Intracellular/ nucle A r receptor signaling
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Transcript of Intracellular/ nucle A r receptor signaling
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
INTRACELLULAR/ NUCLEAR RECEPTOR SIGNALING
Tímea Berki and Ferenc BoldizsárSignal transduction
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
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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
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Mechanism of action• Nuclear 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
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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
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Studying transcription factors
Transcription factor activity:• Luciferase test• Chromatin immunoprecipitation (ChIP)• Electrophoretic Mobility Shift Assay (EMSA)
Transcription factor interaction:• Co-immunoprecipitation
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1 Transfection of the target cell with Luciferase Vector2 Stimulation of cells3 Signaling, TF activation4 Luciferase synthesis5 Light emission
Promoter Reporter gene
RNA polymerase andtranscription factors
Transcription
mRNA
Translation
Reporter protein
Luciferase reporter assay
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Ligands
Lipophilic hormones: bound to transport proteins in the circulationenter through plasma membrane passively/transport protein
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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.
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Intracellular receptorsSteroid hormone rec. Estrogen rec. (ER) Estradiol
Glucocorticoid rec. (GR) CortisolMineralocorticoid rec. (MR)
Aldosterone
Androgen rec. (AR) TestosteroneProgesterone rec. (PR) Progesterone
Thyroid hormone rec. Thyroid hormone rec. (TR)
T3
Retinoid rec. Retinoic acid rec. (RAR) All-trans-retinoic acidRetinoic acid X rec. (RXR)
9-cis-retinoic acid
Vitamin D rec. Vitamin D rec (VDR) 1,25-hydroxy-cholecalciferol
Lipid sensors Liver X rec. (LXR) OxysterolsFarnesoid X rec. (FXR) Bile acids
PPAR Peroxisome proliferator activated rec.
Fatty acids, eicosanoids (eg. LTs, PGs)
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Nuclear receptor superfamily
GR GR
Steroid Recetors
GlucocorticoidMineralocorticoidProgesteroneAndrogen
GRMRPRAR
Dimeric Orphan Receptors
RXRCOUPHNF-4TR2TLXGCNF
9-cis RARXRRXR
Monomeric/Tethered Orphan Receptors
NGFI-BSF-1Rev-erbRORERR
RXR
T3RRARVDRPPARaPPARgEcRFXRCARLXRPXR/SXR
RXR Heterodimers
Thyroid hormoneAll-trans RA1,2,5-(OH)2-VDFatty acids15d-Δ12,14-PGJEcdysoneBile acidsAndrostaneOxysterolXenobiotics
RXR R
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Structural organization of nuclear receptors
AF-1: activation function 1 (ligand-independent)AF-2: activation function 2 (ligand-dependent)
A/B C E FDN-terminal domain Hinge region C-terminal domain
DNA binding domain (DBD) Ligand binding domain (LBD)
Dimerization
70AA highly conserved200-250AA moderately conserved
AF-1 AF-2
50-500AA variable
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Mechanism of steroid receptor action
HSPGRGRGR
HSP
GR
HRE
Co-activatorRNA
polymeraseGRGR
Co-activator
RNApolymerase
Co-activatorRNA
polymeraseRXR R
HRE
Co-activator
RNApolymerase
Co-activatorRNA
polymerase
HRE
RXRRXR
Co-repressor
Hormone
Nucleus
Plasma membrane
Cytoplasm
Co-repressorRXR R
HRE
Transcription Transcription Transcription
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Time scale of GC action
GRE
Milliseconds (?) Seconds-minutes (?)Hours-days
Multiple co-regulators
TFs
Nucleus
Dimerization
BindingMolecular assembly
?
?
Levels ofregulation
CBG bindingin blood
MDR in themembrane
Metabolism andnuclear receptor fate
Transcription
MR/GRSteroid
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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
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Mechanism of steroid receptor action
Nucleus
Plasma membrane
Cytoplasm
Hormone
HSPNR NR NR
HSP
NR
HRE
Co-activatorRNA
polymeraseNRNR
Co-activator
RNApolymerase mRNA
Target gene
mRNA
Protein Changed cell function
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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
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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
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Mechanism of steroid receptor action
Co-activatorRNA
polymeraseRXR R
HRE
Co-repressorCo-activator
RNApolymerase
Nucleus
Plasma membrane
Cytoplasm
mRNA
Target gene
mRNA
Protein
Changed cell function
Hormone
Co-repressorRXR R
HRE
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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
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Nuclear receptor heterodimers
PPR gamma (green) and RXR alpha (cyan) complexed with double stranded DNA (magenta) and NCOA2 co-activator peptide (red)
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DNA bindingDNA 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)
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Genomic action of nuclear receptors
Ligand
LBD
DBDRE
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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.
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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 bind
Transrepression• Without ligand transcription proceeds constitutively,
ligand binding inhibits transcription
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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
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Regulation of nuclear receptorsUp-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
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Regulation of nuclear receptorsDown-regulation of transcriptional activity:• Phosphorylation of the DBD PKC or PKA
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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