Nuclear receptorsNuclear receptors

structure structure and functionand function

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Let’s start from a small fragment..... (?)Let’s start from a small fragment..... (?)

Types of Types of leukemiasleukemias

A.A.

-- LymphoLymphoidid

-- MyeloMyeloidid

B.B.

-- acuteacute(results from a transformation relatively undifferentiated proge(results from a transformation relatively undifferentiated progenitor cells, with only limited nitor cells, with only limited capabilities of differentiation)capabilities of differentiation)

-- chronicchronic(results from a transformation of more differentiated cells, whi(results from a transformation of more differentiated cells, which have capability to mature)ch have capability to mature)

AcuteAcute myolocyticmyolocytic leukemialeukemia

ChronicChronic myolocyticmyolocytic leukemialeukemia

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)-- sustains about sustains about 3% of cancers3% of cancers in humans (15in humans (15--20% all 20% all leukemiasleukemias in adults), in adults), approximately approximately 11--2 cases per 100 000 people2 cases per 100 000 people

-- average ageaverage age of diagnosed patients: of diagnosed patients: 53 years53 years (30% patients is older than 60 years, (30% patients is older than 60 years, less than 10% less than 10% underunder 20 years20 years ofof ageage))

-- in about half of patients the disease is in about half of patients the disease is asympthomaticasympthomatic and is detected during and is detected during routine blood controlroutine blood control

-- untreated is fataluntreated is fatal

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)-- First cancer for which the First cancer for which the geneticalgenetical mutation causing the disease has been identified mutation causing the disease has been identified (1960, Philadelphia)(1960, Philadelphia)

-- Mutated chromosome Philadelphia forms after translocation fragmMutated chromosome Philadelphia forms after translocation fragment of long arm of ent of long arm of chromosome 9 (coding chromosome 9 (coding for for AblAbl kinasekinase) to long arm ) to long arm of chromosome 22 (coding of chromosome 22 (coding for for BcrBcr....) ....) (9 22)(9 22)

-- This mutation is present in 95% patients with This mutation is present in 95% patients with CML; it can also be found in CML; it can also be found in patintspatints with other with other leukemiasleukemias (e.g. in 15(e.g. in 15--30% cases of acute 30% cases of acute lymphoid leukemia)lymphoid leukemia)

-- Translocation leads to formation of hybrid geneTranslocation leads to formation of hybrid geneBcr/AblBcr/Abl and fusion protein of constitutive, and fusion protein of constitutive, unregulated unregulated kinasekinase activityactivity

Chromosome 9 Chromosome 9’

Chromosome 22 Philadelphia

AblBcr

Bcr/Abl

TranslocationTranslocationBCRBCR--AblAbl

Diagnostics of CMLDiagnostics of CML

-- in vast majority of patients Philadelphiain vast majority of patients Philadelphiachromosome can be detected using the chromosome can be detected using the sstandartandar cytogeneticcytogenetic methodsmethods

-- other good tools are FISH or RTother good tools are FISH or RT--PCRPCR

Normal Normal AblAbl tyrosine tyrosine kinasekinase in leukocytes is associated with regulation of cell proliferatioin leukocytes is associated with regulation of cell proliferation n and differentiationand differentiation

BcrBcr--AblAbl kinasekinase, which is constitutively active leads to:, which is constitutively active leads to:-- increased proliferationincreased proliferation-- decreased rate of apoptosisdecreased rate of apoptosis-- decreased expression of decreased expression of adhesinsadhesins (attenuation of adhesion and disturbed signal (attenuation of adhesion and disturbed signal transduction from a bone morrow transduction from a bone morrow microenviromentmicroenviroment))

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)- The most common symptoms at presentation in the chronic phase of CML include:fatigue, weight loss, abdominal fullness, bleeding, and sweating.

- As the disease progresses, bone pain and pain from splenic infarction may be seen, and end stage CML may present with signs and symptoms of acute leukaemia.

Chronic Chronic myelomyeloidid leukemialeukemia(CML)(CML)Therapy:Therapy:

-- Bone mBone maarrowrrow transplantation transplantation (achievable for (achievable for less than 20% of new diagnosed patients)less than 20% of new diagnosed patients)

-- Treatment with interferonTreatment with interferon--α (α (IFNIFNαα))

-- chemotherapychemotherapy (e.g. (e.g. hydroxyureahydroxyurea))

-- GlGleeveevecec

At advanced phase no treatment is effectiveAt advanced phase no treatment is effective

GlGleeeevecvec ((imatinibimatinib))

-- first commercially available inhibitorfirst commercially available inhibitorof tyrosine of tyrosine kinasekinase accepted for a accepted for a clinical useclinical use

-- specifically inhibits specifically inhibits kinaseskinases BcrBcr--AblAbl, c, c--Kit (CD117) and PDGFKit (CD117) and PDGF--RR

-- this way it inhibits proliferation and this way it inhibits proliferation and increasesincreases apoptosis of cells expressing those apoptosis of cells expressing those kinaseskinases

-- causes relatively mild sidecauses relatively mild side--effecteffect

-- significant improvement is observed in 49% significant improvement is observed in 49% patientspatients in the chronic phase of CML, who in the chronic phase of CML, who did not respond to treatment with did not respond to treatment with IFNIFNαα

gleevec

22--phenylaminopyrimidine derivativephenylaminopyrimidine derivative

KinaKinasesses inhibitedinhibited byby glgleeeevecvec

II II phasephase clinicalclinical trialtrial withwith glgleeeevecvec

532 532 patientspatients, 1, 1-- > 5 > 5 yearsyears ofof diagnosisdiagnosis, 400 mg , 400 mg glivecglivec//dayday

HIFHIF--11

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Power Power ofof nuclearnuclear receptorsreceptors…..…..

AmbystomaAmbystoma mexicanummexicanum

Prof. Laura KProf. Laura KaaufmanufmanUniwerytetUniwerytet JagiellońskiJagielloński

19171917

Willson TM and Moore JT

19021902 –– wordword ‘‘hormonehormone’ ’ coinedcoined leadingleading to to conceptconcept ofof chemicalchemicalmessengersmessengers19111911 –– mammalianmammalian thyroidthyroid extractextract inducedinduced amphibianamphibian metamorphosismetamorphosis1919 1919 –– thyroxinthyroxin andand cortisonecortisone purifiedpurified19351935--19451945 –– synthesissynthesis ofof severalseveral hormonshormons andand theirtheir analoguesanalogues19601960 –– ecdysoneecdysone inducedinduced chromosomalchromosomal puffspuffs indicatedindicated genegene activationactivation19611961 –– estrogen estrogen shownshown to to bindbind to to cytoplasmaticcytoplasmatic protein protein andand to to translocatetranslocate to to thethe nucleusnucleus19761976--19791979 –– estrogen receptor estrogen receptor clonedcloned19811981 –– hormon hormon receptorsreceptors shownshown to to interactinteract withwith promoterspromoters ofof targettargetgenesgenes

1988 1988 –– methodmethod for for cloningcloning receptorsreceptors withwith no no knownknown ligandsligands defineddefined((orphanorphan receptorsreceptors))19901990 –– heterodimerizationheterodimerization ofof somesome receptorsreceptors withwith RXRRXR19961996 –– interactioninteraction withwith coco--activatorsactivators andand coco--repressorsrepressors19981998 –– phosphorylationphosphorylation cancan regulateregulate thethe activationactivation ofof nuclearnuclear receptorsreceptors20002000 –– manymany receptorsreceptors existexist as as multiplemultiple subtypessubtypes andand splicingsplicing formsforms......20022002 –– interplayinterplay betweenbetween membranemembrane andand nuclearnuclear signalingsignaling pathwayspathwaysdeterminesdetermines genegene expressionexpression

-- -- Nuclear receptors exist in the all Nuclear receptors exist in the all MetazoaMetazoa sstudiedtudied, , but have not been found in the other organismsbut have not been found in the other organisms

-- Most of receptors are very old and Most of receptors are very old and conservconserveded: mammalian proteins : mammalian proteins have their counterparts in insectshave their counterparts in insects ((exceptionexception: steroid : steroid hormonehormone receptorsreceptors))

-- NNuclear receptors sustain of one uclear receptors sustain of one superfamilysuperfamily, originating perhaps from , originating perhaps from one ancestorone ancestor

-- Primary nuclear receptor acted possibly as a constitutive, Primary nuclear receptor acted possibly as a constitutive, homodimerichomodimerictranscription factortranscription factor

Evolution of nuclear receptorsEvolution of nuclear receptors

-- inin CCaaenorhabditisenorhabditis eleganselegans more than 200more than 200 different different nucleanuclear r receptorreceptor genesgenes have been have been found, but in Drosophila found, but in Drosophila melanogastermelanogaster only 21 (they mediates e.g. action of eonly 21 (they mediates e.g. action of eccdysodysonne)e)

-- in human there are in human there are 4848 nuclear receptor genesnuclear receptor genes

-- atat the protein level the number of receptors is higher the protein level the number of receptors is higher ((alternative alternative splicing,splicing, different promoters,different promoters,postranslationalpostranslational modificationsmodifications))

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Modular structure of nuclear receptorsModular structure of nuclear receptors

A typical nuclear receptor is composed of A typical nuclear receptor is composed of several domainsseveral domains The variable NH2The variable NH2--terminal region (terminal region (region region

A/BA/B) contains the ) contains the ligandligand--independent independent AFAF--11transactivationtransactivation domain.domain.

The conserved DNAThe conserved DNA--binding domain binding domain ((DBD, region CDBD, region C) is responsible for the ) is responsible for the recognition of specific DNA sequencesrecognition of specific DNA sequences

A variable linker A variable linker region Dregion D connects DBD connects DBD to the E/F regionto the E/F region

The conserved The conserved E/FE/F region contains region contains ligandligandbinding domain (binding domain (LBDLBD), ), dimerizationdimerizationstructure and structure and ligandligand--dependenddependend AF2 AF2 transactivationtransactivation domain domain

RRegion A/Begion A/B

The most variable in respect of size and sequence. Very often iThe most variable in respect of size and sequence. Very often it includes AFt includes AF--1. 1.

The alternative splicing occurs usually within this domainThe alternative splicing occurs usually within this domain

Perhaps this domain is responsible for cellPerhaps this domain is responsible for cell--specific action of the nuclear receptorsspecific action of the nuclear receptors

It can be It can be phosphorylatedphosphorylated by different by different kinaseskinases involved in signal transduction involved in signal transduction (MAPK, (MAPK, cyclinecycline--dependent dependent kinaseskinases)), which regulates the activity of the receptor, which regulates the activity of the receptor

RRegion DBDegion DBD (domain C)(domain C)

The most The most conservconserveded domaindomain,, confers the ability to recognize specific targetconfers the ability to recognize specific targetsequences and activate genessequences and activate genes

There are There are 2 2 zinc fingers divided by the fragment of zinc fingers divided by the fragment of 6060--70 70 aminoacidsaminoacids –– in each finger in each finger four four cysteinescysteines coordinates one zinc ioncoordinates one zinc ion

The sequence at the base of the first finger The sequence at the base of the first finger (P box) (P box) recognizes DNA, whereas the recognizes DNA, whereas the sequence at the base of the second finger sequence at the base of the second finger (D box) (D box) is involved in the receptor is involved in the receptor dimerizationdimerization

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

DBD of nuclear receptorDBD of nuclear receptor DBDDBD consists of two zinc fingersconsists of two zinc fingers (60(60--70 70 aminoacidsaminoacids)). . In each zinc finger, four of theIn each zinc finger, four of theinvariable invariable cysteinescysteines coordinate one zinccoordinate one zincion, and both zinc finger modules fold ion, and both zinc finger modules fold together to form atogether to form a compact, compact, interdependent structureinterdependent structure

First finger contains First finger contains P boxP box residues, involved residues, involved in discrimination of response elementin discrimination of response element inin DNADNA

Second finger contains Second finger contains D boxD box, which form a , which form a dimerizationdimerization interfaceinterface

CTE CTE (COOH(COOH--terminal extension) is critical for terminal extension) is critical for monomericmonomeric DNA bindingDNA binding

Fingers form the DBDFingers form the DBDthat recognizes a hemithat recognizes a hemi--sitesiteof the response elementof the response element

HingHingee rregion egion (domain D)(domain D)

Moderately Moderately conservconserveded,, servesserves as a as a hingehinge betweenbetween thethe DBD DBD andand thethe LBD, LBD, allowingallowingrotationrotation ofof thethe DBDDBD

VeryVery oftenoften itit includesincludes a a nuclearnuclear localizationlocalization signalssignals

MutationsMutations withinwithin thisthis region region abolishesabolishes interactioninteraction withwith nuclearnuclear receptor receptor coco--repressorsrepressors

RRegion LBDegion LBD ((domaindomain E)E)ItIt bindsbinds ligand ligand andand mediatesmediates homohomo-- oror heterohetero--dimerdimerizationization andand interactioninteraction withwith heatheat

shockshock proteinsproteins

The The LBDsLBDs are formed by 12 conservedare formed by 12 conserved αα--helical regionshelical regions. . On On thethe 1212thth helisehelise ititcontainscontains anan AFAF--22 domaindomain, , responsibleresponsible for a ligandfor a ligand--dependent dependent transactivationtransactivation. . MutationsMutationswithinwithin AF2 AF2 maymay leadlead to e.g. to e.g. androgenandrogen--insensitivityinsensitivity syndromesyndrome, , oror thyroidthyroid hormoneshormones--insensitivityinsensitivity syndomesyndome..

ThisThis domaindomain includesincludes alsoalso otherother sequencessequences necessarynecessary for for transactivationtransactivation, , disperseddispersed inin thetherestingresting receptor, but receptor, but locatedlocated closelyclosely eacheach otherother uponupon ligand ligand bindingbinding..

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

LBD of nuclear receptorLBD of nuclear receptor

Unligated Ligated

Cylinders represents Cylinders represents αα--helices that are helices that are numbered from 1numbered from 1--12.12.

Note Note thethe different position of different position of thetheCOOHCOOH--terminal helix 12 that contains terminal helix 12 that contains the core AFthe core AF--2 domain2 domain..

The The ligandedliganded structures are more structures are more ccompactompact than the than the unligandedunliganded ones, ones, demonstrating that upon demonstrating that upon ligandligandbinding the receptorsbinding the receptors undergo a undergo a clear conformational change.clear conformational change.

A. LazarA. Lazar

Domain structure of nuclear receptorsDomain structure of nuclear receptors

N-terminal DBD LBD

NNuclearuclear receptorsreceptors regulateregulate transcriptiontranscription throughthrough bindingbinding to consensus to consensus sequencesequence inintargettarget genesgenes. . TheseThese sequencessequences areare usuallyusually locatedlocated withinwithin promoterpromoter, but , but sometimessometimes theytheyareare eveneven severalseveral thousandthousand nucleotidesnucleotides upup-- oror downdown--streamstream ofof thethe start start ofof transcriptiontranscriptionsitesite..

TATA box

TATA box

TATA box

TATA box

consensus consensus sequensesequense

DNADNA

DNADNA

Early primary response

Delayed secondaryresponse

ligandligand

nuclearnuclear receptorsreceptors activateactivateprimaryprimary responseresponse genesgenes

primaryprimary responseresponse proteinsproteinsturnturn on on secondarysecondary responseresponse genesgenes

secondarysecondary responseresponse proteinsproteins

adopted from Deanna Kroetz, University of California, San Francisco

Consensus Consensus sequencesequence consistsconsists ofof 6 6 nunucleotidescleotides: :

AGAGAAAACACA ((recognizedrecognized by by thethe steroid steroid hormonehormone receptorsreceptors))

AGAGGGTCATCA ((recognizedrecognized by by thethe otherother receptorsreceptors))

AGAGTTTCATCA ((recognizedrecognized by by thethe otherother receptorsreceptors))

Consensus Consensus sequencessequences ofof nuclearnuclear receptorsreceptors

fragment fragment ofofacylCoAacylCoA oxidaseoxidasepromoterpromoter

TypTypee II ((homodimerhomodimericic receptorreceptorss ofof steroid steroid hormoneshormones)) –– e.g.e.g. ER,ER, AR, GR, MR.AR, GR, MR.

TypTypee IIII ((hodimerichodimeric orphansorphans) ) –– e.g. e.g. RXRRXR

TTypypee IIIIII ((heterodimersheterodimers) ) –– e.g. e.g. TRTR, , RAR, VDR, PPAR.RAR, VDR, PPAR.

TypTypee IVIV ((monomericmonomeric orphansorphans) ) –– e.g. Nurre.g. Nurr--11

-- on on thethe basisbasis ofof primaryprimary structurestructure nuclearnuclear receptorsreceptors theythey cancan be be classifiedclassified to to 7 7 familiesfamilies, , but one but one familyfamily maymay containcontain receptorsreceptors for for veryvery distinctdistinct ligandsligands, , whereaswhereas thethe same ligand same ligand cancan be be boudboud by by membersmembers ofof differentdifferent familiesfamilies..

ClassificationClassification ofof nuclearnuclear receptorsreceptors

On On thethe basisbasis ofof ligand ligand bindingbinding andand dimerizationdimerization nuclearnuclearreceptorsreceptors cancan be be classifiedclassified to to 4 4 typtypeses::

SomeSome nuclearnuclear receptorsreceptors actact as as monomermonomerssbindingbinding to to hexamerhexamer ofof nucleotidesnucleotides..

Most Most receptorsreceptors actact as as dimersdimers, , bindingbinding to to thethe sequencesequence ofof twotwo hexamershexamers

DimersDimers cancan be be bothboth homohomo-- oror heterodimersheterodimers. In . In thethe latterlatter casecase thethe partner for partner for heterodimerizationheterodimerization isis alwaysalways a a nuclearnuclear receptor receptor RXRRXR

e.g.e.g. NurrNurr--11

homodimershomodimers RXR RXR heterodimersheterodimers

ERERGRGR

RARRARTRTRVDRVDRPPARPPAR

RXRRXR

consensus consensus sequencessequences recognizedrecognized by a by a dimersdimers cancan be:be:

palindrompalindromss ((onlyonly suchsuch sequencessequences cancan be be recognizedrecognized by steroid by steroid hormonehormone receptorsreceptors))

invertedinverted palindromspalindroms

directdirect repetitionsrepetitions (DR1, DR2, DR3 etc.) (DR1, DR2, DR3 etc.)

AGGTCA...TGACCTAGGTCA...TGACCTTCCAGT...ACTGGATCCAGT...ACTGGA

AGGTCA...AGGTCA...AGGTCAAGGTCATCCAGT...TCCAGT...TCCAGTTCCAGT

ACTGGA...TCCAGTACTGGA...TCCAGTTGACCT...AGGTCATGACCT...AGGTCA

Consensus Consensus sequencessequences ofof nuclearnuclear receptorsreceptors

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Binding of receptors toBinding of receptors to theirtheir consensus consensus sequencessequences

Receptors can bind as monomers, Receptors can bind as monomers, homodimershomodimers or RXR or RXR heterodimersheterodimers..

Steroid receptors bind as Steroid receptors bind as homodimershomodimers to to palindromicpalindromicelements spaced by three elements spaced by three nucleotides.nucleotides.

MonomericMonomeric binding requires the binding requires the halfhalf--core motif precede by a 5’core motif precede by a 5’--flanking A/T reach sequence.flanking A/T reach sequence.

HeterodimersHeterodimers can can recognmizerecognmizediverse diverse HREsHREs in which halfin which half--core core motifs can be arranged as motifs can be arranged as polindromespolindromes, direct repeats or , direct repeats or inverted inverted polindromespolindromes

Permissive Permissive heterodimersheterodimers, such as PPAR/RXR, such as PPAR/RXR, FXR/RXR , FXR/RXR oror LXR/RXRLXR/RXR, can , can be activated by be activated by ligandsligands of either RXR or its partner receptor and are of either RXR or its partner receptor and are synergistically activated in the presence of both synergistically activated in the presence of both ligandsligands..

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Permissive and nonPermissive and non--permissive permissive heterodimerheterodimer

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

In In nonnon--permissive permissive heterodimersheterodimers, such as RXR/RAR,, such as RXR/RAR, RXR/RT RXR/RT oror RXR/VDRRXR/VDRheterodimerizationheterodimerization precludes binding of the RXR precludes binding of the RXR ligandligand. .

Binding of Binding of ligandligand to the RAR moiety causes receptor activation and allows to the RAR moiety causes receptor activation and allows binding of the RXR binding of the RXR ligandligand resulting in synergism. resulting in synergism.

Permissive and nonPermissive and non--permissive permissive heterodimerheterodimer

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Mechanism of action of nuclear receptorsMechanism of action of nuclear receptors

LigandLigand can be generated in three different can be generated in three different ways: ways:

11) an active ) an active ligandligand or hormone is synthesized in a or hormone is synthesized in a classical endocrine organ and enters the cell, classical endocrine organ and enters the cell,

22) the ) the ligandligand may be generated from a precursor or may be generated from a precursor or prohormoneprohormone within the target cell, and within the target cell, and

33) the ) the ligandligand may be a metabolite synthesized may be a metabolite synthesized within the target cell.within the target cell.

The The unligandedunliganded receptor may have a nuclear receptor may have a nuclear location. However, some steroid receptors are location. However, some steroid receptors are cytoplasmiccytoplasmic in the absence of in the absence of ligandligand due to their due to their association with a large association with a large multiproteinmultiprotein complex of complex of chaperones, including chaperones, including Hsp90Hsp90 andand Hsp56Hsp56. . LigandLigandbinding induces dissociation of the complex and binding induces dissociation of the complex and nuclear translocation. Once in the nucleus, the nuclear translocation. Once in the nucleus, the receptors regulate transcription by binding, receptors regulate transcription by binding, generally as generally as dimersdimers, to hormone response , to hormone response elements (elements (HREsHREs) normally located in regulatory ) normally located in regulatory regions of target genes.regions of target genes.

Nuclear receptors are Nuclear receptors are activated after activated after ligandligand binding. binding.

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Mechanism of action of nuclear receptorsMechanism of action of nuclear receptors

AlternativeAlternative, , ligandligand--independent pathways for independent pathways for activation of nuclear receptorsactivation of nuclear receptors existexist::

•• Some receptors may be constitutively active, Some receptors may be constitutively active,

•• the activity of others is modulated by other the activity of others is modulated by other means, for instance,means, for instance, phosphorylationphosphorylationmediated by hormones and growth factors mediated by hormones and growth factors that stimulate diverse signal transduction that stimulate diverse signal transduction pathways.pathways.

Nuclear receptors Nuclear receptors cancan actactindependentlyindependently ofof ligandsligands

WhatWhat wouldwould be be profitableprofitable to to rememberremember inin JuneJune::

-- structurestructure ofof nuclearnuclear receptorsreceptors

-- thethe wayway ofof actionaction ofof nuclearnuclear receptorsreceptors

SSlideslides cancan be be foundfound inin thethe librarylibrary andand atat thethe HemeHeme OxygenaseOxygenase Fan Fan ClubClubpagepage::

http://http://biotka.mol.uj.edu.pl/~hemeoxygenasebiotka.mol.uj.edu.pl/~hemeoxygenase

ThankThank youyou andand seesee youyou nextnext weekweek......