REVIEW Physiological functions of the imprinted Gnas locus and its protein variants Gas and XLas ...

22 Pages·2008·0.7 MB·English

193
REVIEW
Physiological functions of the imprinted Gnas locus and its protein
variants Ga and XLa in human and mouse
s s
Antonius Plagge, Gavin Kelsey1 and Emily L Germain-Lee2
PhysiologicalLaboratory,SchoolofBiomedicalSciences,UniversityofLiverpool,CrownStreet,LiverpoolL693BX,UK
1LaboratoryofDevelopmentalGeneticsandImprinting,TheBabrahamInstitute,CambridgeCB223AT,UK
2DivisionofPediatricEndocrinology,DepartmentofPediatrics,SchoolofMedicine,TheJohnHopkinsUniversity,Baltimore,Maryland21287,USA
(CorrespondenceshouldbeaddressedtoAPlagge;Email:[email protected])
Abstract
Thestimulatorya-subunitoftrimericG-proteinsGa,which locusanddescribethephysiologicalrolesofGa andits‘extra
s s
upon ligand binding to seven-transmembrane receptors large’variantXLa atpost-natalandadultstagesasdefinedby
s
activates adenylyl cyclases to produce the second messenger genetic mutations. Opposite and potentially antagonistic
cAMP, constitutes one of the archetypal signal transduction functions of the two proteins in the regulation of energy
molecules that have been studied in much detail. Over homeostasis and metabolism have been identified in Gnas-
the past few years, however, genetic as well as biochemical andGnasxl(XLa)-deficientmice,whicharecharacterisedby
s
approacheshaveledtoarangeofnovelinsightsintotheGa obesityandleannessrespectively.Acomparisonoffindingsin
s
encoding guanine nucleotide binding protein, a-stimulating mice with symptoms of the corresponding human genetic
(Gnas)locus,itsalternativeproteinproductsanditsregulation disease ‘Albright’s hereditary osteodystrophy’/‘pseudohypo-
bygenomicimprinting,whichleadstomonoallelic,parental parathyroidism’indicateshighlyconservedfunctionsaswellas
origin-dependentexpressionofthevarioustranscripts.Here, unresolvedphenotypicdifferences.
wesummarisethemajorcharacteristicsofthiscomplexgene JournalofEndocrinology(2008)196,193–214
The stimulatory G-protein signalling cycle TheinactiveformoftheG-proteinconsistsofatrimercomprising
Ga inassociationwithb-andg-subunitcomplexesattheplasma
s
Heterotrimeric G-proteins that are composed of a, b and membrane,wherebyGa occupiestheGDPnucleotide-bound
s
g-subunits,mediatesignaltransductionfromalargenumberof conformation.Membraneanchorageofthea-andg-subunitsis
activatedseven-transmembranereceptorstodiverseintracellular achievedvialipidmodifications,inthecaseofGa palmitoylation
s
effectorpathways.ManygeneralaspectsofG-proteinsignalling oftheNH -terminus(Kleuss&Krause2003).b-andg-subunits
2
have been covered in recent excellent reviews (Cabrera-Vera form a very tight and stable complex (Wettschureck &
etal.2003,Wettschureck&Offermanns2005).TheG classof Offermanns2005).Aligand-boundG-proteincoupledreceptor
s
a-subunits is characterised by its ability to stimulate adenylyl (GPCR) activates the G-protein through promoting the
s
cyclases (ACs) to produce the second messenger molecule exchangeofGDPforGTPonthea-subunit,whichresultsin
cAMP. Itcomprises two genes,Gnas(GNAS inhuman)and itsdissociationfromthereceptorandtheb-andg-complexes.
guanine nucleotide binding protein, a stimulating, olfactory The free Ga subunit can now interact with and stimulate its
s
type(Gnal),whichencodeGa andGa respectively.While effectorACuntiltheintrinsicGTPaseactivity(hydrolysisofGTP)
s olf
Gnasisgenerallyregardedasaubiquitouslyexpressedgene,Gnal ofthea-subunitreturnsitintotheinactiveGDP-boundform,
expressionislimitedtotheolfactoryepitheliumandafewbrain whichreassociateswiththeb-andg-complexes,toenteranew
regions,inwhichitlargelyreplacesGnasexpressionwithvery cycle(Sunaharaetal.1997,Cabrera-Veraetal.2003).Verylittleis
littleoverlapofthetwoa-subunits(Belluscioetal.1998,Zhuang knownaboutspecificitiesintheinteractionsbetweenGa andthe
s
et al. 2000, Herve et al. 2001). We will focus here on novel 5differentb-subunitsand12g-subunitsthathavebeenidentified,
findings related to the Ga-subunit, its gene locus, variant norwhetherspecificcombinationsofthesesubunitspreferentially
s
proteinisoformsandphysiologicalfunctions. interactwithcertainGPCRs.
G-proteinsundergoacycleofactiveandinactivestatesduring TheGa effectorACcomprisesafamilyofproteinsencoded
s
thesignaltransductionprocessassummarisedforGa inFig.1. byninedifferentgenesinmammaliangenomes,termedtype
s
JournalofEndocrinology(2008)196,193–214 DOI:10.1677/JOE-07-0544
0022–0795/08/0196–193 q2008SocietyforEndocrinology PrintedinGreatBritain Onlineversionviahttp://www.endocrinology-journals.org
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194 APLAGGEandothers Gnasimprintingandfunctions
Figure 1 Scheme of the signalling cycle of the trimeric G-protein. (I) The inactive, trimeric G-protein, consisting of a-, b- and
s s
g-subunits,isassociatedwiththeplasmamembranevialipidmodifications.Thea-subunit,e.g.Ga orXLa,isinitsGDP-bound
s s s
conformation.(II)AgonistbindingtoaG-coupledseven-transmembranereceptor(GPCR)causesaconformationalswitchinthe
s
a-subunit,whichalsoinvolvesanexchangeofGDPforGTP,leadingtoitsactivationanddissociationfromb-andg-subunits.(III)The
active,GTP-boundformofGa/XLa interactswithandactivatestransmembraneadenylylcyclasestypeI–IX,resultinginincreased
s s
formationofthesecondmessengercAMP.(IV)TheintrinsicGTPhydrolysisactivityofGa/XLa,whichcanbestimulatedbyGTPase-
s s
activatingenzymes(GAPs),resultsinitsinactivationandreassociationwithb-andg-subunits.
I–IX, all of which are large transmembrane proteins with GTPaseactivityofGa (Cabrera-Veraetal.2003).Generally,
s
a bipartite catalytic domain (Kamenetsky et al. 2006, the hydrolysis of GTP by a-subunits is stimulated in vivo by
Willoughby & Cooper 2007). Although all transmembrane GTPase-activatingproteins(GAPs).InthecaseofGa,several
s
ACscanbestimulatedbyGa,theyvaryintheirresponsiveness proteins have been demonstrated to exert a GAP function,
s
toadditionalregulators,e.g.Ga,Gb-andg-subunits,Ca2C including regulator of G-protein signalling 2 (RGS2;
i
and protein kinases (Kamenetsky et al. 2006, Willoughby & Abramow-Newerlyetal.2006,Royetal.2006),ACVitself
Cooper2007).MostcelltypesexpressseveralACgenes,but (Scholich et al. 1999), RGS-PX1 (Zheng et al. 2001) and
certain isoforms dominate in specific tissues (Hanoune & cysteine string protein (Natochin et al. 2005). Their
Defer2001,Krumins&Gilman2006,Willoughby&Cooper importanceinGassignallinginvivoremainstobeconfirmed.
2007).Inthecontextofsomeofthephysiologicalfunctionsof
Ga discussedbelow,itisnoteworthy,forexample,thatACIII
s
exertsaspecificroleinbrownadiposetissue(BAT).Inrodents, TheGa variant XLa also stimulates cAMP
s s
AC III expression and AC activity in BAT is transiently signalling from activated receptors
increased during the neonatal period, when offspring are
especiallysensitivetoenvironmentalconditionsandmainten- TheidentificationinPC12cellsof analternative‘extra large’
anceofbodytemperature(Chaudhryetal.1996).Stimulation form of the a subunit, XLa, brought novel aspects to this
s s
ofthissignallingpathwayresultsinincreasedlipolysisandheat signallingpathway(Kehlenbachetal.1994).TheXLa protein
s
production in mitochondria. AC III is strongly upregulated was found to be mostly identical in sequence to Ga, apart
s
upon stimulation by the sympathetic nervous system, e.g. from the NH -terminal domain, which was replaced by a
2
adrenergicreceptorstimulation(Granneman1995). different(w370aminoacid)sequence.Asdetailedbelow,thetwo
ThelaststepoftheG-proteincycle(Fig.1),theinactivation variantsaretranscribedfromalternativepromoters/firstexonsof
oftheGa subunitandre-associationwithb-andg-subunits theGnasgeneandsplicedontoshareddownstreamexonsfrom
s
into the trimeric complex, is triggered by the intrinsic exon2onwards.Thenovel,XL-specificNH -terminusconsists
2
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Gnasimprintingandfunctions APLAGGEandothers 195
ofarepeated,alanine-richmotif,aproline-richdomain,ahighly fullyfunctional G-protein, i.e. bindingofb- andg-subunits,
s
chargedandcysteine-containing regionanda sequencemotif activation of AC and coupling of activated receptors, was
thatincludesastretchofleucinesandishighlyconservedamong established in biochemical assays (Klemke et al. 2000) and in
alla-subunits(Fig.2A;Kehlenbachetal.1994).Whiletherepeat transfections of fibroblasts that lack endogenous G-proteins
s
motifvariesamongmammals(Haywardetal.1998a,Fresonetal. (Bastepeetal.2002,Linglartetal.2006);thecharacteristicsof
2003),theotherXL-specificdomainsarewellconserved.The cAMPsignallingwereidenticalforXLa andGa (forratand
s s
functionoftheproline-richdomainisuncertain;however,the humanversions)inthesetransfectionstudies(Bastepeetal.2002,
cysteine residues serve for lipid anchorage (palmitoylation) to Linglartetal.2006).Neuroendocrinecelllinesthatexpressboth
theplasmamembranesimilartoGa (Ugur&Jones2000),while proteins endogenously have not yet been analysed (see also
s
the leucine-containing motif participates in the binding of Klemkeetal.2000).
G-proteinb-andg-subunits(Kehlenbachetal.1994,Lambright While Ga is regarded as being more or less ubiquitously
s
etal.1996,Klemkeetal.2000).TheabilityofXLa toactasa expressed, XLa shows a much restricted expression pattern,
s s
Figure2 SchemeoftheproteindomainsofGa andXLa encodedbytheirfirstexonsandoftheimprintedGnaslocus.(A)Conserved
s s
proteinregionsencodedbyGnas(Ga)andGnasxl(XLa)firstexons.Thefirstexonsencodeconservedaminoacids(bg)thatcontributeto
s s
thebindingofb-andg-subunits.TheGnasxlspecificexoncontainsfurtherproteinregionsthatareconservedamongmammals,e.g.a
regionwithcysteinesandchargedaminoacids(Cys/chargedAA)thatmediateslipidmembraneanchorage,aproline-richdomain(Pro)
andadomaincontaininganalanine-richrepetitivemotif.TheC-terminusofthetwoproteins,encodedbyexons2–12(exons2–13in
human),isidentical.(B)Theexon–intronstructure(codingexonsfilled),promoteractivitiesandalternativesplicingofthemurine
imprintedGnaslocusaredepicted.Thematernallyandpaternallyinheritedallelesareindicatedinredandbluerespectively.Arrows
indicatethepromotersandtranscriptionaldirectionoftheindividualRNAs.RegionsofdifferentialDNAmethylation(DMRs)aremarked
byMMM;DMRsatNespas/Gnasxlandexon1Arepresentimprintingcontrolregions(ICRs).Splicingpatternsofthetranscriptsand
encodedproteinsareshownaboveandbelowthegenomiclocus.Gnasisexpressedbiallelicallyinmosttissues,butissilencedonthe
paternalalleleinsomecelltypes(hatchedbluebox).Gnasxlshowsexclusivepaternalallele-specificexpressionandissplicedontoexons
2–12ofGnas(exons2–13inhumanGNAS).TheGnasxl-specificfirstexonalsocontainsasecondpotentialopenreadingframe(ORF)for
aproteintermedAlex.Nespisexpressedexclusivelyfromthematernalallele.TheNesp55ORFiscontainedwithinthesecondNesp-
specificexon.Onlyasingle,uninterruptedNesp-specificexonisfoundinhuman.Exon1A(exonA/Binhuman)andNespasproduce
non-coding,regulatoryRNAs;NespastranscriptsexistinmultiplesplicedandunsplicedformsthatextendbeyondtheNespexons.Tissue-
specificsplicingontoexonN1exclusivelyinneuraltissuesleadstoprematuretranscriptionterminationandexpressionofatruncated
XLN1protein(existenceofacorrespondingGaN1proteinisuncertain).
s
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196 APLAGGEandothers Gnasimprintingandfunctions
being mostly confined to neural and endocrine tissues 1999, Peters et al. 1999). The Gnas locus is now known to
(Pasolli et al. 2000, Pasolli & Huttner 2001, Plagge et al. compriseacomplexarrangementofthreeprotein-codingand
2004). At embryonic stages, XLa is already detectable from two non-coding transcripts regulated by imprinting
s
mid-gestationonwardsinregionsofneurogenesisandinearly mechanisms. We will describe the murine locus here, but
differentiating neurons, mainly in areas of the midbrain, most features are conserved in humans. As the mechanisms
hindbrain and spinal cord, including the sympathetic trunk of regulation of the locus by genomic imprinting are
andganglia(Pasolli&Huttner2001).Atlaterembryonicstages currently under much investigation, we will only focus on
expressionwasalsofoundinthehypothalamusandthepituitary themaincharacteristicshere,butseePetersetal.(2006)fora
(adenohypophysisandparsintermedia).Intheneonatalbrain, recentreview.
XLa expressionisconfinedtodistinctregionsofthemidbrain
s
andhindbrain,e.g.thecentreofthenoradrenergicsystemofthe
Theprotein coding transcripts
brain(locuscoeruleus),laterodorsaltegmentalnucleus,motor
nuclei that innervate orofacial muscles (hypoglossal, motor- ThethreeproteintranscriptsGnas,GnasxlandNespeachinitiate
trigeminal and facial nuclei), as well as scattered cells in the at separate promoters/first exons, but share most of the
medulla oblongata (Plagge et al. 2004). Further, sites of downstreamexons(Fig.2B;Plagge&Kelsey2006,Weinstein
expressionincludetheneuroendocrinepituitary(parsanterior etal.2007).TheGa encodingGnastranscriptiscomposedof12
s
and intermedia), the catecholaminergic adrenal medulla and exons(13inhuman,duetoanadditionalintroninterrupting
some peripheral tissues, e.g. white adipose tissue (WAT) and exon9).MostcelltypesexpresstwovariantsoftheGa protein,
s
BAT,pancreas,heart,kidneyandstomach(Plaggeetal.2004). a small (45kDa) and a long 52 (kDa) version, which are
There are indications that this expression pattern changes functionallyequivalent(Grazianoetal.1989,Levis&Bourne
towardsadulthood, as noXLa wasdetected in adult adipose 1992)andaregeneratedthroughalternativesplicingofthe15
s
tissues, kidney and heart, but expression persists in brain, codonscomprisingexon3.BothGa versionscanvaryfurther
s
pancreatic islets, the pituitaryand adrenal glands (Pasollietal. bytheinclusionofasingleserineresidue,addedthroughusage
2000,Xieetal.2006). ofanalternativespliceacceptorsiteatexon4(Brayetal.1986,
Kozasa et al. 1988). The Gnas promoter and exon 1, which
encodesaminoacids1–45ofGa,donotcarryprimarymarksof
s
TheGnaslocus:alternativepromoters,splicingand genomic imprinting (Liu et al. 2000b) and in most tissues
regulationby genomic imprinting transcription occurs equally from both alleles. In a subset of
tissues or cell types, however, expression is monoallelic and
AlthoughthelocationoftheGa encodingGnasgeneonmouse restrictedtothematernalallele,e.g.inproximalrenaltubules,
s
distalchromosome2/humanchromosome20q13.2–q13.3and anterior pituitary, thyroid gland and ovary (Yu et al. 1998,
itsexon–intronstructurehadbeenknownforsometime(Blatt Haywardetal.2001,Germain-Leeetal.2002,2005,Mantovani
etal.1988,Kozasaetal.1988,Gejmanetal.1991,Levineetal. et al. 2002, 2004, Liu et al. 2003); this is relevant to human
1991,Raoetal.1991,Petersetal.1994),anddespitesomeearly inheriteddisordersthatareassociatedwithhormoneresistance
indications for alternative upstream promoters (Ishikawa etal. symptoms, as discussed below. Imprinting of Gnas inadipose
1990,Swaroopetal.1991),thefullcomplexityoftheGnaslocus tissueisstillcontentious,assomestudiesshowedpredominant
was only discovered through work in a different field, i.e. maternalallele-specificexpression(Yuetal.1998,Williamson
genomicimprinting.Imprintingaffectsasmallnumberofgenes etal.2004),whileothersfoundnosuchpreference(Mantovani
in the mammalian genome, currently comprising w90 etal.2004,Chenetal.2005,Germain-Leeetal.2005).Itremains
identified transcription units (see databases: http://igc.otago. tobeclarifiedwhetherthesediscrepantdatareflecttheanalysis
ac.nz/home.html and http://www.mgu.har.mrc.ac.uk/ of different developmental stages, implying a change in the
research/imprinting/index.html). It describes a phenomenon imprintingstatusoftheGnastranscriptinadiposetissueduring
of gene regulation in mammals, whereby one of the two thelifetime.Ingeneral,tissue-specificimprintingofGnashas
chromosomalallelesissilenceddependingonitsparentalorigin. beendifficulttodemonstrate,sinceasmallamountoftranscripts
Thus,animprintedgeneisexpressedfromeitherthepaternally derived from the paternal allele is often detected among the
orthematernallyinheritedchromosome,andthismonoallelic, majority that stems from the maternally inherited allele.
parent of origin-dependent transcription is achieved through Whether this is due to incomplete silencing of the paternal
mechanisms of DNA methylation, as well as chromatin allele or a mixture of cell types with imprinted and
modifications (Reik & Walter 2001, Morison et al. 2005, non-imprinted expression in the tissue samples analysed is
Edwards&Ferguson-Smith2007). unresolved.
Separatescreensforimprintedgenesinhumanandmouse A second promoter and first exon are located w30kb
resultedintheidentificationoftheXLa-specificfirstexonof upstream of Gnas exon 1 and initiates the Gnasxl transcript
s
theGnaslocusandanadditionalexonandpromoter,which (Fig.2B;Haywardetal.1998a,Kelseyetal.1999,Petersetal.
initiates a transcript that also splices onto downstream Gnas 1999),whichissplicedontoexon2–12ofGnas.Thissplice
exonsbutencodesanunrelated,previouslyidentifiedprotein form retains the Gnas open reading frame (ORF) and
termedNesp55(Fig.2B;Haywardetal.1998a,b,Kelseyetal. translatesintotheXLa proteinasaNH -terminalvariantof
s 2
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Gnasimprintingandfunctions APLAGGEandothers 197
Ga (Kehlenbachetal.1994).IncontrasttoGnas,theGnasxl manuscript in preparation) but, in contrast to Ga- and
s s
promoter is silenced on the maternal chromosome and XLa-deficientmice(seebelow),theyexhibitnomajoreffects
s
activates transcription exclusively from the paternal allele. ondevelopment,growthormetabolism.
Apart from the full-length Gnasxl transcript, a prominent
truncated form, encoding the protein XLN1, is found in
Non-coding transcriptsand imprinting marks
neuroendocrinetissuesonly(brain,pituitary,adrenalmedulla;
Klemkeetal.2000,Plaggeetal.2004).Thistruncationisdue The complexity of the Gnas locus is not limited to the
to alternative, neural tissue-specific splicing of exon N1, protein-codingtranscripts,butisincreasedbytheoccurrence
which is located between exons 3 and 4 and contains a of non-coding transcripts and differentially methylated
termination codon and polyadenylation signal. Originally, regions of DNA (DMRs). As noted above, we will only
exonN1wasdescribedascausingneural-specifictruncation briefly describe how these featuresrelateto howimprinting
oftheGnastranscript(Crawfordetal.1993)but,incontrastto inthelocusiscontrolled(seealsoPetersetal.2006).
XLN1 (Klemke et al. 2000), it remains uncertain whether a Two untranslated transcripts are produced from separate
correspondingGaN1proteinisstablyexpressed.Theneural promoters within the locus (Fig. 2B). The paternal allele-
s
N1 proteins retain the residues for membrane anchorage specificexon1Atranscript(exonA/Binhuman)isinitiated
and part of the domain interacting with b- and g-subunits w2.4kb upstream of Gnas exon 1 (Ishikawa et al. 1990,
(Klemke et al. 2000), but lack the major functional domains Swaroopetal.1991,Liuetal.2000b,Petersetal.2006)within
thatareencodedbythedownstreamexonsaswellasfurther a CpG dinucleotide-rich cis-regulatory region that is
residuesforinteractionwithb-andg-complexes(Lambright methylated on the maternal allele (exon 1A DMR). This
etal.1996).ThesignificanceoftheexonN1spliceforms,if transcriptalsosplicesontoexon2ofGnas.Thesecondnon-
any,remainstobedetermined. coding RNA, Nespas, begins w2.1kb upstream of the
ThecomplexityoftheGnasxltranscriptisfurtherincreased Gnasxl-specific exon, but it is transcribed in the opposite
throughthehighlyunusualfeatureinmammalianmRNAsof direction,i.e.antisensetoNesp(Hayward&Bonthron2000,
asecondpotentialORF,whichisshiftedbyC1nucleotide, Wroe etal.2000,Williamson et al.2006),andis transcribed
begins a short distance downstream of the XLa start codon solelyfromthepaternalallelefromwithinaCpG-richDMR
s
and terminates at the end of the Gnasxl-specific exon (methylatedonthematernalallele;Coombesetal.2003).An
(Klemke et al. 2001). This ORF encodes a protein termed increasingevidencepointstowardsaroleforsuchnon-coding
Alex, which is conserved, but unrelated to G-proteins RNA in the regulation of the imprinted, monoallelic
(Klemke et al. 2001, Nekrutenko et al. 2005). Although expressionofthecodingtranscripts(Pauleretal.2007).
AlexwasdetectedinPC12cellsandhumanplatelets(Klemke TheDMRsatexon1AandNespashavebeenshowntobeof
etal.2001,Fresonetal.2003),itsabundance,expressionlevel centralimportancefortheimprintingofthelocus(Williamson
andsignificanceinvivoremainunclarified. et al. 2004, 2006, Liu et al. 2005). At both sites, differential
Asathirdpromoterforaprotein-codingtranscriptwithin methylationofthematernalalleleisestablishedinoocytesand
theGnaslocus,theNesppromoterandfirstexonarelocated maintainedafterfertilisationandintoadulthoodinallsomatic
w15kbupstreamoftheGnasxlexon(Haywardetal.1998b, tissues(Liuetal.2000b,Coombesetal.2003).Suchgermline
Kelsey et al. 1999, Peters et al. 1999). Although the single differences in DNA methylation are characteristic of
humanNESP-specificexonisinterruptedbyashortintronin imprintingcontrolregions(ICRs;Spahn&Barlow2003).A
themousegenome,thedownstreamsplicingontoexons2–12 thirdDMRlocatedattheNesppromoter isunmethylatedin
ofGnasisconservedandoccurssimilarlytoGnasxlandGnas oocytesandsperm,butacquiresmethylationonthepaternal
itself. Nesp is imprinted in an opposite way to Gnasxl being allele during embryonic development (Liu et al. 2000b,
expressed only from the maternally derived allele (Hayward Coombesetal.2003).
etal.1998b,Kelseyetal.1999,Petersetal.1999).TheORF, The roles of the exon 1A and Nespas DMRs have been
which encodes the neuroendocrine secretory protein of M demonstrated through targeted deletion in mice (Williamson
r
55000 (Ischia et al. 1997), is confined to the Nesp-specific etal.2004,2006,Liuetal.2005).Thesestudiesshowthatthe
exon, and the shared downstream exons function as exon1Aregioncontrolsthetissue-specificimprintingofGnas
30-untranslatedsequence.TheNesp55proteinhassimilarities withoutaffectingtheupstreamtranscriptionunits(Williamson
with the chromogranin family, is associated with secretory etal.2004,Liuetal.2005).Deletionoftheexon1ADMRand
vesiclesinneuroendocrinecellsandisregardedasamarkerfor promoteronthepaternal(normallyunmethylated)alleleleadsto
the constitutive secretory pathway (Fischer-Colbrie et al. upregulationincisoftheusuallysilencedexpressionofGnasin
2002). Little is known about its molecular function, but the imprintedtissues.Theexactnatureofthesilencingmechanism
proteinisprocessedintopeptidestovariableextentindifferent exertedbythepaternalexon1AregiononGnastranscriptionis
celltypes(Lovisetti-Scamihornetal.1999).Inagreementwith unknownatpresent(Petersetal.2006).
its predominant expression in the nervous system and Deletion of the Nespas promoter, in contrast, affects the
endocrine tissues (Bauer et al. 1999a,b), mice deficient for imprinting status of all transcripts of the locus (Williamson
Nesp55showabehaviouralphenotype,specificallyanaltered etal.2006),suchthattheNespasDMRcanberegardedasthe
responsetonovelenvironments(Plaggeetal.2005,Islesetal. principalICRforthelocus.Thus,whenNespastranscription
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198 APLAGGEandothers Gnasimprintingandfunctions
is ablated on the paternal allele, Nesp and Gnas become incompatiblewithlifeasembryosdiesoonafterimplantation
derepressed, while Gnasxl and the exon 1A transcript are (Yu et al. 1998, Chen et al. 2005, Germain-Lee et al. 2005).
downregulated. Furthermore, the Nesp DMR loses and the HeterozygousmutationsofthedifferentproteinsoftheGnas
exon 1A DMR gains methylation on the paternal allele locuscausedistinctdysfunctions(Table1).InthecaseofGa
s
(Williamsonetal.2006).Themolecularmechanismsthrough someaspectsofthephenotypevarywiththeparentalorigin
which this ICR controls the imprinted expression of all of the mutation, reflecting its imprinted expression, while
transcriptsoftheGnaslocusremaintobeelucidated. other dysfunctions occur after both maternal and paternal
transmission, indicating haploinsufficiency of Ga in some
s
tissues.HeterozygouslossofGa inmicerecapitulatesmany
s
Physiological functions ofthegene products as aspectsofthehumandisorders,buthaploinsufficiencyeffects
revealed bymutations inmiceandhumans seem to be more prevalent in human than in mice.
Furthermore,theconsequencesoflossofXLa inmicediffer
s
Ithasbeenknownforsometimethatinactivatingmutationsin andareinseveralrespectsoppositetothoseofspecificlossof
the human GNAS gene are associated with the inherited Ga, despite their similar capability to activate the cAMP
s
disorder‘Albright’shereditaryosteodystrophy’(AHO)/‘pseudo- signallingpathway.
hypoparathyroidism’ (PHP; Levine et al. 1980, 1983a, Patten Beforediscussingthephysiologicalandendocrinerolesof
etal.1990,Weinsteinetal.1990,Davies&Hughes1993).Fuller the different proteins and evaluating the (in some aspects
Albright and his colleagues originally described a disorder limited) extent of functional conservation between the two
characterised by hypocalcaemia, hyperphosphataemia andend species(Table1),itshouldbenotedthatactivatingorgainof
organresistance(inproximalrenaltubules)tothemainplasma functionmutationsofGnashavealsobeenidentified.Theseare
Ca2C regulator parathyroid hormone (PTH), and therefore beyond the scope of this review, but have been summarised
namedthediseasePHP(Albrightetal.1942).AsPTHlevelsare elsewhererecently(Haywardetal.2001,Weinsteinetal.2006,
not reduced, but typically elevated, and since GNAS is 2007).Furthermore,aseparatehumandisorderassociatedwith
biallelicallyexpressedinthecalcium-reabsorbingthickascending theGNASlocusisnotduetomutationsaffectingtheprotein-
limbofthekidney,hypercalciuriadoesusuallynotoccurinthese codingsequences,butiscausedbyderegulatedimprintingand
patients. They also described other specific somatic and geneexpressioncontrol.Originally,ithasbeencharacterised
developmentalabnormalitiesinthesepatientsandthedisorder byPTHresistanceonlywithoutclearAHOsymptomsandwas
isnowknowntoincludethefollowingadditionalsymptoms:a thereforetermed‘PHPtypeIb’(PHP-Ib;Bastepe&Ju¨ppner
round face with a ‘short, thickset figure’, early closure of the 2005). Our current understanding of PHP-Ib is briefly
epiphyseswithresultantshorteningofoneormoremetacarpals summarisedtowardstheendofthisreview.
or metatarsals(brachydactyly), s.c. ectopic ossifications, dental
hypoplasia, obesity and cognitive abnormalities of varying
Post-natal physiological functions
degreesfromlearningdisabilitiestosevereretardation(Albright
etal.1942,1952,Weinsteinetal.2001,Levine2002).Albright Allmanipulationsinmicethatleadtolackofmaternalallele-
andcolleaguesalsonoticed patientswho showedmanyof the specific Ga or XLa showan impaired neonatal phenotype
s s
latterphysicalfeatures,buthadnormalcalcium,phosphateand withreducedsurvival(Cattanach&Kirk1985,Yuetal.1998,
PTHlevels(Albrightetal.1952).Theytermedthiscombination Cattanach et al. 2000, Plagge et al. 2004, Chen et al. 2005,
ofsymptoms,whichwasnotassociatedwithhormoneresistance, Germain-Leeetal.2005).
‘pseudopseudohypoparathyroidism’ (PPHP). Both conditions Heterozygous deficiency of Ga in mice, generated
s
arealsoreferredtoasAHO,andidenticalmutationsinGNAS through deletion of Gnas exon 1, results in a neonatal
thataffecttheproteincodingsequencecancauseAHOwithor phenotype on maternal transmission (Chen et al. 2005,
without hormone resistance. It was Davies & Hughes (1993) Germain-Lee et al. 2005). The paternally inherited deletion
whodescribedforthefirsttimetheassociationofthesyndromes has few consequences at this developmental stage, although
with the parental origin of the mutation. Thus, paternal somemortalitywasobservedinaninbredstrainbackground
inheritanceofaGNASexonmutationresultsin(AHO-)PPHP, (Germain-Lee et al. 2005). For exon1mK/pC mice a survival
while maternal inheritance is associated with additional rate toweaning age of 34–51% was observed, againvarying
resistance to PTH (and other hormones, see below; Levine with the genetic background used. Most of the losses occur
et al. 1983a) and is now termed ‘PHP type Ia’ (PHP-Ia; within 3 days after birth, and may result from a severe s.c.
Weinsteinetal.2001).SomeofthetypicalfeaturesofAHOare oedema, which has been described in several mouse models
showninFig.3A–FandaresummarisedinTable1;however,not lacking maternal allele-specific Ga protein (Cattanach &
s
allfeaturesarepresentinallpatients. Kirk 1985,Yuet al.1998,Cattanach et al.2000,Chenetal.
The recent analysis of several mouse models with 2005).Thephysiologicalcauseoftheoedema,whichresolves
deficiencies of the individual protein products has deepened withinafewdaysafterbirth,iscurrentlyunclear,althougha
our understanding of the associated physiological and placental dysfunction has been suggested (Chen et al. 2005,
endocrine functions (Plagge & Kelsey2006, Weinstein et al. Weinstein et al. 2007). Another consequence of loss of Ga
s
2007). Not surprisingly, homozygous deficiency of Ga is expression from the maternal allele is the development of
s
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.
Gnasimprintingandfunctions APLAGGEandothers 199
profound obesity in adulthood (discussed in detail below). withinadayafterbirth(Cattanach&Kirk1985,Williamson
Theincreaseinadiposityarisesalreadyduringthepost-natal etal.1998).Twofurthermutations,adeletionofexon2anda
stage, as has been documented in mice with maternally pointmutationinexon6(termedOed-Sml),affectbothGa
s
inheritedmutationsofexons2and6(Cattanachetal.2000, and XLa upon paternal transmission (Yu et al. 1998, 2000,
s
Yuetal.2000,Plagge&Kelsey2006).Despitetheirincreased Cattanach et al. 2000, Skinner et al. 2002); however, the
lipidaccumulationandadiposetissuemass,thesemiceremain phenotypes of exon2mC/pKmice and Sml mice areidentical
underweightuntilafterweaning. in manyrespectstoGnasxl deficiency (Yu et al. 1998,2000,
Comparativelylittleinformationonpost-natalsymptomsis Cattanachetal.2000,Plagge&Kelsey2006,Weinsteinetal.
available from case studies of AHO/PHP-Ia patients who 2007). The similarity of the phenotypes of these latter two
carry mutations in GNAS exons on the maternal chromo- mutations to the Gnasxl mutation indicates that in mice the
some.Ans.c.oedemahasnotbeendocumented.However,a lossofXLa isdominantoverthesimultaneouslossofpaternal
s
few reports describe an early onset of some symptoms allele-derived Ga. Furthermore, as the paternally inherited
s
characteristic of PHP-Ia at later juvenile or adult stages (see exon6pointmutationdoesnotaffecttheothertwoproteins
also below; Levine et al. 1985, Weisman et al. 1985, Yokoro expressed from the Gnasxl exon (XLN1 and Alex), this
et al. 1990, Scott & Hung 1995, Yu et al. 1999, Riepe et al. indicates that loss of XLa is the main cause for the lack of
s
2005,Gelfandetal.2006).Fromthesestudiesapatternseems paternal function phenotypes (Plagge & Kelsey 2006,
toemergeinwhichabnormalthyroidfunctionandresistance Weinsteinetal.2007).
to thyroid-stimulating hormone (TSH), due to deficient The post-natal phenotype of XLa deficiency improves at
s
receptor signalling via Ga, are among the first symptoms around weaning age; no further premature mortality occurs
s
detectable: typically, TSH levels are elevated in PHP-Ia at fromthisstageonwards,althoughadultsremainlean(seebelow).
birth (Levine et al. 1985, Weisman et al. 1985, Yokoro et al. ItisnotunlikelythatchangesinXLa expressionunderliethese
s
1990, Yu et al. 1999). The s.c. ossifications can also develop phenotypechanges,sinceithasbeenshownforadiposetissue
fromthefirstfewmonthsonwards,whileresistancetoPTH, that Gnasxl expression becomes downregulated during the
hypocalcaemiaandhyperphosphataemiaareusuallydetected secondhalfofthepost-natalperiod(Xieetal.2006).
onlyatlaterstagesofinfancyorjuvenileage(Eddyetal.2000, Itiscurrentlyuncertain whetherXLa hasasimilar rolein
s
Riepe et al. 2005, Gelfand et al. 2006, 2007). Progressive humanneonatalphysiology.Theclassicaldescriptionsofpatients
osseous heteroplasia (POH), a more severe form of with AHO/PPHP do not include comparable symptoms. As
extraskeletal ossification with invasion into deeper tissues, PPHPpatientscarrypaternallyinheritedmutationsinGNAS-
canalsobeginearlyon,andhasbeendescribedinassociation coding exons, similar to exon2mC/pK and Sml mice, XLa
s
with paternally inherited as well as spontaneouslyoccurring functionwouldbeexpectedtobeimpairedanddominantover
GNAS mutations (Eddyet al. 2000, Shoreet al. 2002, Faust lossofpaternallyexpressedGa.However,thesemutationscause
s
et al. 2003, Gelfand et al. 2007). In general, ossification the same common AHO features as in maternally inherited
symptomsareaclassicalAHOfeature,astheycanoccurupon PHP-Ia (plus additional hormone resistances). A conclusive
mutationsofthematernalor paternalallele. human case study, which could distinguish between XLa
s
LossofpaternallyexpressedXLa (throughgenetargeting functions and paternal haploinsufficiency of Ga by analysing
s s
oftheGnasxl-specificexon)causeslethalityininbredmouse paternallyinheritedGNASexon1mutations,hasnotyetbeen
strains, but 15–20% of mutants survive into adulthood if published (Patten et al. 1990, Fischer et al. 1998, Aldred &
maintained on an outbred genetic background (Cattanach Trembath 2000, Mantovani et al. 2000, Long et al. 2007).
etal.2000,Plaggeetal.2004,Xieetal.2006).Deficientpups However,otherraregeneticanomaliesthatdisrupttheGNAS
become distinguishable from wild-type littermates within locusandXLa expression,e.g.largechromosomaldeletionsand
s
1or2daysafterbirth,duetoafailuretothrive,characterised maternaluniparentaldisomies(UPD)ofchromosome20q13.2–
byseveregrowthretardation,poorsuckling,hypoglycaemia, q13.3,havebeenassociatedwithneonatalimpairments.Patients
hypoinsulinaemia,lackofadiposereservesandinertia(Plagge withmaternalUPD20q13.2–q13.3,wholackacorresponding
et al. 2004). This phenotype is most likely related to paternal allele and can be compared with MatDp.dist2 mice
pleiotropic functions of XLa in the central nervous system described above, show pre- andpost-natal growth retardation
s
(CNS, e.g. orofacial motornuclei in the context of suckling (Chudobaetal.1999,Eggermannetal.2001,Salafskyetal.2001,
activity),aswellasperipheraltissues thatareinvolvedinthe Velissariou et al. 2002). The 20q13.2–q13.3 deletions that
maintenance of energy homeostasis (e.g. adipose tissues, include the GNAS locus on the paternal allele, also lead to
pancreas;Plaggeetal.2004).Impairmentinneonatalfeeding, growthretardation,failuretothrive,feedingdifficultiesrequiring
growthandmaintenanceofenergybalanceisfoundnotonly artificial feeding, hypotonia and adipose tissue abnormalities
inmicewithaspecificmutationoftheGnasxlexonbutalso (Aldredetal.2002,Genevieveetal.2005),reminiscentofGnasxl
in other mutants that lack XLa (Plagge & Kelsey 2006, knockoutmice.Althoughthesecasesofchromosomaldeletions
s
Weinsteinetal.2007).Thus,micethatcarrytwocopiesofthe andUPD20srequirecarefulinterpretation,asotherpotentially
maternally inherited gene locus and no paternal copy contributing genes might also be affected, they nevertheless
(MatDp.dist2) show narrow, flat-sided bodies with reduced encourage an investigation of PPHP patients for post-natal
adiposityinBAT,hypoactivity,failuretosuckleandlethality symptoms, as far as this is feasible and records are available.
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.
200 APLAGGEandothers Gnasimprintingandfunctions
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.
Gnasimprintingandfunctions APLAGGEandothers 201
No null mutations for the Gnasxl-specific exon have been accompaniedbyTSHresistanceandnormaltoelevatedTSH
reportedinhumans,butapolymorphismintheXLa domain, plasma levels in mice inheriting a disrupted maternal allele,
s
which results in varied numbers of a 12 amino acid NH - but not in mice with a disrupted paternal allele, similar to
2
terminalrepeatunit,hasbeenassociatedwithsymptomssuchas humans(Yuetal.2000,Chenetal.2005,Germain-Leeetal.
growth retardation, unexplained mental retardation and 2005). Although TSH resistance could contribute to other
brachydactyly(Fresonetal.2001,2003).Furthercharacterisation symptoms observed in AHO/PHP-Ia, e.g. short stature and
of the patients as well as the biochemical functionalityof the obesity(seebelow),itseemsunlikelythatthiswouldbethesole
XLa repeatvariantsisrequired. causeinlightofthemilddegreeofhypothyroidismthatoccurs,
s
as well as the fact that even when patients are successfully
treatedthroughouttheirlifetime,theyareshortasadultsand
Physiological functions inadulthood
alsoobese(Longetal.2007).Inaddition,micewithmaternal
TherolesoftheproteinsoftheGnas-locusatadultstageshave Ga deficiencywereobesewithnormalthyroxinelevels,thus
s
beencharacterisedinmoredetail,bothinhumanandmouse implicating other factors in the development of the obesity
(Table 1). The symptoms common to PHP-Ia and PPHP, (Yuetal.2000,Chenetal.2005,Germain-Leeetal.2005).
whichoccur independentlyofparentaloriginandaredueto
haploinsufficiencyofGa incellswithbiallelicexpression of PTHresistancePTHresistanceinPHP-Iapatientstypically
s
GNAS, as well as the hormone resistances associated with develops over the first several years of life with an elevated
PHP-Ia upon maternal inheritance of mutations, fully PTH usually preceding the hypocalcaemia and hyperpho-
develop towards adulthood. With regard to Ga, many sphataemia(Werderetal.1978,Barretal.1994,Yuetal.1999),
s
parallels have now been described between the human although there are some patients who do not develop
diseasesandcorrespondingmousemodels,althougharoleof hypocalcaemia until late in adulthood (Hamilton 1980) and
XLa inhumansremainsuncertain. otherswhomaintainnormalcalciumlevelsthroughouttheir
s
lifespan (Balachandar etal.1975,Drezner&Haussler1979).
Abnormalitiesincalciumlevelsmostlikelyresultfromlackof
Hormone resistances
PTHsignallinginthekidney,whereitactsonproximalrenal
TSH resistanceMildTSHresistanceoccurs inmost adult tubules as well as distal portions of the nephron. GNAS
patientswith PHP-Ia in addition to the usually pronounced imprintingandpreferentialexpressionfromthematernalallele
PTH resistance described below (Levine et al. 1983a, inthekidneyoccuronlyinproximalrenaltubules,butnotin
Weinstein et al. 2001, Levine 2002). A study using thyroid thethickascendinglimborinthecollectingducts,asbasedon
membranes isolated from a patient with PHP-Ia demon- PHP-Iapatients(Mosesetal.1986,Faulletal.1991)aswellas
stratedthatthedefectliesinthesignaltransductionpathway on results in knockout mouse models (Yu et al. 1998,
forTSH,consistentwithadefectinGa (Malletetal.1982). Ecelbarger et al. 1999, Weinstein et al. 2000, Germain-Lee
s
Threestudiesconfirmedwithstrikinglysimilarresultsthat et al. 2005). Loss of Ga from the maternal allele, therefore,
s
GNASisexpressedpreferentiallyfromthematernalallelein disturbsthePTH-mediatedinhibitionofphosphatereabsorp-
normal human thyroid tissue (mean contribution of the tion and its stimulation of 1,25-dihydroxycholecalciferol
. .
maternal allele: 713–75 7%; Germain-Lee et al. 2002, (activated Vitamin D3) synthesis in the proximal tubules
Mantovani et al. 2002, Liu et al. 2003). The fact that the more than the calcium reabsorption in distal parts of the
imprintinginthethyroidispartial,e.g.incompletesilencing nephron. This combination of effects leads to an imbalance
ofthepaternalGNASallele,mayprovideanexplanationfor characterisedbyreducedexcretionofphosphateandreduced
the mild TSH resistance and hypothyroidism found in 1,25-dihydroxycholecalciferol-mediated uptake of calcium
patients with PHP-Ia. Partial imprinting probably accounts via theintestines, aswell asreducedmobilisation of calcium
for incompletehormonalresistanceinother tissuesaswell. frombone,whereascalciumreabsorptioninthedistalpartsof
ConcurrentstudiesinGnasknockoutmicewithatargeted the kidney remains normal and hypercalciuria is rarely
disruptionofexon1revealedGa imprintinginthethyroid, observedinPHP-Iapatients(Weinsteinetal.2000).Insome
s
Figure3 TypicalfeaturesofAHO.(A)Typicalroundfaceandshort,obesebodyhabitus(althoughextremeobesityhasbeenfoundtobespecific
forPHP-Ia).(B)X-rayofthehandofanAHOpatientshowingthestrikingshorteningofthefourthandfifthmetacarpals.Arrowsarepointingto
multiples.c.ossificationsinthehand.(C)Brachydactylyofthehandswithmarkedshorteningofthefourthphalanxandmetacarpal.(Notthe
samepatientshowninB).Theasymmetryinappearanceofthehandsiscommon.Thearrowpointstotheveryshortleftthumbreferredtoas
‘potter’sthumb’or‘Murder’sthumb.’(D)Asaresultofthebrachymetacarpia,theknucklesareabsentandarereplacedbydimpleswhenthefist
isclenched.Thisisreferredtoas‘Archibald’ssign.’(E)ShorteningofthetoesisfoundcommonlyinAHO.(F)Growthcurvesofthree
GH-deficientpatientswithPHP-Ia(onemale(left)andtwofemales(right))showingthefrequentabsenceofshortstatureinchildhoodwith
resultingshortfinaladultheights.Inaddition,thepubertalgrowthspurtsareabsent.OnepatientwastreatedwithGHfromapproximatelyage
9.5–12years(referredtoasSubject8)priortoreferral.Trianglesrefertoboneage(noboneagedataforSubject7).Reproducedwithpermission
fromGermain-LeeEL,GromanJ,CraneJL,JandeBeurSM&LevineMA2003Growthhormonedeficiencyinpseudohypoparathyroidismtype
1a:anothermanifestationofmultihormoneresistance.(seecomment).JournalofClinicalEndocrinologyandMetabolism884059–4069.
Copyright2003,(TheEndocrineSociety).Signedinformedconsentswereobtainedforthepatientphotographs.
www.endocrinology-journals.org JournalofEndocrinology(2008)196,193–214
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2
0
2
Jo Table1 PhysiologicalfunctionsaffectedbymutationsattheGNAS/Gnasimprintedlocusinhumanandmouse A
urnalo Disorderortypeofphysiologicaldysfunction PLAGG
f E
End Typeofmutation Human Mouse References an
o d
c
rino Protein oth
logy Gaesx(pbrieaslsleiolinc) Mo1udsee:lehtioomnoozryegxoounsG2ndaissruepxtoinon Unknown Embryoniclethality MoGuesrem:aYuin-eLteael.e(t1a9l9.8(2),0C0h5e)netal.(2005)and ers
(20 (a)Post-natalstage .
08) Gas(maternal Human:missenseornonsense AHO/PHP-Ia 49–66%preweaning Human:Levineetal.(1985),Weismanetal.(1985), G
1 allele-specific mutationsinGNASexons1–13; lethality Yokoroetal.(1990),Scott&Hung(1995),Yuetal. n
96, expression) (pointmutations,smalldeletions, †Mildhypothyroidism,early (1999),Eddyetal.(2000),Faustetal.(2003),Chan as
19 splicesitemutations) onsetTSHresistancein †S.c.oedema,resolving etal.(2004),Riepeetal.(2005)andGelfandetal. im
3– thyroidcellsandelevated duringfirstfewdays (2006,2007) p
214 Moduissreu:pdtieolnetioofneoxofnG2n,asmeixssoenns1e,point †ETaSrlHyolenvseeltss.c.ossifications ††RInecdruecaeseddpared-iwpoesaintyingbody Mo(1u9se9:8C),aYtutanetacahl.&(19K9ir8k,(21090805)),,CWatitlalinaamcshoneteatl.al. rintin
mutationinexon6,paternal weight. (2000),Skinneretal.(2002),Chenetal.(2005)and g
a
uniparentalduplicationofdistalchr.2 †Brachydactyly †Alsoreportedinsomemouse Germain-Leeetal.(2005) n
d
models:tremor,imbalance,
hyperactivity,squareshaped fun
body,microcardia ctio
n
s
(b)Adultstage
AHO/PHP-Ia Human:seetext;alsoreviewedin:Aldred&
Trembath(2000),Weinsteinetal.(2001,2006),
†ResistancetoTSHinthyroid †MildandvariableTSH Bastepe&Ju¨ppner(2005),Germain-Lee(2006)
cells,elevatedTSHlevels, resistanceandelevated andMantovani&Spada(2006)
mildhypothyroidism TSHlevels Mouse:Yuetal.(1998,2000,2001),Cattanachetal.(2000),
D †ResistancetoPTHinprox- †ResistancetoPTHinprox- Skinneretal.(2002),Chenetal.(2005)and
o
wn imalrenaltubules,elevated imalrenaltubules,elevated Germain-Leeetal.(2005)
l
o PTHlevels,hypocalcaemia, PTHlevels,hypocalcaemia,
a
de hyperphosphataemia hyperphosphataemia
d †GHRHresistanceinpituitary
f
ro somatotrophcells,GH
m
deficiencyvariable
B
io †Reducedsensitivityto †Reducedfertility
s
c gonadotrophinsLHand
i
en FSH,hypogonadism
ti †Shortstature,brachydactyly †Reducedbodylength
fi w
c w
a. w
com at 02 .endocrino †So.cs.seoosusisfihcaettieorno,plparsoigar(ePsOsivHe) †S.c.ossification (continued)
via/25/202 logy-jou
free a3 12:20 rnals.org
c:
c1
e2
sA
sM