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• W2033728702 abstract "A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Δ371–485)) as a bait, β-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-β-catenin demonstrated that FLAG-β-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5α-dihydrotestosterone, FLAG-β-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen α receptors did not translocate FLAG-β-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate β-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3β, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed β-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of β-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles β-catenin to the nucleus and that nuclear interaction of AR with β-catenin may modulate transcriptional activity in androgen target tissues. A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Δ371–485)) as a bait, β-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-β-catenin demonstrated that FLAG-β-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5α-dihydrotestosterone, FLAG-β-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen α receptors did not translocate FLAG-β-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate β-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3β, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed β-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of β-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles β-catenin to the nucleus and that nuclear interaction of AR with β-catenin may modulate transcriptional activity in androgen target tissues. Androgen receptor (AR) 1The abbreviations used are: ARandrogen receptorPRprogesterone receptorGRglucocorticoid receptorERαestrogen receptor αGSK-3βglycogen synthase kinase-3βGnRHgonadotropin-releasing hormoneTCFT cell-factor5α-DHT5α-dihydrotestosteroneERKextracellular signal-regulated kinasePBSphosphate-buffered salineRTroom temperatureMMTVmurine mammary tumor virusFITCfluorescein isothiocyanaterARrat ARhARhuman ARN-Camino- and carboxyl-terminal is a member of the nuclear steroid receptor superfamily and is vital for normal sexual development in males (reviewed in Ref. 1Lamb D.J. Weigel N.L. Marcelli M. Vitam. Horm. 2001; 62: 199-230Crossref PubMed Google Scholar). In androgen target cells, AR is predominantly localized in the cytoplasmic fraction in the absence of ligand and translocates to the nucleus in the presence of the endogenous androgens, testosterone and 5α-dihydrotestosterone (5α-DHT), where it activates transcription of AR-responsive genes (2Simental 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, 3Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (250) Google Scholar). In addition to the genomic actions of AR, nongenomic signaling mechanisms of androgens have also been described in several cell systems. Through activation of the Src/Shc/ERK signaling pathway, AR was shown to be anti-apoptotic in osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells (4Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). Activation of the ERK mitogen-activated protein kinase pathway by AR has also been demonstrated in prostate cancer cell lines (5Peterziel H. Mink S. Schonert A. Becker M. Klocker H. Cato A.C. Oncogene. 1999; 18: 6322-6329Crossref PubMed Scopus (220) Google Scholar). In bone, androgens have been shown to increase intracellular calcium, diacylglycerol, and inositol 1,4,5-trisphosphate formation (6Lieberherr M. Grosse B. J. Biol. Chem. 1994; 269: 7217-7223Abstract Full Text PDF PubMed Google Scholar). androgen receptor progesterone receptor glucocorticoid receptor estrogen receptor α glycogen synthase kinase-3β gonadotropin-releasing hormone T cell-factor 5α-dihydrotestosterone extracellular signal-regulated kinase phosphate-buffered saline room temperature murine mammary tumor virus fluorescein isothiocyanate rat AR human AR amino- and carboxyl-terminal The genomic actions of AR are dependent upon its nuclear translocation where it then binds to androgen-response elements, some of which conform to the consensus sequence 5′-GG(A/T)ACANNNTGTTCT-3′ (7Roche P.J. Hoare S.A. Parker M.G. Mol. Endocrinol. 1992; 6: 2229-2235Crossref PubMed Scopus (184) Google Scholar), and stimulates the transcription of target genes. Several androgen-response elements are also recognized by the glucocorticoid receptor, and it is believed that protein-protein interactions play a role to discriminate AR- versus GR-mediated effects at these sites (8Adler A.J. Scheller A. Robins D.M. Mol. Cell. Biol. 1993; 13: 6326-6335Crossref PubMed Scopus (93) Google Scholar). Other nuclear factors that modulate AR transcriptional activity include cAMP-response element-binding protein, AP-1, and members of the POU family of homeodomain transcription factors (8Adler A.J. Scheller A. Robins D.M. Mol. Cell. Biol. 1993; 13: 6326-6335Crossref PubMed Scopus (93) Google Scholar, 9Prefontaine G.G. Walther R. Giffin W. Lemieux M.E. Pope L. Hache R.J. J. Biol. Chem. 1999; 274: 26713-26719Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 11Scarlett C.O. Robins D.M. Mol. Endocrinol. 1995; 9: 413-423PubMed Google Scholar). Protein-protein interactions are also involved in the role of AR as a transcriptional repressor independent of DNA binding. Recently, the mechanism of AR repression of the α-glycoprotein hormone subunit gene in the αT3-1 pituitary gonadotrope cell line was found to be through interactions with c-Jun and ATF-2 (12Jorgensen J.S. Nilson J.H. Mol. Endocrinol. 2001; 15: 1496-1504PubMed Google Scholar). Repression of the luteinizing hormone β subunit gene involved protein-protein interactions between AR and SF-1 (13Jorgensen J.S. Nilson J.H. Mol. Endocrinol. 2001; 15: 1505-1516PubMed Google Scholar). In a separate study, AR-mediated repression of the luteinizing hormone β subunit gene was also found to occur through protein-protein interactions with Sp1 and to a lesser degree Egr-1 (14Curtin D. Jenkins S. Farmer N. Anderson A.C. Haisenleder D.J. Rissman E. Wilson E.M. Shupnik M.A. Mol. Endocrinol. 2001; 15: 1906-1917Crossref PubMed Scopus (76) Google Scholar). The yeast two-hybrid system has been used successfully to identify protein partners of AR. Both co-activators and transcriptional repressors have been found. For example, Chang and colleagues (15Fujimoto N. Yeh S. Kang H.Y. Inui S. Chang H.C. Mizokami A. Chang C. J. Biol. Chem. 1999; 274: 8316-8321Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 16Kang H.Y. Yeh S. Fujimoto N. Chang C. J. Biol. Chem. 1999; 274: 8570-8576Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 17Wang X. Yeh S., Wu, G. Hsu C.L. Wang L. Chiang T. Yang Y. Guo Y. Chang C. J. Biol. Chem. 2001; 276: 40417-40423Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 18Yeh S. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5517-5521Crossref PubMed Scopus (530) Google Scholar) have identified the androgen receptor co-activators ARA70, ARA55, ARA54, and ARA267 from yeast two-hybrid screens of prostate cell libraries. Additionally, TIP60, a co-activator of human immunodeficiency virus Tat protein, and TRAM-1, a thyroid receptor co-activator, have been isolated from yeast two-hybrid screens using AR as a bait (19Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 20Tan J.A. Hall S.H. Petrusz P. French F.S. Endocrinology. 2000; 141: 3440-3450Crossref PubMed Scopus (43) Google Scholar). The androgen receptor-specific co-activator FHL2 was also identified by a yeast two-hybrid screen (21Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Crossref PubMed Scopus (288) Google Scholar). The amino-terminal enhancer of split was identified as an AR repressor using a yeast two-hybrid assay (22Yu X., Li, P. Roeder R.G. Wang Z. Mol. Cell. Biol. 2001; 21: 4614-4625Crossref PubMed Scopus (91) Google Scholar). In this study, we performed a yeast two-hybrid screen using a GT1-7 cell cDNA library to identify potential protein partners of AR in GT1-7 GnRH neuronal cells and found β-catenin as an AR-interacting protein. β-Catenin was originally identified at adherins junctions involved in the binding of cadherins with the actin cytoskeleton (23Ozawa M. Baribault H. Kemler R. EMBO J. 1989; 8: 1711-1717Crossref PubMed Scopus (1148) Google Scholar) and therefore is important in the regulation of cell adhesion. However, a role for β-catenin in development and signal transduction was proposed because of sequence similarity with the DrosophilaArmadillo protein that is a component of the wingless signaling pathway (24McCrea P.D. Turck C.W. Gumbiner B. Science. 1991; 254: 1359-1361Crossref PubMed Scopus (506) Google Scholar). In the absence of signaling mechanisms that stabilize cytoplasmic β-catenin, its levels in the cytoplasm are tightly regulated because of phosphorylation by glycogen synthase kinase-3β (GSK-3β) and subsequent proteosomal degradation (25Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2160) Google Scholar, 26Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar). During development, Wnt signaling pathways inhibit β-catenin degradation and cause accumulation of cytoplasmic β-catenin (27van Leeuwen F. Samos C.H. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Subsequently, β-catenin translocates to the nucleus and forms a complex with the T cell factor/lymphoid enhancer factor (TCF/Lef) family of transcription factors and stimulates transcription of target genes. Because it lacks a nuclear localization signal, the mechanisms for β-catenin nuclear translocation are not well understood. In our studies, we found that agonist bound AR but not progesterone receptor (PR), glucocorticoid receptor (GR), or estrogen receptor α (ERα)-induced nuclear translocation of β-catenin. Furthermore, liganded AR repressed β-catenin/TCF activation of β-catenin/TCF-responsive promoter activity. Conversely, β-catenin/TCF expression repressed AR-responsive promoter activity. Our results suggest that interactions between AR and β-catenin may modulate the nuclear localization of β-catenin and transcriptional activity in AR-responsive target tissues. AR (PA1–111A), ERα (PA1–308), GR (PA1–511A), and polyclonal FLAG (PA1–984) antibodies were obtained from Affinity Bioreagents (Golden, CO). Anti-PR (LS434) was a kind gift from Dean Edwards (University of Colorado Health Sciences Center). Anti-Myc (3F10), anti-GSK-3β (clone 7), and monoclonal anti-β-catenin (C19220) were purchased from Transduction Laboratories (Lexington, KY). Polyclonal anti-β-catenin was from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-GSK-3β antibody (9336) was purchased from Cell Signaling Technology (Beverly, MA). Secondary anti-mouse and anti-rabbit Texas Red or FITC-conjugated antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). The yeast Matchmaker 3 system was purchased from CLONTECH (Palo Alto, CA). 5α-DHT was purchased from Steraloids (Wilton, NH). Casodex and hydroxyflutamide were obtained from Zeneca Pharmaceuticals (Wilmington, DE) and Schering Corp. (Kenilworth, NJ), respectively. All other reagents unless otherwise noted were obtained from Sigma. Cell lines were grown in Dulbecco's modified Eagle's medium supplemented with either 5 or 10% fetal calf serum, 100 units of penicillin/ml, 100 μg of streptomycin/ml, and 0.25 μg/ml of amphotericin B at 37 °C in humidified 5% CO2, 95% air. GT1-7 cells are immortalized murine GnRH neuronal cells derived from an SV40 large T antigen-targeted hypothalamic GnRH-producing tumor that displays neuronal specific markers and produce GnRH (28Mellon P.L. Windle J.J. Goldsmith P.C. Padula C.A. Roberts J.L. Weiner R.I. Neuron. 1990; 5: 1-10Abstract Full Text PDF PubMed Scopus (899) Google Scholar). NLT cells are also immortalized murine GnRH neuronal cells that produce GnRH but were derived from an olfactory tumor, thus representing early migrating GnRH neuronal cells (29Radovick S. Wray S. Lee E. Nicols D.K. Nakayama Y. Weintraub B.D. Westphal H. Cutler G.B., Jr. Wondisford F.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3402-3406Crossref PubMed Scopus (197) Google Scholar). COS-7 cells were obtained from the Cancer Center Cell Culture Core facility, University of Colorado Health Sciences Center. pGBKT7-AR-(Δ371–485) was generated by ligating PCR products corresponding to nucleotides 1–1110 of rat AR (rAR) (amino acids 1–370) to nucleotides 1458–2709 (amino acids 486–903) of rAR into pGBKT7. Briefly, the forward 5′-atgggtcgacatggaggtgcagtta-3′ and reverse 5′-tttatgcatcggcccggacagagc-3′ primers were used to generate the 1–1110 fragment. The forward 5′-ggcatgcattatcctggtggagttgtg-3′ and reverse 5′-tataggctgcagtcactgtgtgtggaa-3′ primers were used to generate the 1458–2709 fragment. Both fragments were ligated after restriction enzyme digestion with NsiI. The ligation product was further digested with PstI and then subcloned into pGBKT7 that had been cut with SmaI and PstI. pCMV-rAR, pCMV-human AR (hAR), pCMV-AR-(1–503), pCMV-AR-(1–660), pCMV-AR-R617K618,632,633M, pCMV-AR-K720A, pCMV-AR-E897K and pCMV-AR-V716R were made as described (2Simental 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, 30Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 31Zhou 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, 32He 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 (283) Google Scholar, 33Lubahn D.B. Joseph D.R. Sar M. Tan J. Higgs H.N. Larson R.E. French F.S. Wilson E.M. Mol. Endocrinol. 1988; 2: 1265-1275Crossref PubMed Scopus (471) Google Scholar, 34Tan J.A. Joseph D.R. Quarmby V.E. Lubahn D.B. Sar M. French F.S. Wilson E.M. Mol. Endocrinol. 1988; 2: 1276-1285Crossref PubMed Scopus (230) Google Scholar). pCMV-myc-AR was generated by subcloning hAR into the SfiI and NotI sites of pCMV-myc (CLONTECH). β-Catenin, TCF-4, and pGL3-OT plasmid constructs were kind gifts of Bert Vogelstein and Ken Kinzler (The Johns Hopkins University, Baltimore, MD). pGL3-OT contains a trimerized TCF optimal promoter sequence upstream of the luciferase gene and is a modified version of the TOPFLASH vector (35He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4084) Google Scholar). FLAG-β-catenin was a kind gift of E. R. Fearon (University of Michigan, Ann Arbor, MI). MMTV-luciferase contains sequences −1161 to +102 of the MMTV-long terminal repeat coupled to a luciferase reporter gene (36Chalepakis G. Arnemann J. Slater E. Bruller H.J. Gross B. Beato M. Cell. 1988; 53: 371-382Abstract Full Text PDF PubMed Scopus (152) Google Scholar). The human glucocorticoid receptor (GR) construct RSHGRα was a kind gift from Ron Evans, and human PR (pSG5hPR) and ERα were obtained from Pierre Chambon. Yeast two-hybrid analysis was performed using the CLONTECH Matchmaker 3 system. A cDNA library generated from GT1-7 neuronal cell mRNA was subcloned into the Gal4 activation domain vector pGAD10. Rat AR-(Δ371–485) was subcloned into the bait plasmid pGBKT7 that contains the Gal4 DNA binding domain to generate pGBKT7-AR-(Δ371–485). Yeast AH109 cells were sequentially transformed with the library vector and pGBKT7-AR-(Δ371–485). Approximately 4 × 106 independent clones were plated on synthetic drop-out media (CLONTECH) without histidine, leucine, or tryptophan in the presence of 30 mm3-aminotriazole and 200 nm 5α-DHT. Positive clones were confirmed by replating and selecting clones that grew in the presence, but not absence, of 5α-DHT and by liquid β-galactosidase assays. For the β-galactosidase assays, pGBKT7-AR-(Δ371–485) and potential positive interacting clones were transformed into Y187 cells. Cells were grown to stationary phase in appropriate selection media and then diluted 1:4 into 8 ml of YPD media. Cells were grown an additional 5 h in the absence or presence of 10 nm 5α-DHT. Cells were harvested and lysed by three freeze/thaw cycles in buffer containing 100 mm sodium phosphate, pH 7.0, 10 mm KCl, 1 mm MgSO4. To 0.1 ml of cell lysate was added 0.7 ml of lysis buffer containing 40 mm β-mercaptoethanol, followed by 0.16 ml of lysis buffer containing 4 mg/ml of o- nitrophenyl β-d-galactopyranoside. Samples were incubated at 37 °C, and absorbance values were measured at 420 nm. COS-7 cells were co-transfected with pCMV-myc-AR and FLAG-β-catenin in the presence of 10 nm 5α-DHT. Cells were rinsed with cold PBS and lysed in buffer containing 50 mm HEPES, pH 7.5; 0.5% Triton X-100; 5 mm EDTA; 50 mm NaCl; 10 mm sodium pyrophosphate; 50 mm NaF; 1 mm Na3VO4; 1 mmphenylmethylsulfonyl fluoride; 10 μg/ml each aprotinin, leupeptin, and pepstatin. For immunoprecipitation assays, 300 μg of lysates were incubated with 1 μg anti-Myc antibody for 2 h at 4 °C. Immune complexes were precipitated for 1 h using protein A + G (Oncogene Sciences) and washed three times in lysis buffer prior to analysis by SDS-PAGE and immunoblotting. Proteins were separated using 8% SDS-PAGE, transferred to HyBond-P membranes (Amersham Biosciences), and probed with either anti-Myc or anti-FLAG antibodies (1 μg/ml each). For immunoblot analysis of cell extracts, 20–40 μg of whole cell lysates were separated using 8% SDS-PAGE, transferred to HyBond-P, and incubated with either anti-Myc, anti-FLAG, or β-catenin antibodies. Immunoreactive proteins were visualized using ECL (AmershamBiosciences) following the manufacturer's directions. NLT GnRH neuronal cells were cultured in Dulbecco's modified Eagle's medium containing 5% charcoal-stripped fetal bovine serum for 36–48 prior to transfection. For transfections, ∼15,000 NLT cells were plated on glass coverslips in 24-well plates and allowed to attach overnight. Cells were transfected using 320 ng of FLAG-β-catenin and 80 ng of steroid receptor constructs using LipofectAMINE plus (Invitrogen) and incubated for 16 h. For immunocytochemistry, cells were rinsed with 1 ml PBS at RT and then fixed in 4% paraformaldehyde/PBS for 30 min at RT. Cells were rinsed twice with 1 ml of PBS and then permeabilized with 0.5 ml of 5% bovine serum albumin, 0.2% Triton X-100 in PBS for 90 min at RT. For double label experiments, cells were incubated with either anti-AR (2 μg/ml), anti-PR (1:1000), or anti-GR (2 μg/ml) and either monoclonal anti-FLAG (M2 Sigma; 2 μg/ml for AR and GR) or polyclonal anti-FLAG (2 μg/ml for PR) antibodies overnight at 4 °C in 3% bovine serum albumin in PBS. Cells were then rinsed three times with PBS and incubated with anti-rabbit or mouse secondary antibodies conjugated to either Texas Red or FITC (1:1000) in 3% bovine serum albumin in PBS for 90 min at RT. For immunocytochemistry of endogenous β-catenin, cells were fixed and incubated with polyclonal anti-β-catenin (Santa Cruz Biotechnology) followed by anti-rabbit FITC conjugate (1:1000). Coverslips were coated with 1 mg/ml p- phenylenediamine in 75% glycerol, 0.25× PBS prior to mounting and visualization. Images were viewed using a Zeiss Axioskop II microscope. NLT cells were grown in 5% charcoal-stripped fetal bovine serum in Opti-MEM for 36–48 h prior to transfection. Cells were transfected using LipofectAMINE Plus (Invitrogen) with 1 μg of the luciferase reporter construct (MMTV-LUC or pGL3-OT), 0.5 μg of β-catenin, 0.3 μg of TCF-4, and 0.5 μg of either pCMV-AR or empty control pCMV vector. Cells were also transfected with 0.1 μg of RSVβgal to control for transfection efficiency. Cells were harvested 16–18 h post-transfection and assayed for luciferase and β-galactosidase activities as described previously (37Kepa J.K. Wang C. Neeley C.I. Raynolds M.V. Gordon D.F. Wood W.M. Wierman M.E. Nucleic Acids Res. 1992; 20: 1393-1399Crossref PubMed Scopus (79) Google Scholar). Comparisons between ethanol control, and 5α-DHT-treated samples were analyzed using unpaired t test using GraphPad InStat version 3.02 for Windows, GraphPad Software, San Diego, CA. To identify neuronal protein partners of AR, a cDNA library was prepared from the GT1-7 GnRH cell line. This library was used in a yeast two-hybrid screen using an AR deletion mutant (AR-(Δ371–485)) that contained a truncation of amino acids 371–485 (Fig. 1 A). This construct was designed to possess lower transactivation activity in the yeast two-hybrid system than the wild-type AR, yet still contain AR domains essential for potential protein-protein interactions,i.e. N-C domains and DNA-binding domain. From ∼4 × 106 clones screened in the presence of 5α-DHT, one of the positive clones encoded an open reading frame of 236 amino acids. BLAST analysis against the NCBI protein sequence data base demonstrated that this open reading frame consisted of amino acids 187–423 of murine β-catenin. This region of β-catenin spans the Armadillo repeat region beginning at repeat 2 and ending in repeat 7 (Fig. 1 A). These Armadillo repeats are also necessary for β-catenin binding to cadherins, APC, axin, and TCF transcription factors (38Zhurinsky J. Shtutman M. Ben Ze'ev A. J. Cell Sci. 2000; 113: 3127-3139Crossref PubMed Google Scholar). The dependence of ligand for the interaction between AR-(Δ371–485) and β-catenin-(187–423) was confirmed using the yeast two-hybrid assay. Constructs were co-transformed into the yeast strain Y187 which allowed for quantitative analysis of the interaction by measuring β-galactosidase reporter activity (Fig. 1 B). No measurable β-galactosidase activity was detected in the absence of 5α-DHT with the bait plasmid alone or together with β-catenin-(187–423). In the presence of 5α-DHT, β-galactosidase activity was detected using AR-(Δ371–485) alone because of the ability of this construct to transactivate the β-galactosidase reporter gene. β-Galactosidase activity was increased ∼5-fold, however, when the interaction between AR-(Δ371–485) and β-catenin-(187–423) was assayed in the presence of 5α-DHT, confirming the ligand dependence of this interaction. Because the yeast two-hybrid assay detected an interaction between an AR deletion mutant and the Armadillo repeat region of β-catenin, COS-7 cells were co-transfected with full-length FLAG β-catenin and myc-AR to determine whether AR could interact with β-catenin in mammalian cells. Protein complexes were immunoprecipitated with anti-Myc and immunoblotted with anti-FLAG. Fig. 1 C demonstrates that FLAG-β-catenin was co-immunoprecipitated with AR in cells transfected with both constructs but not from cells transfected with FLAG-β-catenin and control pCMV-myc vector. To confirm the presence of β-catenin in GnRH neuronal cells, endogenous β-catenin was visualized by immunoblot analysis and immunocytochemistry. By immunoblot analysis, β-catenin was present in both GT1-7 and NLT cell lines, with slightly higher expression in NLT cells (Fig. 1 D). Immunocytochemical analysis demonstrated that endogenous β-catenin was predominantly localized to cell membranes, consistent with its cytoskeletal role. Additionally, low levels of diffuse cytoplasmic staining was apparent in both cell lines (not shown). These results confirmed the presence of β-catenin in GT1-7 and NLT GnRH neuronal cell lines and are in agreement with previous studies (39Sun W. Lee H. Choe Y. Cho S. Kim D.H. Kim K. J. Neuroendocrinol. 2001; 13: 249-260Crossref PubMed Scopus (11) Google Scholar) that investigated the presence and distribution of β-catenin in GT1–1 cells during neurite outgrowth. The mechanism of nuclear translocation of β-catenin is not completely understood. To determine whether liganded AR could facilitate nuclear translocation of β-catenin, initial studies were performed using GT1-7 cells that were co-transfected with rat AR and FLAG-β-catenin. To discriminate nuclear and cytoplasmic β-catenin from cytoskeletal components, cells were transfected with FLAG-β-catenin. Co-localization of AR and FLAG-β-catenin in the nucleus was observed in the presence of 5α-DHT (not shown); however, GT1-7 cells adhered poorly to the slides required for immunocytochemical analysis. Therefore, further experiments were performed in the NLT GnRH neuronal cell line. In co-transfected cells, AR and FLAG-β-catenin were distributed between the cytoplasmic and nuclear fractions in the absence of 5α-DHT. In the presence of 5α-DHT, however, both AR and FLAG-β-catenin were exclusively nuclear, suggesting that cytoplasmic β-catenin translocated to the nucleus with liganded AR (Fig. 2 A). In the absence of AR, 5α-DHT alone had no effect on β-catenin localization (data not shown). The time course for nuclear translocation of AR in response to 5α-DHT has been shown previously to be rapid, within 30 min (3Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (250) Google Scholar). In the presence of 5α-DHT, AR translocated to the nucleus in NLT cells within 15–30 min (Fig. 2 B). FLAG-β catenin also localized to the nucleus within 15–30 min in the presence of AR and 5α-DHT (Fig. 2 B), suggesting that a direct interaction between AR and β-catenin was necessary for co-localization. The anti-androgenic compounds casodex and hydroxyflutamide have been shown previously to target AR to the nucleus; however, they do not activate transcription by AR and antagonize the physiological effects of androgens (40Kemppainen J.A. Lane M.V. Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar). To determine whether these anti-androgens could also induce the co-localization of FLAG-β-catenin with AR, NLT cells were co-transfected with AR and FLAG-β-catenin and treated with either 100 nm casodex or hydroxyflutamide. Although these anti-androgens induced nuclear localization of AR, they failed to result in β-catenin translocation (Fig. 2 C). Flutamide, which binds AR with very low affinity (40Kemppainen J.A. Lane M.V. Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar), did not induce nuclear translocation of AR or FLAG-β-catenin (data not shown). The fact that casodex and hydroxyflutamide induced nuclear translocation of AR but not β-catenin suggests that translocation of AR alone is not sufficient to induce complete nuclear localization of β-catenin and that agonist, but not antagonist, -bound AR was required. Because transfected β-catenin is distributed between the nucleus and cytoplasm in the absence of 5α-DHT, an alternative explanation for the nuclear co-localization of AR with β-catenin might be that 5α-DHT treatment induced cytoplasmic β-catenin degradation, thus leaving only the nuclear fraction. The balance between cytoplasmic and nuclear β-catenin during development and Wnt signaling is due to the regulation of degradation and stabilization of cytoplasmic β-catenin (27van Leeuwen F. Samos C.H. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Cytoplasmic β-catenin is rapidly degraded through a proteosomal pathway following phosphorylation by GSK-3β (25Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2160) Google Scholar, 26Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-145" @default.
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• W2033728702 cites W1576763630 @default.
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• W2033728702 cites W2002404925 @default.
• W2033728702 cites W2005088732 @default.
• W2033728702 cites W2007589116 @default.
• W2033728702 cites W2009269691 @default.
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• W2033728702 cites W2019580313 @default.
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• W2033728702 cites W2036476604 @default.
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