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• W2030792950 abstract "The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naïve MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways. The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naïve MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways. The mineralocorticoid receptor (MR) 1The abbreviations used are: MR, mineralocorticoid receptor; GR, glucocorticoid receptor; DBD, DNA-binding domain; LBD, ligand-binding domain; NLS, nuclear localization signal; GFP, green fluorescent protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; FRAP, fluorescence recovery after photobleaching; FLIP, fluorescence loss in photobleaching; WT, wild-type; MMTV, murine mammary tumor virus; CBP, cAMP-responsive element-binding protein-binding protein. exhibits a complex pattern of responsiveness to mineralocorticoids and glucocorticoids. Indeed, MR has a higher affinity for glucocorticoids than for mineralocorticoids and is more sensitive to glucocorticoids than the glucocorticoid receptor (GR) (1Arriza J.L. Simerly R.B. Swanson L.W. Evans R.M. Neuron. 1988; 1: 887-900Abstract Full Text PDF PubMed Scopus (512) Google Scholar, 2Reul J.M. de Kloet E.R. Endocrinology. 1985; 117: 2505-2511Crossref PubMed Scopus (2220) Google Scholar). In epithelial tissues such as the tubules of kidney and distal colon, specificity of MR signaling in response to mineralocorticoids is maintained by the expression of 11β-hydroxysteroid dehydrogenase-2, which converts cortisol or corticosterone to inactive keto metabolites (3White P.C. Mune T. Agarwal A.K. Endocr. Rev. 1997; 18: 135-156Crossref PubMed Scopus (544) Google Scholar). In tissues in which 11β-hydroxysteroid dehydrogenase-2 is not expressed, such as within the brain and vascular system, basal glucocorticoid levels are sufficient to transmit transcriptional responses through MR. By contrast, signaling through GR reflects the diurnal variation of glucocorticoid levels and acute responses to stimulation of the hypothalamic-pituitary axis. Where the potential for signaling through both MR and GR exists, the activation of each receptor communicates distinct physiological outcomes (4Sousa N. Almeida O.F. Rev. Neurosci. 2002; 13: 59-84Crossref PubMed Scopus (110) Google Scholar, 5Roozendaal B. Neurobiol. Learn. Mem. 2002; 78: 578-595Crossref PubMed Scopus (655) Google Scholar). Glucocorticoid signaling in these tissues involves both overlapping targeting of MR and GR in the nucleus and heteromeric interactions between the two receptors (6Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (399) Google Scholar, 7Liu W. Wang J. Sauter N.K. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12480-12484Crossref PubMed Scopus (205) Google Scholar, 8Ou X.M. Storring J.M. Kushwaha N. Albert P.R. J. Biol. Chem. 2001; 276: 14299-14307Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 9Planey S.L. Derfoul A. Steplewski A. Robertson N.M. Litwack G. J. Biol. Chem. 2002; 277: 42188-42196Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10van Steensel B. van Binnendijk E.P. Hornsby C.D. van der Voort H.T. Krozowski Z.S. de Kloet E.R. van Driel R. J. Cell Sci. 1996; 109: 787-792Crossref PubMed Google Scholar). Prior to exposure to steroid, MR and other steroid receptors are associated with a chaperone protein complex anchored by HSP90. Upon binding to steroid, MR dissociates from its chaperone complex and undergoes a conformational change to an active state that allows for the regulation of specific gene expression in collaboration with a variety of transcriptional co-regulatory factors (11Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1537) Google Scholar, 12Cheung J. Smith D.F. Mol. Endocrinol. 2000; 14: 939-946Crossref PubMed Scopus (150) Google Scholar). Although similar in their overall mechanisms of action, steroid hormone receptors exhibit differences in their subcellular localization and the regulation of subcellular localization that are hypothesized to be important for the regulation of steroid responsiveness. Thus, the receptor and progesterone receptors are constitutively nuclear in their naïve state (13Htun H. Holth L.T. Walker D. Davie J.R. Hager G.L. Mol. Biol. Cell. 1999; 10: 471-486Crossref PubMed Scopus (219) Google Scholar), whereas the androgen receptor has been widely reported to localize to the cytoplasm prior to ligand binding (14Georget V. Lobaccaro J.M. Terouanne B. Mangeat P. Nicolas J.C. Sultan C. Mol. Cell. Endocrinol. 1997; 129: 17-26Crossref PubMed Scopus (159) Google Scholar, 15Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar, 16Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (250) Google Scholar). Similarly, GR is almost exclusively cytoplasmic prior to exposure to steroid (17Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (327) Google Scholar, 18Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar, 19Qi M. Hamilton B.J. DeFranco D. Mol. Endocrinol. 1989; 3: 1279-1288Crossref PubMed Scopus (73) Google Scholar, 20Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar) and recycles back to the cytoplasm following the termination of steroidal signaling (21Liu J. DeFranco D.B. Mol. Endocrinol. 2000; 14: 40-51Crossref PubMed Scopus (88) Google Scholar, 22Savory 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 (182) Google Scholar, 23Yang J. Liu J. DeFranco D.B. J. Cell Biol. 1997; 137: 523-538Crossref PubMed Scopus (86) Google Scholar). By contrast, naïve MR is distributed evenly in the nucleus and cytoplasm in most cell types studied (24Fejes-Toth G. Pearce D. Naray-Fejes-Toth A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2973-2978Crossref PubMed Scopus (214) Google Scholar, 25Lombes M. Farman N. Oblin M.E. Baulieu E.E. Bonvalet J.P. Erlanger B.F. Gasc J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1086-1088Crossref PubMed Scopus (105) Google Scholar, 26Nishi M. Ogawa H. Ito T. Matsuda K.I. Kawata M. Mol. Endocrinol. 2001; 15: 1077-1092Crossref PubMed Scopus (84) Google Scholar, 27Sasano H. Fukushima K. Sasaki I. Matsuno S. Nagura H. Krozowski Z.S. J. Endocrinol. 1992; 132: 305-310Crossref PubMed Scopus (57) Google Scholar). The regulation of their distribution between the nucleus and cytoplasm offers an essential control point for the regulation of the activity and stability of many transcription factors. For example, STAT1 (signal transducer and activator of transcription-1) accumulates in the nucleus in response to interferon and rapidly returns to the cytoplasm upon removal of the stimulus (28Begitt A. Meyer T. van Rossum M. Vinkemeier U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10418-10423Crossref PubMed Scopus (130) Google Scholar). Similarly, p53 function is also tightly regulated through nuclear export and sequestration in the cytoplasm (29Lohrum M.A. Woods D.B. Ludwig R.L. Balint E. Vousden K.H. Mol. Cell. Biol. 2001; 21: 8521-8532Crossref PubMed Scopus (195) Google Scholar, 30Nikolaev A.Y. Li M. Puskas N. Qin J. Gu W. Cell. 2003; 112: 29-40Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 31Gu J. Nie L. Wiederschain D. Yuan Z.M. Mol. Cell. Biol. 2001; 21: 8533-8546Crossref PubMed Scopus (77) Google Scholar). Smad proteins accumulate in the nucleus in response to transforming growth factor-β signaling, and the regulation of activated Smad proteins is dependent on communication with cytosolic components of the transforming growth factor-β signaling pathway via continuous nucleocytoplasmic shuttling (32Xu L. Kang Y. Col S. Massague J. Mol. Cell. 2002; 10: 271-282Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 33Inman G.J. Nicolas F.J. Hill C.S. Mol. Cell. 2002; 10: 283-294Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Although the signals that determine MR nuclear import have not been characterized, import of GR in response to steroid occurs through two signals termed NL1 and NL2 (18Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar). NL1 is composed of a cluster of basic amino acids in the C-terminal region of the GR DNA-binding domain (DBD) (22Savory 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 (182) Google Scholar). The NL2 sequence resides within the GR ligand-binding domain (LBD), but has not been mapped to a specific amino acid segment (18Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar). GR and other nuclear hormone receptors have been observed to move continuously between the nucleus and cytoplasm in both their active and naïve states (34Guiochon-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, 35Chandran U.R. DeFranco D.B. Mol. Endocrinol. 1992; 6: 837-844PubMed Google Scholar, 36Guiochon-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, 37Dauvois S. White R. Parker M.G. J. Cell Sci. 1993; 106: 1377-1388Crossref PubMed Google Scholar, 38Madan A.P. DeFranco D.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3588-3592Crossref PubMed Scopus (144) Google Scholar, 39Haché 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, 40Baumann 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). Although regulatory consequences of shuttling have been hypothesized, specific effects largely remain to be discovered. In this study, we performed a detailed examination of the movement of MR between the cytoplasm and nucleus. By contrast to other nuclear receptors, we report that the transfer of MR to the nucleus is essentially unidirectional. Furthermore, the localization of MR to the nucleus in the absence and presence of hormone was dependent upon the complex interplay of three nuclear localization signals (NLSs) of different composition located in the N terminus, DBD, and LBD of the receptor, respectively. The N-terminal MR NLS, which was found to be primarily responsible for the nuclear localization of the naïve receptor, is a novel type consisting of a serine/threonine-rich motif that we show has the potential to be regulated by specific phosphorylation. Plasmids—Plasmids pTLGR, pTLGRNL1–, and pTLBuMR encoding full-length wild-type rat GR, full-length rat GR with the 513NNN515 NL1 mutation, and full-length rat MR with a N-terminal BuGR epitope tag, respectively, have been described previously (22Savory 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 (182) Google Scholar, 41Savory J.G. Prefontaine G.G. Lamprecht C. Liao M. Walther R.F. Lefebvre Y.A. Haché R.J. Mol. Cell. Biol. 2001; 21: 781-793Crossref PubMed Scopus (118) Google Scholar). pTLBuMRNL1– was derived from pTLBuMR by site-directed mutagenesis using the Stratagene QuikChange mutagenesis kit to introduce asparagine substitutions at Lys677, Lys678, and Lys681. The MR LBD sequence encompassing residues 698–981 was PCR-amplified and subcloned into pTL2 to generate pTLMR690C. pTLGGM encodes the GR N terminus and DNA-binding domain (including the GR NL1 sequence) encompassing residues 22–526 cloned in-frame with the MR LBD sequence. pTLGGMNL1– is a derivative of pTLGGM that contains the GR NL1 mutation. pTLGMM encodes the GR N terminus from residues 22 to 437 cloned in-frame with the MR DBD and LBD encompassing residues 600–981. pTLMGG encodes the MR N terminus from residues 1 to 602 cloned in-frame with the GR DBD and LBD encompassing residues 440–795. pTLMRΔ590–602 was cloned by amplification of the entire pTLMR sequence with primers flanking the sequence encoding residues 590–602, followed by ligation of the PCR product. pGFPMR1–602 was derived by cloning the MR N terminus (amino acids 1–602) into pEGFP-C1 (Clontech). pGFPMR encodes full-length rat MR as an N-terminal fusion with green fluorescent protein (GFP). PCR amplification products encompassing MR residues 1–550 and 150–602 were cloned into pEGFP-C1 to generate pGFPMR1–550 and pGFPMR150–602. To mutate the putative Borna disease virus-like NLS at residues 561–570 and 601 (S601D) in the context of GFP-MR, the QuikChange mutagenesis kit was used. The glutathione S-transferase (GST) coding sequence was PCR-amplified and then subcloned into pEGFP-C1. The derivative plasmids of pGSTGFP that express MR or GR peptides C-terminal to the GST-GFP moiety (as indicated in the figure legends) were generated by PCR amplification of the appropriate sequence. In the case of plasmids pGSTGFPNL0592/4/5A, pGSTGFPNL0592/4/5D, pGSTGFPNL0597/8/601A, pGSTGFPNL0597/8/601D, and the derivatives constructed with individual amino acid substitutions at the same positions, the appropriate mutations were introduced using PCR primers. The MR DBD, MR DBDWN, MR DBDNW, and MR DBDNN mutant expression constructs were subcloned by first generating the specified mutation in pTLBuMR using the QuikChange mutagenesis kit, followed by PCR amplification of the mutated sequence and subsequent insertion into pGSTGFP. The pGGMNL1–E959Q plasmid was constructed by replacing the fragment encoding amino acids 923–981 of MR in the GGMNL1– construct with the same fragment containing the point mutation E959Q described previously (17Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (327) Google Scholar). Expression of MR constructs in Sf9 cells was performed using the pIZ/V5-His expression vector (Invitrogen). The pGSTGFPMR constructs for expression in insect cells were prepared by cloning the relevant fragments from the mammalian expression vectors pGFPMR, pGFPMRΔ590–602, pGSTGFPNL0592/4/5A, pGSTGFPNL0592/4/5D, pGSTGFPNL0597/8/601A, pGSTGFPNL1, pGSTGFPNL1–, and pGSTGFPNL0597/8/601D (described above) into pIZ/V5-His. All expression vector inserts were verified by automated sequencing. The expression levels of all constructs were verified by Western analysis following transient transfection in COS-7 cells as described previously (20Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar). Cell Culture and Transient Transfection—COS-7 cells (American Type Culture Collection, CRL-1651) were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and nonessential amino acids. For subcellular distribution, COS-7 cells were transiently transfected using Lipofectamine™ (Invitrogen) according to the manufacturer's protocol. For each dish, between 0.5 and 1.0 μg of DNA was transfected using 8 μl of Lipofectamine reagent. The cells were incubated with the transfection mixture at 37 °C for 16 h. The transfection was stopped by adding an equal volume of phenol red-free Dulbecco's modified Eagle's medium supplemented with 20% charcoal-stripped fetal bovine serum such that the final concentration of fetal bovine serum was 10%. For transcriptional assays, COS-7 cells were seeded in 6-well plates 24 h prior to transfection using Lipofectamine as described above. A total of 0.25 μg of plasmid DNA containing 100 ng of the reporter plasmid pMMTVLuc, 100 ng of pCMVβ-gal (Promega), and 25 ng of the indicated tested plasmids were transfected into each well. 48 h post-transfection, the cells were washed with PBS and lysed in reporter lysis buffer (Promega). 20 μl of extract were assayed for luciferase activity, and 50 μlof extract were assayed for β-galactosidase activity using following the manufacturer's instructions. Luciferase results were normalized for transfection efficiency using the β-galactosidase values for each transfection. The data presented represent an average of three independent experiments done in triplicates. Error bars were calculated as S.D. between the experiments. Sf9 cells were maintained in Trichoplusia ni Medium-Formulation Hink medium at 30 °C and transfected with Cellfectin (Invitrogen) according to the InsectSelect™ system protocol (Invitrogen) for transient expression in insect cells using 1 μg of DNA/60-mm dish and serum-free Grace's insect cell culture medium (Invitrogen) for transfection. Quantification of Subcellular Distribution—For analysis of subcellular distribution in COS-7 and HeLa cells, following transfection, the cells were cultured overnight in complete serum and then seeded onto 22-mm square coverslips. Cells were allowed to attach for 8 h. To restrict the synthesis of new receptor during the course of the experiment, cells were then synchronized in G0 by serum withdrawal for 16 h. Cells were treated with 1 μm cortisol, aldosterone, or spironolactone as indicated with cycloheximide included in some experiments. To initiate steroid withdrawal, cells were rinsed five times (5 min at 37 °C) in 1× PBS containing 5% (w/v) bovine serum albumin and then twice in serum-free medium. The cells were rinsed a final time in serum-free medium and incubated at 37 °C for the indicated withdrawal period. For direct visualization of GFP fluorescence, cells were fixed with 3% paraformaldehyde for 30 min at 4 °C, followed by incubation with PBS containing 0.2 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 scored into five categories from exclusively nuclear to exclusively cytoplasmic as described previously (22Savory 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 (182) Google Scholar). Quantification was performed using double-blind encryption with individual data points derived from a minimum of 1000 cells quantified over three independent experiments performed in duplicate. For indirect immunofluorescence, following fixation in 3% paraformaldehyde and incubation with 0.2 mm glycine in PBS, cells were permeabilized by incubation with 0.5% Triton X-100 in PBS for 30 min at 20 °C. Cells were blocked with 5% normal goat serum in PBS for 1 h at 20 °C and then incubated at 4 °C with primary antibody. Following overnight incubation, the coverslips were washed three times in PBS and then incubated at 20 °C for 45 min with rhodamine red X-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted in PBS (1:150 (v/v)). Cells were mounted onto glass coverslips as described for direct analysis of subcellular distribution. Following double-blind encryption, subcellular distribution was quantified as described previously (20Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar, 22Savory 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 (182) Google Scholar). Fluorescence Recovery after Photobleaching (FRAP) Assays—COS-7 and HeLa cells were seeded into Bioptechs delta T4 culture dishes. Transient transfection with the expression plasmids indicated was carried out using 500 ng of plasmid DNA and 8 μl of Lipofectamine per dish following the manufacturer's protocol. After overnight incubation in Opti-MEM serum-reduced medium, transfection was stopped by replacing the medium with phenol red-free complete medium containing 10% charcoal-stripped fetal bovine serum. Cells were grown in complete medium for 8 h and then withdrawn from the serum for 16 h prior to FRAP or fluorescence loss in photobleaching (FLIP) assays. 20 μg/ml cycloheximide was added 30 min prior to assays to inhibit de novo protein synthesis. Experiments were performed using a Bio-Rad MRC 1024 confocal microscope. For FRAP assays, the signal in the appropriate cellular compartment was ablated using 10–20 laser pulses at full power, and the fluorescence return over time was monitored. For FLIP assays, for each repetition, a portion of each cell was bleached for 5 s at full power, followed by recording an image of the whole cell at 3% laser power. This was repeated at 30-s intervals over a 10-min period. Analysis was performed using Bio-Rad LaserSharp and NIH Image-J software. In FRAP, for nuclear and cytoplasmic bleaching, results are expressed as a percentage of the total initial intensity in the entire cell prior to bleaching. For binuclear cells in which one nucleus was bleached, results are expressed as a percentage of the total initial intensity of fluorescence in both nuclei prior to bleaching. In FLIP, the results are expressed as a percentage of the fluorescence for the appropriate compartment at t = 0. All assays are the average of at least four independent trials. Error bars were calculated as S.D. between the trials. Rapid Nuclear Import of MR in Response to Ligand Is Mediated through a Bipartite NLS—In this study, we sought to examine the nuclear cytoplasmic trafficking of MR over periods of up to 24 h. As synthesis of new receptor during this time would have the potential to mask the behavior of the initial receptor population, we performed our experiments under conditions in which synthesis of new receptor was minimal. In previous experiments with GR, we observed that withdrawing serum from COS-7 cells for 16 h 1 day following transient transfection results in a pool of receptor that is stably maintained for periods of at least 48 h in what become G0-synchronized cells (20Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar). Furthermore, this receptor pool is maintained in the absence of appreciable new receptor synthesis. Expression of MR with an N-terminal BuGR epitope tag in COS-7 cells through a similar protocol yielded a pool of MR whose levels declined only slightly in the 24-h period beginning 16 h following the withdrawal of serum from the tissue culture medium. MR levels were not affected by cycloheximide treatment coincident with serum withdrawal (Fig. 1A), indicating that minimal synthesis of new receptor occurred during this time. Aldosterone treatment induced a reduction in MR levels over the 24-h period, an indication of a destabilization in response to steroid that is common to steroid receptors (42Wallace A.D. Cidlowski J.A. J. Biol. Chem. 2001; 276: 42714-42721Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). This reduction in MR levels was also unaffected by cycloheximide treatment. To quantify MR localization through direct and indirect immunofluorescence, we employed a localization scoring protocol that we (20Sackey F.N. Haché R.J. Reich T. Kwast-Welfeld J. Lefebvre Y.A. Mol. Endocrinol. 1996; 10: 1191-1205PubMed Google Scholar, 22Savory 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 (182) Google Scholar) and others (43Ylikomi T. Bocquel M.T. Berry M. Gronemeyer H. Chambon P. EMBO J. 1992; 11: 3681-3694Crossref PubMed Scopus (254) Google Scholar) have described previously. Using this approach, MR expressed in COS-7 cells was determined to be distributed evenly between the nucleus and cytoplasm in >80% of the cells (Fig. 1B). This was consistent with the results of earlier studies examining the localization of naïve MR (24Fejes-Toth G. Pearce D. Naray-Fejes-Toth A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2973-2978Crossref PubMed Scopus (214) Google Scholar, 25Lombes M. Farman N. Oblin M.E. Baulieu E.E. Bonvalet J.P. Erlanger B.F. Gasc J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1086-1088Crossref PubMed Scopus (105) Google Scholar, 26Nishi M. Ogawa H. Ito T. Matsuda K.I. Kawata M. Mol. Endocrinol. 2001; 15: 1077-1092Crossref PubMed Scopus (84) Google Scholar, 27Sasano H. Fukushima K. Sasaki I. Matsuno S. Nagura H. Krozowski Z.S. J. Endocrinol. 1992; 132: 305-310Crossref PubMed Scopus (57) Google Scholar). Previous experiments with GR have shown that overexpression of the receptor in COS-7 cells results in an artificial shift of the receptor toward the nucleus (44Sanchez E.R. Hirst M. Scherrer L.C. Tang H.Y. Welsh M.J. Harmon J.M. Simons Jr., S.S. Ringold G.M. Pratt W.B. J. Biol. Chem. 1990; 265: 20123-20130Abstract Full Text PDF PubMed Google Scholar, 45Martins V.R. Pratt W.B. Terracio L. Hirst M.A. Ringold G.M. Housley P.R. Mol. Endocrinol. 1991; 5: 217-225Crossref PubMed Scopus (64) Google Scholar). However, this appeared unlikely to explain the partial nuclear accumulation of MR here, as our MR constructs were expressed at levels 4–6-fold lower than similarly tagged GRs (data not shown) that were localized almost exclusively to the cytoplasm in parallel experiments (Fig. 1B). Upon steroid treatment, both MR and GR transferred rapidly and completely to the nucleus (Fig. 1B). Nuclear receptors characteristically possess a major NLS (termed NL1) that is composed of basic amino acids at the C-terminal region of the receptor DNA-binding domain. For rat GR, amino acids 510–517 compose the core of the NLS and are required for NL1 activity (Fig. 1C). Alignment of MR with GR revealed a potentially disruptive two-amino acid (LG) insertion within the analogous basic amino acid cluster of MR. However, a second basic motif between amino acids 488 and 493 that may supplement the NLS activity of the core NL1 motif of GR (46Tang Y. Ramakrishnan C. Thomas J. DeFranco D.B. Mol. Biol. Cell. 1997; 8: 795-809Crossref PubMed Scopus (44) Google Scholar) was precisely represented in MR. Asparagine substitutions at lysine residues in the second basic cluster of GR have been shown previously to completely abrogate GR NL1 activity (GRNL1–) and to restrict overexpressed and antagonist-treated GR to the cytoplasm (22Savory 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 (182) Google Scholar). The analogous substitutions in MR (MRNL1–) (Fig. 1C) had only a modest effect on the localization of naïve MR, with the receptor remaining mostly nuclear or evenly distributed between the nucleus and cytoplasm in ∼65% of the cells (Fig. 1D). Upon steroid treatment, nuclear transfer of MRNL1– was reduced" @default.
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• W2030792950 title "A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus" @default.
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