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

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

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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