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Download The bHLH-PAS protein ARNT - an activator of ER activity
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The bHLH-PAS protein ARNT - effects on ER signalling Biological Effects of Dioxin • Toxic Effects: Wasting, lymph atrophy, endometriosis, etc • Teratogenic Effects • Tumor Promotion • Enzyme Induction • Interference with Hormonal signaling (EDC) The bHLH-PAS proteins Dimerization DNA- and hsp90 binding Examples: Ligand- and hsp90 binding bHLH PAS Domain Metabolism of xenobiotics Dioxin Receptor , AhR Partner for AhR and HIF-1a Sim CNS growth/differentiation Regulation of circadian rhythm Arnt Per A B A B A B A B Activation process of the AhR AhR Nuclear Translocator: ARNT • Obligatory partner for DR and HIF-1a • Ligand DR-ARNT dimers bind to xenobiotic response elements (XREs) of target genes • ARNT is constitutively expressed protein • Nuclear localization • 3 isoforms identified ARNT isoforms • ARNT-1: ubiquitously expressed • ARNT-2: in developing brain, kidney, heart • bMAL/ARNT-3: brain, muscle bHLH PAS1 PAS2 mARNT-1 791 aa 57% 95% 78% mARNT-2 712 aa 34% 63% 39% bMAL/mARNT-3 632 aa Nuclear receptor superfamily A/B AF-1 C DBD D Hinge E LBD AF-2 Ligand regulate receptors like steroid receptors, thyroid hormaone receptors, retinoid acid receptors and Vitamin D Orpan receptors Nuclear receptor signaling HSP 90 NR Complex +ligand NR + HSP 90 Complex transcription cofactors HRE Gene promoter Nuclear receptor co-factors Coactivators: SRC family, CBP/p300, PBP, PCAF… Corepressors: N-CoR, SMRT, DAX-1, SHP… SRC/p160 Co-activator factors bHLH PAS SRC-1 SCR2 (TIF-2) SRC-3 NR-box ARNT regulates multiple pathways p23 hsp90 A B DR A B ARNT p23 hsp90 A HIF-1a B Other nuclear receptors? Other transcription factors? ERa and ARNT interact pSG5-FlagpSG5-Flag-ERa pCMV-ARNT + + + + - E2 + + + + Tam - IP:anti-Flag ARNT WCEs: 10% of input ARNT Western: anti-ARNT ERb and ARNT interact pSG5pSG5-ERb pCMV-ARNT + + + + - - + + + + E2 Tam IP:ERb mAb ARNT WCEs: 10% input ARNT Western: anti-ARNT ARNT stimulates ERa activity HeLa/3xERE-T AT A-luc 500 450 400 350 300 (-) E2 10 nM Tam 1 uM 250 200 150 100 50 0 3xERE pSG5 ERa + ERa + ERa + ERa + ERa + ERa + TATA ERa ARNT ARNT ARNT TIF-2 TIF-2 TIF-2 ARNT TIF-2 ARNT stimulates ERb activity HeLa/3xERE-TATA-luc 2000 1800 1600 1400 (-) E2 10 nM Tam 1 uM 1200 1000 800 600 400 200 0 3xERE TATA luc ERb ERb + ARNT ERb + ARNT ERb + ARNT ARNT ERb + TIF-2 ERb + TIF-2 ERb + TIF-2 TIF-2 ARNT interacts with the LBD of ERa/b The C-Terminal domain of ARNT is required for co-activation ARNT is recruited to the pS2 promoter + E2 PCR products pS2 ERE -353 -54 TCDD and 3-MC inhibit ERs transcriptional activity in HC11 cells 120 E2 DPN PPT 100 % Activity 80 60 40 20 0 - TCDD 3-MC TCDD 3-MC TCDD 3-MC TCDD 3-MC 3-MC but not TCDD activates ERs - E2 3-MC Antiestrogens inhibit 3-MC induced transcriptional activity Hypoxia and TCDD inhibit ER transcriptional activity in HC11 cells Activation of the AhR inhibits recruitment of ARNT to E2 regulated promoters E2+ NH E2 TCDD TCDD Input NH E2 TCDD E2+ TCDD IgG Input a-ERa IgG a-ARNT ARNT c-Fos Lactoferrin ERE UTR ERE Effects of ARNT Si-RNA SUMMARY • ARNT interacts with ligand-activated ERa and ERb • ARNT stimulates both ERa and ERb transcriptional activity • ARNT is recruited to the E2 regulated promoters in an ligand-dependent manner ARNT regulates multiple cellular pathways p23 hsp90 A B DR ERa/b A B ARNT p23 hsp90 A HIF-1a B Other nuclear receptors? Other transcription factors? Pauliina Pentinen / Sari Mäkele University of Turku Juliette Rambaud / Vincent Laudet Ecole Normale Supérieure de Lyon Ingemar Pongratz Katarina Pettersson Sara Brunnberg Elin Swedenborg Wen Cai Petra Berg
