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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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 1–165 of the full-length PRB are deleted in the PRA version of the chimera (see Materials and Methods).

 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GFP-PR Chimeras Activate Mouse Mammary Tumor Virus (MMTV)-Luciferase in a Hormone-Dependent Manner
GFP-PRA and B chimeras (Fig. 1Go) 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. 2Go, 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.

 
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. 3Go, 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.

 
GFP-PRB Responds Like PRB to the Antagonist RU486
GFP-PRB and PRB have similar responsiveness to antagonist as seen in Fig. 4AGo, 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.

 
Antagonist (RU486) Also Induces Translocation for Both GFP-PRA and GFP-PRB
Figure 4BGo 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. 5Go and 6Go). The histogram in Fig. 5AGo shows the distribution of GFP-PRA or GFP-PRB transfected in 1471.1 cells, 24 h after electroporation (no hormone added). Figure 6Go describes the histogram categories in Fig. 5AGo (e.g. an average nuclear intensity, or ANI, of 35% in Fig. 5AGo corresponds to ANI 30–40% in Fig. 6AGo). 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 1Go); 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. 5AGo). 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 6Go 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. 5Go 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. 5Go; images in panel B correspond to category 45, etc.

 

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Table 1. Average Nuclear Intensity (%) for GFP-PRA and GFP-PRB

 
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. 7Go, 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.

 
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. 8Go, lane 1); an extract from 1471.1 cells without GFP-PR or PR constructs transfected was used as a negative control (Fig. 8Go, lane 4). PR antibodies recognize both GFP and non-GFP forms of the PRB; GFP-PRB and PRB were expressed at similar levels (Fig. 8Go, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go), 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go) 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. 1Go). 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 {alpha}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.


    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. Back

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. Back

Received for publication March 11, 1998. Revision received November 17, 1998. Accepted for publication November 19, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  2. 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 56:93–98[CrossRef][Medline]
  3. 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 93:4845–4850[Abstract/Free Full Text]
  4. 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 273:27058–27063[Abstract/Free Full Text]
  5. Guiochon-Mantel A, Lescop P, Christin-Maitre S, Loosfelt H, Perrot-Applanat M, Milgrom E 1991 Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J 10:3851–3859[Abstract]
  6. Vegeto E, Allan GF, Schrader WT, Tsai MJ, McDonnell DP, O’Malley BW 1992 The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. Cell 69:703–713[Medline]
  7. 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 9:1603–1614[Abstract]
  8. 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 57:1147–1154[Medline]
  9. 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 53:449–458[CrossRef][Medline]
  10. 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 271:13308–13316[Abstract/Free Full Text]
  11. 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 11:1114–1128[Abstract/Free Full Text]
  12. 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 24:45–80:45–80[Medline]
  13. King WJ, Greene GL 1984 Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 307:745–747[Medline]
  14. Jans DA 1995 The regulation of protein transport to the nucleus by phosphorylation. Biochem J 311:705–716[Medline]
  15. 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 56:31–37[CrossRef][Medline]
  16. DeFranco DB 1997 Subnuclear trafficking of steroid receptors. Biochem Soc Trans 25:592–597[Medline]
  17. Yang J, Liu J, DeFranco DB 1997 Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export. J Cell Biol 137:523–538[Abstract/Free Full Text]
  18. 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 11:2529–2537[Medline]
  19. 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 192:193–197[Medline]
  20. 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 221–238
  21. Ostrowski MC, Richard-Foy H, Wolford RG, Berard DS, Hager GL 1983 Glucocorticoid regulation of transcription at an amplified, episomal promoter. Mol Cell Biol 3:2045–2057[Medline]
  22. Smith CL, Hager GL 1997 Transcriptional regulation of mammalian genes in vivo: a tale of two templates. J Biol Chem 272:27493–27496[Free Full Text]
  23. 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 272:14227–14235[Abstract/Free Full Text]
  24. 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 14:3496–3509[Abstract]
  25. 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 12:1684–1695[Abstract/Free Full Text]