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Originally published In Press as doi:10.1074/jbc.M501548200 on February 28, 2005

J. Biol. Chem., Vol. 280, Issue 17, 17549-17561, April 29, 2005
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A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus*

Rhian F. Walther{ddagger}§, Ella Atlas¶, Amanda Carrigan{ddagger}, Yanouchka Rouleau{ddagger}, Allison Edgecombe¶, Laura Visentin||, Claudia Lamprecht¶, Gregory C. Addicks¶, Robert J. G. Haché¶||**, and Yvonne A. Lefebvre¶||

From the Departments of Medicine and ||Biochemistry, Microbiology, and Immunology and the {ddagger}Graduate Program in Biochemistry, Ottawa Health Research Institute, Ottawa Hospital, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

Received for publication, February 9, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mineralocorticoid receptor (MR)1 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) (1, 2). 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 11{beta}-hydroxysteroid dehydrogenase-2, which converts cortisol or corticosterone to inactive keto metabolites (3). In tissues in which 11{beta}-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 (4, 5). Glucocorticoid signaling in these tissues involves both overlapping targeting of MR and GR in the nucleus and heteromeric interactions between the two receptors (6–10).

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 (11, 12).

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 (13), whereas the androgen receptor has been widely reported to localize to the cytoplasm prior to ligand binding (14–16). Similarly, GR is almost exclusively cytoplasmic prior to exposure to steroid (17–20) and recycles back to the cytoplasm following the termination of steroidal signaling (21–23). By contrast, naïve MR is distributed evenly in the nucleus and cytoplasm in most cell types studied (24–27).

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 (28). Similarly, p53 function is also tightly regulated through nuclear export and sequestration in the cytoplasm (29–31). Smad proteins accumulate in the nucleus in response to transforming growth factor-{beta} signaling, and the regulation of activated Smad proteins is dependent on communication with cytosolic components of the transforming growth factor-{beta} signaling pathway via continuous nucleocytoplasmic shuttling (32, 33).

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 (18). NL1 is composed of a cluster of basic amino acids in the C-terminal region of the GR DNA-binding domain (DBD) (22). The NL2 sequence resides within the GR ligand-binding domain (LBD), but has not been mapped to a specific amino acid segment (18). GR and other nuclear hormone receptors have been observed to move continuously between the nucleus and cytoplasm in both their active and naïve states (34–40). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (22, 41). 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. pTLMR{Delta}590–602 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 (17).

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, pGFPMR{Delta}590–602, 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 (20).

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 LipofectamineTM (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 pCMV{beta}-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 {beta}-galactosidase activity using following the manufacturer's instructions. Luciferase results were normalized for transfection efficiency using the {beta}-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 InsectSelectTM 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 1x 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 (22). 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 (20, 22).

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (20). 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 (42). This reduction in MR levels was also unaffected by cycloheximide treatment.



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  		FIG. 1.
Partial nuclear localization of naïve MR and characterization of a bipartite ligand-dependent NL1 motif. A, Western analysis of the levels of MR in transiently transfected COS-7 cells incubated in the presence or absence of 20 µg/ml cycloheximide. 16 h post-transfection, cells were withdrawn from serum for 16 h and then incubated in the presence or absence of cycloheximide for 24 h. B, localization of MR and GR expressed in COS-7 cells before and after a 1-h treatment with 1 µM aldosterone (MR) or cortisol (GR) determined by indirect immunofluorescence using antibody BuGR. All quantifications throughout this work are compilations of at least three independent experiments performed in duplicate. Error bars indicate the means ± S.E. Representative micrographs are shown beneath each data set. Scale bars = 10 µm in all photomicrographs. N, nucleus; C, cytoplasm. C, alignment of rat MR (rMR) and rat GR (rGR) indicating the position of the KKK-to-NNN NL1– substitution that abrogates NL1 function in GR. The basic clusters of amino acids within the sequences shown are underlined, and the Lys-to-Asn substitutions made in the respective MRNL1– and GRNL1– constructs are indicated. D, effect of NL1– substitution on the localization of GR and MR before and after a 6-h treatment with steroid. E, transcriptional activation of an MMTV-luciferase reporter gene upon inactivation of NL1. Luciferase activity was assayed 24 h following addition of 10–6 M dexamethasone (GR) or aldosterone (MR) to cells cotransfected with the indicated expression plasmids and the MMTV-luciferase reporter gene and withdrawn from serum for 16 h prior to steroid treatment. Luciferase activity is expressed as -fold induction by steroid over vehicle-treated cells corrected for {beta}-galactosidase activity from a cotransfected standard. Error bars represent the S.E. of a minimum of three independent experiments performed in duplicate. F, effects of amino acid substitutions in basic amino acid clusters on the localization of a synthetic protein composed of amino acids 604–683 of MR fused to the C terminus of GST-GFP. The localization of constructs encoding the WT MR sequence is compared with that of constructs containing the substitutions indicated in the schema between amino acids 676 and 683 (MR DBDWN), between amino acids 650 and 655 (MR DBDNW), or at both positions (MR DBDNN).

 
To quantify MR localization through direct and indirect immunofluorescence, we employed a localization scoring protocol that we (20, 22) and others (43) 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 (24–27).

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 (44, 45). 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 (46) 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 (22). 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 relative to the nuclear localization of wild-type (WT) MR, with the receptor becoming at least mostly nuclear in only 70% of the cells. However, this localization of MRNL1– to the nucleus in response to aldosterone still exceeded the nuclear localization of GRNL1– following cortisol treatment.

For GR, its transcriptional activation potential has been shown to be directly proportional to the extent of its transfer to the nucleus following steroid treatment. To determine whether the NL1– substitution that impeded MR nuclear localization also affected the transcriptional regulatory potential of MR, we compared the ability of WT and NL1– MR and GR to induce transcription through the steroid response elements of mouse mammary tumor virus (Fig. 1E). As observed previously (22), the NL1– substitution in GR reduced transcription from the murine mammary tumor virus (MMTV) promoter. By contrast, MRNL1– induced transcription to the same extent as WT MR, suggesting that the NL1– substitution in MR did not affect localization sufficiently to affect the transactivation potential of the receptor.

Although our data implicated MR Lys677, Lys678, and Lys681 as contributing to the MR NL1 activity, the reduced consequence of substitution at these positions compared with GR suggested that the MR NL1 signal might be more complex than the GR signal. To evaluate the relative importance of the two basic motifs in MR for NL1-mediated nuclear import, we examined the subcellular distribution of a series of MR peptides encompassing the MR DBD to the end of the NL1 region expressed as fusion proteins with GFP coupled to GST (GST-GFP) (Fig. 1F).

Active transport through the nuclear pore is required for proteins >70 kDa in size. Smaller proteins can also gain access to the nucleus through passive diffusion. Thus, at 50 kDa, GST-GFP alone is small enough to have retained the ability to access the nucleus through passive diffusion (47) and thus was observed in our experiments to be equally distributed between the nucleus and cytoplasm of most cells (Fig. 1F).

Addition of the MR DBD peptides to GST-GFP increased the size of the fusion proteins expressed to 70 kDa, a size at which passive diffusion would be expected to be restricted, and the proteins localized predominantly to the cytoplasm in the absence of an NLS. The strong NL1 activity within the WT MR peptide directed the GST-GFP-MR-DBD peptide almost completely to the nucleus (Fig. 1F). Asparagine substitutions in each of the lysine 650–655 and 676–683 clusters (MRNW and MRWN) individually decreased the NLS activity of the MR peptide, resulting in a shift of the fusion proteins toward the cytoplasm, so the GFP signal was equally distributed between the nucleus and cytoplasm in 50–60% of the cells and cytoplasmic in most of the remaining cells. Substitution within both basic clusters at the same time (MR DBDNN) resulted in a further shift in the fusion protein to the cytoplasm, suggesting that each basic cluster on its own retained a small measure of NLS activity. These results indicate that, in contrast to GR, MR NL1 is a bipartite motif in which each of the basic clusters has a similar importance in NLS function. This suggests that the MR and GR NL1 motifs interact differently with the nuclear import machinery and opens the possibility for differential regulation of their activities.

A Serine/Threonine-rich NLS with the Potential to Be Regulated by Phosphorylation Directs Nuclear Localization of Naïve MR—As substitutions within the N-terminal basic cluster of MR NL1 compromise DNA binding by the receptor, it was not possible to directly compare the transcriptional regulatory potential of GR and MR with completely inactivated NL1 motifs at this stage of our study. Comparison of the relative importance of the two NL1 motifs was also likely to be complicated by the potential of an additional NLS in MR that directed the partial nuclear localization of the naïve receptor.

Analysis of the localization of several GR/MR chimeras substantiated this hypothesis and pointed to the presence of a novel NLS in the N terminus of MR (Fig. 2A). A GR/MR chimera consisting of the N terminus and DBD of GR fused to the LBD of MR (GGM) localized to the cytoplasm in the absence of steroid in a manner comparable with unliganded GR. Similarly, a GMM construct composed of the N terminus of GR and the DBD and LBD of MR was also cytoplasmic. By contrast, the reverse MGG construct displayed a partially nuclear localization that was similar to that of MR. All constructs were steroid-responsive and moved completely to the nucleus upon steroid treatment (data not shown).



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  		FIG. 2.
MR contains a hormone-independent NLS (NL0) that requires amino acids 590–602. A, photomicrographs showing the subcellular distribution of MR; GR; and composite receptors MGG, GMM, and GGM determined by indirect immunofluorescence. B, direct fluorescence observation of the subcellular distribution of a series of N-terminal MR peptides expressed as GFP fusion proteins. The amino acid sequences displayed highlight the similarity in the basic amino acid spacing between amino acids 550 and 570 of MR and the NLS of the Borna disease virus (BDV) p10 protein, with the small letters indicating the substitutions in the MR-(1–602)BDV– construct. N, nucleus; C, cytoplasm. C, effect of deletion of amino acids 595–602 on the localization of a fusion protein composed of amino acids 550–602 fused to the C terminus of GST-GFP. D, indirect immunofluorescence determination of the effect of deletion of amino acids 590–602 on the localization of full-length MR before and after a 1-h treatment with 1 µM aldosterone (aldo).

 
Expression of the 602 amino acids composing the N terminus of MR fused to GFP confirmed the presence of a nuclear localization activity that mimicked that observed for naïve WT MR (Fig. 2B). However, examination of the sequence of the N terminus of MR failed to reveal any strong similarities to classical basic NLSs. Thus, to delimit the location of the putative NLS within this region of MR, we initiated a comparison of the localization of the N- and C-terminal deletion constructs of the MR N terminus fused to GFP (Fig. 2B). Intriguingly, the first construct prepared, which lacked the first 150 amino acids of the MR N terminus (MR-(150–602)) was strongly shifted toward the nucleus, suggesting that this region of MR may contain an activity that helps to retain the protein in the cytoplasm. By contrast, deletion of 50 amino acids from the C terminus of MR-(1–602) resulted in a GST-MR fusion protein (MR-(1–550)) that was almost completely cytoplasmic.

Close examination of the amino acids in region 550–602 of MR indicated a match in the spacing between three basic residues in this region and the NLS of Borna disease virus protein p10 (48). However, a substitution within MR (R561N/R562N) analogous to that which abrogated the activity of the p10 NLS failed to compromise the nuclear localization of GFP-MR-(1–602) (Fig. 2B).

Expression of MR amino acids 550–602 fused with GST-GFP confirmed that this region of MR (which we have termed NL0) contains NLS activity (Fig. 2C). Furthermore, this nuclear localization activity was compromised upon deletion of just seven amino acids from the C terminus of the MR peptide (MR-(550–595)), indicating that amino acids 595–602 are important for NLS function.

The striking feature of region 550–602 of MR is a cluster of five serines and one threonine between amino acids 590 and 602 (Fig. 3). This Ser/Thr-rich region was determined to be required for the nuclear localization of naïve MR, as deletion of amino acids 590–602 from full-length MR strongly reduced nuclear occupancy of the receptor in the absence of steroid (Fig. 2D). MR{Delta}590–602 still transferred completely to the nucleus in response to aldosterone, indicating that NL0 is not required for nuclear localization of the liganded receptor.



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  		FIG. 3.
Amino acid charge differentially modulates NL0 activity. A, effect of Ala and Asp substitutions within amino acids 590–602 of MR on the localization of a GST-GFP fusion protein containing MR amino acids 550–602 (GST-GFP-MR-(550–602), abbreviated GST-GFP-NL0) as determined by observation of direct fluorescence. The sequence between amino acids 590–602 of MR is shown with the cluster of the three N-terminal Ala-to-Asp substitutions highlighted by asterisks and the three C-terminal substitutions highlighted by dots. The nomenclature of the individual constructs indicates the positions of the substitutions and the substitution (Ala or Asp) made. N, nucleus; C, cytoplasm. B, effect of single amino acid Ser-to-Asp or Thr-to-Asp substitutions on the localization of GST-GFP-MRNLO. The nomenclature highlights the specific mutations within the parental construct. C, effect of Ser-to-Asp substitution at amino 601 on the localization of full-length GFP-MR. D, MR(S601D) is without effect on the transcriptional activation of the MMTV-luciferase reporter by MR. Luciferase assay of transcriptional activation of the MMTV-luciferase reporter in COS-7 cells transiently transfected as indicated and withdrawn from serum for 16 h prior to treatment with aldosterone or vehicle. Luciferase activity is expressed as -fold induction by steroid over vehicle-treated cells corrected for {beta}-galactosidase activity from a cotransfected standard. Error bars represent the S.E. of a minimum of three independent experiments performed in duplicate.

 
To assess in detail the importance of the Ser/Thr cluster to NL0 and whether NL0 might be susceptible to modulation through modification of one or more of these residues, we compared the subcellular localization of GST-GFP-MR-(550–602) fusion proteins with alanine and aspartic acid substitutions (Fig. 3). Localization of GST-GFP-MR-(550–602) was unaffected by Ser-to-Ala substitution of amino acids 592, 594, and 595 (Fig. 3A). By contrast, Ser-to-Asp substitutions at the same positions significantly enhanced nuclear localization of the fusion protein. Conversely, Ser-to-Ala substitutions at 597, 598, and 601 yielded a fusion protein (GST-GFP-NL0(S597A/S598A/S601A)) that was significantly shifted to the nucleus. Strikingly, mimicking phosphorylation with Ser-to-Asp substitutions at the same positions eliminated NL0 activity, resulting in a fusion protein with localization similar to that of GST-GFP.

Next, we refined our analysis to consider the effects of single amino acid substitution on the localization of GST-GFP-MR-(550–602) (Fig. 3B). Individual substitution of Asp for Ser or Thr at amino acids 592, 594, and 595 had only a modest effect (if any) on the localization of GST-GFP-MR-(550–602), suggesting that the enhancement of NL0 activity observed in Fig. 3A resulted from the accumulation of negative charge at all three positions.

Individual Ser-to-Asp substitutions at amino acids 597 and 598 also had little effect on the NL0 activity. However, Ser-to-Asp substitution at amino acid 601 was sufficient to abrogate nuclear localization of GST-GFP-MR-(550–602). These results suggest that Ser601 of MR might be crucial for NL0 activity and that its modification may provide a means to localize MR to the cytoplasm. By contrast, it appears that potentiation of NL0 activity would require the cumulative modification of at least Ser592, Ser594, and Thr595.

To determine the potential for modification of Ser601 to alter the localization of full-length MR, we assessed the affect of the S601D substitution on the localization of full-length MR expressed as a GFP fusion protein (Fig. 3C). WT GFP-MR was distributed equally between the nucleus and cytoplasm in at least 90% of the cells. By contrast, GFP-MR(S601D) was shifted toward the cytoplasm to the same relative extent as we had observed earlier in Fig. 2D for MR{Delta}590–602. These data highlight that the Ser/Thr cluster between amino acids 590 and 602 of MR is integral to the MR NL0 nuclear localization signal and suggest that modification of Ser601 through phosphorylation may act to restrict naïve MR to the cytoplasm. However, as before with MR{Delta}590–602, the S601D substitution failed to interfere with the transfer of MR to the nucleus in response to aldosterone. Moreover, MR(S601D) stimulated reporter gene transcription from the MMTV promoter to the same extent as WT MR (Fig. 3D).

Although naïve MR has been reported to be distributed throughout the cell in most cell types, the naïve receptor has been reported to be largely cytoplasmic when ectopically expressed in insect cells (49–52). As the activity of most NLSs is conserved between yeast and higher vertebrates (53), these reports suggested that the MR NL0 motif might be specific to mammalian cells.

To determine the potential for NL0 to function in insect cells, we expressed our series of GST-GFP-NL0 constructs in Sf9 cells (Fig. 4). As in mammalian cells, addition of amino acids 550–602 of MR to GST-GFP shifted the fusion protein to the nucleus, indicating that the MR NL0 nuclear transport mechanism was conserved in insect cells. This also suggests that the predominant cytoplasmic localization of naïve MR in insect cells results from an active difference in the receptor rather than the absence of an NL0 nuclear import pathway. Moreover, the Ser-to-Ala/Thr-to-Asp modifications that altered NL0 activity in COS-7 cells were observed to have the same effect in Sf9 cells. Indeed, the stimulatory effect of the S592D/S594D/S595D substitution appeared to be enhanced in Sf9 cells.



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  		FIG. 4.
MR NL0 is functional in insect cells. Shown is the localization of GST-GFP-MR-(550–602) (abbreviated GST-GFPNL0) and derivatives with the indicated Ala and Asp substitutions expressed in Sf9 cells by transient transfection. Localization was determined by direct fluorescence as described in the legend to Fig. 2.

 
MR Exhibits a Steroid-dependent NL2 Activity—In addition to the NL1 NLS, GR and the estrogen receptor have been shown to have a second NLS (termed NL2) that occurs within their LBDs. NL2 is a strictly steroid-dependent NLS that mediates slower and incomplete nuclear transfer to the nucleus (18). NL2-mediated nuclear import of GR has been shown recently to be dependent upon importin-7 (54). NL2 is localized within the LBDs of GR and the estrogen receptor, but does not feature an obvious basic amino acid motif (18, 43). The MR LBD, when expressed alone (MR690C), responded to aldosterone treatment by strongly increasing its localization to the nucleus (Fig. 5A), indicating that the MR LBD also contains an NL2 nuclear localization activity.



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  		FIG. 5.
MR contains an agonist-dependent NL2-like activity that is independent of the ability of MR AF-2 to interact with coactivators. A, examination of the subcellular distribution patterns of MR690C and the GGMNL1– chimeric receptor in the absence of ligand and following treatment with 1 µM aldosterone by indirect immunofluorescence. N, nucleus; C, cytoplasm. B, inactivation of NL1 in GGM reduces transcriptional activation potential. Shown is a comparison of the induction of transcription of the MMTV-luciferase reporter by GGM and GGMNL1– in cells treated with 10–6 M aldosterone or vehicle for 24 h following incubation of the cells in serum-free medium for 16 h. Luciferaseactivity is expressed as -fold induction by steroid over vehicle-treated cells corrected for {beta}-galactosidase activity from a cotransfected standard. Error bars represent the S.E. of a minimum of three independent experiments performed in duplicate. C, translocation of GGMNL1– to the nucleus of COS-7 cells following treatment with 1 µM aldosterone analyzed by indirect immunofluorescence. D, localization of GGMNL1– during a 6-h treatment with 1 µM spironolactone (spirono; upper) and of GFP-MR following a 1-h spironolactone treatment (lower). E, comparison of the localization of GGMNL1– and GGMNL1–E959Q containing a glutamate-to-glutamine substitution at position 959 that abrogates the interaction of MR AF-2 with p160 coactivators and p300/CBP before and after a 6-h treatment with aldosterone.

 
The complex nature of the MR NL1 motif and the presence of NL0 in the N terminus rendered validation of NL2 within the context of full-length MR lacking any additional NLS activity difficult. However, coupling of the LBD of MR to the N terminus and DBD of GR containing the NL1– substitution, which has been shown previously to lack any NLS activity (22), provided a mimic for the full-length receptor in which nuclear localization would be dependent on NL2. Transfer of GGMNL1– to the nucleus in COS-7 cells occurred equivalently to the transfer of the MR LBD alone. Transfer of GGMNL1– to the nucleus in COS-7 cells occurred equivalently to the transfer of the MR LBD alone, indicating that MR NL2 is certain to function as an NLS in the context of full-length MR and that NL2 has the ability to function independently from NL1 and NL0. Furthermore, the steady-state accumulation of GGMNL1– following steroid treatment closely resembled the accumulation of GRNL1– seen in the experiments shown in Fig. 1. By contrast to MR(S601D) and MRNL1–, which activated transcription of an MMTV reporter gene similarly to WT MR, GGMNL1– exhibited a reduced transcriptional activation potential compared with GGM (Fig. 5B). This reduction in transcriptional activation potential was similar to the difference seen previously between GR and GRNL1– (22), suggesting a threshold relationship between nuclear localization and transcriptional activation by MR rather than a strictly linear correlation.

NL1-mediated nuclear import of WT MR and WT GGM occurred rapidly upon exposure to steroid, being largely complete with 10 min of treatment (data not shown), as is typical for NL1-mediated nuclear import of steroid receptors (18, 20, 24, 55). By contrast, NL2-mediated nuclear import occurred more slowly (Fig. 5C), with localization of GGMNL1– reaching steady state in just over 1 h.

Although MR NL2 was similarly responsive to aldosterone, cortisol, and the synthetic glucocorticoid dexamethasone (data not shown), the response appeared to be almost completely dependent on steroid agonist, as the mineralocorticoid antagonist spironolactone stimulated little if any transfer of GGMNL1– to the nucleus (Fig. 5D). By contrast, spironolactone strongly stimulated the transfer of MR to the nucleus in these experiments. Spironolactone also effectively competed the transcriptional response of GGMNL1– and MR to steroid agonist (data not shown).

The strict agonist dependence of the MR NL2 activity suggested the possibility that this activity reflect the cotransport of MR to the nucleus with transcriptional coactivators that interact with the steroid-dependent AF-2 transactivation function in the MR LBD. To assess this possibility, we tested the effect of a substitution in GGMNL1– (GGMNL1–E959Q) that has been shown recently to abrogate AF-2 function without affecting steroid binding (56, 57). Localization of GGMNL1–E959Q exactly paralleled localization of GGMNL1–, with aldosterone treatment inducing a similar accumulation of the two proteins in the nucleus (Fig. 5E). This supports NL2 being an inherent property of the MR LBD rather than a reflection of the cotransport of MR by coactivator factors.

Localization of the naïve receptors was also similar in this experiment, although, in this series, both GGMNL1– and GGMNL1–E959Q were somewhat shifted to the cytoplasm in the absence of steroid compared with the experimental series shown in Fig. 5C. The reason for this variation in initial receptor distribution remains to be clarified, but appears to correlate with variations in the serum employed to maintain the cells.

Transfer of MR between the Cytoplasm and Nucleus Is Essentially Unidirectional—A complex aspect of steroid receptor action is the degree to which steroid hormone receptors exchange or shuttle between the nucleus and cytoplasm. We demonstrated recently that nuclear export of WT GR occurs only very slowly with a t1/2 of several hours under normal cell culture conditions (58). This slow rate of nuclear export was observed both for liganded GR and for receptor following the withdrawal of steroid treatment. Other nuclear receptors exhibit similar slow nuclear export under at least some conditions (55). The slow export of GR from the nucleus appears to result from active retention of the receptor within the nucleus through a process that involves a signal that overlaps with NL1.2 Thus, the NL1– substitution in GR results in a receptor that is lost from the nucleus upon the withdrawal of steroid treatment with a half-time of ~1–2 h (22).

By contrast to GR, transfer of MR between the cytoplasm and nucleus unexpectedly appeared to be essentially unidirectional in COS-7 cells (Fig. 6). In the first instance, treatment of cells with 10 µM aldosterone, a concentration of steroid sufficient to saturate both MR and GR, induced the rapid and complete nuclear transfer of both receptors to the nucleus (Fig. 6A). Steroid withdrawal was accomplished through extensive washing of the cells in the presence of bovine serum albumin, which has steroid binding activity.



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  		FIG. 6.
MR is retained in the nucleus upon hormone withdrawal by a sequence that does not overlap with NL1. A, localization of GR and MR following withdrawal of cells from a 1-h treatment with 1 µM aldosterone (Ald.) for the times indicated. N, nucleus; C, cytoplasm. B, localization of MRNL1– following withdrawal (w/d) of cells from a 1-h treatment with 1 µM aldosterone (aldo) for the times indicated. C, transfer of a synthetic GST-GFP fusion protein containing an SV40 NLS and an human immunodeficiency virus Rev nuclear export signal (NES) from the donor to acceptor nucleus in a FRAP experiment performed in multinucleated COS-7 cells as described under "Experimental Procedures." Transfer was measured over a 30-min time course following irreversible ablation of GFP fluorescence in the acceptor nucleus. Error bars represent the S.D. over the course of a minimum of four experiments. D, transfer of GFP-MR from the donor to acceptor nucleus in a FRAP experiment performed in multinucleated COS-7 cells treated with 1 µM aldosterone for 1 h.

 
Upon steroid withdrawal, GR returned to the cytoplasm over a 16-h period (Fig. 6A), in the same way GR has been observed previously to return to the cytoplasm in cells withdrawn from cortisol or the synthetic steroid dexamethasone (21, 22, 38). By contrast, we were unable to detect redistribution of MR from the nucleus over the same period. This did not appear to be simply a result of more active nuclear retention coincident with MR NL1. Substitution of Lys677, Lys678, and Lys681 in MR, which is the substitution directly analogous to the GRNL1– substitution that dramatically accelerates GR nuclear export, had only a marginal effect in rescuing nuclear export of MR following steroid withdrawal (Fig. 6B). Compared with GRNL1–, where export to the cytoplasm is complete within 1–2 h (22), only ~20% of MRNL1– redistributed from the nucleus to the cytoplasm over 24 h following the withdrawal of aldosterone.

To study the movement of proteins between the nucleus and cytoplasm in greater detail, we recently described a FRAP procedure in which GFP-tagged protein trafficking between the nuclei of multinucleated cells is recorded (58). Many tissue culture cell lines contain a significant number of cells that stably maintain two or more nuclei. These cells offer the potential to follow the shuttling of a protein between the nucleus and cytoplasm by tracking the movement of fluorescently tagged proteins from one nucleus to a second nucleus in which the initial fluorescent signal has been irreversibly ablated by photobleaching. Using this approach, we have shown that nuclear export of liganded WT GR occurs slowly in naïve cells with kinetics indistinguishable from those for the redistribution of GR to the cytoplasm upon steroid withdrawal. Moreover, we demonstrated that the more rapid nuclear export of liganded GR observed in heterokaryon fusion assays results from the transient release of calreticulin from the endoplasmic reticulum during cell fusion (58).

In COS-7 cells expressing a synthetic GFP control protein in which nuclear import is directed by the SV40 NLS and nuclear export is directed by the human immunodeficiency virus Rev nuclear export signal, re-equilibration of GFP fluorescence between the nuclei of multinucleated cells following photobleaching of one nucleus approached completion within 30 min of FRAP (Fig. 6C). By contrast, no significant recovery of fluorescence was observed for GFP-MR in aldosterone-treated cells over 1 h following photobleaching (Fig. 6D).

To determine whether these results reflect the static localization of MR within the nucleus, we performed FLIP experiments in which the mobility of MR within the nucleus and cytoplasm was measured. First, GFP-MR was observed to be highly mobile within the nucleus, as photobleaching of the GFP-MR signal in one corner of the nucleus of aldosterone-treated cells at 30-s intervals led to the nearly complete depletion of the GFP-MR signal from the nucleus within 10 min (Fig. 7A). A similar rapid mobility was also observed for naïve GFP-MR in both the nucleus and cytoplasm, with ablation of fluorescence within a box covering equal sections of the nucleus and cytoplasm, also resulting in the loss of GFP-MR fluorescence over 10 min (Fig. 7B).



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  		FIG. 7.
MR moves freely within the nucleus and cytoplasm, and naïve MR moves continuously to the nucleus. A, FLIP analysis of GFP-MR in the nucleus. COS-7 cells were treated with 1 µM aldosterone for 1 h. Photobleaching was performed at 30-s intervals over 10 min as described under "Experimental Procedures." The area ablated is boxed. Error bars represent the S.D. over the course of a minimum of four experiments. B, simultaneous FLIP analysis of the movement of naïve GFP-MR in the cytoplasm. The area ablated is boxed. C, FRAP analysis of the transfer of naïve GFP-MR from the nucleus to the cytoplasm. Transfer was recorded over a 2-h period following ablation of the GFP-MR signal from the cytoplasm of COS-7 cells. D, FRAP analysis of the transfer of naïve GFP-MR from the cytoplasm to the nucleus. Transfer was recorded over a 2-h period following ablation of the GFP-MR signal from the nuclei of COS-7 cells.

 
FRAP analysis of the cytoplasm of cells containing naïve GFP-MRs distributed equally between the nucleus and cytoplasm demonstrated that naïve nuclear GFP-MR was also not exported from the nucleus over 2 h following photobleaching (Fig. 7C). Similarly, treatment of naïve cells with the export inhibitor leptomycin B had no effect on the subcellular localization of MR (data not shown). In striking contrast, however, nuclear GFP-MR fluorescence was recovered over a 2-h period following photobleaching of the naïve GFP-MR signal from the nucleus (Fig. 7D). These results identify the half-time for the nuclear import of naïve MR as being ~1 h, but leave open the question of why naïve MR never accumulates fully in the nucleus in the absence of nuclear export.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding the regulation of the movement of transcription factors between the nucleus and cytoplasm is central to understanding their activation, turnover, and regulation by post-translational modification. Most nuclear receptors have been shown to exchange between the nucleus and cytoplasm. For some, including the glucocorticoid and androgen receptors, this exchange appears to be integrally linked with the regulation of steroidal signaling. Unexpectedly, we have observed that MRs differ from other nuclear receptors in moving continuously toward the nucleus without apparent means to return to the cytoplasm.

Nuclear import is mediated through the binding of NLSs to one of at least 20 importin nuclear transport receptors (53). Movement of MR into the nucleus was determined to be mediated through three nuclear import signals. Each signal is uniquely regulated, with one signal being ligand-independent (NL0), one being agonist-specific (NL2), and the third being responsive to both steroid agonists and antagonists (NL1). The signals are also biochemically distinct, with NL1 being composed of a bipartite basic motif, NL2 lacking an obvious basic motif and being dependent on LBD conformation, and NL0 requiring a serine/threonine motif that represents a new class of NLS. Determining whether these three NLSs mediate nuclear import through the same pathways or whether they reflect the use of distinct nuclear transport receptors, as might be predicted by their biochemistry, will be key to understanding the potential to differentially manipulate the three NLS activities.

Although protein phosphorylation is known to regulate the access of trafficking receptors to nuclear localization and nuclear export signals alike, the serine/threonine motif in NL0 represents the first instance where the serines/threonines are integral to the trafficking sequence. Amino acid substitution experiments revealed that NL0 activity has the potential to be both activated and repressed by phosphorylation, depending on the residues modified. Most notably, conversion of Ser601 to Asp blocks NL0 activity. Analysis of the amino acid sequence through the NL0 region with NetPhos Version 2.0 (59) indicates that Ser601 has a particularly high probability of being phosphorylated. PhosphoBase Version 2.0 analysis (60) suggests that protein kinase A and casein kinase-1 as likely kinases for Ser601.

Recent studies have indicated that naïve cytoplasmic MR preferentially colocalizes with 11{beta}-hydroxysteroid dehydrogenase-2 on the external surface of the endoplasmic reticulum (61, 62). This colocalization serves as a means of providing maximal protection of MR from activation by glucocorticoids in tissues such as the kidney. Thus, regulating NL0 activity through phosphorylation of Ser601 and other residues has the potential to directly impact on the responsiveness of MR to glucocorticoids in 11{beta}-hydroxysteroid dehydrogenase-2-containing tissues.

Despite the high degree of overall similarity between MR and GR and the overlap in steroid responsiveness, the signals directing the import of these two receptors have clear differences. Although the cluster of basic amino acids at positions 511–517 in GR is necessary and sufficient for NL1 activity, the MR NL1 sequence is clearly bipartite, dependent in equal part on two basic amino acid clusters. A 2-bp insertion with the potential to weaken the strength of the NLS activity of the C-terminal basic motif in MR is one likely explanation for the increased importance of the N-terminal basic cluster for MR NL1 activity. These differences in the MR and GR NL1 motifs provide the potential for a second means of differentially regulating the nuclear import of MR and GR by separating their interactions with the nuclear import machinery.

A number of instances of transcription factors gaining access to the nucleus through cotransport have been reported (66, 67). Both p160 coactivators and p300/CBP have potent NLSs and interact strongly with steroid receptors in an agonist-specific manner. However, neither appears to be important for NL2-mediated nuclear import of MR because a mutation abrogating MR interaction with these factors failed to affect NL2 activity. Although it is still possible that NL2 reflects cotransport of MR through coactivator factors that interact differently with the LBD, our results provide direct evidence that NL2 function is inherent to the LBD. This also is consistent with a recent report suggesting that GR NL2 has a specific ability to interact with importin-7 (54).

Although nuclear hormone receptors are known to be exported from the nucleus, their rate of export appears to be generally much slower than is usually observed for active nuclear export pathways. Our recent results indicated that the nuclear export of GR (and likely other nuclear receptors) may be opposed by an active nuclear retention activity that overlaps with the receptor NL1 motif.3 Our results here with MR were even more striking, with essentially no movement of the receptor to the cytoplasm being detected within 24 h of steroid withdrawal. Furthermore, little difference was observed with a substitution in MR analogous to the nuclear retention-disrupting substitution in GR, suggesting that the control of nuclear localization of MR may be very rigid indeed.

By contrast to reports that nuclear receptor retention in the nucleus results from the stable interaction of the spent receptor with the nuclear matrix (23), our FLIP results indicate that MR remains highly mobile within the nucleus following steroid withdrawal. Furthermore, the naïve receptor is similarly mobile in both the nucleus and cytoplasm. It will be important to determine whether the absence of MR redistribution to the cytoplasm following steroid withdrawal affects secondary responses to steroid.

It has been shown recently that the transient release of calreticulin from the endoplasmic reticulum dramatically accelerates the export of many nuclear receptors, including GR, from the nucleus (63, 64). Calreticulin is a calcium-binding chaperone protein localized to the lumen of the endoplasmic reticulum. Calreticulin-mediated receptor nuclear export depends upon physical interaction with the first finger of nuclear receptor DBDs (65), a region of MR that is exactly conserved in GR and is highly conserved in other nuclear receptors whose export has also been shown to be induced through this pathway. Thus, induction of MR nuclear export subsequent to activation of the calreticulin pathway seems likely. The physiological conditions under which the calreticulin export pathway functions remain to be identified, but our recent studies indicate that calreticulin-mediated nuclear export occurs as a specific response to stressors that induce the transient release of calreticulin from the endoplasmic reticulum (58).

In summary, the tight control of the nuclear import and stability of MR highlights a nuclear receptor dependent upon exquisite regulatory control of its localization in the cells. Delimitation of context-specific utilization of and regulation of individual NLSs within MR may be expected to provide significant insight into the regulation of differential steroidal signaling through this complex receptor.


   FOOTNOTES
 
* This work was supported in part by Operating Grant MOP 53142 from the Canadian Institutes of Health Research (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

** Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: Ottawa Health Research Inst., Ottawa Hospital, 725 Parkdale Ave., Ottawa, Ontario K1Y 4E9, Canada. Tel.: 613-761-5142; Fax: 613-761-5036; E-mail: rhache{at}ohri.ca.

1 The 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. Back

2 R. F. Walther, A. Carrigan, E. Atlas, and R. J. G. Haché, manuscript in preparation. Back

3 R. F. Walther, A. Carrigan, E. Atlas, and R. J. G. Haché, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank S. Kato for the MR(E959Q) expression construct.



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