicates that in 1471.1 cells, the antagonist RU486 (30
nM) causes import of both GFP-PRA and B chimeras into the
nucleus, again excluding the nucleoli. These results are consistent
with previous data from our laboratory showing agonist (dexamethasone)
or antagonist (RU486)-induced nuclear localization of a GFP-tagged
glucocorticoid receptor (GR) (3). Agonist or antagonist RU486 would be
expected to induce nuclear translocation of PR or GR (3, 8). The
antagonist RU486 is thought to act as an antagonist at steps after
nuclear localization (9).
Unliganded GFP-PRA and GFP-PRB Are Differentially Localized in
Living Cells
Transfection of GFP-PRA and B in mammalian cells (1471.1 cells)
results in distinct subcellular localization patterns not previously
described. Using fluorescence microscopy, we observe that without
hormone, GFP-PRA localizes primarily in the nucleus while GFP-PRB
distributes between both the nucleus and the cytoplasm (Figs. 5 and 6).
The histogram in Fig. 5A shows the distribution of GFP-PRA
or GFP-PRB transfected in 1471.1 cells, 24 h after electroporation
(no hormone added). Figure 6 describes the histogram categories in Fig. 5A (e.g. an average nuclear intensity, or ANI, of 35% in
Fig. 5A corresponds to ANI 30–40% in Fig. 6A). For
GFP-PRA-transfected cells, the mean value of the ANI is 82.2%
(SD 12.5) compared with 56% (SD 10.9) for
GFP-PRB transfected cells (Table 1);
these values are statistically different (P < 0.01,
unpaired Student’s t test). In broader terms, approximately
88% of cells transfected with GFP-PRA had an ANI of greater than 70%
nuclear compared with only 7% of cells transfected with GFP-PRB (Fig. 5A). Furthermore, no GFP-PRA-transfected cells displayed an ANI of less
than 50% compared with GFP-PRB-transfected cells, where none displayed
an ANI of less than 31%. Figure 6 shows the distribution of
localization of GFP-PRA or GFP-PRB in individual cells. There is a
clear difference in subcellular localization for uninduced cells.
GFP-PRA is predominantly located in the nucleus, whereas GFP-PRB
partitions between the cytoplasmic and nuclear compartments.
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, the unsubstituted
receptor manifests a pattern of localization similar to that of
GFP-PRB. These findings suggest that the presence of the GFP tag has
little effect on the localization of PRB.
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GFP-PRB Is Expressed at Similar Levels Compared with PRB; No PRA Is
Synthesized from Either the GFP-PRB or PRB Vectors
Since the GFP-PR and PR expression vectors are different, it was
of importance to determine the expression levels of these vectors via
Western blotting. Whole-cell extracts prepared from affinity-sorted
cells transfected with GFP-PRB or PRB constructs were used for Western
blotting. A T47D cell extract, containing high amounts of PRA and B,
was used as a positive control (Fig. 8, lane 1); an extract from 1471.1 cells without GFP-PR or PR constructs
transfected was used as a negative control (Fig. 8, lane 4). PR
antibodies recognize both GFP and non-GFP forms of the PRB; GFP-PRB and
PRB were expressed at similar levels (Fig. 8, lanes 2 and 3; the
GFP-tagged PRB runs slower due to the presence of the 30-kDa GFP tag).
Additionally, neither GFP-PRB nor PRB vectors express detectable
amounts of PRA, despite the presence of the PRA translation start site
in the PRB (but not the GFP-PRB) vector. The PR antibody used in these
studies was a mixture of several antibodies that recognize either PRB
alone, or both PRA and PRB. It is apparent that despite the difference
in parental vectors, GFP-PRB and PRB are expressed at similar
levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have examined the subcellular distribution of the two forms of the human PR in living cells using GFP-tagged versions of the two receptors. Previous localization studies on the PR have focused only on PRB or a mixture of the two forms. The localization of unliganded PR was originally thought to be cytoplasmic (12), but later studies suggested the receptor was primarily in the nucleus (13).
This study is the first to characterize the distribution of PR in living cells and to compare directly localization of the PRA and PRB forms of the receptor. We show that the two forms are differentially distributed between the nuclear and cytoplasmic compartments in the unliganded state. This finding is surprising, given that the receptors appear to have indistinguishable nuclear translocation signals (5), as well as identical known chaperone interaction domains.
What features of the PRA and PRB receptor forms cause the proteins to
adopt altered distribution profiles? Differences in localization are
unlikely to result from size differences between the two forms of the
receptor. Both the 94-kDa A form and the 120-kDa B form are well above
the exclusion size of approximately 45 kDa for free diffusion across
the nuclear pore complex (separating the nucleus from the cytoplasm)
(14). Also, both forms of the receptor contain the same known nuclear
localization signals, including a constitutive nuclear localization
signal (NLS) in the junction between the DNA-binding domain (DBD) and
the hinge regions (Fig. 1), and a hormone-dependent NLS found in the
DBD (5). Finally, hormone-independent differential localization of PRA
and PRB is unlikely to be mediated by heterodimerization. Addition of
an excess of PRA has no effect on the localization of GFP-PRB, and
excess PRB has no effect on the localization of GFP-PRA (data not
shown).
We suggest several mechanisms for the differential subcellular distribution observed. First, since the PRB contains an extra 164-amino acid N-terminal domain, these extra amino acids may interact with a factor(s) that allows PRB to shuttle more actively between the nucleus and the cytoplasm. A variety of PR-associated proteins have been characterized (hsp90, hsp70, p23, p60) (15); some of these proteins (or others not yet identified) may be responsible for the differential PRA/PRB localization. This protein could possibly be a cytoplasmic retention factor (14) that keeps PRB from migrating back to the nucleus. Second, the extra amino acids in the PRB form may cause the PRB to adopt a tertiary conformation that leads to a less effective NLS. Guiochon-Mantel et al. (5) have suggested than a less effective NLS would lead to an increased residency time of the PRB in the cytoplasm, which, in turn, would lead to an apparent distribution between the nucleus and the cytoplasm. Alternatively, a difference in overall conformation of the A form of the receptor may allow for a more effective NLS. Additionally, there may be a yet-undiscovered nuclear export signal in the N-terminal region of the PR. If there is such an export signal, altered kinetics between import into and export from the nucleus could explain the differential localization. A recent report by Guiochon-Mantel and co-workers (25) suggests that a nuclear export signal is not involved . Finally, the amino-terminal end of PRA may induce a conformation of the A receptor form that causes a specific interaction with some factor(s) in the nucleus, and thus leads to an increased PRA nuclear distribution.
The findings presented here represent the first documentation for a differential localization of the A and B forms of PR. The majority of cells transfected with GFP-PRB in our studies are still mostly nuclear (79%) with a smaller fraction being more cytoplasmic (21%). However, all of the GFP-PRA transfected cells are at least 50% nuclear, with the majority being more than 70% nuclear. While these findings are not in direct contradiction to previous studies (8), we demonstrate here a differential localization of PRA and PRB. Factors that could account for differences between our results and those of Guiochon-Mantel et al. (1989) on the localization of exogenously added PR include cell type (1471.1 cells in our study vs. Cos-7 cells in their study), the amount of DNA used for transfection (0.5 µg vs. 30 µg), the transfection method (electroporation vs. calcium phosphate), promoter differences (CMV vs. SV40), method of determining localization (GFP fluorescence in living cells vs. immunolocalization), live or fixed cells, and the type of PR used (human vs. rabbit).
Differences between the findings presented here and previous work are not likely due to cell type, since a similar differential pattern of localization was seen for PRA and PRB in three other cell lines, including Cos-1 (data not shown). The amount of DNA transfected could have an impact if a nuclear export system that transports PR out of the nucleus were in place, as suggested by Guiochon-Mantel et al. 1991 (5). An excess of PR could potentially saturate nuclear export systems and hence cause the receptor to appear uniquely nuclear. Fixation differences probably do not account for differences between our studies and theirs because paraformaldehyde fixation of GFP-PR-transfected cells yielded the same results as live, nonfixed cells (data not shown). In addition, we show that indirect immunofluorescence of unsubstituted PRB results in a similar pattern of distribution compared with GFP-PRB. However, it should be noted that the fixation methods/immunolocalization methods previously (8) involved many steps including several temperature changes and long incubations. The utilization of different receptor types (human vs. rabbit) could contribute to alternate findings, especially if nuclear export signals are present in the N-terminal portion. Although the human and rabbit PR are 87% identical, the majority of the differences in amino acid sequence lie in the first 164 amino acids (44 of 164 amino acids are different).
The TR was previously thought to be found in the nucleus in the absence of ligand. However, a recent study using GFP-TRß1 (ß1 subtype) showed that TRß1 was partially cytoplasmic, with a nuclear to cytoplasmic ratio of 1.5 with no hormone added. When hormone was added, the nuclear to cytoplasmic ratio increased to 5.5 (4). These results for the TR are similar to ours for the PR.
We should note that the results presented here were obtained with transiently introduced PR forms. Most of the literature on steroid receptor localization and function has been carried out with transiently transfected molecules. We have discussed elsewhere (22) that the properties of transiently introduced receptors can differ substantially from those of their endogenous counterparts. It will be important to extend the findings presented here to a comparable analysis of molecules stably expressed in replicating cells.
Intranuclear targets for the nuclear receptor family are poorly understood. It is often assumed that nuclear accumulation is equivalent to chromatin/hormone response element targeting. However, results from a number of recent investigations suggest a more complex set of intranuclear interactions (3, 16, 17). New experimental approaches will be required to elucidate the details of intracellular and intranuclear trafficking by members of the receptor superfamily. It is clear from our findings that GFP-PRA and GFP-PRB chimeras will serve as useful tools for future studies on cellular distribution, colocalization with other factors, and potential direct interactions between receptors and nuclear structures. These approaches will contribute to elucidating the differential role of PRA, PRB, and their associated factors in transcriptional activation/ repression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The plasmids pGFP-PRB and pGFP-PRA (GFP linked to the amino terminus of
PR, Fig. 1) were constructed from pCI-nGFP-C656G (3) and phPRB. To
create the pGFP-PRB plasmid, site-directed mutagenesis (Chameleon
double-stranded site-directed mutagenesis kit, Stratagene, La Jolla,
CA) of phPRB using the mutagenesis primer
5'-CCTTCAGCTCAGTCAACGCGTCTGGACTCCCCTTTT-3'was used to
replace the first ATG (start) site with a MluI site to
create phPRBmut5. The plasmid phPRBmut5 was then digested to completion
with KpnI. A resulting 4-kb fragment was then partially
digested with MluI. A purified 2.8-kb fragment was ligated
to a phosphorylated hairpin linker and then cut with NotI.
The resulting fragment was ligated into pCI-nGFP-C656G, which had been
cut with NotI and BssH II (compatible with
MluI) to remove the glucocorticoid receptor cDNA.
To create the pGFP-PRA plasmid, site directed mutagenesis of phPRB
replaced the second ATG site with a MluI site to create
phPRBmut1 using mutagenesis primer:
5'-ACCCGGACCGGCTCAACGCGTGGGACAACACCCGCT-3'. The
plasmid phPRBmut1 was digested to completion with MluI, and
the resulting 0.6-kb fragment was dephosphorylated. A 1.7-kb fragment
was obtained from digesting pGFP-PRB with MluI and
NotI. The plasmid pCI-nGFP-C656G was cut with
NotI and BssHII (compatible with
MluI), and the resulting 4.8-kb fragment was ligated to the
0.6-kb fragment above and the 1.7-kb fragment to yield pGFP-PRA (Fig. 1). Both plasmids were sequenced to verify their identity using
sequencing primers 5'-GCATTCTAGTTGTGGTT-3' and
5'-CAACGAAAAGAGAGACC-3'.
Cell Lines and Cell Culture
1471.1 cells (a C127-derived mouse mammary tumor line, stably
integrated with multiple copies of MMTV-chloramphenicol
acetyltransferase; does not express endogenous PR) (20) were used for
the majority of the studies. Also, 3134 cells (a C127- derived mouse
mammary tumor line with a 200-copy MMTV ras tandem array) (21), HeLa
cells (human cervical adenocarcinoma line, ATCC, Manassas, VA), and
Cos-1 cells (African green monkey kidney line, SV40 transformed, ATCC)
were used. All cells were maintained with DMEM (GIBCO BRL, Grand
Island, NY) with 10% FBS (Atlanta Biologicals, Norcross, GA)
plus antibiotics (100 U/ml penicillin and streptomycin, 0.5 mg/ml
gentamycin; GIBCO) and L-glutamine, 2 mM
(GIBCO) in a 5% CO2 incubator at 37 C.
Transfections, Luciferase Assay, and ß- Galactosidase
Assay
Cells (2 x 107) were transfected with 10 µg
pLTRluc, 1 µg pRSVßgal (internal control), 5 µg pCMVIL2R
(included in cell sorting experiments only), and 0.5 µg of one of the
following PR plasmids: pGFP-PRA, phPRA, pGFP-PRB, or phPRB.
Transfections were performed in 0.25 ml cold DMEM by electroporation at
250 V and 800 microfarads. After a 10-min recovery, electroporated
cells were plated into six-well plates (at a density of 
1.4 x
106 total cells per well) for luciferase and
ß-galactosidase assays or two 150-mm dishes (1 x
107 cells per dish) for cell sorting experiments. All cells
were plated with DMEM containing 10% charcoal/dextran-treated FBS
(Hyclone Laboratories, Logan UT) plus L-glutamine and
antibiotics. After cells were incubated for 18 h in a 37 C
CO2 incubator, the medium was refreshed. Cells were then
induced with hormone (R5020) or antihormone (RU486) for 6 h (see
figures for hormone concentrations).
For the luciferase assay, cells were harvested after hormone treatment by scraping in 100 mM potassium phosphate buffer (pH 7.8) with 1 mM dithiothreitol. Cell extracts (supernatants) were prepared by three freeze-thaw cycles and were centrifuged to remove cellular debris. Luciferase assays were performed as previously using 10 µl (containing 5–15 µg protein) of supernatant (18, 22) except that the values for luciferase activity were normalized to ß-galactosidase levels (using the Galacto-Light kit from Tropix, Inc., Bedford, MA) instead of protein.
Microscopy
For fluorescence microscopy, cells were electroporated with 0.5
µg GFP-PRA or GFP-PRB and 9.5 µg pUC18 as carrier DNA, except that
clean coverslips were placed in the bottom of each six-well plate
before plating 8 x 105 cells per well. After
incubation and hormone induction, cells on coverslips were rinsed with
PBS, inverted onto microscope slides, and immediately viewed on a Zeiss
Axiophot microscope (Carl Zeiss, Thornwood, NY) using a standard
fluorescein isothiocyanate filter set. Cells were photographed
using a Zeiss 35-mm camera and Kodak Elite 200 color slide film
(Eastman Kodak, Rochester, NY). Confocal images were collected on a
Zeiss laser scanning instrument (model LSM 510).
Indirect Immunofluorescence
PRB (7.5 µg) was transfected via electroporation into 1471.1
cells and plated onto coverslips in a six-well dish in
charcoal-stripped, phenol red-free media. The following day, cells were
fixed with 3.5% formaldehyde and then permeabilized with 0.5%
Triton-X in PBS. Cells were first washed with PBS, and then with 10%
FBS/0.1% Tween in PBS before incubating with primary antibody (40
µg/ml of 
PR6, Affinity Bioreagents, Golden, CO) for 1 h at
room temperature. Cells were then washed twice with FBS/Tween in PBS
and then incubated with secondary antibody (rhodamine-conjugated goat
antimouse antibody, Calbiochem, La Jolla, CA). Finally, cells were
washed twice with FBS/Tween in PBS, once with PBS, and once with water
before mounting on slides (using SlowFade Antifade kit, Molecular
Probes, Inc., Eugene, OR). Confocal images were collected using a Leica
TCS SP laser scanning instrument.
Cell Analysis
Of the 97 cells photographed and analyzed for this study, 54
cells were transfected with GFP-PRA while 43 cells were transfected
with GFP-PRB. For analysis, slide photographs of cells were scanned
using a ScanMaker III (Microtek, Redondo Beach, CA) scanner
and Adobe Photoshop 4.0.1. (Adobe Systems, Inc., San Jose, CA). Scanned
images were analyzed using the Optimas 6 software (Media Cybernetics,
L.P., Silver Spring, MD) by converting green fluorescent images to
grayscale and analyzing the mean scaled luminance gray value, excluding
any contained areas (mArGVFore), of the nucleus and cytoplasm of each
cell. To create the histogram, ANI (%) was calculated by dividing the
mean scaled luminance values of the nucleus (mArGVForeN) by
the total mean scaled luminance (of the nucleus and cytoplasm,
mArGVForeN + mArGVForeC), and expressing
the result as a percentage. Background values were subtracted
from mArGVForeN and mArGVForeC before
ratios were calculated. GraphPad Prism 2.0 was used to create
histograms.
Western Analysis and Cell Sorting
Magnetic affinity sorting was performed as described previously
(23). Whole-cell extracts from sorted cells for Western analysis were
prepared as described previously (24). PR-containing cellular extracts
(50 µg total protein each) were run on an 7.5% SDS-polyacrylamide
gel (5% stacking, 7.5% separating gels) at 25 mA for 15 min and then
at 45 mA for 45 min. Proteins were then transferred to a Hybond ECL
nitrocellulose membrane (Amersham Life Science, Buckinghamshire, U.K.)
in Transblot buffer (25 mM Tris, 192 mM
glycine) at 4 C for 1 h at 100 V. Membranes were then blocked with
2% nonfat milk in TBS (20 mM Tris, pH 7.5, 140
mM NaCl) overnight, and incubated with an anti-PR primary
antibody cocktail mixture diluted 1:1000 (Ab-1, Ab-2, Ab-3, and Ab-6,
mouse monoclonal antibodies, Lab Vision Corp./Neomarkers, Freemont, CA)
for 2 h at room temperature. Ab-1 and Ab-3 recognize both the A
and B forms of the progesterone receptor; Ab-2 and Ab-6 recognize only
the B form. Membranes were then washed three times for 10 min each with
0.1% Tween-20 in TBS and incubated with a 1:2500 dilution of secondary
antibody (peroxidase-conjugated AffiniPure goat antimouse IgG + IgM,
Jackson ImmunoResearch Labs, West Grove, PA) for 1 h at room
temperature. Next, membranes were washed three times (10 min each) with
0.1% Tween 20 in TBS. The signal was detected using chemiluminescence
(SuperSignal Ultra Chemiluminescent kit, Pierce, Rockford, IL)
according to the manufacturer’s protocol. Exposure time was 15
sec.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Pharmacology Research Associate Training Program
(PRAT) Fellowship from the National Institute of General Medical
Sciences. 
2 Current Address: Departments of Obstetrics & Gynecology, and
Molecular & Medical Pharmacology, 27–139 CHS, University of
California Los Angeles School of Medicine, 10833 Le Conte Avenue, Los
Angeles, California 90095-1740.
Received for publication March 11, 1998. Revision received November 17, 1998. Accepted for publication November 19, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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P. Maruvada, C. T. Baumann, G. L. Hager, and P. M. Yen Dynamic Shuttling and Intranuclear Mobility of Nuclear Hormone Receptors J. Biol. Chem., March 28, 2003; 278(14): 12425 - 12432. [Abstract] [Full Text] [PDF]
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C. Ballare, M. Uhrig, T. Bechtold, E. Sancho, M. Di Domenico, A. Migliaccio, F. Auricchio, and M. Beato Two Domains of the Progesterone Receptor Interact with the Estrogen Receptor and Are Required for Progesterone Activation of the c-Src/Erk Pathway in Mammalian Cells Mol. Cell. Biol., March 15, 2003; 23(6): 1994 - 2008. [Abstract] [Full Text] [PDF]
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J. L. Ruas, L. Poellinger, and T. Pereira Functional Analysis of Hypoxia-inducible Factor-1alpha -mediated Transactivation. IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR TRANSCRIPTIONAL ACTIVATION AND/OR INTERACTION WITH CREB-BINDING PROTEIN J. Biol. Chem., October 4, 2002; 277(41): 38723 - 38730. [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. 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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 C. Elbi, T. Misteli, and G. L. Hager Recruitment of Dioxin Receptor to Active Transcription Sites Mol. Biol. Cell, June 1, 2002; 13(6): 2001 - 2015. [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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S. Mackem, C. T. Baumann, and G. L. Hager A Glucocorticoid/Retinoic Acid Receptor Chimera That Displays Cytoplasmic/Nuclear Translocation in Response to Retinoic Acid. A REAL TIME SENSING ASSAY FOR NUCLEAR RECEPTOR LIGANDS J. Biol. Chem., November 30, 2001; 276(49): 45501 - 45504. [Abstract] [Full Text] [PDF]
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K. L. Sunn, T.-A. Cock, L. A. Crofts, J. A. Eisman, and E. M. Gardiner Novel N-Terminal Variant of Human VDR Mol. Endocrinol., September 1, 2001; 15(9): 1599 - 1609. [Abstract] [Full Text] [PDF]
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R. H. Price Jr., C. A. Butler, P. Webb, R. Uht, P. Kushner, and R. J. Handa A Splice Variant of Estrogen Receptor {beta} Missing Exon 3 Displays Altered Subnuclear Localization and Capacity for Transcriptional Activation Endocrinology, May 1, 2001; 142(5): 2039 - 2049. [Abstract] [Full Text]
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