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• W2022707915 abstract "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. 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. 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 karyopherinα/β pathway (1Carey K.L. Richards S.A. Lounsbury K.M. Macara I.G. J. Cell Biol. 1996; 133: 985-996Crossref PubMed Scopus (133) Google Scholar, 2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar). How nuclear receptor export is accomplished is less clear, although recent reports have suggested an integral role for calreticulin (CRT) 1The 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. (3Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (224) Google Scholar, 4Black B.E. Holaska J.M. Rastinejad F. Paschal B.M. Curr. Biol. 2001; 11: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), a calcium binding protein localized to the lumen of the endoplasmic reticulum (5Krause K.H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). 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 (6Cheung J. Smith D.F. Mol. Endocrinol. 2000; 14: 939-946Crossref PubMed Scopus (150) Google Scholar). Upon ligand binding, the chaperone complex is dissociated, and the receptor moves rapidly to the nucleus to regulate specific gene transcription (7McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar). Within the nucleus, the receptor becomes localized to specific sites but exchanges very rapidly with chromatin and remains highly mobile (8McNally J.G. Muller W.G. Walker D. Wolford R. Hager G.L. Science. 2000; 287: 1262-1265Crossref PubMed Scopus (647) Google Scholar). Ligand binding and transcriptional regulation are transient events, with molecular chaperones also being involved in the disassembly of regulatory complexes (9Freeman B.C. Felts S.J. Toft D.O. Yamamoto K.R. Genes Dev. 2000; 14: 422-434PubMed Google Scholar, 10Freeman B.C. Yamamoto K.R. Science. 2002; 296: 2232-2235Crossref PubMed Scopus (350) Google Scholar). Heterokaryon fusion assays have indicated that, while localized to the nucleus, nuclear receptors, including liganded GR, traffic continuously and transiently to the cytoplasm (11Guiochon-Mantel A. Loosfelt H. Lescop P. Sar S. Atger M. Perrot-Applanat M. Milgrom E. Cell. 1989; 57: 1147-1154Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 12Guiochon-Mantel A. Lescop P. Christin-Maitre S. Loosfelt H. Perrot-Applanat M. Milgrom E. EMBO J. 1991; 10: 3851-3859Crossref PubMed Scopus (248) Google Scholar, 13Dauvois S. White R. Parker M.G. J. Cell Sci. 1993; 106: 1377-1388Crossref PubMed Google Scholar, 14Madan A.P. DeFranco D.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3588-3592Crossref PubMed Scopus (144) Google Scholar, 15Haché R.J. Tse R. Reich T. Savory J.G. Lefebvre Y.A. J. Biol. Chem. 1999; 274: 1432-1439Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Baumann C.T. Maruvada P. Hager G.L. Yen P.M. J. Biol. Chem. 2001; 276: 11237-11245Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 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 (17DeFranco D.B. Qi M. Borror K.C. Garabedian M.J. Brautigan D.L. Mol. Endocrinol. 1991; 5: 1215-1228Crossref PubMed Scopus (123) Google Scholar, 18Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar, 19Liu J. DeFranco D.B. Mol. Endocrinol. 2000; 14: 40-51Crossref PubMed Scopus (88) Google Scholar). 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 (15Haché R.J. Tse R. Reich T. Savory J.G. Lefebvre Y.A. J. Biol. Chem. 1999; 274: 1432-1439Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). 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 (20Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (326) Google Scholar, 21Dedhar S. Rennie P.S. Shago M. Hagesteijn C.Y. Yang H. Filmus J. Hawley R.G. Bruchovsky N. Cheng H. Matusik R.J. Giguère V. Nature. 1994; 367: 480-483Crossref PubMed Scopus (309) Google Scholar, 22Michalak M. Burns K. Andrin C. Mesaeli N. Jass G.H. Busaan J.L. Opas M. J. Biol. Chem. 1996; 271: 29436-29445Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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 (3Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (224) Google Scholar, 4Black B.E. Holaska J.M. Rastinejad F. Paschal B.M. Curr. Biol. 2001; 11: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Moreover, the redistribution of GR to the cytoplasm following steroid withdrawal is compromised in CRT-deficient cells (3Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (224) Google Scholar). Nuclear receptor binding and the stimulation of nuclear export by CRT appear to be dependent on calcium binding (23Holaska J.M. Black B.E. Rastinejad F. Paschal B.M. Mol. Cell Biol. 2002; 22: 6286-6297Crossref PubMed Scopus (93) Google Scholar). 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 (3Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (224) Google Scholar, 24Holaska J.M. Paschal B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14739-14744Crossref PubMed Scopus (38) Google Scholar). 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. Plasmids—pGFPGR, pGFP-GRNL1-, pDM128, pRevRex, pRevIκ Bα1–72, pRSVβ-gal, and pNESGFPPKNLS are described elsewhere (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar, 25Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar, 26Préfontaine G.G. Lemieux M.E. Giffin W. Schild-Poulter C. Pope L. LaCasse E. Walker P. Haché R.J. Mol. Cell Biol. 1998; 18: 3416-3430Crossref PubMed Scopus (85) Google Scholar, 27Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (623) Google Scholar). 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. (28Lee S. Neumann M. Stearman R. Stauber R. Pause A. Pavlakis G.N. Klausner R.D. Mol. Cell Biol. 1999; 19: 1486-1497Crossref PubMed Google Scholar). 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 (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar). 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 (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar) as a template. Mutations were confirmed by restriction digest and sequencing. pRevGR was derived from pRevΔ NES (25Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar). 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 pRevΔ 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 LipofectAMINE™ (Invitrogen). Following overnight incubation in Opti-MEM™ 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 (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar). 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 LipofectAMINE™ 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 (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar, 18Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar). 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. (25Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar). Cells were cotransfected by calcium phosphate with 1 μg of the indicated RevΔ NES fusion constructs (IκB NES, HIV Rex NES, and GR), 200 ng of pRSV β-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 (29Seed B. Sheen J.Y. Gene (Amst.). 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar) and normalized to β-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. (30Groulx I. Bonicalzi M.E. Lee S. J. Biol. Chem. 2000; 275: 8991-9000Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). COS7 cells were transiently transfected using LipofectAMINE™ 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. (31Du X. Stoops J.D. Mertz J.R. Stanley C.M. Dixon J.L. J. Cell Biol. 1998; 141: 585-599Crossref PubMed Scopus (22) Google Scholar). 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. 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 (32Ullman K.S. Powers M.A. Forbes D.J. Cell. 1997; 90: 967-970Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), 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). 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 (2Savory J.G. Hsu B. Laquian I.R. Giffin W. Reich T. Haché R.J. Lefebvre Y.A. Mol. Cell Biol. 1999; 19: 1025-1037Crossref PubMed Scopus (183) Google Scholar, 3Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (224) Google Scholar, 18Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar, 19Liu J. DeFranco D.B. Mol. Endocrinol. 2000; 14: 40-51Crossref PubMed Scopus (88) Google Scholar). 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) (8McNally J.G. Muller W.G. Walker D. Wolford R. Hager G.L. Science. 2000; 287: 1262-1265Crossref PubMed Scopus (647) Google Scholar). 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 RevΔNES fusion protein·RNA complex mediates the export of an unspliced RNA message containing the CAT coding sequence (33Kim F.J. Beeche A.A. Hunter J.J. Chin D.J. Hope T.J. Mol. Cell Biol. 1996; 16: 5147-5155Crossref PubMed Scopus (84) Google Scholar). In this instance, the rapid nuclear export of the RevΔNES·RNA complex directed by the IκB or HIV Rex CRM1-dependent NESs occurred rapidly prior to RNA splicing as reflected by strong CAT activity. By contrast, a RevΔNES-GR fusion protein failed to induce CAT activity in the same assay (Fig. 2A), even though the RevΔNES-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). 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 (30Groulx I. Bonicalzi M.E. Lee S. J. Biol. Chem. 2000; 275: 8991-9000Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 34Yang J. Liu J. DeFranco D.B. J. Cell Biol. 1997; 137: 523-538Crossref PubMed Scopus (88) Google Scholar). 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. (34Yang J. Liu J. DeFranco D.B. J. Cell Biol. 1997; 137: 523-538Crossref PubMed Scopus (88) Google Scholar). 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 (t½ < 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. 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 (4Black B.E. Holaska J.M. Rastinejad F. Paschal B.M. Curr. Biol. 2001; 11: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (326) Google Scholar), 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 s" @default.
• W2022707915 created "2016-06-24" @default.
• W2022707915 creator A5011928374 @default.
• W2022707915 creator A5021519896 @default.
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• W2022707915 date "2003-09-01" @default.
• W2022707915 modified "2023-10-12" @default.
• W2022707915 title "Nuclear Export of the Glucocorticoid Receptor Is Accelerated by Cell Fusion-dependent Release of Calreticulin" @default.
• W2022707915 cites W1213045642 @default.
• W2022707915 cites W1600052472 @default.
• W2022707915 cites W1764126564 @default.
• W2022707915 cites W1900565974 @default.
• W2022707915 cites W1963612767 @default.
• W2022707915 cites W1967171371 @default.
• W2022707915 cites W1967658150 @default.
• W2022707915 cites W1972783510 @default.
• W2022707915 cites W1973341497 @default.
• W2022707915 cites W1977558343 @default.
• W2022707915 cites W1981593440 @default.
• W2022707915 cites W1990529844 @default.
• W2022707915 cites W2004753972 @default.
• W2022707915 cites W2007620508 @default.
• W2022707915 cites W2008412361 @default.
• W2022707915 cites W2008494281 @default.
• W2022707915 cites W2017800749 @default.
• W2022707915 cites W2018095466 @default.
• W2022707915 cites W2022174079 @default.
• W2022707915 cites W2026203588 @default.
• W2022707915 cites W2031267915 @default.
• W2022707915 cites W2037404178 @default.
• W2022707915 cites W2037709017 @default.
• W2022707915 cites W2038256325 @default.
• W2022707915 cites W2052059014 @default.
• W2022707915 cites W2054402423 @default.
• W2022707915 cites W2059132145 @default.
• W2022707915 cites W2061964177 @default.
• W2022707915 cites W2072931419 @default.
• W2022707915 cites W2080080632 @default.
• W2022707915 cites W2083028663 @default.
• W2022707915 cites W2084028629 @default.
• W2022707915 cites W2088134840 @default.
• W2022707915 cites W2096594220 @default.
• W2022707915 cites W2108538978 @default.
• W2022707915 cites W2118316073 @default.
• W2022707915 cites W2125099281 @default.
• W2022707915 cites W2132251976 @default.
• W2022707915 cites W2135913162 @default.
• W2022707915 cites W2143103923 @default.
• W2022707915 cites W2146437475 @default.
• W2022707915 cites W2147673480 @default.
• W2022707915 cites W2156056701 @default.
• W2022707915 cites W2156437614 @default.
• W2022707915 cites W2163491939 @default.
• W2022707915 cites W2170645024 @default.
• W2022707915 cites W2207800914 @default.
• W2022707915 cites W270887500 @default.
• W2022707915 cites W4238224698 @default.
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