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J. Biol. Chem., Vol. 278, Issue 43, 41998-42005, October 24, 2003
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From the
Departments of Urology and ¶Molecular Pharmacology and Biological Chemistry, the ||Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
Received for publication, March 10, 2003 , and in revised form, August 1, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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100 kDa in size, must be actively transported through the nuclear pore complex to enter or leave the nucleus. AR is localized to the cytoplasm in the absence of ligand and translocates into the nucleus in the presence of ligand (5–7). Likewise, nuclear AR can be exported to the cytoplasm upon ligand withdrawal (8). These observations indicate that AR contains both a nuclear localization signal (NLS) and nuclear export signal (NES) and that their activities are regulated by androgen either directly or indirectly. Because nuclear localization is necessary for steroid receptors to transactivate their target genes, the ligand-dependent nuclear import/export represents a critical mechanism by which steroids regulate the activity of their cognate receptors.
The signal involved in the transport of steroid receptors from the cytoplasm to the nucleus has been studied extensively. Mutagenesis studies of the human AR have defined a bipartite NLS in the DBD and hinge region at amino acids 617–633 (9). This NLS is composed of two clusters of basic amino acids (underlined) separated by 10 amino acid residues: RKCYEAGMTLGARKLKK. Similar NLS were found in other steroid receptors. In addition to the NLS in the DBD and hinge region, Picard and Yamamoto (10) demonstrated the existence of a ligand-dependent NLS present in the LBD of GR. When fused to 
-galactosidase or to N-terminal fragments of the GR lacking the NLS, the LBD of GR conferred ligand-dependent nuclear localization to the fusion proteins. Similarly, a ligand-dependent NLS also exists in the LBD of AR, which is capable of inducing nuclear import in the absence of the NLS in the DBD and hinge region (11, 12).
Studies on sequences responsible for transportation of steroid receptors from the nucleus to the cytoplasm are limited. The best characterized nuclear export mechanism uses the export receptor CRM1 that binds to proteins containing a leucine-rich NES to direct protein export from the nucleus. Steroid receptors lack any leucine-rich sequence that would resemble a classical NES (13). Studies with progesterone receptor (PR) showed that nuclear export of PR involves its NLS and is not mediated by CRM1/exportin 1 (13, 14). PR export does not involve Ran GTP and does not depend upon the hydrolysis of GTP. Recent studies with GR showed that the DBD of steroid receptors can function as an NES and that the nuclear export of GR requires the binding of calreticulin (15, 16). Because the calreticulin-binding site and NLS are in the DBD and hinge region rather than in the LBD, it remains unclear how steroids regulate the nuclear/cytoplasmic shuttling of steroid receptors by means of the calreticulin-binding site and NLS.
The fact that AR intracellular localization is regulated by androgen has led to the hypothesis that the LBD is involved in the regulation of AR nuclear/cytoplasmic shuttling. Through mutagenesis studies, we have identified NESAR, a region in the LBD that is necessary for AR nuclear export and is dominant over the reported NLS in the DBD and hinge region in the absence of ligand. When hormone is present, NESAR is repressed, and the NLS directs nuclear localization of the AR. This ligand-regulated NES is also present in MR and ER and contains many amino acid residues in the immediate vicinity of the bound ligand. Our studies provide insights into the mechanism by which steroids regulate intracellular localization of steroid receptors.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Once the NES sequence was identified in the LBD region corresponding to AR (742–817), we tried to amplify the cDNA coding for AR (742–817). We were unable to amplify this region; however, we were able to amplify the cDNA coding for AR (743–817) and clone it into pEGFP-C1 and pEGFP-AR (1–665) vectors, resulting in expression vectors pEGFP-NESAR (743–817) and pEGFP-AR (1–665)-NESAR (743–817). We also generated the GFP-AR(
NESAR) expression vector; the deletion of NESAR was PCR-based. Substitution mutagenesis of pEGFP-NESAR (743–817) was accomplished to introduce nucleotide substitutions over a conserved region corresponding to amino acids YFAPDAR (762–766). The substituted nucleotides encoded the amino acid sequence GPLGS. This NESAR mutant was cloned into pEGFP-C1 to produce pEGFP-NESAR-GPLGS762. The regions in estrogen receptor (ER) and MR corresponding to the NESAR were cloned into pEGFP-C1 to generate pEGFP-NESER (386–455) and pEGFP-NESMR (809–883). Furthermore, the peptide sequence of GSNELALKLAGLDINKTGGC, corresponding to the NES of the heat-stable protein kinase inhibitor (13, 17), was cloned into pEGFP-C1 to generate pEGFP-NESPKI.
Cell Culture and Transfection—The human prostate cancer cell line PC3 was obtained from American Type Culture Collection (Manassas, VA). PC3 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37 °C in the presence of 5% CO2 in a humidified incubator.
The various AR deletion constructs were transfected into PC3 cells using FuGENE 6 according to the manufacturer's protocol (Roche Applied Science). PC3 cells were transfected at >60% confluence in 6-well plates in phenol red-free cFBS-RPMI. To induce nuclear translocation of GFP-AR and GFP-LBD, 10–8 M dihydrotestosterone was added at the time of transfection. The localization of GFP fusion proteins was visualized 16 h after transfection with fluorescence microscopy using either a Leica DM-IL microscope or a Nikon TE 2000U inverted microscope. Cytoplasmic localization in transfected cells was defined when the GFP fluorescence in the cytoplasm was greater than in the nuclei. Likewise, nuclear localization was defined when the GFP fluorescence in the nuclei was greater than in the cytoplasm. Even distribution indicates that GFP fluorescence was evenly distributed between the nucleus and cytoplasm in transfected cells.
Inhibition of CRM-1-mediated nuclear export by leptomycin B (see Fig. 5, LMB) was based on experiments described previously (8). Four hours after transfection of PC3 cells, the media were replaced with cFBS-RPMI containing 15 ng/ml LMB. Localization of GFP fusion proteins was observed through fluorescence microscopy 12 h after LMB addition. GFP-NESPKI was used as a control in parallel experiments.
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To test whether liganded nuclear GFP-LBD fusion protein undergoes nuclear export upon ligand withdrawal, PC3 cells were transfected with GFP-LBD (see Fig. 2). Four hours after transfection, the cells were treated with 10–8 M mibolerone (mib) for 16 h to induce nuclear localization of GFP-LBD, which was confirmed by fluorescence microscopy. Then, cells were washed five times with cFBS-RPMI and cultured in cFBS-RPMI under androgen-free conditions for 4 h to induce export of nuclear GFP-LBD in the presence or absence of LMB (see Fig. 2). The protein synthesis inhibitor cycloheximide (50 µg/ml) was added 2 h prior to the ligand withdrawal to ensure that cytoplasmic GFP-LBD was exported from the nucleus rather than derived from de novo synthesis in the cytoplasm (8). The localization of GFP-LBD in transfected cells was analyzed by fluorescence microscopy 24 h after transfection. GFP-NESPKI was used as a control in parallel experiments.
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Quantification of cells exhibiting cytoplasmic localization of GFP-tagged fusion proteins was carried out by counting 20–200 transfected cells/dish in at least two dishes in each experiment. All of the experiments were reproduced at least once.
ClustalW Analysis—To determine homology, the amino acid sequences of NESAR, NESER, and NESMR were entered into the ClustalW program accessed from the European Bioinformatics Institute web site. ClustalW generated a multiple sequence alignment and graded amino acid identity and similarity, distinguishing between highly conserved residues and semi-conserved residues.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We separately transfected GFP-AR and GFP-LBD expression vectors into the human prostate cancer cell line, PC3. PC3 cells were chosen as a model in this study because high transfection efficiency is easily obtained in these cells. As expected, unliganded AR is localized predominantly to the cytoplasm and translocates into the nuclei in the presence of dihydrotestosterone (DHT, Fig. 1). Interestingly, the LBD alone is also localized to the cytoplasm in the absence of ligand and undergoes nuclear translocation in the presence of dihydrotestosterone. This observation demonstrates the importance of the LBD in androgen regulation of AR nuclear/cytoplasmic shuttling. Further, it indicates the existence of a signal in the LBD that specifies cytoplasmic localization in the absence of ligand. In the presence of hormone, GFP-LBD translocates to the nucleus, suggesting that this signal is repressed upon ligand binding. This result also confirms the existence of an NLS in the liganded LBD.
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Cytoplasmic Localization of GFP-LBD Is Due to CRM1-independent Nuclear Export—To determine whether the cytoplasmic localization of GFP-LBD was due to nuclear export, hormone-withdrawal experiments were conducted. Briefly, PC3 cells were transfected with GFP-LBD. The cells were treated with androgen agonist mibolerone to induce nuclear import of GFP-LBD, resulting in nuclear localization of GFP-LBD (Fig 2A). As expected, GFP-LBD was localized to the cytoplasm after hormone withdrawal. The cytoplasmic GFP-LBD was exported from the nucleus rather than derived from de novo synthesis in the cytoplasm because the presence of the protein synthesis inhibitor cycloheximide did not affect intracellular localization of GFP-LBD after hormone withdrawal (Fig. 2A). This observation suggests the existence of a novel NESAR in the LBD of AR.
To examine whether the nuclear export of GFP-LBD depends upon CRM1, the hormone withdrawal experiments were repeated in the presence or absence of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export (Fig. 2B). Cycloheximide was again used to prevent new protein synthesis in the cytoplasm. LMB did not prevent nuclear export of GFP-LBD fusion protein after hormone withdrawal (Fig. 2B), suggesting that GFP-LBD nuclear export is CRM1-independent. In parallel control experiments, LMB inhibited the nuclear export of the GFP-tagged NES of the heat stable protein kinase inhibitor NESPKI, a CRM1-dependent nuclear export signal sensitive to LMB inhibition (13), resulting in even localization or predominantly nuclear localization of GFP-NESPKI in the majority of the transfected cells (Fig. 2B). Our finding is in agreement with a previous report demonstrating that nuclear export of the AR was insensitive to LMB inhibition (8). Taken together, our observations indicate the existence of a CRM1-independent, ligand-regulated NESAR in the LBD.
Identification of NESAR in the LBD—Figs. 1 and 2 show that cytoplasmic localization of GFP-LBD in the absence of ligand is caused by the presence of an NESAR in GFP-LBD. A mutant GFP-LBD with NESAR disrupted should be evenly distributed between the cytoplasm and nucleus. On the other hand, if NESAR were not deleted, the mutant GFP-LBD construct would predominantly localize to the cytoplasm. Based on the above rationale, we employed deletion mutagenesis to map the NESAR in the LBD. Nine N-terminal (n1–n9) and nine C-terminal (c1–c9) LBD deletion mutants (Fig. 3E) were generated and cloned into the pEGFP-C1 expression vector.
Fig. 3A shows the effect of N-terminal deletion on the intracellular localization of GFP-LBD. The n1–n3 mutants exhibit cytoplasmic localization in 97% of the transfected cells. The n4 and n5 deletion mutants are localized to the cytoplasm in 65 and 20% of the transfected cells, respectively. The n6–n9 deletion mutants are evenly distributed between the nuclei and cytoplasm in more than 90% of the transfected cells. This result indicates that the N-terminal boundary of the NESAR is around the n3 position at amino acid residue 742 in AR.
Fig. 3B shows the effect of C-terminal deletion on the intracellular localization of GFP-LBD. The c7–c9 deletion mutants are localized to cytoplasm in >93% of the transfected cells. In contrast, the c1–c3 mutants are evenly distributed in >97% of the transfected cells. The c4, c5, and c6 mutants are localized to the cytoplasm in 42, 60, and 85% of the transfected cells, respectively. This observation indicates that the C-terminal boundary of the NESAR is around the c6 position at amino acid residue 817 in the AR. The results from both N- and C-terminal deletion analyses suggest that this NESAR is localized within the region of amino acid residues 742–817 of AR.
The above experiments identified the region containing NESAR from the LBD in the absence of the N-terminal region, DBD, and hinge region of AR. It is well established that the DBD and hinge region contain an NLS; we sought to determine whether NESAR can specify cytoplasmic localization in the presence of other AR domains. To address this question, amino acid residues 1–665 of AR, consisting of the N-terminal region, DBD, and hinge region, were inserted between GFP and the various LBD deletion mutants in our expression vectors. This procedure resulted in a series of new expression vectors for tripartite fusion proteins consisting of GFP, AR (1–665), and various deletion mutants of LBD, denoted N1–N9 or C1–C9 (Fig. 3E). The inclusion of AR (1–665) in the fusion protein did not inhibit the activity of the NESAR in the LBD deletion mutants (Fig. 3, C and D).
Fig. 3C shows the result of N-terminal deletion. The N1, N2, and N3 deletion mutant fusion proteins are localized to the cytoplasm in the transfected cells. In contrast, the N6, N7, N8, and N9 deletion mutant fusion proteins, which lack the region in the LBD containing NESAR, are localized to the nuclei of >90% transfected cells, presumably because of the NLS in the DBD and hinge region in AR (1–665). N4 and N5 deletion mutant fusion proteins, which only partially contain NESAR, are localized to cytoplasm in 51 and 27% of the transfected cells, respectively. The above observation suggests that the NESAR in LBD deletion mutants is dominant over the NLS in DBD and hinge region of the AR.
The studies with the GFP, AR (1–665), and C-terminal deletion mutant tripartite fusion proteins also argue that the NESAR in the LBD is dominant over the NLS in the DBD and hinge region (Fig. 3D). As expected, C1, C2, and C3 mutant fusion proteins are localized to the nuclei in >95% of the transfected cells, presumably because of the NLS in AR (1–665). C4, C5, and C6 mutants are either predominantly localized to the cytoplasm or nuclei, with C4, C5, and C6 having cytoplasmic localization in 29.5, 57.5, and 74.5% of the transfected cells, respectively. C7, C8, and C9 deletion mutants, all of which contain the entire NESAR, were localized to the cytoplasm in >93% of the transfected PC3 cells.
NESAR Is Necessary and Sufficient for AR Cytoplasmic Localization—Our mutagenesis studies identified NESAR in the LBD. To determine whether NESAR is necessary for cytoplasmic localization of AR, we have generated an expression vector for GFP-tagged AR with the deletion of NESAR. In the absence of NESAR, the mutant AR is predominantly localized to the nuclei of the transfected cells (Fig. 4). This observation argues that NESAR is necessary for nuclear export of AR. To determine whether NESAR is sufficient to confer cytoplasmic localization, we cloned the amino acid sequence corresponding to 743–817 of the AR into the pEGFP-C1 expression vector. Transient transfection of the expression vector into the PC3 cells showed that NESAR is sufficient to specify cytoplasmic localization of the tagged GFP reporter protein (Fig. 4). The above observations indicate that NESAR is necessary and sufficient for AR cytoplasmic localization.
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NESAR Is Dominant over the NLS in the DBD and Hinge Region of AR—Results in Fig. 3, C and D suggest that NESAR in the LBD is dominant over the NLS in the DBD and hinge region. To substantiate the above finding, we generated expression vector for fusion protein GFP-AR (1–665)-NESAR consisting of GFP at the N terminus, AR (1–665) in the middle, and NESAR at the C terminus. The percentage of cells exhibiting cytoplasmic localization of GFP-AR (1–665)-NESAR is virtually identical to that of GFP-NESAR (Fig. 4), suggesting that NESAR is dominant over the NLS in the DBD and hinge region within AR (1–665).
NESAR Is Insensitive to Leptomycin B Inhibition—Nuclear export of steroid receptors including AR is insensitive to LMB inhibition (8, 13, 15, 18). Thus, NESAR-mediated nuclear export should also be insensitive to LMB. As expected, LMB had a minimal effect on the cytoplasmic localization of GFP-NESAR, GFP-LBD, and GFP-AR fusion proteins (Fig. 5). In our control experiment, leptomycin B prevented predominant cytoplasmic localization of GFP-tagged NESPKI, which is known to be LMB-sensitive (13).
The NES Is Functionally Conserved in the Steroid Receptor Superfamily—Because the LBD is conserved in the steroid receptor superfamily, the NES is also, not surprisingly, evolutionarily conserved. We cloned the regions in ER (amino acids 386–455) and MR (amino acids 809–883) corresponding to NESAR into the pEGFP-C1 vector, generating pEGFP-NESER (386–455) and pEGFP-NESMR (809–883). Fig. 6A shows the sequence similarities, as determined by ClustalW analysis (19), between NESAR, NESER, and NESMR. The three NES share 18.7% identity, with an additional 25% of the residues being highly conserved. Although the amino acid sequence homology for the NES is moderate, the structure of NES is very similar, consisting of helix 5 through helix 8 of the LBD and a highly conserved -turn between helix 5 and helix 6. This NES contains at least seven amino acid residues in the immediate vicinity of the ligand (20, 21), suggesting that the NES activity in the LBD can be regulated by ligand binding. As expected, NESMR and NESER are sufficient to cause cytoplasmic localization of the fusion proteins in transfected PC3 cells (Fig. 6B). The GFP-NESER and GFP-NESMR fusion proteins are localized to the cytoplasm of 78 and 100% of the transfected PC3 cells, respectively.
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The high sequence identity in the -turn (Fig. 6A) suggests that these amino acid residues may be important for nuclear export activity of NESAR. Adopting a mutagenesis strategy used to characterize the NLS in the DBD and hinge region of AR (9), we substituted residues YFAPDAR (762–766) of NESAR with the sequence GPLGS. When cloned into pEGFP-C1, we designated the resulting fusion protein GFP-NESAR-GPLGS762. In transient transfection experiments with PC3 cells, the percentage of transfected cells exhibiting cytoplasmic localization of GFP-NESAR-GPLGS762 is only slightly less than that of GFP-NESAR (Fig. 6, B and C). This finding suggests that cytoplasmic localization of GFP-NESAR does not depend upon the identity of the conserved amino acid residues YFAPDAR (762–766) in the -turn region (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The observation that GFP-LBD fusion protein is localized to the cytoplasm in the absence of androgen and to the nuclei in the presence of androgen (Fig. 1) indicates that the LBD contains a ligand-regulated nuclear export signal, NESAR. The NESAR is active in the absence of androgen and is repressed in the presence of androgen. The androgen withdrawal experiment in Fig. 2 confirms the existence of NESAR and indicates that NESAR is insensitive to LMB. According to our deletion mutagenesis studies (Fig. 3), NESAR consists of residues in helix 5 through helix 8 of the LBD and has at least seven amino acid residues in the immediate vicinity of the bound ligand (Fig. 6). Thus, it is not surprising that ligand binding can regulate the activity of the NES in the intact LBD. The identification of NESAR also provides an explanation for previous findings that deletion of regions overlapping NESAR tended to reduce the cytoplasmic localization of the AR mutants (9, 22).
Figs. 1 and 2 demonstrate that the LBD of AR performs nuclear export activity in the absence of ligand and nuclear import activity in the presence of ligand. Some peptide sequences are bidirectional signals, exhibiting either NES or NLS activity (23). Thus, an interesting possibility is that the NES in the intact LBD could function as an NLS upon ligand binding. This would explain the nuclear import of GFP-LBDAR in the presence of androgen.
Our studies showed that the NESAR is necessary and sufficient to cause AR nuclear export (Fig. 4). GFP-AR(NESAR) is localized to the nuclei of the transfected cells, demonstrating the necessity of NESAR in AR nuclear export. On the other hand, GFP-NESAR exhibited cytoplasmic localization in >80% of the transfected cells, indicating that NESAR is sufficient to cause nuclear export.
Our observations suggest that the mechanism of AR nuclear export is identical to that of NESAR-mediated nuclear export. Previous studies showed that nuclear export of steroid receptors, including AR, GR, and PR, is insensitive to LMB, an inhibitor of CRM1/exportin 1 (8, 13, 15, 18). This observation is consistent with the finding that NESAR does not share sequence similarity with the leucine-rich NES, which directs export mediated by the CRM1/exportin 1 pathway. If NESAR is truly the nuclear export signal responsible for AR nuclear export, the NESAR-mediated nuclear export should also be insensitive to LMB. As expected, LMB had virtually no influence on cytoplasmic localization of GFP-NESAR (Fig. 5).
Our studies indicate the existence of this NES in the LBD of at least two other steroid receptors. We have demonstrated that GFP-NESER and GFP-NESMR are localized to the cytoplasm of 78 and 100% of transfected cells, respectively (Fig. 6). This NES is likely to be present in additional steroid receptors. For example, rat GR mutants with deletions protruding into the region which would correspond to NESGR also show impaired cytoplasmic localization (10). Although the amino acid sequences of the NES are only 18.6% identical, the structure of the NES is very similar, consisting of helices 5–8 of the LBD and the highly conserved -turn region between helix 5 and helix 6 (Fig. 6A). Interestingly, when five amino acids in the middle of the -turn region (YFAPDAR (762–766)) were replaced with the sequence GPLGS, only a small decrease in the cytoplasmic localization of this construct was observed (Fig. 6, B and C). Because the -turn region is the most highly conserved section of the NES (Fig. 6A), the minimal effect of the GPLGS substitution presents the possibility that the three dimensional structure of the NES rather than its exact amino acid composition may be critically important for its nuclear export activity.
AR has a well characterized NLS in the DBD and hinge region. Our studies showed that, in the absence of ligand, NESAR in the LBD is dominant if both NESAR and NLS are present in the same peptide. This result provides an explanation for androgen regulation of AR nuclear/cytoplasmic shuttling. In the absence of ligand, NESAR in the LBD is active and dominant, causing nuclear export of AR. In contrast, the binding of ligand inhibits NESAR in the LBD, induces NLS activity in the LBD, and alleviates the NESAR-dependent repression of the NLS in the DBD and hinge region, leading to efficient nuclear import of AR.
Our observations argue strongly that NESAR is a nuclear export signal, although we were unable to directly demonstrate that with nuclear microinjection. This was caused by our inability to purify fusion proteins containing NESAR for nuclear injection, because the GST fusion proteins containing NESAR are present only in the insoluble fraction. Nevertheless, our data show that NESAR is required for AR cytoplasmic localization.
In summary, we have identified NESAR, a region in the LBD of AR which specifies cytoplasmic localization. NESAR is active and dominant over the NLS in the absence of ligand. The binding of ligand to AR inhibits NESAR and simultaneously releases repression of the NLS, leading to AR nuclear localization. This NES is also present in other steroid receptors. Our findings provide a mechanistic explanation for the ligand-dependent nuclear import/export of AR and other steroid receptors.
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FOOTNOTES |
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Supported by NCI, National Institutes of Health-sponsored training Grant T32 CA80621 from the Robert H. Lurie Comprehensive Cancer Center.
** O'Conner Family Research Professor of Urology. To whom correspondence should be addressed: Dept. of Urology, Tarry 11-715, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-908-2264; Fax: 312-908-7275; E-mail: wangz{at}northwestern.edu.
1 The abbreviations used are: AR, androgen receptor; DBD, DNA-binding domain; LBD, ligand-binding domain; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; NLS, nuclear localization signal; NES, nuclear export signal; PR, progesterone receptor; CRM1, chromosome region maintenance 1; ER, estrogen receptor ; GFP, green fluorescent protein; pEGFP, plasmid enhanced GFP; FBS, fetal bovine serum; LMB, leptomycin B; cFBS-RPMI, phenol red-free RPMI 1640 medium containing 10% charcoal-stripped FBS, 1% glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin; mib, mibolerone.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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