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

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Intracellular/ nucle A r receptor signaling

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Intracellular nucle a r receptor signaling

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen

Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011


Intracellular nucle a r receptor signaling

Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

Tímea Berki and Ferenc Boldizsár

Signaltransduction

Intracellular/nucleAr receptor signaling


History

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 andthyroxine

  • 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

Mechanism of action

  • Nuclearreceptorsareproteinsfoundwithincellsthatareresponsibleforsensingsteroid and thyroid hormonos and certainotherlipophilicmolecules

  • Ligandbindingto a nuclear receptor resultsin a conformationalchangeinthe receptor, whichafteractivationbehaveastranscriptionfactors

  • The activation of the receptor resultsinup-regulationordown-regulation of geneexpression


Transcription factors

Transcription factors

  • Transcriptionfactors: 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

Studying transcription factors

  • Transcription factor activity:

  • Luciferase test

  • Chromatin immunoprecipitation (ChIP)

  • ElectrophoreticMobility Shift Assay (EMSA)

  • Transcription factor interaction:

  • Co-immunoprecipitation


Luciferase reporter assay

Luciferase reporter assay

  • 1Transfection of the target cell with Luciferase Vector

  • 2Stimulation of cells

  • 3Signaling, TF activation

  • 4Luciferase synthesis

  • 5Light emission

RNA polymerase and

transcriptionfactors

Promoter

Reportergene

Transcription

mRNA

Translation

Reporter protein


Ligands

Ligands

  • Lipophilic hormones: bound to transport proteins in the circulation

  • enter through plasma membrane passively/transportprotein


Species distribution of nrs

Species distribution of NRs

  • Nuclearreceptorsarespecifictoanimals and arenotfoundinalgaefungiorplants

  • 270 known receptors in C. elegans

  • NOTE: several orphan receptors

  • Humans, mice, and ratshave 48, 49, and 47 nuclearreceptorseach, respectively.


Intracellular receptors

Intracellular receptors


Nuclear receptor superfamily

Nuclear receptor superfamily

GR

GR

Steroid Recetors

RXR Heterodimers

GR

MR

PR

AR

T3R

RAR

VDR

PPARa

PPARg

EcR

FXR

CAR

LXR

PXR/SXR

Glucocorticoid

Mineralocorticoid

Progesterone

Androgen

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

Dimeric Orphan Receptors

Monomeric/Tethered Orphan Receptors

RXR

COUP

HNF-4

TR2

TLX

GCNF

9-cis RA

NGFI-B

SF-1

Rev-erb

ROR

ERR

RXR

RXR

RXR


Structural organization of nuclear receptors

Structuralorganization of nuclearreceptors

50-500AA variable

Dimerization

Hinge region

C-terminaldomain

N-terminaldomain

A/B

C

D

E

F

Ligand binding domain (LBD)

DNA binding domain (DBD)

AF-1

AF-2

70AA highly conserved

200-250AA moderately conserved

AF-1: activation function 1 (ligand-independent)

AF-2: activation function 2 (ligand-dependent)


Mechanism of steroid receptor action

Mechanism of steroid receptor action

Hormone

Plasmamembrane

Cytoplasm

HSP

RXR

RXR

GR

GR

GR

GR

GR

RXR

RXR

R

R

GR

HSP

Co-repressor

Nucleus

Co-activator

Co-activator

Co-activator

Co-activator

Co-activator

RNA

polymerase

RNA

polymerase

RNA

polymerase

RNA

polymerase

RNA

polymerase

Co-repressor

HRE

HRE

HRE

HRE

Transcription

Transcription

Transcription


Time scale of gc action

Time scale of GC action

Levels of

regulation

Milliseconds (?)

Hours-days

Seconds-minutes (?)

CBG binding

inblood

MDR in the

membrane

?

Molecular

assembly

Binding

Metabolism and

nuclear receptor fate

?

TFs

Multipleco-regulators

Dimerization

Nucleus

Transcription

GRE

Steroid

MR/GR


Types of nrs

Types of NRs

  • Class I nuclearreceptorsincludemembers of subfamily 3, suchastheandrogen receptor, estrogenreceptors, glucocorticoidreceptor, and progesterone receptor

  • Type II nuclearreceptorsincludeprincipallysubfamily 1, forexampletheretinoicacid receptor, retinoid X receptor and thyroidhormone receptor


Mechanism of steroid receptor action1

Mechanism of steroid receptor action

Hormone

Plasmamembrane

HSP

Cytoplasm

NR

NR

NR

NR

NR

NR

HSP

Protein

Changedcellfunction

mRNA

Co-activator

Nucleus

Co-activator

RNA

polymerase

RNA

polymerase

mRNA

HRE

Targetgene


Type i nrs

Type I NRs

  • Class I NRsintheabsence of ligandarelocatedinthecytosol

  • Hormonebindingtothe NR triggersdissociation of heatshockproteins, dimerization, and translocationtothenucleus

  • Inthenucleustheybindto a specificsequence of DNA knownas a hormoneresponseelement (HRE)

  • The nuclear receptor DNA complexinturnrecruitsotherproteinsthatareresponsiblefortranscription and translationinto protein, whichresultsin a changeincellfunction


Cytoplasmic receptor complex

Cytoplasmic receptor complex

  • Hsp90, 70, 40 + co-chaperone p23 + immunophilineg. FKBP52 – links the complex to dynein

  • Dynamic assembly-disassembly

  • Ligand-bound receptors are transported to the nuclear pores along microtubules


Mechanism of steroid receptor action2

Mechanism of steroid receptor action

Hormone

Plasmamembrane

Cytoplasm

RXR

R

R

RXR

Changedcellfunction

Protein

Co-repressor

mRNA

Nucleus

Co-repressor

Co-activator

Co-activator

RNA

polymerase

mRNA

RNA

polymerase

HRE

HRE

Targetgene


Type ii nrs

Type II NRs

  • Theyareretainedinthenucleusregardless of theligandbinding status and inadditionbindashetero-dimers (usuallywith RXR) to DNA

  • Intheabsence of ligand, type II nuclearreceptorsareoftencomplexedwithco-repressorproteins


Nuclear receptor heterodimers

Nuclear receptor heterodimers

  • PPR gamma (green) and RXR alpha (cyan) complexedwithdoublestranded DNA (magenta) and NCOA2 co-activatorpeptide (red)


Dna binding

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

    Genomicaction of nuclearreceptors

    Ligand

    LBD

    RE

    DBD


    Structure of dbd

    Structure of DBD

    • Structure of the human progesterone receptor DNA-bindingdomaindimer (cyan and green) complexedwithdoublestranded DNA (magenta). Zincatomsaredepictedasgreyspheres.


    Gene regulation

    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/histoneacetylation)

    • Ligand binding →co-repressor dissociation → co-activators bind

    • Transrepression

    • Withoutligandtranscription proceeds constitutively, ligand binding inhibits transcription


    Transrepression and selectivity of ligands

    Transrepression and selectivity of ligands

    • Somenuclearreceptorsnotonlyhavetheabilitytodirectlybindto DNA, butalsotoothertranscriptionfactors. Thisbindingoftenresultsindeactivation of thesecondtranscriptionfactor

    • Certain GR ligandsknownasSelectiveGlucocorticoid Receptor Agonists (SEGRAs) areabletoactivate GR insuch a waythat GR more stronglytrans-repressesthantrans-activates

    • Thisselectivityincreasesthepossibilitytodevelopligandswichareabletoseparatelycausedesiredanti-inflammatoryeffects and there is less undesiredmetabolicsideeffects of theseselectiveGCs


    Regulation of nuclear receptors

    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: Srcin ER


    Regulation of nuclear receptors1

    Regulation of nuclear receptors

    Down-regulation of transcriptional activity:

    • Phosphorylation of the DBD PKC or PKA


    Therapeutic implications hormone analogues

    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

    • VitamineA/D deficiency


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