at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
INTRACELLULAR/
NUCLEAR RECEPTOR SIGNALING
Tímea Berki and Ferenc Boldizsár Signal transduction
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
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
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
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
Studying transcription factors
Transcription factor activity:
• Luciferase test
• Chromatin immunoprecipitation (ChIP)
• Electrophoretic Mobility Shift Assay (EMSA) Transcription factor interaction:
• Co-immunoprecipitation
1 Transfection of the target cell with Luciferase Vector 2 Stimulation of cells
3 Signaling, TF activation 4 Luciferase synthesis 5 Light emission
Promoter Reporter gene RNA polymerase and
transcription factors
Transcription mRNA
Translation
Reporter protein
Luciferase reporter assay
Ligands
Lipophilic hormones: bound to transport proteins in the circulation enter through plasma membrane passively/transport protein
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.
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 sensors Liver X rec. (LXR) Oxysterols
Farnesoid X rec. (FXR) Bile acids
PPAR Peroxisome proliferator
activated rec.
Fatty acids, eicosanoids (eg. LTs, PGs)
Nuclear receptor superfamily
GR 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 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-Δ12,14-PGJ Ecdysone Bile acids Androstane Oxysterol Xenobiotics
RXR R
Structural organization of nuclear receptors
AF-1: activation function 1 (ligand-independent) AF-2: activation function 2 (ligand-dependent)
A/B C D E F
N-terminal domain Hinge region C-terminal domain
DNA binding domain (DBD) Ligand binding domain (LBD) Dimerization
70AA highly conserved 200-250AA moderately conserved
AF-1 AF-2
50-500AA variable
action
HSP GR GR
GR
HSP
GR
HRE
Co-activator polymerase RNA GR
GR
Co-activator
RNA polymerase
Co-activator polymerase RNA RXR R
HRE
Co-activator
RNA polymerase
Co-activator polymerase RNA
HRE RXR RXR Co-repressor
Hormone
Nucleus Plasma membrane
Cytoplasm
Co-repressor RXR R
HRE
Transcription Transcription Transcription
Time scale of GC action
GRE
Milliseconds (?) Hours-days Seconds-minutes (?)
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/GR Steroid
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
Mechanism of steroid receptor action
Nucleus Plasma membrane
Cytoplasm Hormone
NR HSP NR NR
HSP
NR
HRE
Co-activator polymerase RNA NR
NR Co-activator
RNA
polymerase mRNA
Target gene
mRNA
Protein Changed cell function
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
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
action
Co-activator polymerase RNA RXR R
HRE
Co-repressor Co-activator
polymerase RNA
Nucleus Plasma membrane
Cytoplasm
mRNA
Target gene
mRNA Protein Changed cell function Hormone
Co-repressor RXR R
HRE
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
Nuclear receptor heterodimers
PPR gamma (green) and RXR alpha (cyan) complexed with double stranded DNA (magenta) and NCOA2 co-activator peptide (red)
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)
Genomic action of nuclear receptors
Ligand
LBD
DBD RE
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.
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/histone acetylation)
• Ligand binding → co-repressor dissociation → co-activators bind
Transrepression
• Without ligand transcription proceeds constitutively, ligand binding inhibits transcription
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
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
Regulation of nuclear receptors
Down-regulation of transcriptional activity:
• Phosphorylation of the DBD PKC or PKA
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
