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Originally published In Press as doi:10.1074/jbc.M306356200 on July 17, 2003

J. Biol. Chem., Vol. 278, Issue 39, 37858-37864, September 26, 2003
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Nuclear Export of the Glucocorticoid Receptor Is Accelerated by Cell Fusion-dependent Release of Calreticulin*

Rhian F. Walther {ddagger} § , Claudia Lamprecht {ddagger}, Andrew Ridsdale {ddagger}, Isabelle Groulx ||, Stephen Lee || **, Yvonne A. Lefebvre {ddagger} {ddagger}{ddagger} and Robert J. G. Haché {ddagger} § {ddagger}{ddagger} §§

From the {ddagger}The Ottawa Health Research Institute and the Departments of {ddagger}{ddagger}Medicine and of §Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1Y 4E9 and the ||Department of Cellular and Molecular Medicine and the Kidney Research Center, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

Received for publication, June 16, 2003 , and in revised form, July 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear hormone receptors are dynamic transcription factors that move rapidly through the nucleus and that, based on the results of heterokaryon fusion assays, are believed to shuttle or exchange rapidly between nucleus and cytoplasm. Shuttling offers the potential for rapid modulation of receptor function in response to cytoplasmic signaling pathways. Nuclear receptors are imported into the nucleus through the karyopherin{alpha}/{beta} pathway (1, 2). How nuclear receptor export is accomplished is less clear, although recent reports have suggested an integral role for calreticulin (CRT)1 (3, 4), a calcium binding protein localized to the lumen of the endoplasmic reticulum (5).

The naïve glucocorticoid receptor (GR) is a cytoplasmic protein, which is held in a chaperone complex anchored by hsp90 and containing hsp70, immunophilins, and other factors, including p23, where it is poised to bind ligand (6). Upon ligand binding, the chaperone complex is dissociated, and the receptor moves rapidly to the nucleus to regulate specific gene transcription (7). Within the nucleus, the receptor becomes localized to specific sites but exchanges very rapidly with chromatin and remains highly mobile (8). Ligand binding and transcriptional regulation are transient events, with molecular chaperones also being involved in the disassembly of regulatory complexes (9, 10).

Heterokaryon fusion assays have indicated that, while localized to the nucleus, nuclear receptors, including liganded GR, traffic continuously and transiently to the cytoplasm (11–16). Upon withdrawal of steroid, shuttling continues for GR as the receptor reassembles into chaperone complexes and slowly reaccumulates in the cytoplasm over a period of several hours (17–19). Depending on the cell type, the time required for reaccumulation of GR in the cytoplasm following steroid withdrawal varies from 6 to 24 h. By contrast, we have reported that treatment of cells with the glucocorticoid antagonist RU486 results in a receptor that appears to remain permanently localized to the nucleus upon the withdrawal of treatment despite continuous nucleocytoplasmic shuttling, suggesting differential effects of agonists and antagonists on GR that communicate differences in localization subsequent to loss of ligand (15). How rapid shuttling of GR is reconciled with slow redistribution to the cytoplasm remains to be determined.

CRT has been identified as a repressor of transcriptional activation by GR and other nuclear receptors (20–22). It has been shown in vitro and in transient heterokaryon fusion assays that nuclear export of these nuclear receptors is mediated through direct contact between CRT and the receptor DNA binding domain (DBD) through a region of the DBD that includes the DNA recognition helix (3, 4). Moreover, the redistribution of GR to the cytoplasm following steroid withdrawal is compromised in CRT-deficient cells (3). Nuclear receptor binding and the stimulation of nuclear export by CRT appear to be dependent on calcium binding (23). However, it remains unclear given the localization of CRT to the lumen of the endoplasmic reticulum (ER), how CRT accesses the receptor in vivo One explanation proposed is that there may be sufficient CRT normally present in the cytoplasm to allow for a role in nuclear export (3, 24).

In the present study we have determined, in a series of assays in live cells, that the transfer of GR across the nuclear membrane to the cytoplasm occurs only very slowly in native cells through a process that is independent of direct binding to CRT. By contrast CRT-mediated GR nuclear export is an inducible pathway for the rapid transfer of GR to the cytoplasm that is transiently activated during polyethylene glycol (PEG)-mediated cell fusion. These results change our understanding of the accessibility of nuclear hormone receptors to cytoplasmic signaling molecules. Additionally, our results provide an important caution for all reports of rapid nuclear export independent of the CRM1 pathway relying on heterokaryon fusions and suggest that CRT-mediated nuclear export is a specific response to breaches in the integrity of the ER.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—pGFPGR, pGFP-GRNL1-, pDM128, pRevRex, pRevI{kappa} B{alpha}1–72, pRSV{beta}-gal, and pNESGFPPKNLS are described elsewhere (2, 25–27). pGSTGFPNLS expresses a fusion protein comprised of GST and GFP, with the sequence of the SV40 NLS at the C terminus. This expression vector was derived from the FVHL-GFP vector described by Lee et al. (28). The sequence encoding von Hippel-Lindau was removed by restriction digest and replaced with an oligonucleotide linker. The GST cDNA was amplified by PCR using the proofreading polymerase Vent (New England Biolabs) and then cloned N-terminal to the GFP coding sequence to produce pGSTGFPNLS. pNESGSTGFPNLS was directly derived from pGSTGFPNLS by inserting the HIV Rev NES N-terminal to the GST coding sequence by linker tailing. Sequencing was performed to ensure that the reading frame of each construct was correct, and Western blotting was performed using the GFP antibody JL8 (Clontech) to verify the expected size of each expression product.

pGFPGRF463,4A, pGFPGRR496H, and pGFPGRC500Y were derived from pGFPGR (2). Oligonucleotides encoding the desired mutations were synthesized, and the mutations were introduced using the Stratagene QuikChange mutagenesis kit. Similarly, pGFPGRNL1-/F463,4A was cloned using the Stratagene QuikChange mutagenesis kit using the pGFP-GRNL1- construct (2) as a template. Mutations were confirmed by restriction digest and sequencing.

pRevGR was derived from pRev{Delta} NES (25). The full-length GR coding sequence was PCR-amplified with Vent polymerase (New England Biolabs) using primers encoding BglII and XbaI restriction sites and inserted into pRev{Delta} NES to produce pRevGR. Sequencing of the N-terminal junction was performed to verify that the GR cDNA was cloned in the correct reading frame. Western blotting using the GR MAI-510 antibody (Affinity Bioreagents) was performed to verify that the expression product was of the correct size. Transcriptional activity of the pRevGR expression construct in 293T cells was verified using the pMMTVCAT reporter.

FRAP and Quantification of Subcellular Distribution—COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and non-essential amino acids. Cells were seeded onto 40-mm round coverslips (Bioptechs) and transfected with 1 µg of the indicated expression construct using 10 µl of LipofectAMINETM (Invitrogen). Following overnight incubation in Opti-MEMTM reduced serum media (Invitrogen) the transfection was stopped by addition of charcoal-stripped serum. Cells were cultured in complete serum for 8 h and then in serum-free medium for 16 h prior to treatment. 20 µg/µl cycloheximide was then added 1 h prior to FRAP. For FRAP experiments, treatment with ligand at 1 µM or leptomycin B at 10 nM was initiated 1 h prior analysis. Steroid withdrawal was performed as previously described (2). Coverslips were visualized on a Bio-Rad MRC 1024 confocal microscope in a Bioptechs FCS2 environmental chamber maintained at 37 °C. Nuclei were photobleached by using 5–10 rapid laser pulses at full power. Each experiment included a minimum of three independent trials performed over several months with each involving 10–50 individual repetitions of FRAP. Recovery of the fluorescent signal within the bleached nucleus was quantified using the LaserSharp software package (Bio-Rad). The average pixel intensity within both nuclei was quantified and corrected for background fluorescence. Signal recovery in the bleached nucleus was calculated by expressing the fluorescent signal within the bleached nucleus as a percentage of the total fluorescent signal within both nuclei.

For direct analysis of subcellular distribution, COS7 cells were transfected using LipofectAMINETM as described for FRAP experiments. Following transfection cells were cultured in complete serum overnight and then seeded onto 22-mm square coverslips. Cells were allowed to attach for 8 h and then withdrawn from serum for 16 h. Cells were treated with ligand at 1 µM for the indicated time period, and the ligand was then removed for the specified time period. Following ligand withdrawal, cells were fixed with 3% paraformaldehyde for 30 min at 4 °C followed by incubation with PBS containing 0.1 M glycine for 10 min at 20 °C. Coverslips were mounted onto microscope slides, overlaid with 50% glycerol in PBS, and sealed with nail polish. Cells were visualized on a Nikon TE300 microscope and were scored into five categories ranging from exclusively nuclear (N) to exclusively cytoplasmic (C) as previously described (2, 18). Quantification was performed using double-blind encryption with individual data points derived from a minimum of 1000 cells quantified over four independent experiments performed in duplicate.

HIV Rev Complementation Assay—Analysis of HIV Rev cotransport of CAT encoding RNA in 293T cells was performed as described by Johnson et al. (25). Cells were cotransfected by calcium phosphate with 1 µg of the indicated Rev{Delta} NES fusion constructs (I{kappa}B NES, HIV Rex NES, and GR), 200 ng of pRSV {beta}-galactosidase and 200 ng of the pDM128 CAT reporter. Cells were treated with 1 µM dexamethasone as indicated for 24 h. CAT activity was measured by liquid scintillation counting (29) and normalized to {beta}-galactosidase activity to account for potential variations in transfection efficiency. Error bars represent the standard error of the mean of a minimum of three independent experiments performed in duplicate.

Digitonin Permeabilization Export Assay—Digitonin permeabilization was performed essentially as described by Groulx et al. (30). COS7 cells were transiently transfected using LipofectAMINETM for 16 h with the indicated expression constructs as for FRAP assay. The cells were maintained in complete media for a further 24 h and then treated with 1 µM cortisol. The cells were then rinsed three times in ice-cold transport buffer (20 mM HEPES, pH 7.3, 110 mM KOAc, 5 mM NaOAc, 2 mM Mg(OAc)2, 1 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 1.0 µg/ml aprotinin). Digitonin was added to a final concentration of 50 µg/ml, and the cells were incubated for 5 min at 4 °C. Cells were then rinsed three times in transport buffer, incubating 5 min at 4 °C between each wash. HeLa cell lysate containing 20 units/ml creatine phosphokinase, 5 mM creatine phosphate, 2 mM ATP, and 2 mM GTP was then added to each plate. Cells were placed at 20 °C, and export was monitored on a Zeiss Axiovert S100TV microscope equipped with an Empix digital charge-coupled device camera using Northern Eclipse software.

Homokaryon Cell Fusion—COS7 cells were separately transfected with the dsRed-C1 expression construct or the indicated GFP constructs. Following transfection, cells were cultured in complete serum overnight and harvested by trypsinization. Cells expressing dsRed-C1 were mixed 1:1 with cells expressing the indicated GFP construct and seeded onto 40-mm round coverslips at high density. Cells were allowed to attach for 8 h and then withdrawn from serum for 16 h. Cell fusion was initiated by incubation with 50% w/v PEG-4000 in Ca2+/Mg2+ free Hanks' balanced salt solution for 2 min at 37 °C. Cells were then washed five times in Ca2+/Mg2+ free Hanks' balanced salt solution and incubated at 37 °C with serum-free Dulbecco's modified Eagle's medium for 1 h. The coverslips were then placed in a Bioptechs FCS2 environmental chamber maintained at 37 °C, and the cells were visualized on a Nikon TE300 microscope. Live cell images were obtained using an Orca ER camera (Hamamatsu) and SimplePCI software (Compix).

Analysis of Calreticulin and Calnexin Exposure—Selective permeabilization experiments were performed using a modification of the protocol of Du et al. (31). Cells were plated onto 22-mm square coverslips, treated as described, and then fixed with 3% paraformaldehyde in PBS for 30 min at 4 °C, followed by incubation with PBS containing 0.1 M glycine for 10 min at 20 °C. Permeabilization with Triton X-100 (0.5% in PBS) was for 30 min at 20 °C. For streptolysin O (SLO) permeabilization, cells were incubated with 250 units/ml SLO in BBII buffer (25 mM HEPES, pH 7.5, 75 mM KOAc) for 15 min on ice. Cells were washed once in cold BBII to remove unbound SLO and then incubated at 37 °C for 15 min. Following permeabilization, cells were pre-blocked with 5% IgG-free bovine serum albumin in PBS for 1 h at 20 °C. Cells were then incubated overnight at 4 °C with primary antibodies to calreticulin (C-17, Santa Cruz Biotechnologies) or calnexin (H-70, Santa Cruz Biotechnologies) diluted 1/150 in PBS containing 5% IgG-free bovine serum albumin. Following three washes with PBS, cells were incubated at 20 °C for 45 min with rhodamine red X-conjugated donkey-anti-goat antibody (1/150, Jackson ImmunoResearch Laboratories). Cells were washed three times in PBS and then mounted on microscope slides. Images were recorded with an Orca ER camera (Hamamatsu) on a Nikon TE300 microscope using SimplePCI software (Compix). For each primary antibody the optimal exposure time required to record an image following Triton X-100 permeabilization was determined. This exposure time was used to gather all subsequent images within that repetition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Liganded GR Is Statically Localized to the Nucleus in Live and Digitonin-permeabilized Cells—To examine the mechanism for nuclear export of GR in situ we developed a fluorescence recovery after photobleaching (FRAP) assay that takes advantage of the significant proportion of cells in many established mammalian tissue culture cell lines that are stably maintained in a multinucleated state (Fig. 1). As demonstrated by examination of the trafficking of a series of synthetic control proteins tagged with green fluorescent protein (GFP), nucleocytoplasmic protein shuttling in this assay is reflected by the rapid reappearance of fluorescence in the photobleached nucleus of a multinucleated cell (Fig. 1A). Thus for a GSTGFP fusion protein with an SV40 nuclear localization sequence (NLS) and an HIV Rev nuclear export sequence (NES) that mediates nuclear export by the CRM1 pathway (32), recovery of fluorescence in photobleached nuclei of multinucleated COS7 cells occurred within 10 min. This recovery was prevented by the CRM1-inhibitor leptomycin B and was dependent on the Rev NES. Similar results were obtained in other cell lines including HeLa cells (data not shown).



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  		FIG. 1.
FRAP analysis of GR nuclear export. A and B, FRAP analyses of GR shuttling in COS7 cells. Cells expressing GFPGR and GFPGST proteins containing an SV40 NLS and HIV Rev NES were treated with cycloheximide and the indicated steroid for 1 h prior to FRAP. Scale bar = 10 µm. For the NESGSTGFPNLS construct, fluorescence recovery was complete within 10 min, whereas in either the presence of 10 nM LMB or for a construct lacking the Rev NES, less than 5% recovery was observed at 30 min or 1 h, respectively. Fluorescence recovery values for cortisol-treated, RU486-treated, and ligand-withdrawn GFPGRwt were calculated at 19 ± 6%, 19 ± 2%, and 12 ± 3%, respectively. C, FLIP ablation of nuclear GR. GFPGR-transfected cells were treated as described in A and B and exposed to a single laser scan at full power at 30-s intervals over the area marked by the red boxes. Images were recorded immediately after the laser pulses.

 

By contrast, we were unable to detect rapid nucleocytoplasmic exchange of GR in this assay (Fig. 1B). In the presence of cortisol or the steroid antagonist RU486, modest movement of GFPGR between nuclei was observed, with less than 20% transfer of fluorescent GFPGR detected 4 h after the initial ablation of signal from the acceptor nucleus. Steroid withdrawal also failed to stimulate rapid nuclear export, initiating only a slow redistribution of the receptor to the cytoplasm. Again we obtained the same results in HeLa cells (data not shown), emphasizing that the rate of GR export was not cell type-dependent. Although a contrast to the rapid nuclear export of GR observed previously in heterokaryon fusion assays, the slow rate of GR export obtained by FRAP closely matched the export rates for GR obtained previously in indirect immunofluorescence assays (2, 3, 18, 19).

The inclusion of cycloheximide and controls demonstrating that GFP fluorescence was not recovered following photobleaching of mononucleated cells, verified the significance of the slow transfer of GR observed with FRAP. The observation of similar slow movement of GR between nuclei in the absence of cycloheximide excluded nonspecific effects resultant from the inhibition of protein synthesis (data not shown). Receptor movement was also unaffected by the level of GFPGR expression or the positioning of the GFP at the N or C terminus of GR. Furthermore, a GFP-mineralocorticoid receptor fusion protein exhibited similarly restricted movement (data not shown).

Although GFPGR transferred only very slowly between nuclei, directed ablation of fluorescence from a small portion of the nucleus in fluorescence loss in photobleaching experiments (FLIP) confirmed that GFPGR was highly mobile within the nucleus (Fig. 1C) (8). Furthermore, FLIP of the GFP signal within one nucleus of a multinucleated cell also failed to induce a reduction of the GFPGR signal from the second nucleus (Fig. 1C, middle row).

The slow rate of nuclear export observed for GR in FRAP was confirmed in a HIV Rev-RNA cotransport assay (Fig. 2A). In this assay, which reports the rapid nuclear export of proteins in live cells, CAT activity is detected when the nuclear export of an HIV Rev{Delta}NES fusion protein·RNA complex mediates the export of an unspliced RNA message containing the CAT coding sequence (33). In this instance, the rapid nuclear export of the Rev{Delta}NES·RNA complex directed by the I{kappa}B or HIV Rex CRM1-dependent NESs occurred rapidly prior to RNA splicing as reflected by strong CAT activity. By contrast, a Rev{Delta}NES-GR fusion protein failed to induce CAT activity in the same assay (Fig. 2A), even though the Rev{Delta}NES-GR fusion protein was expressed at levels comparable to the control Rev fusion proteins and was transcriptionally active in response to steroid (data not shown).



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  		FIG. 2.
Slow nuclear export of GR in intact and digitonin cells. A, NES-dependent nuclear export of pDM128 RNA prior to splicing. CAT activity correlates directly with rate of RNA export and is expressed as a percentage of the maximum induced activity and normalized to {beta}-galactosidase. B, nuclear export from digitonin-permeabilized cells. COS7 cells were transiently transfected using LipofectAMINETM to express GFPGR or GFP pyruvate kinase (PK) containing an NES and NLS. Cells were treated with 1 µM cortisol for 1 h as indicated. Digitonin permeabilization was performed as described previously (30).

 

In a third assay (Fig. 2B), digitonin treatment to permeabilize cell membranes allows for observation of the rapid export of proteins from nuclei incubated with cytosolic extracts by allowing for nuclear export in the absence of re-import (30, 34). Thus fluorescence from a control protein containing pyruvate kinase fused to the HIV Rev NES is lost to the cytosolic extract within 5 min of treatment in a manner that is blocked by leptomycin B treatment. By contrast, GFPGR again failed to export from the nucleus, remaining nuclear over at least 45 min, in agreement with previous observations by Yang et al. (34).

Rapid Nucleocytoplasmic Exchange of GR Is Induced through Transient Exposure of CRT following Cell Fusion—To ensure that the difference between our results and previous reports of nucleocytoplasmic shuttling of GR was not due to our reagents or the nature of the systems employed in the other studies, we assessed the nuclear export of GR in a homokaryon fusion assay in which COS7 cells expressing dsRed were fused using polyethylene glycol (PEG) with cells expressing nuclear GFP fusion proteins (Fig. 3). Nuclear export of GSTGFP, marked by the rapid transfer (t1/2 < 30 min) of green fluorescence to a red fluorescent nucleus was again observed to be dependent on the HIV Rev NES. However, in this instance we also observed accelerated nuclear export of liganded GFPGR, with receptor transfer between heterokaryon nuclei also occurring with of t1/2 of less than 30 min in over 80% of the cells examined.



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  		FIG. 3.
Shuttling of GR subsequent to cell fusion. COS7 cells were transfected separately using LipofectAMINETM with 1 µg of dsRed2-C1 plasmid and the indicated GFP constructs. Nuclei from the GFP-containing cells (arrowheads) failed to rapidly take up dsRed owing to the size of the dsRed tetramer.

 

One possibility suggested by these findings was that PEG mediated cell fusion affected the endoplasmic reticulum in a way that promoted the mobilization of CRT for nuclear export. To begin to test this hypothesis, we evaluated the effect of mutations in GR (F463, 464A) that abrogate CRT-GR binding and CRT-dependent transport of GR (4, 20), on the redistribution of GR to the cytoplasm upon withdrawal of steroid (Fig. 4A). To control for the loss of GR DNA binding that also results from the F463A,F464A substitution, we also tested two additional DNA binding mutants of GR (GRR496H and GRC500Y) that were not expected to affect recognition by CRT.



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  		FIG. 4.
GR relocalization to the cytoplasm following ligand withdrawal in the absence of calreticulin binding. A and B, analysis of the redistribution WT GR and the indicated mutants from the nucleus upon steroid withdrawal performed by direct visualization of GFP fluorescence. The display is a compilation of four independent experiments performed in duplicate scoring a minimum of 200 cells for each data point in each experiment. Error bars indicate the mean ± S.E.

 

In COS7 cells, redistribution of GR, GRR496H, and GRC500Y from the nucleus was 50–60% complete 24 h following steroid withdrawal, and this redistribution was not affected by the F463A,F464A substitution. We have previously reported that this slow redistribution of GR to the cytoplasm is accelerated upon mutation of the receptor's strong NLS, NL1, and suggests the possibility that NL1 contributes to the retention of GR in the nucleus (2). Although GRNL1-accumulates to a lower level in the nucleus than WT GR in response to steroid, its return to the cytoplasm approaches completion within 4 h of withdrawal from steroid (Fig. 4B). This more rapid nuclear export of GRNL1- was also unaffected by the F463A,F464A substitution. Similarly in FRAP, the F463A,F464A substitution failed to block the modest transfer of GR to the photobleached nuclei at 4 h observed for GRC5000Y (Fig. 5A) and GRR496H (data not shown).



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  		FIG. 5.
Dependence of CRT-mediated GR nuclear export on cell fusion. A, trafficking of GFPGRF463,4A and GFPGRC500Y visualized by FRAP. Fluorescence recovery values for GFPGRwt, GFPGRF463,4A, and GFPGRC500Y were calculated at 19 ± 6%, 30 ± 6%, and 36 ± 6%, respectively. B, trafficking of GFPGRR496H and GFPGRF463,4A 1 h subsequent to COS7 homokaryon fusion. Nuclei from the GFP-containing cells are indicated with an arrowhead. C, trafficking of GFPGR visualized by FRAP 1 h subsequent to COS7 homokaryon fusion.

 

By contrast, the F463A,F464A substitution completely prevented the transfer of GFPGR between nuclei in homokaryon fusion experiments, whereas GFPGRR496H transferred rapidly between nuclei (Fig. 5B), consistent with the results of Black et al. (4). This increased rate of transfer of GR between nuclei occurred only transiently following fusion. FRAP analysis performed 1 h subsequent to cell fusion (Fig. 5C) produced the same result observed in our original FRAP experiments with multinucleated cells, in which the transfer of GR between nuclei occurred only very slowly (Fig. 1).

These results indicated that PEG-mediated cell fusion mobilized or activated the CRT-dependent nuclear export pathway and suggested the possibility that cell fusion affected the integrity of the ER in a manner that released or activated CRT to participate in nuclear export. To investigate this possibility we probed for exposure of CRT from the ER following PEG treatment using to detergents selectively permeabilize cellular membranes to provide access to specific antibodies (Fig. 6). Triton X-100 treatment of cells gently fixed with paraformaldehyde permeabilizes ER membranes to expose CRT and calnexin, which extends into the lumen of the ER from the inner membrane (Fig. 6A). By contrast, streptolysin O permeabilizes the cell membrane to allow for antibody penetration into the cell without affecting the ER as revealed by the lack of signal obtained with CRT and calnexin antibodies (Fig. 6B). However, PEG treatment induced a change in the integrity of the ER, exposing both CRT and calnexin to their respective antibodies in streptolysin O-permeabilized cells (Fig. 6, C and D), thereby confirming specific effects of PEG on the integrity of the ER. Furthermore, this PEG-dependent effect on the ER was transient, and resistance to CRT/calnexin antibodies was recovered within 1 h of PEG removal (Fig. 6, E–G), confirming our previous observation that intranuclear trafficking of GR in homokaryons ceased by 1 h post-fusion (Fig. 5C). Interestingly, PEG treatment alone was not sufficient to activate GFPGR export (data not shown), indicating that steps in addition to simple exposure of the CRT may be required to fully mobilize the CRT nuclear export pathway.



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  		FIG. 6.
Transient PEG-mediated disruption of the endoplasmic reticulum. Indirect immunofluorescence of endogenous calreticulin and calnexin in COS7 cells. Cells were treated with 50% (w/v) PEG-4000 and fixed following incubation at 37 °C for the indicated times. Selective permeabilization was performed with either streptolysin O or Triton X-100. For each antibody the optimal exposure time required for visualization of signal following Triton X-100 permeabilization was determined, and this exposure time was used to capture all subsequent images within that experiment.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have determined that export of GR across the nuclear membrane to the cytoplasm occurs only very slowly and is mediated through a process that does not involve the direct interaction of GR with CRT. Rather, the rapid CRT-dependent nuclear export of GR in our assays occurred as an induced reaction to PEG-induced cell fusion. These results suggest that CRT-mediated rapid export of nuclear hormone receptors from the nucleus may be an inducible response to some acute forms of cellular stress.

Redistribution of GR to the cytoplasm from the nucleus following the withdrawal of steroidal stimuli has long been known to be a process that occurs very slowly over a period of many hours. This contrasts the nuclear import of GR following steroid treatment, which is estimated to occur within 30 s following dissociation of GR from the chaperone complex (35). The demonstration in transient heterokaryon assays of rapid nuclear export for GR and other nuclear receptors via what was subsequently shown to be a CRT-dependent nuclear export pathway, provided impetus for models of nuclear receptor function being directly affected by transient forays into the cytoplasm that could allow for specific post-translational modification and specific cotransport of other cellular factors that could modify receptor function (36).

Rapid exchange between cellular compartments has been documented for several other transcriptional regulatory proteins and has been shown to allow for regulation of nuclear activity through posttranslational modification in the cytoplasm or regulated sequestration of the factors from the nucleus. For example, phosphorylation of HMGN1, a histone-like chromatin binding protein, inactivates its nuclear localization sequence to promote its accumulation in the cytoplasm (37). By contrast, nuclear import of the shuttling proteins Smad2/3 is activated by phosphorylation induced by transforming growth factor {beta} signaling in the cytoplasm (38, 39), whereas nuclear export of HDAC4/5 is activated in the nucleus by phosphorylation adjacent to their nuclear export signals (40).

Our present results demonstrate that rapid nucleocytoplasmic shuttling is unlikely to represent a default state for the movement of nuclear receptors in the cell. Since its initial report as a rapid nuclear exporter of GR, cytoplasmic CRT has been shown to have the ability to direct the nuclear export of several additional nuclear receptors in a manner that is consistent with a direct interaction between CRT and the first zinc finger of the nuclear receptor DBD (4). However, one challenge has been to understand the relationship between the localization of CRT to the ER and the export of nuclear receptors from the nucleus. Through a combination of approaches, our results show that rapid nuclear export of GR is an induced response to the mobilization of CRT in a manner that leads to its exposure from the lumen of the ER. This result is consistent with all previous data marking cytoplasmic CRT as an efficient nuclear transporter.

An intriguing potential extension to these results is that CRT-mediated rapid nuclear export of nuclear receptors may be a mechanism for cells to rapidly clear nuclear receptors and other proteins from the nucleus as part of normal physiology. For example, disassembly of the nuclear envelope, which is contiguous with the ER, at the onset of mitosis might directly activate the CRT export pathway. Indeed, many nuclear factors have been demonstrated to be lost from the nucleus in the time immediately preceding mitosis (41). Moreover, it has previously been reported that GR is unable to sustain its presence in the nucleus during G2 as the cell prepares for mitosis (42). Alternatively, it is also feasible that the CRT nuclear export pathway provides a specific response to acute forms of cellular stress that impact on the integrity of the ER or natural processes such as phagocytosis that are known to include dynamic changes in ER (43).

But what of the measurable, but slow nuclear export of GR to the cytoplasm that we observed both in our FRAP assays and in direct and indirect immunofluorescence experiments with steroid-withdrawn GR and that have been the hallmark of receptor behavior in many studies? Although it remains possible that a small amount of CRT remains active in the cytoplasm, the slow export by GRF463,4A, which contains an amino acid substitution that abrogates CRT binding, suggests it more likely that this export of GR occurs through a classic nuclear export pathway. Indeed, preliminary results from ongoing analyses have demonstrated that nuclear occupancy of the GFPGRNL1- is dramatically enhanced by inhibition of CRM1-dependent nuclear export through LMB treatment (2)2 in a manner that is exactly consistent with the effects reported previously for LMB on shuttling proteins such as BRCA1 and Smad1 that are distributed equally between nucleus and cytoplasm (45, 46). It is also consistent with a previous study suggesting a direct role for CRM1 in the export of nuclear receptors from the nucleus (2, 47). Intriguingly, the increased nuclear export rate for GFPGRNL1- also suggests a specific role for the GR NL1 NLS in inhibiting receptor nuclear export, something that has not been reported previously for a basic NLS.

One unresolved issue between our results and reports describing CRT-dependent transport is the observation that in CRT–/– mouse embryo fibroblasts GR failed to redistribute to the cytoplasm over a period of 9 h following steroid withdrawal (3). Although more study will be required to resolve this issue, CRT plays a major role in regulating calcium homeostasis (48), and thus its absence may be expected to have multiple effects on cellular function. One or more of these effects might directly or indirectly impact on the rate on nuclear protein export. Furthermore, we note that the persistent nuclear localization of steroid-withdrawn GR in CRT–/– cells strongly resembles the persistent nuclear localization of GR in cells withdrawn from steroid antagonists (15). This suggests that an agonist-dependent event may be required for nuclear export of the spent receptor and is somehow compromised in CRT–/– cells. Possibilities include post-translational modification or an alteration in the reassociation of GR with its molecular chaperones following ligand withdrawal. Such an effect would also be consistent with the affects on GR function observed upon CRT overexpression (49).

Nuclear receptors have been shown in a number of assays to have the ability to cotransport cytoplasmic factors to the nucleus. In particular, the cotransport of nuclear import-deficient nuclear receptors to the nucleus by wild type partners has been used to demonstrate that nuclear receptors dimerize in solution subsequent to ligand binding (11, 44, 50). In the absence of rapid nucleocytoplasmic shuttling, our results would imply that these receptor interactions occur more efficiently than has previously been appreciated. Because our results indicate that receptors shuttle only slowly, the interaction in the cytoplasm between receptors must be very efficient. For GR this implies that dimerization of the receptor in the cytoplasm (51) is a rapid event that occurs in the short period between ligand binding and nuclear import.

Lastly, the heterokaryon fusion assay has long been considered to be a definitive assay for demonstrating nucleocytoplasmic protein trafficking. Although our results suggest it to be valid for rapid nuclear export through the CRM1 pathway, they identify a need to revaluate descriptions of nuclear export independent of CRM1 that were based solely on this assay.


   FOOTNOTES
 
* This work was supported in part by a grant from the Canadian Institutes of Health Research (CIHR) (to Y. A. L. and R. J. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by a studentship from the Government of Ontario. Back

** A New Investigator of the CIHR. Back

§§ An Investigator of the CIHR. To whom correspondence should be addressed: The Ottawa Health Research Institute, Hormones, Growth, and Development Program, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 16283); Fax: 613-761-5036; E-mail: rhache{at}ohri.ca.

1 The abbreviations used are: CRT, calreticulin; CAT, chloramphenicol acetyltransferase; ER, endoplasmic reticulum; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GR, glucocorticoid receptor; GST, glutathione S-transferase; hsp, heat shock protein; LMB, leptomycin B; NES, nuclear export sequence; NLS, nuclear localization sequence; PEG, polyethylene glycol; SLO, streptolysin O; PBS, phosphate-buffered saline. Back

2 R. F. Walther and R. J. G. Haché, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank M. Michalak and X. Zha for reagents and advice, T. J. Hope for reagents, and X. Zha and A. Edgecombe for technical support.



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