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

2. INTRACELLULAR/ NUCLEAR RECEPTOR SIGNALING Tímea Berki and Ferenc Boldizsár Signal transduction Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

3. TÁMOP-4.1.2-08/1/A-2009-0011 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 and thyroxine 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. TÁMOP-4.1.2-08/1/A-2009-0011 Mechanism of action Nuclear receptorsNuclear receptors are proteins found within cells that are responsible for sensing steroid and thyroid hormonos and certain other lipophilic molecules Ligand binding to a nuclear receptor results in a conformational change in the receptor, which after activation behave as transcription factors The activation of the receptor results in up-regulation or down-regulation of gene expression

5. TÁMOP-4.1.2-08/1/A-2009-0011 Transcription factors Transcription factors: Transcription factors: 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. TÁMOP-4.1.2-08/1/A-2009-0011 Studying transcription factors Transcription factor activity: Luciferase test Chromatin immunoprecipitation (ChIP) Electrophoretic Mobility Shift Assay (EMSA) Transcription factor interaction: Co-immunoprecipitation

7. TÁMOP-4.1.2-08/1/A-2009-0011 1 1Transfection of the target cell with Luciferase Vector 2 2Stimulation of cells 3 3Signaling, TF activation 4 4Luciferase synthesis 5 5Light emission Promoter Reporter gene RNA polymerase and transcription factors Transcription mRNA Translation Reporter protein Luciferase reporter assay

8. TÁMOP-4.1.2-08/1/A-2009-0011 Ligands Lipophilic hormones: Lipophilic hormones: bound to transport proteins in the circulation enter through plasma membrane passively/transport protein

9. TÁMOP-4.1.2-08/1/A-2009-0011 Species distribution of NRs Nuclear receptors are specific to animals and are not found in algae fungi or plants 270 known receptors in C. elegans NOTE: several orphan receptors Humans, mice, and rats have 48, 49, and 47 nuclear receptors each, respectively.

10. TÁMOP-4.1.2-08/1/A-2009-0011 Intracellular receptors Steroid hormone rec.Estrogen rec. (ER)Estradiol Glucocorticoid rec. (GR) Cortisol Mineralocorticoid rec. (MR) Aldosterone Androgen rec. (AR)Testosterone Progesterone rec. (PR) Progesterone Thyroid hormone rec.Thyroid hormone rec. (TR) T3 Retinoid rec.Retinoic acid rec. (RAR) All-trans-retinoic acid Retinoic acid X rec. (RXR) 9-cis-retinoic acid Vitamin D rec.Vitamin D rec (VDR)1,25-hydroxy- cholecalciferol Lipid sensorsLiver X rec. (LXR)Oxysterols Farnesoid X rec. (FXR) Bile acids PPARPeroxisome proliferator activated rec. Fatty acids, eicosanoids (eg. LTs, PGs)

11. TÁMOP-4.1.2-08/1/A-2009-0011 Nuclear receptor superfamily GR Steroid Recetors Glucocorticoid Mineralocorticoid Progesterone Androgen GR MR PR AR Dimeric Orphan Receptors RXR COUP HNF-4 TR2 TLX GCNF 9-cis RA RXR Monomeric/Tethered Orphan Receptors NGFI-B SF-1 Rev-erb ROR ERR RXR T3R RAR VDR PPARa PPARg EcR FXR CAR LXR PXR/SXR RXR Heterodimers Thyroid hormone All-trans RA 1,2,5-(OH)2-VD Fatty acids 15d-Δ 12,14 -PGJ Ecdysone Bile acids Androstane Oxysterol Xenobiotics RXR R

12. TÁMOP-4.1.2-08/1/A-2009-0011 Structural organization of nuclear receptors AF-1: AF-1: activation function 1 (ligand-independent) AF-2: AF-2: activation function 2 (ligand-dependent) A/BCEFD N-terminal domain Hinge regionC-terminal domain DNA binding domain (DBD) Ligand binding domain (LBD) Dimerization 70AA highly conserved 200-250AA moderately conserved AF-1AF-2 50-500AA variable

13. TÁMOP-4.1.2-08/1/A-2009-0011 Mechanism of steroid receptor action HSP GR HSP GR HRE Co-activator RNA polymerase RNA polymerase GR Co-activator RNA polymerase RNA polymerase Co-activator RNA polymerase RNA polymerase RXRR HRE Co-activator RNA polymerase RNA polymerase Co-activator RNA polymerase RNA polymerase HRE RXR Co-repressor Hormone Nucleus Plasma membrane Cytoplasm Co-repressor RXRR HRE TranscriptionTranscriptionTranscription

14. TÁMOP-4.1.2-08/1/A-2009-0011 Time scale of GC action GRE Milliseconds (?)Seconds-minutes (?)Hours-days Multiple co- regulators TFs Nucleus Dimerization Binding Molecular assembly ? ? Levels of regulation CBG binding in blood MDR in the membrane Metabolism and nuclear receptor fate Transcription MR/GRSteroid

15. TÁMOP-4.1.2-08/1/A-2009-0011 Types of NRs Class I nuclear receptors include members of subfamily 3, such as the androgen receptor, estrogen receptors, glucocorticoid receptor, and progesterone receptor Type II nuclear receptors include principally subfamily 1, for example the retinoic acid receptor, retinoid X receptor and thyroid hormone receptor

16. TÁMOP-4.1.2-08/1/A-2009-0011 Mechanism of steroid receptor action Nucleus Plasma membrane CytoplasmHormone HSP NR HSP NR HRE Co-activator RNA polymerase RNA polymerase NR Co-activator RNA polymerase RNA polymerase mRNA Target gene mRNA Protein Changed cell function

17. TÁMOP-4.1.2-08/1/A-2009-0011 Type I NRs Class I NRs in the absence of ligand are located in the cytosol Hormone binding to the NR triggers dissociation of heat shock proteins, dimerization, and translocation to the nucleus In the nucleus they bind to a specific sequence of DNA known as a hormone response element (HRE) The nuclear receptor DNA complex in turn recruits other proteins that are responsible for transcription and translation into protein, which results in a change in cell function

18. TÁMOP-4.1.2-08/1/A-2009-0011 Cytoplasmic receptor complex Hsp90, 70, 40 + co-chaperone p23 + immunophilin eg. FKBP52 – links the complex to dynein Dynamic assembly-disassembly Ligand-bound receptors are transported to the nuclear pores along microtubules

19. TÁMOP-4.1.2-08/1/A-2009-0011 Mechanism of steroid receptor action Co-activator RNA polymerase RNA polymerase RXRR HRE Co-repressor Co-activator RNA polymerase RNA polymerase Nucleus Plasma membrane Cytoplasm mRNA Target gene mRNA Protein Changed cell function Hormone Co-repressor RXRR HRE

20. TÁMOP-4.1.2-08/1/A-2009-0011 Type II NRs They are retained in the nucleus regardless of the ligand binding status and in addition bind as hetero- dimers (usually with RXR) to DNA In the absence of ligand, type II nuclear receptors are often complexed with co-repressor proteins

21. TÁMOP-4.1.2-08/1/A-2009-0011 Nuclear receptor heterodimers PPR gamma (green) and RXR alpha (cyan) complexed with double stranded DNA (magenta) and NCOA2 co-activator peptide (red)

22. TÁMOP-4.1.2-08/1/A-2009-0011 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. TÁMOP-4.1.2-08/1/A-2009-0011 Genomic action of nuclear receptors Ligand LBD DBDRE

24. TÁMOP-4.1.2-08/1/A-2009-0011 Structure of DBD Structure of the human progesterone receptor DNA-binding domain dimer (cyan and green) complexed with double stranded DNA (magenta). Zinc atoms are depicted as grey spheres.

25. TÁMOP-4.1.2-08/1/A-2009-0011 Gene regulationTransactivation 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/histone acetylation) Ligand binding → co-repressor dissociation → co-activators bindTransrepression Without ligand transcription proceeds constitutively, ligand binding inhibits transcription

26. TÁMOP-4.1.2-08/1/A-2009-0011 Transrepression and selectivity of ligands Some nuclear receptors not only have the ability to directly bind to DNA, but also to other transcription factors. This binding often results in deactivation of the second transcription factor Certain GR ligands known as Selective Glucocorticoid Receptor Agonists (SEGRAs) are able to activate GR in such a way that GR more strongly trans-represses than trans- activates This selectivity increases the possibility to develop ligands wich are able to separately cause desired anti-inflammatory effects and there is less undesired metabolic side effects of these selective GCs

27. TÁMOP-4.1.2-08/1/A-2009-0011 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: Src in ER

28. TÁMOP-4.1.2-08/1/A-2009-0011 Regulation of nuclear receptors Down-regulation of transcriptional activity: Phosphorylation of the DBD PKC or PKA

29. TÁMOP-4.1.2-08/1/A-2009-0011 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 Vitamine A /D deficiency