Transcript nucleAr receptor signaling

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
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
Tímea Berki and Ferenc Boldizsár
Signal transduction
INTRACELLULAR/
NUCLEAR RECEPTOR
SIGNALING
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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
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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: sequencespecific 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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Luciferase reporter assay
1 Transfection of the target cell with Luciferase Vector
2 Stimulation of cells
3 Signaling, TF activation
4 Luciferase synthesis
5 Light emission
RNA polymerase and
transcription factors
Promoter
Reporter gene
Transcription
mRNA
Translation
Reporter
protein
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Ligands
Lipophilic hormones: bound to transport proteins in
the circulation
enter through plasma membrane passively/transport
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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 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-hydroxycholecalciferol
Lipid sensors
Liver X rec. (LXR)
Oxysterols
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Nuclear receptor
superfamily
Steroid Recetors
RXR Heterodimers
GR
Glucocorticoid
T3R
Thyroid hormone
MR
Mineralocorticoid
RAR
All-trans RA
PR
Progesterone
VDR
1,2,5-(OH)2-VD
AR
Androgen
PPARa
Fatty acids
PPARg
15d-Δ12,14-PGJ
EcR
Ecdysone
FXR
Bile acids
CAR
Androstane
LXR
Oxysterol
PXR/SXR
Xenobiotics
GR
GR
Dimeric Orphan Receptors
RXR
COUP
HNF-4
TR2
TLX
GCNF
RXR
Monomeric/Tethered Orphan Receptors
9-cis RA
RXR
RXR
NGFI-B
SF-1
Rev-erb
ROR
ERR
RXR
R
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Structural organization of
nuclear receptors
50-500AA variable
Dimerization
Hinge region
N-terminal domain
A/B
AF-1
C
D
DNA binding domain (DBD)
C-terminal domain
E
Ligand binding domain (LBD)
F
AF-2
70AA highly conserved
200-250AA moderately conserved
AF-1: activation function 1 (ligand-independent)
AF-2: activation function 2 (ligand-dependent)
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Mechanism of steroid
receptor action
Hormone
Plasma membrane
Cytoplasm
HSP
GR
GR
GR
HSP
GR
Nucleus
RXR
R
Co-repressor
Co-activator
RNA
polymerase
Co-activator
HRE
RNA
polymerase
Co-repressor
Co-activator
GR
RNA
Transcription
polymerase
GR
HRE
RXR
HRE
R
Co-activator
RNA
Transcription
polymerase
RXR
Co-activator
RXR
RNA
Transcription
polymerase
HRE
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Time scale of GC action
Levels of
regulation
Milliseconds (?)
Hours-days
Seconds-minutes (?)
CBG binding
in blood
MDR in the
membrane
Metabolism and
nuclear receptor fate
?
Molecular
assembly
?
Binding
TFs
Multiple
coregulators
Dimerization
Nucleus
Transcription
GRE
Steroid
MR/GR
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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
Mechanism of steroid
receptor action
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Hormone
Plasma membrane
Cytoplasm
HSP
NR
HSP
Protein
NR
NR
Changed cell function
NR
mRNA
Nucleus
Co-activator
RNA
polymerase
mRNA
NR
NR
Co-activator
RNA
polymerase
HRE
Target gene
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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
Cytoplasmic receptor
complex
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• 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
Mechanism of steroid
receptor action
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Hormone
Plasma membrane
Cytoplasm
Changed cell function
Protein
mRNA
Nucleus
Co-activator
Co-repressor
RNA
polymerase
RXR
R
HRE
Co-repressor
mRNA
RXR
R
Co-activator
RNA
polymerase
HRE
Target gene
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Type II NRs
• They are retained in the nucleus
regardless of the ligand binding
status and in addition bind as heterodimers (usually with RXR) to DNA
• In the absence of ligand, type II
nuclear receptors are often complexed
with co-repressor proteins
Nuclear receptor
heterodimers
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PPR gamma (green) and RXR alpha (cyan) complexed with
double stranded DNA (magenta) and NCOA2 co-activator
peptide (red)
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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)
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Genomic action of nuclear
receptors
Ligand
LBD
RE
DBD
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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 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
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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 transactivates
• This selectivity increases the possibility
to develop ligands wich are able to
separately cause desired anti-inflammatory
effects and there is less undesired
Regulation of nuclear
receptors
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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
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Down-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