Differential Localization and Activity of the A- and B-Forms of the Human Progesterone Receptor Using Green Fluorescent Protein Chimeras
Carol S. Lim1,
Christopher T. Baumann,
Han Htun2,
Wenjuan Xian,
Masako Irie,
Catharine L. Smith and
Gordon L. Hager
Laboratory of Receptor Biology and Gene Expression National
Cancer Institute Bethesda, Maryland 20892-5055
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ABSTRACT
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Subcellular localization and transcriptional
activity of green fluorescent protein-progesterone receptor A and B
chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells.
Both GFP-PRA and B chimeras were found to be similar in transcriptional
activity compared with their non-GFP counterparts. GFP-PRA and PRA were
both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold
induction using a reporter gene containing the full-length mouse
mammary tumor virus long-terminal repeat linked to the
luciferase gene (pLTRluc). Using fluorescence microscopy,
nuclear/cytoplasmic distributions for the unliganded and hormone
activated forms of GFP-PRA and GFP-PRB were characterized. The two
forms of the receptor were found to have distinct intracellular
distributions; GFP-PRA was found to be more nuclear than GFP-PRB in
four cell lines examined. The causes for and implications of this
differential localization of the A and B forms of the human PR are
discussed.
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INTRODUCTION
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The progesterone receptor (PR) is a member of the nuclear receptor
superfamily, which includes steroid, thyroid, vitamin D, and retinoic
acid receptors. The human PR exists as two isoforms (A and B forms) in
most cell types. The A form, PRA, is a 164-amino acid N-terminally
truncated version of the B form, PRB (Fig. 1
). 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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Figure 1. GFP-PR Plasmid
The pGFP-PRA (or B) expression vector contains the S65T version of GFP
fused to the N terminus of the receptor through a 15-amino acid peptide
linker (3 ). Amino acids 1165 of the full-length PRB are deleted in
the PRA version of the chimera (see Materials and
Methods).
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The gene targeting activity of steroid/thyroid receptors is partially
controlled by subcellular compartmentalization of the receptor. The
glucocorticoid receptor is found exclusively in the cytoplasm in the
unliganded state and translocates rapidly to the nucleus when activated
with hormone (3). In contrast, members of the retinoic acid
receptor/thyroid group of nuclear receptors are generally found
in the nucleus, both in the hormone-induced and uninduced state.
However, a recent study has shown, in the absence of ligand, the
thyroid receptor ß1 subtype (TRß1) is also somewhat cytoplasmic,
with a nuclear to cytoplasmic ratio of 1.5 (4). Previous studies have
also shown the PR to be primarily nuclear in the absence of ligand (5).
This paper demonstrates a differential localization of PRA and PRB with
the A form being more nuclear than the B form.
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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GFP-PR Chimeras Activate Mouse Mammary Tumor Virus
(MMTV)-Luciferase in a Hormone-Dependent Manner
GFP-PRA and B chimeras (Fig. 1
) 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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Figure 2. Dose-Response Curve
Hormone-induced activity of GFP-PRA, PRA, GFP-PRB, and PRB on
MMTV-luciferase. Fold inductions shown were calculated from normalized
luciferase activities of hormone-treated samples relative to untreated
controls. The concentration of R5020 ranged from 10-12
M to 10-7 M. Experiments were
performed three times in duplicate. A representative example is
shown.
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Induction with Agonist Induces Translocation for Both GFP-PRA and
GFP-PRB
As expected for PRA and PRB, with a 6-h hormone induction (using
30 nM R5020), both GFP-PRA and B also become completely
nuclear but are excluded from nucleoli (compare Fig. 3
, panels A and B, induced, with panels C
and D, uninduced).

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Figure 3. GFP-PRA and GFP-PRB Localization in 1471.1 Cells
Intracellular distribution of PR was examined by confocal microscopy
(shown are projections of a complete z-series for each condition).
Panels A and B show GFP-PRA and GFP-PRB, respectively, induced for
6 h with R5020, and panels C and D present GFP-PRA and GFP-PRB,
respectively, with no hormone treatment.
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GFP-PRB Responds Like PRB to the Antagonist RU486
GFP-PRB and PRB have similar responsiveness to antagonist as seen
in Fig. 4A
, 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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Figure 4. Treatment with Antagonist, RU486
A, The effect of the antagonist RU486 on hormone- induced GFP-PRB
and PRB activation of MMTV-luciferase. Cells were treated with 10
nM R5020, 10 nM RU486, or 10 nM
R5020 + 100 nM RU486 for 6 h. Fold inductions shown
were calculated from normalized luciferase activities of
hormone-treated samples relative to untreated controls. Experiments
were performed twice in duplicate; one example is shown. B, RU486
treatment of GFP-PRA- and GFP-PRB-transfected 1471.1 cells. The effect
of RU486 treatment (6-h induction) on the intracellular distribution of
GFP-PRA (left panel) and GFP-PRB (right
panel) is shown.
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Antagonist (RU486) Also Induces Translocation for Both GFP-PRA and
GFP-PRB
Figure 4B
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 3040% 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 Students 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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Figure 5. Distribution of GFP-PRA and GFP-PRB
A, Histogram of the relative frequency distribution of cells with a
given ANI (expressed as a percentage). ANI (%) is defined as the mean
scaled luminance in the nucleus, divided by the sum of the mean scaled
luminance in the nucleus and cytoplasm, multiplied by 100. This value
was corrected for background. 1471.1 cells were transfected with 0.5
µg of the respective GFP-PR construct and observed 24 h after
electroporation (not induced with hormone). B, The data in panel A are
presented as a scatter plot.
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Figure 6. Examples of Cells Falling within Histogen
Categories
Distribution profiles representative for each of the categories
described in Fig. 5 are shown; left image in each panel
shows GFP fluorescence, and the right image shows phase contrast of the
same field. Images in panel A correspond to histogram category 35 in
Fig. 5 ; images in panel B correspond to category 45, etc.
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While utilization of the GFP tag confers the considerable advantage of
monitoring receptor localization in living cells, it is conceivable
that the added GFP moiety could affect distribution of the receptors
differentially. To compare the distribution of the unlabeled PRB
receptor with the GFP form, we performed indirect immunofluorescence
studies of PRB after transient introduction of the unlabeled PRB. As
shown in Fig. 7
, 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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Figure 7. Indirect Immunofluorescence, PRB Transfected Cells
Cells were transfected with unsubstituted PRB and the distribution of
receptor was examined by indirect immunofluorescence. Three fields
showing the representative distribution of PRB are shown.
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Although these studies were performed 24 h after electroporation,
the same pattern of expression and localization could be seen as early
as 8 h and as late as 48 h after electroporation (data not
shown). In titration studies, as little as 0.1 µg and up to 10 µg
pGFP-PR chimeras can be successfully transfected and visualized. In
other cell lines tested (3134, mouse; HeLa, human; and Cos-1, monkey
cells) GFP-PR chimeras show similar trends in localization patterns
24 h after electroporation (data not shown).
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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Figure 8. Western Blot Using anti-PR Antibodies
The molecular size and expression level for GFP-PRB was characterzied
by Western blot analysis. Lane 1 contains T47D extract (positive
control; contains both PRA and PRB); lanes 2 and 3 contain extracts
from GFP-PRB- and PRB-transfected 1471.1 cells, respectively. Lane 4
presents a negative control (no PR constructs transfected), and lane 5
contains molecular weight (M.W.) markers.
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DISCUSSION
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Variant forms for the human PR have been recognized for some time.
The biological significance of these alternate receptor forms is
unclear. Many differences between the A and B forms have been reported,
including their structural variation, differences in response to
agonist and antagonists (10), and altered cell- and promoter-dependent
activity (1). PRA has been shown to exert a dominant negative effect on
PRB in some systems (1), and variation in dimerization efficiencies
have been reported (11) with the formation of AA dimers more likely
than AB dimers, and BB dimers being the least efficient. Also,
differences in extent of phosphorylation for the two forms have been
observed (10), and altered PRA to PRB ratios in some breast cancers
have been described (2).
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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Construction of Plasmids
The plasmids phPRA and phPRB have been described previously (1, 6). Briefly, phPRA and phPRB contain cDNAs for the A and B forms of the
human progesterone receptor, respectively, under the control of the
SV40 enhancer/human metallothionine II promoter. The plasmid pLTRluc
(18) contains full-length MMTV driving the luciferase gene. The
pCMVIL2R plasmid (19) expresses the interleukin 2 (IL-2) receptor (used
for cell sorting).
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 515 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 manufacturers protocol. Exposure time was 15
sec.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Nancy Weigel for phPRA and B
plasmids, and Dawn Walker and Ron Wolford for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Gordon L. Hager, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Building 41, Room B602, Bethesda, Maryland 20892-5055. E-mail: hagerg{at}dce41.nci.nih.gov
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, 27139 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.
 |
REFERENCES
|
---|
-
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]
-
Graham JD, Yeates C, Balleine RL, Harvey SS, Milliken JS,
Bilous AM, Clarke CL 1996 Progesterone receptor A and B protein
expression in human breast cancer. J Steroid Biochem Mol Biol 56:9398[CrossRef][Medline]
-
Htun H, Barsony J, Renyi I, Gould DJ, Hager GL 1996 Visualization of glucocorticoid receptor translocation and intranuclear
organization in living cells with a green fluorescent protein chimera.
Proc Natl Acad Sci USA 93:48454850[Abstract/Free Full Text]
-
Zhu X, Hanover JA, Hager GL, Cheng S-Y 1998 Hormone-induced translocation of thyroid hormone receptors in
living cells visualized using a receptor-green fluorescent protein
chimera. J Biol Chem 273:2705827063[Abstract/Free Full Text]
-
Guiochon-Mantel A, Lescop P, Christin-Maitre S, Loosfelt H,
Perrot-Applanat M, Milgrom E 1991 Nucleocytoplasmic shuttling of the
progesterone receptor. EMBO J 10:38513859[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]
-
Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer
H, Chambon P 1990 Two distinct estrogen-regulated promoters generate
transcripts encoding the two functionally different human progesterone
receptor forms A and B. EMBO J 9:16031614[Abstract]
-
Guiochon-Mantel A, Loosfelt H, Lescop P, Sar S, Atger M,
Perrot-Applanat M, Milgrom E 1989 Mechanisms of nuclear localization of
the progesterone receptor: evidence for interaction between monomers.
Cell 57:11471154[Medline]
-
Edwards DP, Altmann M, DeMarzo A, Zhang Y, Weigel NL, Beck CA 1995 Progesterone receptor and the mechanism of action of progesterone
antagonists. J Steroid Biochem Mol Biol 53:449458[CrossRef][Medline]
-
Takimoto GS, Hovland AR, Tasset DM, Melville MY, Tung L,
Horwitz KB 1996 Role of phosphorylation on DNA binding and
transcriptional functions of human progesterone receptors. J Biol
Chem 271:1330813316[Abstract/Free Full Text]
-
Tetel MJ, Jung S, Carbajo P, Ladtkow T, Skafar DF, Edwards DP 1997 Hinge and amino-terminal sequences contribute to solution
dimerization of human progesterone receptor. Mol Endocrinol 11:11141128[Abstract/Free Full Text]
-
Gorski J, Toft D, Shyamala G, Smith D, Notides A 1968 Hormone
receptors: studies on the interaction of estrogen with the uterus.
Recent Prog Horm Res 24:4580:4580[Medline]
-
King WJ, Greene GL 1984 Monoclonal antibodies localize
oestrogen receptor in the nuclei of target cells. Nature 307:745747[Medline]
-
Jans DA 1995 The regulation of protein transport to the
nucleus by phosphorylation. Biochem J 311:705716[Medline]
-
Johnson J, Corbisier R, Stensgard B, Toft D 1996 The
involvement of p23, hsp90, and immunophilins in the assembly of
progesterone receptor complexes. J Steroid Biochem Mol Biol 56:3137[CrossRef][Medline]
-
DeFranco DB 1997 Subnuclear trafficking of steroid receptors.
Biochem Soc Trans 25:592597[Medline]
-
Yang J, Liu J, DeFranco DB 1997 Subnuclear trafficking of
glucocorticoid receptors in vitro: chromatin recycling and
nuclear export. J Cell Biol 137:523538[Abstract/Free Full Text]
-
Lefebvre P, Berard DS, Cordingley MG, Hager GL 1991 Two
regions of the mouse mammary tumor virus LTR regulate the activity of
its promoter in mammary cell lines. Mol Cell Biol 11:25292537[Medline]
-
Giordano T, Howard TH, Coleman J, Sakamoto K, Howard BH 1991 Isolation of a population of transiently transfected quiescent and
senescent cells by magnetic affinity cell sorting. Exp Cell Res 192:193197[Medline]
-
Archer TK, Cordingley MG, Marsaud V, Richard-Foy H, Hager GL 1989 Steroid transactivation at a promoter organized in a
specifically-positioned array of nucleosomes. In: Gustafsson JA,
Eriksson H, Carlstedt-Duke J (eds) Proceedings: Second International
CBT Symposium on the Steroid/Thyroid Receptor Family and Gene
Regulation. Birkhauser Verlag AG, Berlin, pp 221238
-
Ostrowski MC, Richard-Foy H, Wolford RG, Berard DS, Hager GL 1983 Glucocorticoid regulation of transcription at an amplified,
episomal promoter. Mol Cell Biol 3:20452057[Medline]
-
Smith CL, Hager GL 1997 Transcriptional regulation of
mammalian genes in vivo: a tale of two templates. J
Biol Chem 272:2749327496[Free Full Text]
-
Smith CL, Htun H, Wolford RG, Hager GL 1997 Differential
activity of progesterone and glucocorticoid receptors on mouse mammary
tumor virus templates differing in chromatin structure. J Biol
Chem 272:1422714235[Abstract/Free Full Text]
-
Kitabayashi I, Eckner R, Arany Z, Chiu R, Gachelin G,
Livingston DM, Yokoyama KK 1995 Phosphorylation of the adenovirus
E1A-associated 300 kDa protein in response to retinoic acid and E1A
during the differentiation of F9 cells. EMBO J 14:34963509[Abstract]
-
Tyagi RK, Amazit L, Lescop P, Milgrom E, Guiochon-Mantel A 1998 Mechanisms of progesterone receptor export from nuclei: role of
nuclear localization signal, nuclear export signal, and ran guanosine
triphosphate. Mol Endocrinol 12:16841695[Abstract/Free Full Text]