Identification and Characterization of a Ligand-regulated Nuclear Export Signal in Androgen Receptor

Abstract

Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor α and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors. Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor α and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors. Androgen receptor (AR) 1The 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; ERα, estrogen receptor α; 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. is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner (1Mainwaring W. Gross F. Grumbach M. Labhart A. Lipsett M. Mann T. Samuels L. Zander J. The Mechanism of Action of Androgen. Springer Verlag, New York1977: 10Google Scholar, 2Zhou Z. Wong C. Sar M. Wilson E. Recent Progress in Hormone Research. 1994; 49: 249-274PubMed Google Scholar). AR is necessary for male accessory organ development and is involved in prostate cancer progression. The human AR contains 918 amino acids and consists of the N-terminal transactivation domain (1–555), DNA-binding domain (DBD) (556–623), hinge region (624–665), and C-terminal ligand-binding domain (LBD) (666–918) (3Chang C.S. Kokontis J. Liao S.T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7211-7215Crossref PubMed Scopus (459) Google Scholar). The DBD, hinge region, and LBD of AR share a high degree of homology with other steroid receptors. Intracellular localization of AR and other steroid receptors such as glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is ligand-dependent. The limit for passive diffusion across the nuclear pore complex has been suggested to fall within the range of 20–40 kDa (4Marte B. Nat. Cell. Bio. 2001; 3: E135Crossref PubMed Scopus (3) Google Scholar). Larger proteins such as AR, which is ∼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 (5Georget 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, 6Simental 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, 7Roy A.K. Tyagi R.K. Song C.S. Lavrovsky Y. Ahn S.C. Oh T.S. Chatterjee B. Ann. N. Y. Acad. Sci. 2001; 949: 44-57Crossref PubMed Scopus (104) Google Scholar). Likewise, nuclear AR can be exported to the cytoplasm upon ligand withdrawal (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar). 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 (9Zhou Z.X. Sar M. Simental J.A. Lane M.V. Wilson E.M. J. Biol. Chem. 1994; 269: 13115-13123Abstract Full Text PDF PubMed Google Scholar). 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 (10Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar) demonstrated the existence of a ligand-dependent NLS present in the LBD of GR. When fused to β-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 (11Jenster G. van der Korput J.A. Trapman J. Brinkmann A.O. J. Steroid Biochem. Mol. Biol. 1992; 41: 671-675Crossref PubMed Scopus (64) Google Scholar, 12Poukka H. Karvonen U. Yoshikawa N. Tanaka H. Palvimo J.J. Janne O.A. J. Cell Sci. 2000; 113: 2991-3001Crossref PubMed Google Scholar). 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 (13Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar). Studies with progesterone receptor (PR) showed that nuclear export of PR involves its NLS and is not mediated by CRM1/exportin 1 (13Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar, 14Guiochon-Mantel A. Delabre K. Lescop P. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7179-7183Crossref PubMed Scopus (79) Google Scholar). 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 (15Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (225) Google Scholar, 16Black B.E. Holaska J.M. Rastinejad F. Paschal B.M. Curr. Biol. 2001; 11: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). 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 ERα 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. Expression Vector Construction—Cloning into the expression vector pEGFP-C1 (Clontech) results in fusion proteins with GFP at the N terminus. The GFP tag allows for convenient visualization by fluorescence microscopy. We generated pEGFP-AR to express GFP-AR fusion protein, pEGFP-LBDAR (666–918) to express GFP-LBD fusion protein, and pEGFP-AR (1–665) to express the GFP-AR (1–665) fusion protein. AR (1–665) consists of the N-terminal region, DBD, and hinge region. We also generated one set of expression vectors for fusion proteins with GFP at the N terminus and LBD deletion mutants at the C terminus and another set for fusion proteins with GFP at the N terminus, AR (1–665) in the middle, and LBD deletion mutants at the C terminus (see Fig. 3E). Various AR mutants were generated by anchor PCR using the full-length human AR cDNA as the template, which was kindly provided by Dr. Liao (3Chang C.S. Kokontis J. Liao S.T. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7211-7215Crossref PubMed Scopus (459) Google Scholar). expression vectors were by The were for the NES sequence was identified in the LBD region to AR we to the cDNA for AR We were to this however, we were to the cDNA for AR and it into pEGFP-C1 and pEGFP-AR (1–665) in expression vectors and pEGFP-AR We also generated the expression the deletion of NESAR was mutagenesis of was to over a conserved region to amino acids The the amino acid sequence This NESAR was into pEGFP-C1 to The in estrogen receptor α and MR to the NESAR were into pEGFP-C1 to and the sequence of to the NES of the protein (13Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar, W. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google was into pEGFP-C1 to Cell and human prostate cancer was from were in RPMI 1640 medium with 10% fetal bovine 1% glutamine, 100 units/ml penicillin, and 100 μg/ml at in the presence of in a The AR deletion were into using to the were at in in phenol red-free nuclear of GFP-AR and was at the of The localization of GFP fusion proteins was with fluorescence using either a or a localization in was defined the GFP fluorescence in the cytoplasm was than in the Likewise, nuclear localization was defined the GFP fluorescence in the nuclei was than in the that GFP fluorescence was the nucleus and cytoplasm in of nuclear export by leptomycin (see Fig. was on (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar). of the were with containing of GFP fusion proteins was through fluorescence was used as a in nuclear GFP-LBD fusion protein nuclear export upon ligand were with GFP-LBD (see Fig. the were with for to nuclear localization of which was by fluorescence microscopy. were with and in for to export of nuclear GFP-LBD in the presence or absence of (see Fig. The protein was to the ligand withdrawal to that GFP-LBD was exported from the nucleus rather than from in the cytoplasm (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar). The localization of GFP-LBD in was by fluorescence was used as a in of localization of fusion proteins was by in at two in of the were at the amino acid sequences of NESAR, and were into the from the generated a sequence and amino acid and conserved residues and Androgen Regulation of of the of the of the LBD of AR in nuclear import and we the full-length AR or the LBD acids with a green fluorescent protein AR is from the AR (5Georget 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, R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar). The GFP tag to the intracellular localization of the AR and AR We GFP-AR and GFP-LBD expression vectors into the human prostate cancer were as a in this high is in AR is localized to the cytoplasm and translocates into the nuclei in the presence of Fig. the LBD is also localized to the cytoplasm in the absence of ligand and nuclear in the presence of This the of the LBD in androgen regulation of AR nuclear/cytoplasmic shuttling. it the existence of a signal in the LBD that localization in the absence of ligand. In the presence of GFP-LBD translocates to the suggesting that this signal is repressed upon ligand binding. This also the existence of an NLS in the of GFP-LBD to the localization of GFP-LBD was to nuclear were were with The were with androgen to nuclear import of in nuclear localization of GFP-LBD GFP-LBD was localized to the cytoplasm hormone The GFP-LBD was exported from the nucleus rather than from in the cytoplasm the presence of the protein not intracellular localization of GFP-LBD hormone withdrawal This the existence of a NESAR in the LBD of AR. the nuclear export of GFP-LBD upon CRM1, the hormone withdrawal were in the presence or absence of leptomycin an of nuclear export was used to protein in the not nuclear export of GFP-LBD fusion protein hormone withdrawal suggesting that GFP-LBD nuclear export is In the nuclear export of the NES of the protein a nuclear export signal to (13Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google in localization or nuclear localization of in the of the Our is in with a that nuclear export of the AR was to (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar). observations indicate the existence of a ligand-regulated NESAR in the of NESAR in the 1 and that localization of GFP-LBD in the absence of ligand is by the presence of an NESAR in A GFP-LBD with NESAR be the cytoplasm and nucleus. the other NESAR were not the GFP-LBD would to the on the we deletion mutagenesis to the NESAR in the N-terminal and C-terminal LBD deletion mutants were generated and into the pEGFP-C1 expression Fig. the of N-terminal deletion on the intracellular localization of The mutants localization in of the The and deletion mutants are localized to the cytoplasm in and of the The deletion mutants are the nuclei and cytoplasm in than of the This that the N-terminal of the NESAR is the at amino acid in AR. Fig. the of C-terminal deletion on the intracellular localization of The deletion mutants are localized to cytoplasm in of the In the mutants are in of the The and mutants are localized to the cytoplasm in and of the This that the C-terminal of the NESAR is the at amino acid in the AR. The results from both and C-terminal deletion suggest that this NESAR is localized within the region of amino acid residues of AR. The identified the region containing NESAR from the LBD in the absence of the N-terminal region, DBD, and hinge region of AR. is that the DBD and hinge region contain an we to NESAR can localization in the presence of other AR this amino acid residues of AR, of the N-terminal region, DBD, and hinge region, were GFP and the LBD deletion mutants in expression This in a of expression vectors for fusion proteins of GFP, AR and deletion mutants of LBD, or 3E). The of AR (1–665) in the fusion protein not the activity of the NESAR in the LBD deletion mutants C and Fig. the of N-terminal The and deletion fusion proteins are localized to the cytoplasm in the In the and deletion fusion which lack the region in the LBD containing NESAR, are localized to the nuclei of of the NLS in the DBD and hinge region in AR and deletion fusion which contain NESAR, are localized to cytoplasm in and of the The 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 and C-terminal deletion fusion proteins also that the NESAR in the LBD is dominant over the NLS in the DBD and hinge region and fusion proteins are localized to the nuclei in of the of the NLS in AR and mutants are either localized to the cytoplasm or with and localization in and of the and deletion of which contain the NESAR, were localized to the cytoplasm in of the NESAR and for AR mutagenesis studies identified NESAR in the NESAR is necessary for localization of AR, we have generated an expression vector for AR with the deletion of In the absence of NESAR, the AR is localized to the nuclei of the This that NESAR is necessary for nuclear export of AR. NESAR is to we the amino acid sequence to of the AR into the pEGFP-C1 expression of the expression vector into the showed that NESAR is to localization of the GFP protein The observations indicate that NESAR is necessary and for AR NESAR over the NLS in the DBD and of in Fig. C and suggest that NESAR in the LBD is dominant over the NLS in the DBD and hinge region. the we generated expression vector for fusion protein GFP-AR of GFP at the N terminus, AR (1–665) in the middle, and NESAR at the C terminus. The of localization of GFP-AR is to that of suggesting that NESAR is dominant over the NLS in the DBD and hinge region within AR NESAR to export of steroid receptors AR is to (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar, R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar, J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (225) Google Scholar, J. Mol. Endocrinol. 2000; 14: PubMed Scopus Google Scholar). nuclear export also be to a on the localization of and GFP-AR fusion proteins In leptomycin localization of which is to be (13Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar). The NES in the Steroid the LBD is conserved in the steroid receptor the NES is not We the in ERα acids and MR acids to NESAR into the pEGFP-C1 and Fig. the sequence as by 1994; PubMed Scopus Google NESAR, and The NES share with an of the residues the amino acid sequence homology for the NES is the of NES is of through of the LBD and a conserved and This NES contains at amino acid residues in the immediate vicinity of the ligand J.M. W. V. P. D. H. Nat. Biol. 3: PubMed Scopus Google Scholar, P. M. C. S. N. S. P. A. S. M. U. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google suggesting that the NES activity in the LBD can be regulated by ligand binding. and are to localization of the fusion proteins in The and fusion proteins are localized to the cytoplasm of and of the The high sequence in the that amino acid residues be for nuclear export activity of a mutagenesis used to the NLS in the DBD and hinge region of AR (9Zhou Z.X. Sar M. Simental J.A. Lane M.V. Wilson E.M. J. Biol. Chem. 1994; 269: 13115-13123Abstract Full Text PDF PubMed Google we residues of NESAR with the sequence When into we the fusion protein In with the of localization of is than that of and This that localization of does not depend upon the of the conserved amino acid residues in the region This the and of a NES that AR NESAR is dominant over the NLS in the DBD and hinge region in the absence of hormone and is repressed by ligand binding. This NES is also conserved in the LBD of MR and Our findings provide insights into the mechanism of ligand-dependent nuclear/cytoplasmic shuttling of steroid receptors. The that GFP-LBD fusion protein is localized to the cytoplasm in the absence of androgen and to the nuclei in the presence of androgen that the LBD contains a ligand-regulated nuclear export The NESAR is active in the absence of androgen and is repressed in the presence of The androgen withdrawal in Fig. the existence of NESAR and that NESAR is to to deletion mutagenesis studies NESAR consists of residues in through of the LBD and has at amino acid residues in the immediate vicinity of the bound ligand it is not that ligand binding can regulate the activity of the NES in the The of NESAR also an for findings that deletion of NESAR to the localization of the AR mutants (9Zhou Z.X. Sar M. Simental J.A. Lane M.V. Wilson E.M. J. Biol. Chem. 1994; 269: 13115-13123Abstract Full Text PDF PubMed Google Scholar, G. Trapman J. Brinkmann A.O. Biochem. J. PubMed Scopus Google Scholar). 1 and that the LBD of AR nuclear export activity in the absence of ligand and nuclear import activity in the presence of ligand. sequences are either NES or NLS activity S. G. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). an is that the NES in the LBD function as an NLS upon ligand binding. This would the nuclear import of in the presence of Our studies showed that the NESAR is necessary and to AR nuclear export is localized to the nuclei of the the of NESAR in AR nuclear the other localization in of the that NESAR is to nuclear Our observations suggest that the mechanism of AR nuclear export is to that of nuclear studies showed that nuclear export of steroid AR, GR, and PR, is to LMB, an of CRM1/exportin 1 (8Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (251) Google Scholar, R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (72) Google Scholar, J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (225) Google Scholar, J. Mol. Endocrinol. 2000; 14: PubMed Scopus Google Scholar). This is with the that NESAR does not share sequence with the leucine-rich NES, which directs export mediated by the CRM1/exportin 1 NESAR is the nuclear export signal responsible for AR nuclear the nuclear export also be to on localization of Our studies indicate the existence of this NES in the LBD of at two other steroid receptors. We have demonstrated that and are localized to the cytoplasm of and of This NES is to be present in steroid receptors. GR mutants with into the region which would to also localization (10Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (724) Google Scholar). the amino acid sequences of the NES are the of the NES is of of the LBD and the conserved region and amino acids in the of the region were with the sequence a in the localization of this was and Because the region is the conserved of the NES the of the the that the of the NES rather than its amino acid be for its nuclear export AR has a characterized NLS in the DBD and hinge region. Our studies showed in the absence of NESAR in the LBD is dominant both NESAR and NLS are present in the This an for androgen regulation of AR nuclear/cytoplasmic shuttling. In the absence of NESAR in the LBD is active and nuclear export of AR. In the binding of ligand NESAR in the LBD, NLS activity in the LBD, and the of the NLS in the DBD and hinge region, to nuclear import of AR. Our observations that NESAR is a nuclear export we were to directly that with nuclear This was by to fusion proteins containing NESAR for nuclear the fusion proteins containing NESAR are present in the that NESAR is for AR In we have identified NESAR, a region in the LBD of AR which NESAR is active and dominant over the NLS in the absence of ligand. The binding of ligand to AR NESAR and of the NLS, to AR nuclear This NES is also present in other steroid receptors. Our findings provide a for the ligand-dependent nuclear import/export of AR and other steroid receptors. We Liao for kindly providing full-length AR for and for critical and for the