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Intracellular/ nucle A r receptor signaling

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Intracellular/ nucle A r receptor signaling

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signaltransduction Intracellular/nucleAr receptor signaling

  3. History • Scottish surgeon G.T. Beatson: inoperable breast tumors showed regression after ovaryectomy • Castration of animals improves meat • Ancient Chinese medicine used placental extracts • 1926 Kendall and Reichstein cortisone andthyroxine • Butenandt / Doisy estrogen (urine of pregnant women) • Androsteron and progesteron (first isolated from the corpus luteum of pigs) followed • “estrus” ~ “oistros” (Greek) = gadfly • 1961 Jensen: estrogen receptor • 1980s: cloning of ER, GR, TR by Chambon, Evans and Vennström

  4. Mechanism of action • Nuclearreceptorsareproteinsfoundwithincellsthatareresponsibleforsensingsteroid and thyroid hormonos and certainotherlipophilicmolecules • Ligandbindingto a nuclear receptor resultsin a conformationalchangeinthe receptor, whichafteractivationbehaveastranscriptionfactors • The activation of the receptor resultsinup-regulationordown-regulation of geneexpression

  5. Transcription factors • Transcriptionfactors: sequence-specific DNA-binding factors • Control the transmission of genetic information from DNA to mRNA • Act as activators (=promote gene expression) or repressors (=inhibit gene expression) by affecting the recruitment of RNA Polymerase

  6. Studying transcription factors • Transcription factor activity: • Luciferase test • Chromatin immunoprecipitation (ChIP) • ElectrophoreticMobility Shift Assay (EMSA) • Transcription factor interaction: • Co-immunoprecipitation

  7. Luciferase reporter assay • 1Transfection of the target cell with Luciferase Vector • 2Stimulation of cells • 3Signaling, TF activation • 4Luciferase synthesis • 5Light emission RNA polymerase and transcriptionfactors Promoter Reportergene Transcription mRNA Translation Reporter protein

  8. Ligands • Lipophilic hormones: bound to transport proteins in the circulation • enter through plasma membrane passively/transportprotein

  9. Species distribution of NRs • Nuclearreceptorsarespecifictoanimals and arenotfoundinalgaefungiorplants • 270 known receptors in C. elegans • NOTE: several orphan receptors • Humans, mice, and ratshave 48, 49, and 47 nuclearreceptorseach, respectively.

  10. Intracellular receptors

  11. Nuclear receptor superfamily GR GR Steroid Recetors RXR Heterodimers GR MR PR AR T3R RAR VDR PPARa PPARg EcR FXR CAR LXR PXR/SXR Glucocorticoid Mineralocorticoid Progesterone Androgen Thyroid hormone All-trans RA 1,2,5-(OH)2-VD Fatty acids 15d-Δ12,14-PGJ Ecdysone Bile acids Androstane Oxysterol Xenobiotics RXR R Dimeric Orphan Receptors Monomeric/Tethered Orphan Receptors RXR COUP HNF-4 TR2 TLX GCNF 9-cis RA NGFI-B SF-1 Rev-erb ROR ERR RXR RXR RXR

  12. Structuralorganization of nuclearreceptors 50-500AA variable Dimerization Hinge region C-terminaldomain N-terminaldomain A/B C D E F Ligand binding domain (LBD) DNA binding domain (DBD) AF-1 AF-2 70AA highly conserved 200-250AA moderately conserved AF-1: activation function 1 (ligand-independent) AF-2: activation function 2 (ligand-dependent)

  13. Mechanism of steroid receptor action Hormone Plasmamembrane Cytoplasm HSP RXR RXR GR GR GR GR GR RXR RXR R R GR HSP Co-repressor Nucleus Co-activator Co-activator Co-activator Co-activator Co-activator RNA polymerase RNA polymerase RNA polymerase RNA polymerase RNA polymerase Co-repressor HRE HRE HRE HRE Transcription Transcription Transcription

  14. Time scale of GC action Levels of regulation Milliseconds (?) Hours-days Seconds-minutes (?) CBG binding inblood MDR in the membrane ? Molecular assembly Binding Metabolism and nuclear receptor fate ? TFs Multipleco-regulators Dimerization Nucleus Transcription GRE Steroid MR/GR

  15. Types of NRs • Class I nuclearreceptorsincludemembers of subfamily 3, suchastheandrogen receptor, estrogenreceptors, glucocorticoidreceptor, and progesterone receptor • Type II nuclearreceptorsincludeprincipallysubfamily 1, forexampletheretinoicacid receptor, retinoid X receptor and thyroidhormone receptor

  16. Mechanism of steroid receptor action Hormone Plasmamembrane HSP Cytoplasm NR NR NR NR NR NR HSP Protein Changedcellfunction mRNA Co-activator Nucleus Co-activator RNA polymerase RNA polymerase mRNA HRE Targetgene

  17. Type I NRs • Class I NRsintheabsence of ligandarelocatedinthecytosol • Hormonebindingtothe NR triggersdissociation of heatshockproteins, dimerization, and translocationtothenucleus • Inthenucleustheybindto a specificsequence of DNA knownas a hormoneresponseelement (HRE) • The nuclear receptor DNA complexinturnrecruitsotherproteinsthatareresponsiblefortranscription and translationinto protein, whichresultsin a changeincellfunction

  18. Cytoplasmic receptor complex • Hsp90, 70, 40 + co-chaperone p23 + immunophilineg. FKBP52 – links the complex to dynein • Dynamic assembly-disassembly • Ligand-bound receptors are transported to the nuclear pores along microtubules

  19. Mechanism of steroid receptor action Hormone Plasmamembrane Cytoplasm RXR R R RXR Changedcellfunction Protein Co-repressor mRNA Nucleus Co-repressor Co-activator Co-activator RNA polymerase mRNA RNA polymerase HRE HRE Targetgene

  20. Type II NRs • Theyareretainedinthenucleusregardless of theligandbinding status and inadditionbindashetero-dimers (usuallywith RXR) to DNA • Intheabsence of ligand, type II nuclearreceptorsareoftencomplexedwithco-repressorproteins

  21. Nuclear receptor heterodimers • PPR gamma (green) and RXR alpha (cyan) complexedwithdoublestranded DNA (magenta) and NCOA2 co-activatorpeptide (red)

  22. DNA binding • DNA binding sites (=Response Elements): • 2x6 base pairs • Steroid receptors (homodimers): palindromic, inverted repeats separated by 3bp spacer (IR3) • GR, MR, PR, AR: 5’-AGAACA-3’ • ER: 5’-AGGTCA-3’ • Non-steroid receptors: direct repeats of 5’-AGGTCA-3’ (DRn, n=number of spacers) • homodimers (eg. TR, VDR) • heterodimers (eg. TR, VDR, RAR, LXR, FXR, PXR, CAR, PPAR)

  23. Genomicaction of nuclearreceptors Ligand LBD RE DBD

  24. Structure of DBD • Structure of the human progesterone receptor DNA-bindingdomaindimer (cyan and green) complexedwithdoublestranded DNA (magenta). Zincatomsaredepictedasgreyspheres.

  25. Gene regulation • Transactivation • Ligand-bound receptor recruits co-activators→ up-regulation of transcription: interaction with the general transcription factors + chromatin has to be “opened up” (ATP-dependent chromatin remodeling/histoneacetylation) • Ligand binding →co-repressor dissociation → co-activators bind • Transrepression • Withoutligandtranscription proceeds constitutively, ligand binding inhibits transcription

  26. Transrepression and selectivity of ligands • Somenuclearreceptorsnotonlyhavetheabilitytodirectlybindto DNA, butalsotoothertranscriptionfactors. Thisbindingoftenresultsindeactivation of thesecondtranscriptionfactor • Certain GR ligandsknownasSelectiveGlucocorticoid Receptor Agonists (SEGRAs) areabletoactivate GR insuch a waythat GR more stronglytrans-repressesthantrans-activates • Thisselectivityincreasesthepossibilitytodevelopligandswichareabletoseparatelycausedesiredanti-inflammatoryeffects and there is less undesiredmetabolicsideeffects of theseselectiveGCs

  27. Regulation of nuclear receptors • Up-regulation of transcriptional activity: • Phosphorylation: • Ser residues in the N-terminal A/B domains; • Cyclin-dependent kinases • PKC, PKA • ERK • PKB/Akt • JNK/SAPK • p38-MAPK • AF-1: CDK, ERK, JNK, p38-MAPK, PKB • AF-2: Srcin ER

  28. Regulation of nuclear receptors Down-regulation of transcriptional activity: • Phosphorylation of the DBD PKC or PKA

  29. Therapeutic implications – hormone analogues • Glucocorticoids: anti-inflammatory, immunosuppressive therapy (eg. autoimmune diseases, transplantation, some leukemias) • Sex steroids: substitution therapy (endocrine diseases), birth control, breast cancer • Thyroxin: substitution therapy after thyroidectomy • VitamineA/D deficiency

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