Ligand- and Coactivator-mediated Transactivation Function (AF2) of the Androgen Receptor Ligand-binding Domain Is Inhibited by the Cognate Hinge Region
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m009916200
ISSN1083-351X
AutoresQi Wang, Jinhua Lu, Eu‐Leong Yong,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoTransactivation functions (AF2) in the ligand-binding domains (LBD) of many steroid receptors are well characterized, but there is little evidence to support such a function for the LBD of the androgen receptor (AR). We report a mutant AR, with residues 628–646 in the hinge region deleted, which exhibited transactivation activity that was more than double that of the wild type (WT) AR. Although no androgen-dependent AF2 activity could be observed for the WT ARLBD fused to a heterologous DNA-binding domain, the mutant ARLBD(Δ628–646) was 30–40 times more active than the WT ARLBD. In the presence of the p160 coactivator TIF2, AR(Δ628–646) was significantly more active than similarly treated WT AR. Deletion of residues 628–646 also enhanced TIF2-ARLBD activity 8-fold, an effect not present when the LBD-interacting LXXLL motifs of TIF2 were mutated, suggesting that the negative modulatory activity of residues 628–646 were exerted via coactivator pathways. Although the AP-1 (c-Jun/c-Fos) system and NcoR have been reported to interact with and repress the activity of some steroid receptors, c-Jun, c-Fos, c-Jun/c-Fos, nor NcoR function was consistently affected by the absence or presence of residues 628–646, implying that the AR hinge region exerts its silencing effects in a manner independent of these corepressors. Our data provide evidence for the novel finding that strong androgen-dependent AF2 exists in the ARLBD and is the first report of a negative regulatory domain in the AR. Because mutations in this region are commonly associated with prostate cancer, it is important to characterize the mechanisms by which the hinge region exerts its repressor effect on ligand-activated and coactivator-mediated AF2 activity of the ARLBD. Transactivation functions (AF2) in the ligand-binding domains (LBD) of many steroid receptors are well characterized, but there is little evidence to support such a function for the LBD of the androgen receptor (AR). We report a mutant AR, with residues 628–646 in the hinge region deleted, which exhibited transactivation activity that was more than double that of the wild type (WT) AR. Although no androgen-dependent AF2 activity could be observed for the WT ARLBD fused to a heterologous DNA-binding domain, the mutant ARLBD(Δ628–646) was 30–40 times more active than the WT ARLBD. In the presence of the p160 coactivator TIF2, AR(Δ628–646) was significantly more active than similarly treated WT AR. Deletion of residues 628–646 also enhanced TIF2-ARLBD activity 8-fold, an effect not present when the LBD-interacting LXXLL motifs of TIF2 were mutated, suggesting that the negative modulatory activity of residues 628–646 were exerted via coactivator pathways. Although the AP-1 (c-Jun/c-Fos) system and NcoR have been reported to interact with and repress the activity of some steroid receptors, c-Jun, c-Fos, c-Jun/c-Fos, nor NcoR function was consistently affected by the absence or presence of residues 628–646, implying that the AR hinge region exerts its silencing effects in a manner independent of these corepressors. Our data provide evidence for the novel finding that strong androgen-dependent AF2 exists in the ARLBD and is the first report of a negative regulatory domain in the AR. Because mutations in this region are commonly associated with prostate cancer, it is important to characterize the mechanisms by which the hinge region exerts its repressor effect on ligand-activated and coactivator-mediated AF2 activity of the ARLBD. androgen receptor transactivation domain DNA-binding domain ligand-binding domain thyroid hormone -2, activation functions 1 and 2 glucocorticoid receptor progesterone receptor peroxisome proliferator-activated receptor estrogen receptor androgen response element nuclear localization signal wild-type polymerase chain reaction dihydrotestosterone kilobase(s) relative light unit(s) The androgen receptor (AR),1 a member of the steroid-hormone superfamily of nuclear transcription factors, mediates male sexual differentiation in utero, sperm production at puberty, and prostate growth in the adult. Like other members of the steroid/nuclear receptor superfamily, the AR has four major functional regions: the N-terminal transactivation domain (TAD), a central DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD), and a hinge region connecting the LBD and the DBD (see Fig. 1below). In the absence of hormone, nuclear receptors are maintained in a repressed state by association with heat shock proteins and/or corepressors (1McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1658) Google Scholar). In the thyroid hormone (THR) and retinoic acid receptors, ligand-independent repression of gene transcription occurs and is mediated by nuclear receptor corepressor proteins such as NcoR (2Dowell P. Ishmael J.E. Avram D. Peterson V.J. Nevrivy D.J. Leid M. J. Biol. Chem. 1999; 274: 15901-15907Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 3Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google Scholar) and SMRT (4Lavinsky R.M. Jepsen K. Heinzel T. Torchia J. Mullen T.M. Schiff R. Del-Rio A.L. Ricote M. Ngo S. Gemsch J. Hilsenbeck S.G. Osborne C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2920-2925Crossref PubMed Scopus (585) Google Scholar). Upon exposure to hormone, steroid receptors are released from their inactive states and the receptor-ligand complexes translocate to the nucleus and bind to and activate hormone response elements of target genes. Activation function 1 (AF1) and AF2 are located in the TAD and the LBD, respectively, of the steroid receptors, and activity of these is dependent on the recruitment of coactivator molecules to form active preinitiation sites for gene transcription (5Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. 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Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar, 14Berrevoets C.A. Doesburg P. Steketee K. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1998; 12: 1172-1183Crossref PubMed Google Scholar, 15He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 16He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Although p160 coactivators, like SRC1 and TIF2, can bind to the ARLBD in a hormone-dependent manner (6Onate S.A. Boonyaratanakornkit V. Spencer T.E. Tsai S.Y. Tsai M.J. Edwards D.P. O'Malley B.W. J. Biol. Chem. 1998; 273: 12101-12108Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 17Ghadessy F.J. Lim J. Abdullah A.A. Panet-Raymond V. Choo C.K. Lumbroso R. Tut T.G. Gottlieb B. Pinsky L. Trifiro M.A. Yong E.L. J. Clin. Invest. 1999; 103: 1517-1525Crossref PubMed Scopus (71) Google Scholar, 18Lim J. Ghadessy F.J Abdullah A.A. Pinsky L. Trifiro M. Yong E.L. Mol. Endocrinol. 2000; 14: 1187-1197Crossref PubMed Scopus (36) Google Scholar), the ARLBD itself demonstrates very little activation function when fused to heterologous DNA-binding domains (5Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar, 14Berrevoets C.A. Doesburg P. Steketee K. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1998; 12: 1172-1183Crossref PubMed Google Scholar, 15He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 16He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Deletion of the AR TAD results in an LBD fragment that can bind ligand and androgen response elements (ARE) but is relatively inactive in reporter gene assays in human cells. In contrast, the AR TAD fragment alone has a hormone-independent AF1 that is almost equal to the ligand-activated full-length AR. The very existence of an AF2 in the AR has been questioned, and it has been proposed that AR transactivation function is dependent on the strong AF1 activity of the TAD, consequent to ligand-activated interactions between the TAD and the LBD (5Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar, 16He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 19Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar, 20Ikonen T. Palvimo J.J. Janne O.A. J. Biol. Chem. 1997; 272: 29821-29828Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Crystal structures of all LBDs solved to date indicate that activation of the apo-receptor by ligand involves a structural change, where C-terminal helix 12 of the LBD is positioned over the ligand-binding pocket to complete the AF2 surface (21Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Ruff M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 22Wurtz J.M. Bourguet W. Renaud J.P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (685) Google Scholar). Helix 12 and helices 3, 4, and 5 form opposite ends of a hydrophobic cleft for binding leucine-rich motifs of nuclear-receptor-interacting domains of coactivator molecules (23Feng W. Ribeiro R.C. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (517) Google Scholar, 24McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Crossref PubMed Scopus (529) Google Scholar, 25Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO, J. 1998; 17: 507-519Crossref PubMed Scopus (432) Google Scholar). Considering the high similarity in crystal structure of the AR LBD (21Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Ruff M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) to that of the PPARδ (11Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1700) Google Scholar), vitamin D receptor (26Rochel N. Wurtz J.M. Mitschler A. Klaholz B. Moras D. Mol. Cell. 2000; 5: 173-179Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar), ER (27Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar), and PR (28Tanenbaum D.M. Wang Y. Williams S.P. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5998-6003Crossref PubMed Scopus (597) Google Scholar), it is puzzling as to why the prominent AF2 activity present in other steroid receptors cannot be elicited in the ARLBD. Although the LBD and the DBD of the AR have been characterized in some detail (29Quigley C.A. De Bellis A. Marschke K.B. el-Awady M.K. Wilson E.M. French F.S. Endocr. Rev. 1995; 16: 271-321Crossref PubMed Google Scholar), the hinge region in between these major domains, defined by residues 628–669, is less well understood (Fig.1). Although this region is poorly conserved among steroid receptors, several lines of evidence indicate that key functional elements may reside in this AR domain. For example, 7 of 10 reported amino acid substitutions affecting residues 619–672 in the AR hinge region are associated with the androgen-dependent tumor, prostate cancer (30Gottlieb B. Lehvaslaiho H. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Nucleic Acids Res. 1998; 26: 234-238Crossref PubMed Scopus (144) Google Scholar). Furthermore, a sequence located between amino acids 628 and 657 within the hinge region contains a short stretch of basic amino acids that resemble the nuclear targeting signals of the glucocorticoid receptor and the SV-40 large T antigen (31Simental 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) and has been described to form part of a bipartite nuclear localization signal (NLS) (32Zhou Z.X. Sar M. Simental J.A. Lane M.V. Wilson E.M. J. Biol. Chem. 1994; 1269: 13115-13123Abstract Full Text PDF Google Scholar). In addition, transactivation activity of the ARLBD in yeast, but not in mammalian cells, appears to be modulated when the hinge region is attached (33Moilanen A. Rouleau N. Ikonen T. Palvimo J.J. Janne O.A. FEBS Lett. 1997; 412: 355-358Crossref PubMed Scopus (49) Google Scholar), and coexpression of Ubc9, a ubiquitin-conjugating enzyme that interacts with the hinge region, can enhance AR transactivation activity (34Poukka H. Aarnisalo P. Karvonen U. Palvimo J.J. Janne O.A. J. Biol. Chem. 1999; 274: 19441-19446Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In this study we explored the effects of deleting key residues in the hinge region and observed that the ligand-activated and coactivator-augmented transcriptional activity of the deletion mutant was unexpectedly higher than the WT. Experiments were performed to elucidate the mechanism whereby the deleted residues exert an inhibitory effect on ARLBD function. The full-length AR deletion mutant, AR(Δ628–646), was created by site-directed mutagenesis using two internal primers: Sense (dF), 5′-gaagcagggatgactctgggcagcaccaccagccccact-3′ and the antisense (dR), 5′-cagtggggctggtggtgctgcccagagtcatccctgcttc-3′. The external primers were sense (DNA-B), 5′-gacttcaccgcacctgatgtg-3′ and antisense (QE3), 5′-cctggagttgacattggtgaa-3′. Initially, two primary PCRs were performed using the AR expression plasmid pSV-ARas the template. Primers DNA-B and dR were used to generate fragment 1, and primers dF and QE3 for fragment 2. These overlapping primary amplification products were then denatured and allowed to anneal together to produce a heteroduplex product with overlapping ends. The recessed ends of the heteroduplexes were extended with Pfu DNA polymerase to produce a fragment that is the sum of the two overlapping products. A subsequent reamplification was performed for 30 cycles using primers DNA-B and QE3 to generate the cDNA fragment with a deletion of AR codons 628–646 (Δ628–646). The secondary PCR product was double-digested with HindIII andXhoI and ligated into pSV-AR, with the equivalent fragment excised, to generate AR (Δ628–646).The pGAL4DBD-ARLBD chimeric vector comprised theAR hinge and LBD regions (codons 628–919) fused in-frame to the GAL4DBD residues 1–147 (23Feng W. Ribeiro R.C. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (517) Google Scholar).pGAL4DBD-ARLBDΔ628–646 was generated by usingAR (Δ628–646) as the template and amplifying the cDNA fragment encoding theARLBD(Δ628–646) with the forward primer 5′-cagcaccaccagccccactgaggagac-3′ and the reverse primer 5′-gtttccaaagcttcactgggtgtggaa-3′. The PCR product, including the stop codon in exon 8 was digested with HindIII and ligated in-frame into the SmaI/HindIII site of pm containing the Gal4DNA-binding domain. The plasmid pSV-AR (N727K,M886V), containing the double mutations M886V and N727K, was created by placing theXhoI-EcoRI segment containing the N727K mutation (17Ghadessy F.J. Lim J. Abdullah A.A. Panet-Raymond V. Choo C.K. Lumbroso R. Tut T.G. Gottlieb B. Pinsky L. Trifiro M.A. Yong E.L. J. Clin. Invest. 1999; 103: 1517-1525Crossref PubMed Scopus (71) Google Scholar) into the M886V AR expression vector (18Lim J. Ghadessy F.J Abdullah A.A. Pinsky L. Trifiro M. Yong E.L. Mol. Endocrinol. 2000; 14: 1187-1197Crossref PubMed Scopus (36) Google Scholar).pGAL4DBD-ARLBD(N727K,M886V) was generated by using pSV-AR (N727K,M886V) as the template and amplifying theLBD with the forward primers 5′-ggcccggaagctgaagaaactt-3′ and the reverse primer 5′-gtttccaaagcttcactgggtgtggaa-3′. The PCR product, including the stop codon in exon 8, was digested withHindIII and ligated in-frame into theSmaI/HindIII site of pM containing the GAL4DNA-binding domainpGAL4DBD-ARLBD(Δ628–646), andpGAL4DBD-ARLBD(Δ628–646,N727K,M886V) were generated by amplifying the relevant fragments of pSV-AR or pSV-AR (N727K,M886V) and fused in-frame intoGAL4DBD. pVP16-NcoRC′ was generated by digesting the cDNA encoding NcoR (kind gift of Dr. G. Jenster, Erasmus University, Rotterdam) with EcoRI andBamHI to obtain a 1.9-kb fragment containing residues 1818–2453 of the C-terminal fragment of NcoR, and ligating it in-frame to the VP16 transactivation domain. This NcoRC′ fragment have both repression domains removed, but included amino acids 1859–2142 and 2239–2300 of steroid-receptor-interacting domains I (3Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google Scholar) and II (35Seol W. Mahon M.J. Lee Y.K. Moore D.D. Mol. Endocrinol. 1996; 10: 1646-1655PubMed Google Scholar), respectively. The pCMV-cJun encoding human c-Jun driven by the cytomegalovirus promoter (36Baichwal V.R. Tjian R. Cell. 1990; 63: 815-825Abstract Full Text PDF PubMed Scopus (142) Google Scholar), and the pRSV-c-fos containing rat c-fos driven by the RSV-LTR promoter (37Turner R. Tjian R. Science. 1989; 243: 1689-1694Crossref PubMed Scopus (425) Google Scholar) were kind gifts of Dr. R. Tjian (University of California, Berkeley, CA). pSG5-TIF2 and pSG5-mTIF2, encoding full-length TIF2 and mutant TIF2 (in which all three LXXLL nuclear-receptor-interacting motifs were mutated to LXXAA), respectively, were provided by Dr. H. Gronemeyer (Institut de Génetique et de Biologie Moléculaire et Cellulaire, Strasbourg) (25Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO, J. 1998; 17: 507-519Crossref PubMed Scopus (432) Google Scholar). The reporter vectors pARE-TATA-Luc and pGAL4-TATA-Luc,contained five GAL4 DNA-binding sites and two tandem copies of the ARE from the aminotransferase gene, respectively, driving the luciferase reporter gene (17Ghadessy F.J. Lim J. Abdullah A.A. Panet-Raymond V. Choo C.K. Lumbroso R. Tut T.G. Gottlieb B. Pinsky L. Trifiro M.A. Yong E.L. J. Clin. Invest. 1999; 103: 1517-1525Crossref PubMed Scopus (71) Google Scholar). All constructs were sequenced to confirm the fidelity of enzymatic manipulations. HeLa cells were maintained in RPMI 1640 medium and COS cells in Dulbecco's modified Eagle's medium. 1.5–1.8 × 104 cells were seeded into each well of 24-well plates 20 h prior to transfections. For Western blotting, COS-7 cells were seeded on P60 Petri dishes 29 h before transfection. All transient transfections were performed using LipofectAMINE (38Wang Q. Ghadessy F.J. Trounson A. Kretser D. McLachlan R. Ng S.C. Yong E.L. J. Clin. Endocrinol. Metab. 1998; 83: 4303-4309Crossref PubMed Scopus (43) Google Scholar), and appropriate amounts of empty parent vector were added to the replicates, if indicated, to normalize the amount of total DNA in each well to prevent general squelching. Immunoblotting was performed as previously described (17Ghadessy F.J. Lim J. Abdullah A.A. Panet-Raymond V. Choo C.K. Lumbroso R. Tut T.G. Gottlieb B. Pinsky L. Trifiro M.A. Yong E.L. J. Clin. Invest. 1999; 103: 1517-1525Crossref PubMed Scopus (71) Google Scholar). Transfected COS-7 cells were lysed, and 10 μg of protein from the cell lysate was resolved on 8% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto Hybond-C nitrocellulose membranes, and AR protein was detected using the rabbit polyclonal antibody, NH27, which recognizes amino acids 360–564 of the AR (gift of Dr. A. Mizokami, Kitakyushu, Japan). COS-7 cells were seeded on 15-mm diameter coverslips on 12-well pates and transfected with LipofectAMINE. Five hours after transfection, the cells received fresh medium with 10% charcoal-treated fetal bovine serum and were cultured for an additional 20 h in the presence or absence of increasing doses of DHT. Cells were fixed in 4% paraformaldehyde in phosphate buffer saline and permeabilized with 1% Brij. AR protein was detected with the antibody NH27 (1:50 dilution). Fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody was used for visualization of the receptor protein under a confocal laser scanning biological microscope (Olympus Fluoview IX70, Tokyo, Japan). To test the consequences of deleting amino acids 628–646 from full-length AR, WT AR or mutant AR(Δ628–646) was expressed in the HeLa cells and transactivation activity measured with a multimeric ARE promoter linked to a luciferase reporter gene (Fig. 2). WT AR activity at 0.001 nm DHT was 2-fold higher than replicates not exposed to hormone and reached a maximum of 1600-fold increase in activity at 10 nm (Fig. 2 A). Further increases in androgen dose did not raise AR activity above this maximum, indicating that saturating doses of hormone had been reached. However, the transactivation response of mutant AR was biphasic and differed from WT. Low doses of androgen (0.001–0.01 nm) did not increase mutant AR activity significantly, resulting in WT AR activity 10- to 44-fold higher than mutant AR. Surprisingly, this pattern was reversed for doses of androgen of 0.1 nm. The AR mutant, despite having its hinge region and the associated NLS deleted, exhibited AR activity that was more than double that of the WT AR for doses of DHT and mibolerone between 0.1 and 1000 nm(Fig. 2). These differences in transactivation function were not due to changes in protein expression, because both WT AR and deletion mutants were present in approximately equal amounts in the cells, as indicated by immunoblotting (Fig. 2 C). To measure activation function of the ARLBD, a chimeric construct, comprising the GAL4DBD fused in-frame to theARLBD, was coexpressed with the GAL4-TATA-Lucreporter gene (Fig. 3). WT ARLBD chimeric protein did not demonstrate significant transactivation activity with, or without androgen, consistent with previous studies indicating that very little transactivation function resides in the AR LBD (5Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar, 15He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 19Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar). Unexpectedly, deletion of residues 628–646 from the ARLBD increased androgen-dependent transactivation activity markedly. The ARLBD(Δ628–646) fragment was about 30–40 times more active than the intact WT LBD. Whereas no androgen-dependent increase in AF2 activity was observed for the WT ARLBD, dose-dependent augmentation of ARLBD(Δ628–646) AF2 activity was observed for doses of DHT and mibolerone from 0.01 to 1000 nm. This suggests that amino acids 628–646 may serve to inhibit the transactivation potential of AF2 of the ARLBD. A short stretch of basic amino acids located within amino acids 628–646 of the hinge region forms part of a bipartite NLS, the other portion located 10 residues upstream in the DBD (Fig. 1) (31Simental 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). To determine whether nuclear localization of the AR is affected by the hinge deletion, immunofluorescence confocal microscopy was performed (Fig.4). In the absence of androgen, transfected AR was located in both cytoplasm and the nucleus (Fig. 4,top panels). With low doses of DHT (0.01 nm), WT AR was observed increasingly in the nucleus. At doses of DHT >1 nm, WT AR protein was localized mainly in the nucleus, and at 100 nm the WT AR signal was observed almost exclusively in the nucleus (Fig. 4, bottom panels). The deletion mutant surprisingly behaved in a similar manner, with most of the AR being located in the nucleus in the presence of androgen doses >1 nm, except that the nuclear signals were marginally less intense. Thus deletion of the hinge portion of the bipartite NLS in the mutant AR did not prevent its localization to the nucleus, suggesting that the intact portion located in the DBD was sufficient for this purpose (32Zhou Z.X. Sar M. Simental J.A. Lane M.V. Wilson E.M. J. Biol. Chem. 1994; 1269: 13115-13123Abstract Full Text PDF Google Scholar). The effects of coactivator on mutant AR were next investigated, because AF2 is dependent on their efficient recruitment to the ARLBD. Of the three steroid coactivators identified to date, TIF2 interacts the strongest with AR (39Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar). Consistent with this observation, TIF2 augmented full-length WT AR activity by over 100% (Fig. 5). Remarkably, the AR(Δ628–646) mutant, was observed to display even greater TIF2-augmented activity, being 70% higher than similarly treated WT AR. Thus the synergistic activity of TIF2 on full-length AR function was present when the hinge region was deleted. To further define this effect, a chimeric construct consisting of the GAL4DBD linked in-frame to ARLBD was coexpressed with TIF2, and transactivation activity was measured with a GAL4 reporter gene (Fig.6 A). In the absence of TIF2, no AF2 function of the WT LBD was observed, whereas deletion of the 628–646 region resulted in significant ARLBD AF2 activity. The presence of TIF2 augmented the AF2 function of the WT LBD by more than 80-fold. Strikingly, the activity of LBD(Δ628–646) fusion protein with TIF2 was 8-fold higher than that of the corresponding TIF2-stimulated WT LBD fragment, and 40-fold higher than that observed with mutant LBD alone. In contrast, a TIF2 mutant, with three LXXLL nuclear-receptor-interacting motifs mutated, was not able to synergize with either WT LBD or LBD(Δ628–646) transactivation function, indicating that residues 628–646 exert their repressive actions via LXXLL motifs of steroid receptor coactivators. To further delineate the sites of action of TIF2, LBD mutants incorporating two substitutions, N727K and M886V, were constructed. The N727K,M886V mutations have previously been demonstrated to have partially defective interactions with TIF2 (17Ghadessy F.J. Lim J. Abdullah A.A. Panet-Raymond V. Choo C.K. Lumbroso R. Tut T.G. Gottlieb B. Pinsky L. Trifiro M.A. Yong E.L. J. Clin. Invest. 1999; 103: 1517-1525Crossref PubMed Scopus (71) Google Scholar,18Lim J. Ghadessy F.J Abdullah A.A. Pinsky L. Trifiro M. Yong E.L. Mol. Endocrinol. 2000; 14: 1187-1197Crossref PubMed Scopus (36) Google Scholar), resulting in minimal androgen insensitivity and male infertility. The WT ARLBD chimeric protein did not have any activity in the absence of hormone, but displayed strong androgen-dependent activity in the presence of TIF2 (Fig. 6 B). As expected, the LBD(N727K,M886V) fusion protein was partially defective in the presence of TIF2, having only half the activity of the WT. Nevertheless, deletion of residues 628–646 resulted in TIF2-dependent augmentation of mutant AR activity, such that triply mutated LBD(Δ628–646,N727K,M886V) was more than 3-fold stronger than the doubly mutated LBD(N727K,M886V) fragment. Thus mutations in AR residues 727 and 886, unl
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