Characterization of the intracellular signaling mechanisms activated by ghrelin through GHSR-1a: role of beta-arrestins
Abstract
La ghrelina, una hormona peptídica de 28 aminoácidos con una modificación post-traduccional de O-n-octanoylation en el residuo de la serina 3, es el ligando natural del receptor de secretagogos de hormona de crecimiento tipo 1a (GHSR-1a). Además de regular la secreción de hormona de crecimiento y la ingesta, la ghrelina también controla la homeostasis energética, la actividad pancreática, el metabolismo de la glucosa y procesos proliferativos y antiproliferativos. Algunas de estas funciones suponen la activación de diferentes rutas de señalización como la ruta de las MAPK (mitogen-activated protein kinase) o la de Akt, que están mediadas por las proteínas G. Sin embargo, los estudios recientes sugieren que los receptores acoplados a proteínas G (GPCR) se pueden acoplar a las β-arrestinas. Las β – arrestinas constituyen una pequeña familia de sólo cuatro miembros que fueron inicialmente descubiertas en los procesos de desensibilización de los GPCR. Actualmente, también juegan un papel importante en la endocitosis de los receptores y en la señalización intracelular, actuando como proteínas adaptadoras multifuncionales. Este trabajo pone de manifiesto que en la línea celular HEK-GHSR-1a, como modelo para estudiar la interacción entre proteínas, la ghrelina activa tanto la ruta de las MAPK como la de Akt a través de 2 vías de señalización diferentes, dependientes tanto de las proteínas G como de las β-arrestinas. En el caso de la activación de MAPK, ésta se consigue por la interrelación de 3 vías diferentes: la primera está mediada por las β-arrestinas 1 y 2 y requiere la presencia del GHSR-1a en un complejo multiproteico junto con las β-arrestinas, Raf-1, cSrc, ERK 1/2 y quizás otros componentes de la ruta de las MAPK. La segunda depende de una proteína Gq/11 e implica la activación de una PKC dependiente de calcio (PKC α/β) y de la proteína cSrc. La tercera ruta depende de una proteína Gi/o e implica la activación de PKC ε y cSrc. Respecto al mecanismo de activación de Akt, ésta se activa a través de dos rutas: una de ellas es dependiente de las subunidades βγ de las proteínas G e implica la activación de la PI3K; la otra está mediada por las β-arrestinas 1 y 2 y requiere la entrada del receptor en un complejo multiproteico. Hay que destacar por lo tanto, el papel de las β-arrestinas en los procesos de señalización, actuando como proteínas adaptadoras y transductoras de señales. También es destacable el papel clave que juega la proteína cSrc en la activación de ambas rutas. Asimismo se ha identificado a SHP-1 como uno de las proteínas reguladoras negativas de la activación de Akt mediada por la ghrelina. La activación de SHP-1 es dependiente de cSrc, lo que supone la interrelación de las rutas dependientes de proteínas G y de las β-arrestinas, y se ejerce a través de la defosforilación de la PI3K y cSrc. Se encontró también una expresión de SHP-1 en células 3T3-L1, tanto en preadipocitos como adipocitos maduros, y en tejido adiposo de ratones obtenido de mantenidos con dieta normal o dieta alta en grasa. Los resultados obtenidos muestran que el tejido adiposo omental muestra un aumento de la expresión de SHP-1 comparado con el tejido subcutáneo en condiciones normales. Sin embargo, en condiciones de dietas altas en grasa, hay una mayor expresión de SHP-1 en el tejido adiposo subcutáneo comparado con el omental. En base a estos resultados se puede especular que la acción de la ghrelina en el tejido adiposo está, al menos en parte, mediada por la acción de SHP-1: un aumento de expresión de SHP-1 en el tejido subcutáneo resulta en una atenuación de la señalización dependiente de Akt con la consecuente reducción de la sensibilidad de la ghrelina. Por contra, la down-regulación de SHP-1 en el tejido omental reforzaría la sensibilidad a la ghrelina, promoviendo el almacenamiento de grasa. Además, también se ha determinado una interacción funcional potencial entre la cortistatina 17 (CST-17) y el sistema de la ghrelina, proponiendo dos hipótesis. Por una parte, la CST-17 podría unirse al GHSR-1a actuando como un ligando potencial del mismo; por otra parte, la CST-17 también se podría unir al SSTR2, que formaría un dímero con el GHSR-1a, afectando de este modo a la señalización mediada por la ghrelina.
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UNIVERSIDADDESANTIAGODECOMPOSTELA FACULTADDEMEDICINA DEPARTAMENTODEFISIOLOGÍA LABORATORIODEENDOCRINOLOGÍAMOLECULAR Characterizationoftheintracellularsignaling mechanismsactivatedbyghrelinthroughGHSR‐1a: roleofβ‐arrestins MARÍALODEIROPOSE SantiagodeCompostela,mayode2011 ISBN 978-84-9887-794-6 (Edición digital PDF)
UNIVERSIDADDESANTIAGODECOMPOSTELA FACULTADDEMEDICINA DEPARTAMENTODEFISIOLOGÍA LABORATORIODEENDOCRINOLOGÍAMOLECULAR Characterizationoftheintracellularsignaling mechanismsactivatedbyghrelinthroughGHSR‐1a: roleofβ‐arrestins MemoriaqueparaoptaralGradodeDoctorenBiología porlaUniversidaddeSantiagodeCompostelapresenta: MaríaLodeiroPose SantiagodeCompostela,mayode2011
Lamemoriaadjuntatitulada“Characterizationoftheintracellular signalingmechanismsactivatedbyghrelinthroughGHSR‐1a:roleofβ‐ arrestins”queparaoptaralGradodeDoctorenBiologíapresentaDña. MaríaLodeiroPose,hasidorealizadabajonuestradirecciónenelÁreade EndocrinologíaMolecularyCelulardelInstitutodeInvestigaciónSanitaria deSantiago(ComplejoHospitalarioUniversitariodeSantiagode Compostela). ConsiderandoqueconstituyetrabajodeTesisDoctoral,autorizamos supresentaciónenlaUniversidaddeSantiagodeCompostela. Yparaqueasíconste,firmamoslapresenteenSantiagode Compostelaenmayode2011. Dr.JesúsPérezCamiña InvestigadorSERGAS Prof.Dr.FelipeCasanuevaFreijo CatedráticodeMedicina SantiagodeCompostela,mayode2011
Gustaríameexpresaromeuagradecementoa: ÓProfesorFelipeCasanuevaFreijo,directordestetraballo,pordarmeaoportunidadede iniciarmenocampodainvestigacióndentrodoseugrupo. ÓDr.JesúsPérezCamiña,co‐directordotraballo,polasúaaxudanarealizacióndomesmo. AMaryLage,polasúacalidezeapoioduranteestesanos,quefanaunsentirseagustoeque nonsempresevalora. AosmeuscompañeirosnolaboratorioMarco,Carlos,Maribel,María,BegoetaménaSara,cos quecompartínunpoucodetodoequesempreestiveroneestánaídispostosabotarunha man. ÓGrupodoDr.RoyGSmith,directordoDepartmentofMetabolismandAging,TheScripps ResearchInstitute,Florida,USA,poracollermenoseulaboratorio,eáxentequealícoñecín, especialmenteaJorgeeaKarimaquemefixeronsentircomoencasa. Ámiñafamilia. AJose,graciaspolatúaaxuda,ánimoeapoiodurantetodoestetempo. AIria,Elena,MónicaeAndrea,porestaraísempre,apoiándomeincondicionalmentee sabendoquesempresepoderácontarconvósparaoquesexa.Gracias. ALúa,Sabela,AbeleParcero….poraturarmetodosestosanos,coascousasboasemalas,por estarsempreaíeporapoiarmeeanimarmenosmomentosmáisdifíciles. AMerceeMaría,poresasquedadaseconversasnosas(surrealistasásveces),queespero continúenpormoitosanosmáis. Atodosaquelesquecoñecínólongodestesanos,quemeaxudarondunxeitoououtroeque merecentaménunrecoñecemento:Pedro,Ramón,Sabela,Bruno,Toño,Raquel,Leonor… gracias!
“Sinoestásdispuestoaequivocarte,nuncallegarásanada”. KenRobinson
Abbreviations 17 7TM:seventransmembane 7TMR:seventransmembranereceptor AC:adenylatocyclase ACTH:adrenocorticotropehormone AG:acylatedghrelin Akt:serine/threoninekinase(ProteinkinaseB) AMP:adenosinemonophosphate AMPK:AMP‐activatedproteinkinase ANOVA:analysisofvariance AP‐2:adaptorprotein2 AT1AR:angiotensinIItype1Areceptor AT2R:angiotensinIItype2receptor ATM:ataxiatelangengiectasiamutatedgeneproduct β1AR:β‐1adrenergicreceptor β2AR:β‐2adrenergicreceptor BHK:babyhamsterkidneycells BSA:bovineserumalbumin cAMP:cyclicadenosinemonophosphate cGMP:cyclicguanosinemonophosphate CONA:concanavalinA CPM:countsperminute CPT‐1:carnitinepalmitoyltransferase1 CST:cortistatin DAG:diacylglycerol DEX:dexamethasone DMEM:Dulbecco´sModifiedEagle´sMedium DNAPK:double‐strandedDNA‐dependentproteinkinase
Abbreviations 18 ER:endoplasmicreticulum ERK1/2:extracellularsignal‐regulatedkinases1/2 FAS:fattyacidsynthase FBS:fetalbovineserum FURA‐2AM:fura‐2acetoxymethylester GC:guanylylciclase GH:growthhormone GHRH:growthhormonereleasinghormone GHRH‐R:growthhormonereleasinghormonereceptor GHS:growthhormonesecretagogues GHS‐R:growthhormonesecretagoguereceptor GHSR‐1a:growthhormonesecretagoguereceptortype1a GHSR‐1b:growthhormonesecretagoguereceptortype1b GOAT:ghrelinO‐acyl‐transferase GPCR:G‐protein‐coupledreceptor GPR39:G‐protein39‐coupledreceptor GRB2:growthfactorreceptor‐boundprotein2 GRK:G‐protein‐coupledreceptorkinase HEK:humanembrionarykidneycells HFD:highfatdiet IBMX:3‐isobutyl‐1‐methylxanthine ILB:immunoprecipitationlysisbuffer ILK:integrin‐linkedkinase IGF‐1:insulin‐likegrowthfactor1 IHC:immunohistochemistry IP3:inositol(1,4,5)‐triphosphate IRS‐1:insulinreceptorsubstrate1
Abbreviations 19 KDa:KiloDaltons KRH:Krebs‐RingersHenseleit MAPK:mitogen‐activatedproteinkinase mRNA:messengerRNA MBOAT:membrane‐boundO‐acyltransferase MrgX2:massrelatedgeneX2 mTOR:mammaliantargetofrapamycin mTORC2:mammaliantargetofrapamycincomplex2 NO:nitricoxide NOS:nitricoxidesynthase PAM:peptidylglycineα‐amidatingmonooxygenase PAMP12:proadrenomedullinN‐terminalpeptide12 PC1/3:proproteinconvertase1/3 PCR:polymerasechainreaction PDK‐1:3‐phosphoinositide‐dependentkinase‐1 PH:pleckstrinhomologydomain PHLPP1:PHdomainleucine‐richrepeatproteinphosphatase1 PHLPP2:PHdomainleucine‐richrepeatproteinphosphatase2 PI3K:phosphatidylinositol3´‐kinase PI‐PLC:phosphatidylinositol‐specificphospholipaseC PKA:proteinkinaseA PKC:proteinkinaseC PMA:phorbol12‐myristate13‐acetate PP2A:proteinphosphatase2A PRL:prolactin PTH1R:parathyroidhormonetype1receptor PTP:proteintyrosinephosphatase
Abbreviations 20 PTX:pertussistoxin qRT‐PCR:quantitativerealtimePCR RNA:ribonucleicacid RTK:receptortyrosinekinase SDS:sodiumdodecylsulfate SHP‐1:Srchomology‐2domaincontainingphosphatase‐1 SHP‐1dn:SHP‐1dominantnegative siRNA:smallinterferingRNA SST:somatostatin SSTR:somatostatinreceptor TCA:trichloroaceticacid UAG:unacylatedghrelin V2R:vasopressintype2receptor VMH:ventromedialnucleusofthehypothalamus VIP:vasoactiveintestinalpeptide WAT:whiteadiposetissue WORT:wortmannin WT:wildtype
1. OBJECTIVES
Objectives 23 Theghrelin/ghrelinreceptor(GHSR‐1a)systemdirectsdiversearrayofphysiologicalresponses andhencehasbroadrelevancetonumerousdiseases,amongthemobesityandanorexia. RecentstudiessuggestthatsignalingthroughGPCRisfarmorediversethanoriginalthought, asGPCRcancoupletomultipleG‐proteinsaswellasotheradaptorproteins,amongthemthe β‐arrestinfamily.Basedonit,theworkhypothesisforthisThesiswasthat,forghrelin/GHSR‐1a system,β‐arrestinsconstitutearelevantandseparablesignalingarmfromtheclassical heterotrimericG‐proteinsinadditiontotheirroleintheendingofG‐protein/GHSR‐1a signaling. Theglobalobjectiveistodelineatetheβ‐arrestinsignalingfortheghrelinreceptorGHSR‐1a. Thisgeneralobjectiveisdividedintothefollowingspecificobjectives: 1.Todeterminetheproteinpartnersofbothβ‐arrestin1and2underbasalandghrelin‐ stimulatedconditionsintheregulationofmetabolism,apoptosis,transcriptionandcell‐cycle. Thispointisdividedin: 1.1.Todeterminetheroleofβ‐arrestinsonERK1/2‐dependentsignalingpathwaythat mediatetheeffectofghrelinoncellsurvival,growthandproliferation. 1.2.Todeterminetheroleofβ‐arrestinsonAkt‐dependentsignalingpathwaythat mediatetheeffectofghrelinonmetabolism,apoptosis,transcriptionandcell‐cycle. 2.TodefinethemechanismforregulatingGHSR‐1a‐associatedAktactivity,withemphasisin theimplicationofthecytoplasmicproteinSrchomology‐2domaincontainingphosphatase‐1 (SHP‐1). 3.Todeterminetheroleofβ‐arrestinsonAkt‐andERK1/2‐dependentsignalingpathwaysthat mediatetheeffectsofCST‐17throughGHSR‐1a. ThesemolecularmechanismswereinvestigatedinHEK293cells,stablyexpressingtheGHSR‐ 1a(HEK‐GHSR‐1a)asmodelsystemtofollowtheinteractionofspecificproteinsandtheroles, ifany,playedbyG‐proteinsandβ‐arrestins.Inaddition,someresultswereconfirmedin3T3‐L1 cellsandwhiteadiposetissue.
2. INTRODUCTION
Introduction 32 Figure3.Post‐translationalprocessingandacylationofthepro‐ghrelinpeptide.Afterthesignal sequenceiscleavedbyasignalpeptidepeptidasetheacylationofpro‐ghrelinoccurs.Forthispurpose, GOAT,whichislocatedattheERcompartment,mediatesthetranslocationoftheoctanoyl‐CoAfrom thecytosolicside.Later,inthetrans‐Golgicompartment,thepro‐ghrelinprecursoriscleavedbyPC1/3 proproteinconvertase,packagedinvesicles,andreleasedtotheblood.Differentformsofghrelincanbe foundintothecirculation:acylated(AG),unacylated(UAG),andothershorterformswhoseroleisstill unknown.(FigureextractedfromEurJEndocrinol.2010;163:1‐8).
Introduction 33 roleofGOAThasbeenstudiedunderdifferentconditionssuchasnutritionalstatus,age,sex andlactatingstatus.21,22Thissuggeststhattheghrelin‐GOATsystemactasalipidicsensorthat informsthebrainaboutthehighfatmealavailabilitytoachieveanoptimalenergeticstorage. 20Thisalsosuggeststhatthephysiologicalfunctionofghrelinmaynotnecessarily,oratleast notexclusively,beahungersignalreflectinganemptystomach.BecauseGOATistheunique enzymethatacylatesghrelininahighlyconservedmanner,itonlybindsandactivatesits receptorGHSR‐1awhenacylated,anditsinhibitionorstimulationwouldnotaffect physiologicalprocessesotherthanghrelinacylation.ItisalsoimportanttomentionthatGOAT hasimportantimplicationsintermsofdevelopingdrugstotargettheacylationprocessand, consequently,thephysiologicaleffectsofAG.19 In2005,usinggenomiccomparativeanalysisandbioinformaticpredictions,apeptideencoded bytheghrelingenewasdiscoveredanditwascalledobestatin,acontractionofobese,from theLatin"obedere,"meaningtodevour,and"statin,"denotingsuppression.23Thisnew peptidederivesfromproghrelinandithas23aminoacidswithaglycineresidueonthe carboxyterminaldomainthatcanbeamidated(Figure4).Itwasoriginallyisolatedfromrat stomach,showingtobeacirculatingpeptidewhosesecretionispulsatileanddisplaysan ultradianrhythmicitysimilartoghrelinandgrowthhormonesecretion,andthenitwasshown thatitwasalsofoundinothertissueslikeduodenum,pancreas,spleen,mammaryglandsand plasma.24,25ItwasoriginallyreportedtobetheligandfortheorphanreceptorGPR39,which belongstothefamilyoftheghrelinreceptorGHSR‐1aandthemotilinreceptor.24Despitethe initialenthusiasmaboutthepotentialofthismoleculeasaphysiologicalopponentofghrelin, severalobservationsrelatedtothispointhavesetitseffectivenessintoquestion.26,27 21GonzálezCR,VázquezMJ,LópezM,DiéguezC.InfluenceofchronicundernutritionandleptinonGOAT mRNAlevelsinratstomachmucosa.JMolEndocrinol.2008;41:415‐21. 22Al‐MassadiO,CrujeirasAB,GonzálezRC,PardoM,DiéguezC,CasanuevaFF,SeoaneLM.Age,sex,and lactatingstatusregulateghrelinsecretionandGOATmRNAlevelsfromisolatedratstomach.AmJ PhysiolEndocrinolMetab.2010;299:E341‐50. 23ZhuX,CaoY,VoogdK,SteinerDF.Ontheprocessingofproghrelintoghrelin.JBiolChem.2006;281: 38867‐70. 24ZhangJV,RenPG,Avsian‐KretchmerO,LuoCW,RauchR,KleinC,HsuehAJ.Obestatin,apeptide encodedbytheghrelingene,opposesghrelin'seffectsonfoodintake.Science.2005;310:996‐9. 25GrönbergM,TsolakisAV,MagnussonL,JansonET,SarasJ.Distributionofobestatinandghrelinin humantissues:immunoreactivecellsinthegastrointestinaltract,pancreas,andmammaryglands.J HistochemCytochem.2008;56:793‐801. 26NogueirasR,PflugerP,TovarS,ArnoldM,MitchellS,MorrisA,Perez‐TilveD,VázquezMJ,WiedmerP, CastañedaTR,DiMarchiR,TschöpM,SchurmannA,JoostHG,WilliamsLM,LanghansW,DiéguezC. Effectsofobestatinonenergybalanceandgrowthhormonesecretioninrodents.Endocrinology.2007; 148:21‐6. 27ZhangJV,JahrH,LuoCW,KleinC,VanKolenK,VerDonckL,DeA,BaartE,LiJ,MoecharsD,HsuehAJ. Obestatininductionofearly‐responsegeneexpressioningastrointestinalandadiposetissuesandthe mediatoryroleofGprotein‐coupledreceptor,GPR39.MolEndocrinol.2008;22:1464‐75.
Introduction 34 Consequently,thestate‐of‐knowledgeonobestatinsuffersfromseriousgaps,especiallyfor thelackofreproducibilityofitscentralactivities.28,29 Figure4.Proposedpost‐translationalprocessingofpreproghrelintomatureghrelinormature obestatin.ThehumanGHRLgeneconsistsoffourexonsandthreeintrons.FormationofmRNAand furthertranslationyieldsapolypeptideof117aminoacidscalledpreproghrelin.Thispolypeptide containsasignalpeptideof23aminoacidsattheaminoterminus,whichisfirstcleavedbyaputative signalpeptidaseresultinginproghrelinwith94aminoacids.Furtherprocessingofproghrelintoghrelin involvesproteolyticcleavagebyproproteinconvertase.Thenghrelinisacylatedattheserine3byGOAT enzyme.Obestatinresultsfromproteolysisofproghrelinattwocleavagesites.Afterthecleavage,the carboxy‐terminalleucineresidueisamidatedbyabifunctionalenzyme,peptidylglycineα‐amidating monooxygenase(PAM).(FigureextractedfromJClinEndocrinolMetab.2007;92:3396‐8.) 28SeoaneLM,Al‐MassadiO,PazosY,PagottoU,CasanuevaFF.Centralobestatinadministrationdoes notmodifyeitherspontaneousorghrelin‐inducedfoodintakeinrats.JEndocrinolInvest.2006;29: RC13‐RC15. 29ChartrelN,Alvear‐PerezR,LeprinceJ,IturriozX,Reaux‐LeGoazigoA,AudinotV,ChomaratP,CogeF, NosjeanO,RodriguezM,GalizziJP,BoutinJA,VaudryH,Llorens‐CortesC.Commenton"Obestatin,a peptideencodedbytheghrelingene,opposesghrelin'seffectsonfoodintake".Science.2007;315:766.
Introduction 35 Keepingasideitscontroversialanorexigenicactivity,therearedatasuggestingarelevant biologicalrole,likethemitogeniceffectdescribedin3T3‐L1preadipocyte,27humangastric carcinoma30,31andpancreaticβ‐cells.32Furthermoreobestatininducedc‐fosexpressionin gastrointestinalandwhiteadiposetissuesthroughbindingtoGPR39.27Ofinterest,GPR39 expressioninwhiteadiposetissueofratswasup‐regulatedduringfastingwhereasGPR39 levelsweredecreasedinculturedmouseembryonicfibroblastcelllinesduringadipogenesis.33 Inhumanadiposetissue,decreasedGPR39expressionwasfoundinpatientswithobesity‐ associatedtype2diabetesmellitus.34 Recentlyaroleforobestatinasaregulatorofadipocytemetabolismandadipogenesiswas proposed,pointingtoaputativeroleofobestatininthepathogenesisofmetabolicsyndrome throughanautocrine/paracrinemanner.35Itisalsoshownthatobestatinstimulates preadipocyteproliferation,adipocytedifferentiation,andfattyaciduptakemeanwhileit inhibitslipolysisin3T3‐L1adipocytes,suggestingthatobestatincouldstimulateadiposetissue hyperplasiaandhypertrophybyvariousmechanisms.36Itisalsoknownthatobestatinprotects cardiaccellsagainstmyocardialinjuryandapoptosisinducedbyischemia‐reperfusion.These effectsarelikelytobeinitiatedbyspecificobestatinbindingtoreceptorspresenton cardiomyocytesandinvolvetheactivationofPI3KandERK1/2pathways.Thisfactsuggests thatcirculatingobestatinalsomightplayacrucialroleinprotectingthemyocardiumfrom 30PazosY,AlvarezCJ,CamiñaJP,CasanuevaFF.Stimulationofextracellularsignal‐regulatedkinasesand proliferationinthehumangastriccancercellsKATO‐IIIbyobestatin.GrowthFactors.2007;25:373‐81. 31AlvarezCJ,LodeiroM,TheodoropoulouM,CamiñaJP,CasanuevaFF,PazosY.ObestatinstimulatesAkt signallingingastriccancercellsthroughβ‐arrestin‐mediatedepidermalgrowthfactorreceptor transactivation.EndocrRelatCancer.2009;16:599‐611. 32GranataR,SettanniF,GalloD,TrovatoL,BianconeL,CantaluppiV,NanoR,AnnunziataM,Campiglia P,ArnolettiE,GhèC,VolanteM,PapottiM,MuccioliG,GhigoE.Obestatinpromotessurvivalof pancreaticβ‐cellsandhumanisletsandinducesexpressionofgenesinvolvedintheregulationofβ‐cell massandfunction.Diabetes.2008;57:967‐79. 33EgerodKL,HolstB,PetersenPS,HansenJB,MulderJ,HökfeltT,SchwartzTW.GPR39splicevariants versusantisensegeneLYPD1:expressionandregulationingastrointestinaltract,endocrinepancreas, liver,andwhiteadiposetissue.MolEndocrinol.2007;21:1685‐8. 34CatalánV,Gómez‐AmbrosiJ,RotellarF,SilvaC,GilMJ,RodríguezA,CienfuegosJA,SalvadorJ, FrühbeckG.Theobestatinreceptor(GPR39)isexpressedinhumanadiposetissueandisdown‐regulated inobesity‐associatedtype2diabetesmellitus.ClinEndocrinol.2007;66:598‐601. 35Gurriarán‐RodríguezU,Al‐MassadiO,Roca‐RivadaA,CrujeirasAB,GallegoR,PardoM,SeoaneLM, PazosY,CasanuevaFF,CamiñaJP.Obestatinasaregulatorofadipocytemetabolismandadipogenesis.J CellMolMed.2010doi:10.1111/j.1582‐4934.2010.01192.x. 36MiegueuP,StPierreD,BroglioF,CianfloneK.Effectofdesacylghrelin,obestatinandrelatedpeptides ontriglyceridestorage,metabolismandGHSRsignalingin3T3‐L1adipocytes.JCellBiochem.2011;112: 704‐14.
Introduction 36 prolongedandexcessivestress,havingpotentiallyimportantimplicationsinclinicalconditions ofcardiodegenerativediseaseand/orischemicinjury.37 2.1.2. Ghrelinreceptor Thegrowthhormonesecretagoguereceptor(GHS‐R)wasoriginallydiscoveredin1997andit wasanorphanreceptoruntilthediscoveryofghrelin,itsnaturalligand.38Itismainly expressedinthepituitaryandhypothalamus,whereghrelinactsreleasingGHandmodulating foodintakerespectively.6Itisalsodetectedinotherorgans39andtissues40,41presentinga broaderexpressionasforexampleinthyroid,pancreas,spleen,myocardium,adrenalglands, testis,ovary,placentaandstomach.ThehumanGHSR‐1ageneislocatedonthecromosome position3q26.2.42TherearetwotypesofcDNAafteranalternativesplicingmechanism, namelyreceptortype1aand1b(Figure5).43Thetype1acDNAcodesa366aminoacids receptorwithseventransmembranedomains;ithasamolecularweightof41KDaand presentshighaffinityandspecifityforghrelinandthegrowthhormonesecretagogues(GHS). Ontheotherhand,thetype1bcDNAonlypresentsfivetransmembranedomainsand289 aminoacidsanditdoesnotallowligandbinding.44 37AlloattiG,ArnolettiE,BassinoE,PennaC,PerrelliMG,GhéC,MuccioliG.Obestatinaffords cardioprotectiontotheischemic‐reperfusedisolatedratheartandinhibitsapoptosisinculturesof similarlystressedcardiomyocytes.AmJPhysiol.2010;299:H470‐81. 38KarenKuljuMcKee,OksanaC.Palyha,ScottD.Feighner,DonnaL.Hreniuk,CarinaP.Tan,MichaelS. Phillips,RoyG.Smith,LexH.T.VanderPloegandAndrewD.Howard.Molecularanalysisofratpituitary andhypothalamicgrowthhormonesecretagoguereceptors.MolecularEndocrinology.1997;11:415‐ 423. 39GaytanF,BarreiroML,ChopinLK,HeringtonAC,MoralesC,PinillaL,CasanuevaFF,AguilarE,Diéguez C,Tena‐SempereM.Immunolocalizationofghrelinanditsfunctionalreceptor,thetype1agrowth hormonesecretagoguereceptor,inthecyclichumanovary.JClinEndocrinolMetab.2003;88:879‐87. 40GaytanF,BarreiroML,CaminosJE,ChopinLK,HeringtonAC,MoralesC,PinillaL,PaniaguaR,NistalM, CasanuevaFF,AguilarE,DiéguezC,Tena‐SempereM.Expressionofghrelinanditsfunctionalreceptor, thetype1agrowthhormonesecretagoguereceptor,innormalhumantestisandtesticulartumors.JClin EndocrinolMetab.2004;89:400‐9. 41KageyamaH,FunahashiH,HirayamaM,TakenoyaF,KitaT,KatoS,SakuraiJ,LeeEY,InoueS,DateY, NakazatoM,KangawaK,ShiodaS.Morphologicalanalysisofghrelinanditsreceptordistributioninthe ratpancreas.RegulPept.2005;126:67‐71. 42SmithRG,LeonardR,BaileyAR,PalyhaO,FeighnerS,TanC,MckeeKK,PongSS,GriffinP,HowardA. Growthhormonesecretagoguereceptorfamilymembersandligands.Endocrine.2001;14:9‐14. 43McKeeKK,PalyhaOC,FeighnerSD,HreniukDL,TanCP,PhillipsMS,SmithRG,VanderPloegLH, HowardAD.Molecularanalysisofratpituitaryandhypothalamicgrowthhormonesecretagogue receptors.MolEndocrinol.1997;11:415‐23. 44HowardAD,FeighnerSD,CullyDF,ArenaJP,LiberatorPA,RosenblumCI,HamelinM,HreniukDL, PalyhaOC,AndersonJ,ParessPS,DiazC,ChouM,LiuKK,McKeeKK,PongSS,ChaungLY,ElbrechtA, DashkeviczM,HeavensR,RigbyM,SirinathsinghjiDJ,DeanDC,MelilloDG,PatchettAA,NargundR, GriffinPR,DeMartinoJA,GuptaSK,SchaefferJM,SmithRG,VanderPloegLH.Areceptorinpituitary andhypothalamusthatfunctionsingrowthhormonerelease.Science.1996;273:974‐7.
Introduction 37 GhrelinbindingtoGHSR‐1apromotesimportantchangesinthetransmmebranealpha‐helix, affectingtoitsconformationandfacilitatingtheG‐proteinbindingsiteanditsinteraction.10 WithregardtoGHSR‐1aexpressionlevel,bothGHandleptinhormonesinhibititsexpression meanwhileghrelinandgrowthhormonereleasinghormone(GHRH)increaseit.45,46Inthis way,ghrelinandleptinhavecomplementaryroleswithregardtofoodintakecontrolandbody energetichomeostasis.47 Figure5.Ghrelinreceptorsstructure.Afteranalternativesplicingmechanism,twotypesofcDNA encodingghrelinreceptoraregenerated,namedtype1aand1b.(Figureextractedfrom Neuropharmacology.2010;58:2‐16). 45BennettPA,ThomasGB,HowardAD,FeighnerSD,vanderPloegLH,SmithRG,RobinsonIC. Hypothalamicgrowthhormonesecretagogue‐receptor(GHS‐R)expressionisregulatedbygrowth hormoneintherat.Endocrinology.1997;138:4552‐7. 46NassR,GilrainJ,AndersonS,GaylinnB,DalkinA,DayR,PeruggiaM,ThornerMO.Highplasmagrowth hormone(GH)levelsinhibitexpressionofGHsecretagoguereceptormessengerribonucleicacidlevelsin theratpituitary.Endocrinology.2000;141:2084‐9. 47NogueirasR,TovarS,MitchellSE,RaynerDV,ArcherZA,DieguezC,WilliamsLM.Regulationofgrowth hormonesecretagoguereceptorgeneexpressioninthearcuatenucleioftheratbyleptinandghrelin. Diabetes.2004;53:2552‐8.
Introduction 38 GHSR‐1alevelsarealsodown‐regulatedbyGHSinthepituitary,butinthearcuatusnucleus, ghrelinisabletoup‐regulatethereceptorexpressionlevels.48,49 ItisknownthatGHSR‐1aisinternalizatedafterligandbindingpromotingitsdesensitization. TheseexperimentswereshowninHEK293celllinestablytransfectedwiththeghrelin receptorthroughradioligandbindingassaysandconfocalmicroscopy.50Theresultsindicate thatGHSR‐1aismainlylocalizedattheplasmamembraneunderunstimulatedconditionsand rapidlydesensitizesafterstimulation.Theghrelin/GHSR‐1acomplexprogressivelydisappears fromtheplasmamembraneafter20minutesofexposuretoghrelinandaccumulatesinthe perinuclearregionafter60minutesviaclathrin‐coatedpits.InadditionGHSR‐1ashowslow recyclingandinthisway,itsinternalizationmayexplainthecharacteristicphysiological responsesmediatedbythisreceptor.50 TheghrelinreceptorbelongstoafamilythatincludestheGPR39aswellasreceptorsforthe peptidesmotilinandneurotensin.Forthesereceptorsahighagonist‐independentactivitywas displayedresultingfromspontaneousadoptionofanactivatedseventransmembranereceptor conformation(7TM).Forexampleghrelinreceptorsignalswith50%,dependingonthesignal transductionpathway,ofitsmaximalsignalingcapacitywithoutthepresenceofanyhormone. 51Thisphenomenonwasshownnotonlyinheterologousexpressionssystemsbutalsofor endogenousGHSR‐1a,detectedbyspecificantibodiesinbothcentralandgastrointestinal neurons,endothelialcellsandastrocytomacells.Constitutiveactivityhasbeendemonstrated formany7TMRinvitro,butwhetherornotconstitutivesignalinghasphysiologicalrelevancein vivoremainsunclear.52,53However,thisfactwasrecentlyclarifiedwiththeidentificationof naturallyoccurringhumanmutationswhichselectivelyeliminateitsconstitutiveactivity withoutaffectingtheaffinity,potency,orefficacyoftheghrelinhormone.Importantly,this 48BrescianiE,NassR,TorselloA,GaylinnB,AvalloneR,LocatelliV,ThornerMO,MüllerEE.Hexarelin modulatestheexpressionofgrowthhormonesecretagoguereceptortype1amRNAatHypothalamic andPituitarySites.Neuroendocrinology.2004;80:52‐9. 49KinemanRD,KamegaiJ,FrohmanLA.Growthhormone(GH)‐releasinghormone(GHRH)andtheGH secretagogue(GHS),L692,585,differentiallymodulateratpituitaryGHSreceptorandGHRHreceptor messengerribonucleicacidlevels.Endocrinology.1999;140:3581‐6. 50CamiñaJP,CarreiraMC,ElMessariS,Llorens‐CortesC,SmithRG,CasanuevaFF.Desensitizationand endocytosismechanismsofghrelin‐activatedgrowthhormonesecretagoguereceptor1a. Endocrinology.2004;145:930‐40. 51HollidayND,HolstB,RodionovaEA,SchwartzTW,CoxHM.Importanceofconstitutiveactivityand arrestin‐independentmechanismsforintracellulartraffickingoftheghrelinreceptor.MolEndocrinol. 2007;21:3100‐12. 52HolstB,MokrosinskiJ,LangM,BrandtE,NygaardR,FrimurerTM,Beck‐SickingerAG,SchwartzTW. Identificationofanefficacyswitchregionintheghrelinreceptorresponsibleforinterchangebetween agonismandinverseagonism.JBiolChem.2007;282:15799‐811. 53HolstB,CygankiewiczA,JensenTH,AnkersenM,SchwartzTW.Highconstitutivesignalingofthe ghrelinreceptor‐‐identificationofapotentinverseagonist.MolEndocrinol.2003;17:2201‐10.
Introduction 39 mutation,whichselectivelyeliminatedtheconstitutivesignaling,segregatedwiththe developmentofshortstatureandthedevelopmentofobesity.54Thehighconstitutive signalingactivityoftheGHSR‐1aindicatesthatacompoundactingasanefficientinverse agonistcouldbeaninterestingantiobesityagentandmanyeffortsarefollowedinthisway.55 Becausemany7TMRundergointernalizationandintracellulartraffickingafteragonist exposure,ofteninitiatedbyreceptorphosphorylationandsubsequentrecruitmentofβ‐ arrestinproteins,isimportanttounderstandhowconstitutiveactivityaffectsGHSR‐1a signaling,andhowtheeffectsofagonistsandinverseagonistsareinfluencedbyprocessesthat regulatereceptoractivity.Inthissense,itisknownthatGHSR‐1aundergoesbothconstitutive andagonist‐inducedreceptorinternalizationandthemoleculardeterminantsofthetrafficking profilesresidewithinthereceptorC‐terminaldomainsandthatconstitutiveactivitydrives GHSR‐1aendocytosis,inpartbyβ‐arrestinindependentmechanisms.51 AnotherinterestingpointwithregardtoGHSR‐1aistheexistenceofalternativeligandsforthis receptor.TheGHSR‐1atransducesinformationprovidednotonlybyghrelinbutalsobythe groupofGHSnotrelatedstructurallytoghrelin.44,56Thisfactisexplainedbytheexistenceofa commonbindingdomaindemonstratedbymolecularmodelingandsite‐directedmutagenesis studiesdevelopedwithGHSpeptideandnonpeptideagonists.57Thisbindingsitemight determinethataconservedstructureofagonistsrecognizesacomplementaryconserved bindingpocketwhichdirectsthevariablepartoftheligandandinteractswithspecificagonist‐ associatedregionsdeterminingandoverlappingintheagonist‐bindingsite.58 AfewyearsagoaroleforadenosineasendogenousligandforGHSR‐1awasproposed, showinganintracellularcalciumresponseinHEK293andbabyhamsterkidney(BHK)cells whichexpressedtheGHSR‐1abutwithoutanyeffectwithregardtoGHreleasefrompituitary 54PantelJ,LegendreM,CabrolS,HilalL,HajajiY,MorissetS,NivotS,Vie‐LutonMP,GrouselleD,de KerdanetM,KadiriA,EpelbaumJ,LeBoucY,AmselemS.Lossofconstitutiveactivityofthegrowth hormonesecretagoguereceptorinfamilialshortstature.JClinInvest.2006;116:760‐8. 55HolstB,SchwartzTW.Ghrelinreceptormutations‐‐toolittleheightandtoomuchhunger.JClinInvest. 2006;116:637‐41. 56CassoniP,PapottiM,GhèC,CatapanoF,SapinoA,GrazianiA,DeghenghiR,ReissmannT,GhigoE, MuccioliG.Identification,characterization,andbiologicalactivityofspecificreceptorsfornatural (ghrelin)andsyntheticgrowthhormonesecretagoguesandanalogsinhumanbreastcarcinomasandcell lines.JClinEndocrinolMetab.2001.86:1738‐45. 57BennettKA,LangmeadCJ,WiseA,MilliganG.Growthhormonesecretagoguesandgrowthhormone releasingpeptidesactasorthostericsuper‐agonistsbutnotallostericregulatorsforactivationoftheG proteinGαo1bytheghrelinreceptor.MolPharmacol.2009;76:802‐11. 58BondensgaardK,AnkersenM,ThøgersenH,HansenBS,WulffBS,BywaterRP.Recognitionof privilegedstructuresbyG‐proteincoupledreceptors.JMedChem.2004;47:888‐99.
Introduction 40 cellsinvitro.59,60Howeverthisideawasfinallyrejectedalthougharoleforadenosinesignaling incontrollingthereleaseofghrelinfromthemousestomachhasbeenrecentlyproposed.61 AnotherproposedligandfortheGHSR‐1aiscortistatin.62,63Cortistatin(CST)isahormone originallydescribedintherat,mouse,andhumancerebralcortexthatdisplaysstructuraland functionalsimilaritiestosomatostatin(SST).Itbindstoallfivesomatostatinreceptors(SSTR) and,differentlyfromSST,alsobindstoMrgX2,whichhasrecentlybeenidentifiedasitsspecific receptor.64Amongitsfunctions,CST inhibitsGHrelease fromhumanfetalandadenoma pituitarycellsandprolactin(PRL)secretionfromculturedprolactinomas.65CSTalsoposses otherendocrineactionslikeinhibitionofGHandinsulinsecretioninphysiologicalconditions andinacromegalyandasimilarinhibitoryeffectonPRLandadrenocorticotropichormone (ACTH)secretionwasshowninacromegaly,prolactinomaorinCushing'sdisease.66Recently,a roleforCSTastherapeuticagenttoautoimmunediseaseshasbeenproposed.67 AnothercandidateasGHSR‐1aligandisgrowthhormonereleasinghormone(GHRH).GHRH,a 44‐aminoacidhypothalamicpeptide,isakeyregulatorofGHsecretion(fromthepituitary glandtogetherwithsomatostatin)inwhichghrelinisalsoinvolved.68,69GhrelinstimulatesGH releasebothdirectly,actingattheleveloftheanteriorpituitarygland,andbyenhancingGHRH release.70TheGH‐releasingactivityofghrelinislowerthanthatofGHRHhoweverghrelinand 59SmithRG,GriffinPR,XuY,SmithAG,LiuK,CalacayJ,FeighnerSD,PongC,LeongD,PomésA,ChengK, VanderPloegLH,HowardAD,SchaefferJ,LeonardRJ.Adenosine:Apartialagonistofthegrowth hormonesecretagoguereceptor.BiochemBiophysResCommun.2000;276:1306‐13. 60TullinS,HansenBS,AnkersenM,MøllerJ,VonCappelenKA,ThimL.Adenosineisanagonistofthe growthhormonesecretagoguereceptor.Endocrinology.2000;141:3397‐402. 61YangGK,YipL,FredholmBB,KiefferTJ,KwokYN.Involvementofadenosinesignalingincontrolling thereleaseofghrelinfromthemousestomach.JPharmacolExpTher.2011;336:77‐86. 62DeghenghiR,PapottiM,GhigoE,MuccioliG.Cortistatin,butnotsomatostatin,bindstogrowth hormonesecretagogue(GHS)receptorsofhumanpituitarygland.JEndocrinolInvest.2001;24:RC1‐3. 63DeghenghiR,BroglioF,PapottiM,MuccioliG,GhigoE.Targetingtheghrelinreceptor:orallyactive GHSandcortistatinanalogs.Endocrine.2003;22:13‐8. 64AlliaE,TarabraE,VolanteM,CerratoM,GhigoE,MuccioliG,PapottiM.Expressionofcortistatinand MrgX2,aspecificcortistatinreceptor,inhumanneuroendocrinetissuesandrelatedtumours.JPathol. 2005;207:336‐45. 65RubinfeldH,HadaniM,BarkaiG,TaylorJE,CullerMD,ShimonI.Cortistatininhibitsgrowthhormone releasefromhumanfetalandadenomapituitarycellsandprolactinsecretionfromcultured prolactinomas.JClinEndocrinolMetab.2006;91:2257‐63. 66BroglioF,GrottoliS,ArvatE,GhigoE.Endocrineactionsofcortistatin:invivostudies.MolCell Endocrinol.2008;286:123‐7. 67Gonzalez‐ReyE,Delgado‐MarotoV,SouzaMoreiraL,DelgadoM.Neuropeptidesastherapeutic approachtoautoimmunediseases.CurrPharmDes.2010;16:3158‐72. 68MüllerEE,LocatelliV,CocchiD.Neuroendocrinecontrolofgrowthhormonesecretion.PhysiolRev. 1999;79:511‐607. 69TannenbaumGS,LingN.Theinterrelationshipofgrowthhormone(GH)‐releasingfactorand somatostatiningenerationoftheultradianrhythmofGHsecretion.Endocrinology.1984;115:1952‐7. 70AndersonLL,JeftinijaS,ScanesCG.Growthhormonesecretion:molecularandcellularmechanisms andinvivoapproaches.ExpBiolMed.2004.229:291‐302.
Introduction 41 GHRHshowanadditiveorsynergisticeffecttostimulateGHsecretionwhenadministeredin vivo71.Moreover,ithasbeenreportedthatforthefullGH‐releasingeffectofghrelin,the vagusnerveisessentialandthatalsorequiresafunctionalhypothalamus‐pituitaryconnection. 72Thegrowthhormonereleasinghormonereceptor(GHRH‐R)isaseventransmembrane receptorof52KDathatbelongstothesecretin/glucagon/vasoactiveintestinalpeptide(VIP) subfamilyofGPCR.73ItstimulatesadenylylcyclasethroughaGsproteintoincreasethe productionofthecellularsecondmessengercAMPwhichwillstimulatePKAactivation.74In addition,inpituitarycellsfrompigs,ghrelinrequiresactivationoftheNOS/NOroute,andits subsequentGC/cGMPsignaltransductionpathway,asnecessarystepstoinduceGHsecretion. 75Recently,ithasbeenshownthatGHRHmaydirectlyactivatetheghrelinreceptor modulatingtheghrelin‐associatedintracellularsignalingpathways,whichopennew possibilitiesonthestudyofthemolecularcircuitsinvolvedintheregulationofGHreleaseand otherghrelin‐mediatedactions.76 2.1.3. Functionsoftheghrelin/GHSR‐1asystem. Withregardtothesignalingsystemsevokedbyghrelin/GHSR‐1asystem,theGHreleaseisthe moststudied.Inthisparticularcase,itiswellknownthatafterligandbindingthe phosphatidylinositol‐specificphospholipaseC(PI‐PLC)isactivated,43,44producinginositol(1,4, 5)‐triphosphate(IP3)anddiacylglycerol(DAG).TheIP3activatescalciumreleasefromIP3‐ sensitivestoresattheERwhereasDAGinducestheactivationofproteinkinaseC(PKC)inthe plasmamembrane.ThisPKCinhibitsthepotassiumchannelscausingadepolarizationonthe plasmamembranepromotinganopeningofthevoltage‐dependentL‐and‐ T‐typecalcium channels.77LatelytheincreaseoftheintracellularcalciumpromotesthereleaseofGHtothe 71HatayaY,AkamizuT,TakayaK,KanamotoN,AriyasuH,SaijoM,MoriyamaK,ShimatsuA,KojimaM, KangawaK,NakaoK.Alowdoseofghrelinstimulatesgrowthhormone(GH)releasesynergisticallywith GH‐releasinghormoneinhumans.JClinEndocrinolMetab.2001.86:4552‐55. 72Al‐MassadiO,TrujilloML,SeñarisR,PardoM,CastelaoC,CasanuevaFF,SeoaneLM.Thevagusnerve asaregulatorofgrowthhormonesecretion.RegulPept.2011;166:3‐8. 73MayoKE,MillerTL,DeAlmeidaV,ZhengJ,GodfreyPA.Thegrowth‐hormone‐releasinghormone receptor:signaltransduction,geneexpression,andphysiologicalfunctioningrowthregulation.AnnNY AcadSci.1996;805:184‐203. 74 LabrieF,GagnéB,LefèvreG.Growthhormone‐releasingfactorstimulatesadenylatecyclaseactivityin theanteriorpituitarygland.LifeSci.1983;33:2229‐33. 75Rodríguez‐PachecoF,LuqueRM,Tena‐SempereM,MalagónMM,CastañoJP.Ghrelininducesgrowth hormonesecretionviaanitricoxide/cGMPsignallingpathway.JNeuroendocrinol.2008;20:406‐12. 76CasanuevaFF,CamiñaJP,CarreiraMC,PazosY,VargaJL,SchallyAV.Growthhormone‐releasing hormoneasanagonistoftheghrelinreceptorGHS‐R1a.ProcNatlAcadSciUSA.2008;105:20452‐57. 77ChenC,ZhangJ,VincentJD,IsraelJM.Sodiumandcalciumcurrentsinactionpotentialsofrat somatotrophs:theirpossiblefunctionsingrowthhormonesecretion.LifeSci.1990;46:983‐9.
Introduction 48 arrestins,inadditiontodesensitizeGPCR,actasaintermediaryendocyticadaptorproteins thattargetGPCRtocoatedpitsviatheirassociationwithbothadaptorprotein2(AP‐2)and clathrin.97Theinteractionbetweenβ‐arrestin,clathrinandAP‐2andalsowith phosphoinositides,definestheplaceswhereclathrincoatedpitswillbemakeup.Inthelast step,theactionofdynaminisrequired.102 Theendocyticfunctionofβ‐arrestin1isregulatedbyphosphorylation,being dephosphorylatedwhenistranslocatedtotheplasmamembrane.Ontheotherhand,β‐ arrestin2isregulatedbypost‐transductionalsmodificationsafteritsbindingtothereceptor. 103Onceinternalizated,GPCRcanbedegradatedinlysosomesordephosphorylatedand recycledbacktotheplasmamembraneasfullyfunctionalreceptors.104Ithastobementioned thatthereexistatightrelationshipbetweenthisfactandβ‐arrestinsubiquitination.100β‐ arrestinsmodulatepost‐endocyticGPCRtraffickingpatternsandappeartobespecificinGPCR recycling;therefore,β‐arrestinsareinvolved,notonlyinterminatingreceptorG‐protein couplingbutalsoininitiatingprocessesthatregulatere‐establishmentofreceptor responsiveness. 2.2.2.3. Down‐regulation Down‐regulationisaprocesscharacterizedbyadecreaseofthetotalnumberofbindingsites evokedbyprolongedexposuretoagonist.ThelatelypurposeofdownregulationistheGPCR resensitization,forwhichdephosphorylationandliganddissociationarerequired.Thereare evidencesthatinternalizationmayalsoberequired.105Inaphysiologicalapproach,itisnot probablethatcellsarecontinuouslyexposedtohormonesduetheexistenceofmechanisms thatavoidit.However,thissituationcanoccurinpathophysiologicalstates,ascontinuous hormonereleaseintumoralprocess.106Thedown‐regulationmechanisminvolvestwo 102Barki‐HarringtonL,RockmanHA.β‐arrestins:multifunctionalcellularmediators.Physiology.2008;23: 17‐22. 103ShenoySK,LefkowitzRJ.Traffickingpatternsofβ‐arrestinandGprotein‐coupledreceptors determinedbythekineticsofβ‐arrestindeubiquitination.JBiolChem.2003;278:14498‐506. 104AnborghPH,SeachristJL,DaleLB,FergusonSS.Receptor/β‐arrestincomplexformationandthe differentialtraffickingandresensitizationofβ2‐adrenergicandangiotensinIItype1Areceptors.Mol Endocrinol.2000;14:2040‐53. 105LuttrellLM.TransmembranesignalingbyGprotein‐coupledreceptors.MethodsMolBiol.2006;332: 3‐49. 106CollinsS,BouvierM,LohseMJ,BenovicJL,CaronMG,LefkowitzRJ.Mechanismsinvolvedin adrenergicreceptordesensitization.BiochemSocTrans.1990;18:541‐4.
Introduction 49 differentmechanisms:pre‐existingreceptorsdegradationanddecreasingandblockingof receptorsfromdenovosynthesis.107 2.2.2.4. Signaling Inrecentyears,apreviouslyunappreciatedmechanismhasbeenelucidatedforβ‐arrestins.In aβ‐arrestin‐dependentmechanism,receptorinternalizationinitiatesnewsequencesofevents fromreceptordesensitizationandinternalizationtoscaffoldingofkinasestocontrolling intracellulartraffickingofGPCRfollowingendocytosisandsignaling.Thatis,β‐arrestinswould actastransducerssignals(Figure8).102,108 Figure8.NewparadigmsignalinginGPCR.Thebindingofagonisttoitscognatereceptorinitiatesnot onlyclassicalG‐protein‐mediatedsignallingviasecondmessengers,butalsoGRKandβ‐arrestin dependentsignals.Newpathwayswithβ‐arrestinactivatingproteinssuchasERKandSrcaffectcell responsesincludinganti‐apoptoticsignalling,cardiaccontractility,anddopaminergicbehavioursamong others.TheserolesareinadditiontothehistoricallydefinedrolesfortheGRKandβ‐arrestinsin7TM receptordesensitizationandinternalization.(FigureextractedfromActaPhysiol.2007;190:9‐19). 107BöhmSK,GradyEF,BunnettNW.RegulatorymechanismsthatmodulatesignallingbyG‐protein‐ coupledreceptors.BiochemJ.1997;322:1‐18. 108GetherU.UncoveringmolecularmechanismsinvolvedinactivationofGprotein‐coupledreceptors. EndocrRev.2000;21:90‐113.
Introduction 50 Itiswellknownthatβ‐arrestinscanformcomplexeswhitdifferentsignalingproteinsincluding cSrcfamilytyrosinkinasesandcomponentsofMAPKpathway.97,109Inthisway,β‐arrestinsact asadaptorproteinsthatprovideGPCR‐mediatedMAPKactivation,increaseefficiencysignaling betweenthedifferentkinasesofthepathwayandtheyalsodirectthemtospecificlocations insidethecells.Thisfactsupposesimportantfunctionalroles. Awelldocumentedexampleisthereceptortyrosinkinase–mediatedERK1/2activation.110In thisexample,afterligandbinding,thereceptortyrosinkinase(RTK)reclutesSosproteinto plasmamembrane,whereitactivatesRasandthisoneactivatesRaf‐1,beingtranslocatedto plasmamembrane.Lately,MAPKareactivatedandERK1/2istranslocatedtothenucleus, wheremitogenesiswillbestimulated,orwillberetainintothecytosol,whereitwill phosphorylatedifferentcytosolictargets.111Thereceptortyrosinkinaseactivationalso initiatestheclathrincoatedpitsformation,allowingtherecyclingofthereceptororits degradation.TherearemultipleexampleswheremanyGPCRactivateMAPKsignaling pathwaysandwhereendocytosisisamechanismrequired.112,113Nevertheless,therearealso otherreportswhereMAPKactivationcanoccurinanindependentwaytotheendocytosis mechanism.112Theexplanationtothisquestionisexposedbytwohypothesescloselyrelated. Thefirstonesuggeststhatendocytosiswouldaddanadditionalspecificinformationlayerto signalingpathwaysthroughitscompartmentalization.Thesecondoneproposesthat endocytosiswouldallowtransportingthesignalingcomplexestospecificlocationsinsidethe cells. TheG‐protein‐mediatedsignalingischaracterizedbyarapidandtransientmechanismwhich involvesnucleartranslocationofERK1/2.Ontheotherhand,β‐arrestin‐mediatedsignalingisa slow,persistentonewhichinvolvesthecytosolicretentionofERK1/2.Thissupposesdifferent physiologicalconsequences.102Inaddition,itiswellestablishedthatactivationofaGPCRcan promoteG‐protein‐independent/β‐arrestin‐mediatedsignaling,suggestingthatreceptorscan assumedistinctconformationalstatesthatinitiatemultiplesignalingpathways.Thisrepresents 109AhnS,ShenoySK,WeiH,LefkowitzRJ.Differentialkineticandspatialpatternsofβ‐arrestinandG protein‐mediatedERKactivationbytheangiotensinIIreceptor.JBiolChem.2004;279:35518‐25. 110FergusonSS.Receptortyrosinekinasetransactivation:fine‐tuningsynaptictransmission.Trends Neurosci.2003;26:119‐22. 111PeyssonnauxC,EychèneA.TheRaf/MEK/ERKpathway:newconceptsofactivation.BiolCell.2001; 93:53‐62. 112KimSJ,KimMY,LeeEJ,AhnYS,BaikJH.Distinctregulationofinternalizationandmitogen‐activated proteinkinaseactivationbytwoisoformsofthedopamineD2receptor.MolEndocrinol.2004;18:640‐ 52. 113QianH,PipoloL,ThomasWG.Associationofβ‐Arrestin1withthetype1AangiotensinIIreceptor involvesphosphorylationofthereceptorcarboxylterminusandcorrelateswithreceptorinternalization. MolEndocrinol.2001;15:1706‐19.
Introduction 51 anopportunityfordevelopmentofnoveltherapeuticstargetingselectivebiologicaleffectsofa givenreceptor.Moreover,itnowappearsthat,forsomereceptors,multipleligandscan differentiallydirectsignalingdownβ‐arrestininsteadofG‐proteinsignalingpathways.4This conceptiscalledthebiasedagonism.Biasedagonismisapropertyoftheligand‐receptor complexandso,aligandorareceptormaybebiased.Abiasedligandfavorsoneresponse overanother(eitherG‐proteinorβ‐arrestin)comparedwiththeendogenousligand,whichis consideredtobeneutral.Abiasedreceptorisonlycapableofsignalingthrougharestricted subsetofpathwaysthataretypicallyavailabletothatclassofreceptor.102,114Forexample, somereceptorssuchasAT1R,severalchemokinereceptorsandμ‐opioidreceptorshave ligandsthatarebiased,thatis,compoundsthatstimulateonlyβ‐arrestin‐dependent pathways.115,116Suchligandsareofconsiderableinterestinphysiologicalandpathological settingsbecauseoftheirpotentialbenefittodrivereceptorsignalingindesireddirections. Althoughbiasedagonismwasrecentlyproposed,therearestillgapsintheunderstandingof biasandwithregardtothedifferentreceptorconformationsthatareresponsibleforsignaling toG‐proteinsandβ‐arrestins.SomehypothesispointtoaroleforGRKorcofactorscontrolling G‐proteinversusβ‐arrestinsignalingbutmorestudiesarerequired.117Theseoneswillserveto abetterunderstandingofthedifferentcontributionsofspecificpathwaystotheregulationof physiologicalfunctionsandalsoastargetedagentsforclinicaltherapies.4Inasimilarwayitis unclearwhethersuchselectiveG‐proteinuncouplingiscausedbyalackofabilitytointeract withG‐proteinsorratherbyanincreasedabilityofthereceptortorecruitβ‐arrestins(Figure 9).SinceuncouplingofG‐proteinsbyincreasedabilitytorecruitβ‐arrestinscouldleadto differentcellularorinvivooutcomesthanlackofabilitytointeractwithG‐proteins,itis essentialtodistinguishbetweenthesetwomechanisms.Thesefindingshaveimportant implicationsfordrugdiscoveryand7TMRbiologyandillustratethenecessityofuncoveringthe exactmoleculardeterminantsforG‐proteincouplingandβ‐arrestinrecruitment,respectively. 118 114ViolinJD,LefkowitzRJ.β‐arrestin‐biasedligandsatseven‐transmembranereceptors.Trends PharmacolSci.2007;28:416‐22. 115WhistlerJL,vonZastrowM.Morphine‐activatedopioidreceptorseludedesensitizationbyβ‐arrestin. ProcNatlAcadSciUSA.1998;95:9914‐9. 116WeiH,AhnS,ShenoySK,KarnikSS,HunyadyL,LuttrellLM,LefkowitzRJ.Independentβ‐arrestin2 andGprotein‐mediatedpathwaysforangiotensinIIactivationofextracellularsignal‐regulatedkinases1 and2.ProcNatlAcadSciUSA.2003;100:10782‐7. 117WhalenEJ,FosterMW,MatsumotoA,OzawaK,ViolinJD,QueLG,NelsonCD,BenharM,KeysJR, RockmanHA,KochWJ,DaakaY,LefkowitzRJ,StamlerJS.Regulationofβ‐adrenergicreceptorsignaling byS‐nitrosylationofG‐protein‐coupledreceptorkinase2.Cell.2007;129:511‐22. 118BondeMM,HansenJT,SanniSJ,HaunsøS,GammeltoftS,LyngsøC,HansenJL.Biasedsignalingofthe angiotensinIItype1receptorcanbemediatedthroughdistinctmechanisms.PlosOne.2010;5:e14135.
Introduction 52 Figure9.Schematicpresentationofpossiblemechanismunderlyingdifferentialactivation. Hypothetically,areceptormutantselectivelyactivatingβ‐arrestininducedpathwayscouldeitherbe impairedinG‐proteincoupling(right)orshowverystronginteractionwithβ‐arrestins(left).(Figure extractedfromPlosOne,2010;5:e14135).
3. METHODS
Methods 55 3.1.Materials HumanghrelinwasobtainedfromGlobalPeptide(FortCollins,CO,USA).Pertussistoxin(PTX), concanavalinA(ConA)andPhorbol12‐myristate13‐acetate(PMA)werefromSigma(St.Louis, MO,USA).Wortmannin,BAPTA‐2AM,calphostinC,Gö6976,PP2,PP3,genisteinand cortistatin‐17werepurchasedfromCalbiochem(SanDiego,CA,USA).Fura‐2 acetoxymethylester(FURA‐2AM)wasobtainedfromMolecularProbes(Eugen,OR,USA). Anti‐phospho‐p44/42MAPK,anti‐p44/42MAPKrabbitpolyclonal,anti‐phospho‐PKCα/βrabbit polyclonal,anti‐phospho‐PKCμrabbitpolyclonal,anti‐phospho‐cSrc(Tyr416)rabbit polyclonal,anti‐phosphocSrc(Tyr527)rabbitpolyclonal,anti‐pAktHM(S473),anti‐pAktA‐ loop(T308),anti‐Aktrabbitpolyclonal,anti‐Rictorrabbitpolyclonal,anti‐mTORrabbit polyclonalandanti‐pPDK‐1(S241)rabbitpolyclonalantibodieswerefromCellSignaling Technology(Beverly,MA,USA).Anti‐β‐arrestin1goatpolyclonal,anti‐β‐arrestin2rabbit monoclonal,anti‐GHSR(H‐80)rabbitpolyclonal,anti‐Raf‐1rabbitpolyclonal,anti‐cSrcrabbit polyclonal,anti‐PI3K(p85)rabbitpolyclonal,anti‐phospho‐PKCεrabbitpolyclonal,anti‐ phospho‐PKCδrabbitpolyclonal,anti‐phospho‐PKCζrabbitpolyclonal,anti‐SHP‐1(H‐65) mousepolyclonal,anti‐pSHP‐1(Y536)rabbitpolyclonal,anti‐actin(H‐300)rabbitpolyclonal antibodiesandproteinA/GagarosewerefromSantaCruzBiotechnology(SantaCruz,CA, USA).Anti‐phospho‐tyrosinerabbitpolyclonalantibodywasfromUpstateTechnology(Lake Placed,NY,USA).β‐arrestin1siRNA,β‐arrestin2siRNA,cSrcsiRNAandsiRNAcontrolwere likewisefromSantaCruzBiotechnology(SantaCruz,CA,USA).SSTR2siRNAwasprovidedby Dharmacon(CO,USA).RictorsiRNAwassynthesizedbyCellSignaling(Beverly,MA,USA).Anti‐ rabbithorseradishperoxidasewasfromGE‐Amersham(Buckinghamshire,UK),whileanti‐goat horseradishperoxidasewasfromSantaCruz(SantaCruz,CA,USA).Rabbitanti‐ratβ‐arrestin 1C‐terminal(A1CT)antiserumwasprovidedbyProf.R.J.Lefkowitz(DukeUniversityMedical Center,Durham,NC,USA). ProteaseandphosphataseinhibitorcocktailswereobtainedfromSigma(St.Louis,Mo,USA). Pro‐QDiamondphosphoproteingelstain,SYPRO®RubyandLipofecamine2000werefrom Invitrogen(Carlsbad,CA,USA). [125I]‐ghrelinandQuickPrepTMMicromRNAPurificationKitwerefromGEHealthcare AmershamPharmacia(AilingtonHeghts,IL,USA).[125I]‐CST‐17wasfromPhoenix Pharmaceutical(Burlingame,CA,USA).
Methods 56 3.2.Cellculture HEK293cells,whichstablyexpressthehumanghrelinreceptor1a(HEK‐GHSR‐1a),were culturedin100‐mmdiameterdishesinhighglucoseDMEMcontaining10%(v/v)fetalcalf serumplus500μg/mlgeneticinG‐418to70–80%confluencefor3daysasdescribed previously.50NontransfectedHEK293wereseededin100‐mmdishesandculturedto80% confluencefor2daysinDMEMsupplementedwith10%(v/v)fetalcalfserum.Mediawere supplementedwithpenicillinG(100U/ml)andstreptomycinsulfate(100μg/ml).Cellswere grownunderahumidifiedatmosphereof95%air,and5%CO2at37°C. 3T3‐L1preadipocytecellswereobtainedfromAmericanTypeCultureCollectionand maintainedinDMEMcontaining10%calfserum,100U/mlpenicillin,and100U/ml streptomycin.Cellsweregrownunderahumidifiedatmosphereof95%air,and5%CO2at 37°C.Confluent3T3‐L1cellsweretreatedwith0.5mMisobutylmethylxantine(IBMX),25μM dexamethasone(DEX),and861nM(5μg/mL)insulinfor3daysandmaintainedinDMEM containing10%FBS,100U/mLpenicillin,100U/mLstreptomycinandsupplementedwith172 nM(1μg/mL)insulinfor10daysafterthebeginningofdifferentiationasdescribed previously.35 3.3.Immunoblottinganalysis Serum‐starvedcellswerestimulatedfortheindicatedtimeperiodat37°C.Themediawasthen aspiratedandthecellswerelysedinice‐coldRIPAbuffer[Tris‐HCl(pH7.2),50mM;NaCl,150 mM;EDTA,1mM;NP‐40,1%(v/v);Na‐deoxycholate,0.25%(w/v);proteaseinhibitorcocktail; phosphataseinhibitorcocktail].Thesolubilizedlysatesweretransferredintocentrifugetubes andleftat4°Cfor15minutes,thenpre‐clearedbycentrifugingat13,000gfor15minutes. ProteinconcentrationwasevaluatedwiththeQuantiProTMBCAassaykit.Subsamples(same amountofprotein)ofeachsamplewereseparatedon10%SDS‐polyacrylamidegelsand transferredtonitrocellulosemembranes.Theblotswereincubatedwith5%non‐fatdrymilkin TBST[Tris‐HCl(pH8.0),20mM;NaCl,150mM;Tween‐20,0.1%(v/v);usedforallincubation andwashingsteps]for1hour.Next,blotswereincubatedfor1hourwiththecorresponding antibodiesaccordingtothemanufacturer’sinstructions.Blotsweresubsequentlyincubated withthecorrespondingperoxidase‐conjugatedIgGantibody.Afterwashing,signalswere visualizedusinganenhancedchemiluminescencedetectionsystem.
Methods 57 3.4.[125I]‐ghrelininternalizationassays ConfluentHEK‐GHSR‐1amonolayercells(70‐80%)wereresuspendedinbindingbuffer [containing,DMEM(pH7.4)plus1%(w/v)BSA],centrifugedat500gfor3minutesatroom temperature,washedtwiceandthenresuspended(1x100‐mmplate/1.5mL;5‐6x105cells peraliquot)inbindingbuffersupplementedwith100.000cpm/aliquotof[125I]‐ghrelinfor2 hoursat4°C.Themediacontaininglabeledghrelinwereremovedandcellswerewashedtwice withice‐coldbindingbuffer.Freshbindingbuffer(0.5mL)wasaddedandcellswereincubated at37°Cforperiodsfrom0to60minutes.Ateachtimepoint,cellswerepelletedandthe incubationmediumwastreatedwith10%(w/v)trichloroaceticacid(TCA)at4°Cfor1hour. TheTCA‐insolublematerialswerepelletedbycentrifugationat10,000gfor10minutesat4°C. Supernatantandinsolublefractionswerecountedtodeterminepostactivationreleasablelabel andTCA‐insolublematerial,respectively.Cell‐surface‐boundghrelinwasremovedby resuspendingthecellsinice‐coldacidbufferfor15‐20minutesat4°C.Thecellswerepelleted bycentrifugationandthesupernatants,correspondingtocell‐surface‐boundghrelin,were counted.Finally,thepelletobtainedwassolubilizedinlysisbuffer(NonidetP‐40,1%;TritonX‐ 100,0.5%andNaOH,1M)andradioactivity,correspondingtointernalizedghrelin,was measuredaspreviouslydescribed.50 3.5.AssaysinK+‐depletedmedium HEK‐GHSR‐1acellswereresuspendedinHEPES‐bufferedsaline[HEPES(pH7.4),100mM;NaCl, 120mM;MgSO4,1.2mM;EDTA,1mM;CH3COONa,15mM;CaCl2,1mM;glucose,10mM] complementedwith1%(w/v)BSA.Intracellularpotassiumdepletionwascarriedoutby incubatingthecellsfor5minutesat37°Cinhypotonicmedium[HEPES‐bufferedsaline:water, 1:1(v/v)],followedby60minutesofincubationinHEPES‐bufferedsalinesupplementedwith (control)orwithout10mMK+at37°C.For[125I]‐ghrelininternalizationexperiments,cellswere thenwashedandresuspendedinHEPES‐bufferedsalinesupplementedwith100,000 cpm/aliquotof[125I]‐ghrelinfor2hoursat4°C.Afterincubation,cellswerewashedandthen incubatedat37°Cforvariousperiodsoftime.Ateachtimepoint,cellswerepelletedandthe internalizedghrelinwasmeasuredaspreviouslydescribed.50Forimmunoblottinganalysis, cellswerepreincubatedinmediumwithorwithoutK+andthenthecellswerestimulatedwith ghrelinat37°Cforthetimeperiodsindicated,lysedandanalyzedbyimmunoblottinganalysis.
CHAPTER1
Chapter1 67 Stimulationbyghrelinofp42/p44mitogen‐activatedproteinkinasethroughthe GHSR‐1areceptor:roleofG‐proteinsandβ‐arrestins. SincethetemporalpatternofERK1/2activationisoneofthekeydeterminantsthatestablish itsbiologicalresponses,firstofallwewantedtoinvestigatethepatternofERK1/2activation inducedbyghrelinthroughthebindingtoitsreceptor,theGHSR‐1a.Forthispurpose,we examinatedthetime‐courseofghrelin‐inducedERK1/2activation(100nM)atdifferenttime pointsinHEK‐GHSR‐1acells.TheresultobtainedisshowninFigure1wherewecanobservea maximalERK1/2phosphorylationwithin5‐10minutesofghrelinstimulation,decreasingto approximately50%ofthemaximumby60minutesafterstimulation.Thisresultsuggestthe existenceofdifferentpathwaysbywhichghrelin,throughGHSR‐1a,activatesERK1/2,pointing toarolefortheG‐proteinsandtothereceptorinternalizationprocess. Toassesstheroleofreceptorinternalizationintheghrelin‐inducedERK1/2activationwefirst testedtheeffectofpotassium‐depletedmedium,aconditionthatsignificantlyaffectsthe formationofclathrin‐coatedpitsandinternalizationthroughthispathway.Figure2Ashows thisconditioninhibitedtheinternalizationprocessof[125I]‐ghrelin.Underthesameconditions theimpactofGHSR‐1ainternalizationonERK1/2activationwasalsoevaluated,showingan inhibitionofghrelin‐inducedERK1/2phosporylation(Figure2B),demonstratingthatthe internalizationprocesscontributesprimarilytothelatetimecomponentofERK1/2activation. Bysubtractingthecontrolcurve(withpotassium;blueline)fromtheinternalization‐ independentcurve(withoutpotassium;redline)anestimationoftheinternalization‐ dependentpathwaycanbeobtained(dottedcurve,greenline). Additionally,theimpactofinternalizationofGHSR‐1aonERK1/2activationwascheckedusing twoindependentexperimentaltreatmentspreviouslydemonstratedtoblockinternalizationby clathrincoatedpitpathways,pretreatmentwithhypertonicsucroseorConcanavalinA(ConA). Hypertonicsucrosepretreatmentwasanon‐viablestrategyinthiscellline,sinceitcausedERK 1/2activationwithoutghrelintreatment(datanotshown).Thesecondapproach, pretreatmentwithConA(250mg/ml,30min),whichblocksreceptorclustering,inhibitedthe ghrelin‐inducedERK1/2phosphorylation(45%inhibition;datanotshown).Theseresults suggesttheexistenceofatleasttwopathwaysbywhichGHSR‐1amightactivateERK1/2,one mediatedbyG‐proteinsandtheotheronerelatedtoGHSR‐1aendocytosis.
Chapter1 68 Figure1.Time‐courseoftheeffectofghrelinonphosphorylationofERK1/2.Serum‐starvedHEK‐ GHSR‐1acellswerestimulatedwithghrelin(100nM)forthetimeperiodsindicated.Cellswerelysedand analyzedbyimmunoblottingusingspecificantibodiesagainstphospho‐ERK1/2andERK1/2.ERK1/2 phosphorylationwasquantifiedbydensitometryandexpressedasapercentageofthemaximal phosphorylation(mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments.
Chapter1 69 Figure2.Effectofpotassiumdepletionontheinternalizationof[125I]‐ghrelinandonthe phosphorylationofERK1/2.A.Cellswerepreincubatedwith[125I]‐ghrelinfor2hat4°Candthen incubatedat37°CinmediumwithorwithoutK+forthetimeperiodsindicated.Ateachtimepoint,cells werewashedandsurface[125I]‐ghrelinwasremovedbyacidstripping,andtheremainingcellswerethen solubilizedinNaOH([125I]‐ghrelininternalized).B.Cellswerepreincubatedinmediumwithorwithout K+,thenstimulatedwithghrelin(100nM)forthetimeperiodsindicatedandanalyzedby immunoblottingusingspecificantibodiesagainstphospho‐ERK1/2andERK1/2.ERK1/2 phosphorylationwasquantifiedbydensitometryandexpressedasapercentageofthemaximal phosphorylationofERK1/2(mean±SE).Immunoblotsarerepresentativeofthreeindependent experiments. A B
Chapter1 70 AcandidateforamoleculeconnectingtheGHSR‐1ainternalizationtotheactivationofERK1/2 mightbeβ‐arrestins.Tothisend,weusedRNAinterferencetoreducetheexpressionof endogenousβ‐arrestinsinHEK‐GHSR‐1acells.IntheseexperimentssiRNAtargetingβ‐arrestin 1orβ‐arrestin2reducedtheirexpressionby48%and54%,respectively(Figure3A).Inthe presenceofanon‐targetingcontrolsiRNA,ERK1/2phosphorylationmeasuredat10minutes afterghrelintreatmentwasidenticaltothatobservedwithoutanytransfection(datanot shown).β‐arrestin1andβ‐arrestin2siRNAreducedERK1/2phosphorylationat10minutesby 65%and55%,respectively(Figure3B),suggestingthatpartoftheERK1/2activationrequires thesimultaneouspresenceofbothisoforms.Takingtogether,theresultsconfirmthatthe ghrelin‐inducedactivationofERK1/2involvesGHSR‐1ainternalization,andthattheobserved reductionintheghrelin‐inducedactivationfollowingβ‐arrestindepletionreflectsthe requirementofβ‐arrestinsforthisinternalization. Next,weevaluatedtheroleofG‐protein‐dependentpathways.Forthispurpose,wefirst investigatedthepossibleroleofGi/o‐proteinsbymeansofpretreatmentwithpertussistoxin (PTX,100ng/mL,12h).AsFigure4Ashows,PTXreducedtheghrelin‐inducedactivationofERK 1/2by50%approximatelyat5‐10minutesofstimulationwithghrelin,correspondingtothe earlytimecomponent.Wealsoobservedthatatlatertimepoints,theeffectofPTXonERK1/2 activationwasmoresensitive.Subtractionofthetime‐courseobtainedfromthestimulation withghrelininthepresenceofPTX(greenline)fromthecontrolcurve(blueline)predictsthe time‐coursefortheGi/o‐dependentactivationofERK1/2(dottedcurve;redline). Wealsotestedtheeffectsonghrelin‐inducedERK1/2activationoftheCa+2chelatorBAPTA‐ AM.Figure4BshowsthatthepretreatmentwithBAPTA‐AM(30μM,30min)reducedtheERK 1/2activationinducedbyghrelin(100nM,5min)by65%.TheeffectofthePI3Kinhibitor wortmanninwasalsoassayed.Figure4Bshowsthatpretreatmentwithwortmannin(1μM,30 min)decreasedtheghrelin‐inducedERK1/2activationby78%.
Chapter1 71 Figure3.EffectofsiRNAdepletionofβ‐arrestin1orβ‐arrestin2onghrelin‐inducedERK1/2 phosphorylation.HEK‐GHSR‐1acellstransfectedwithβ‐arrestin1orβ‐arrestin2siRNAwereserum‐ starvedandthenstimulatedwithghrelin(100nM,5min).Afterstimulation,cellextractswereprepared asdescribedinmethods.Equalamountsofproteinineachsamplewereusedtoassesstheexpressionof β‐arrestin1orβ‐arrestin2(A)orERK1/2phosphorylation(B)byimmunoblottinganalysis.Expressionof β‐arrestin1orβ‐arrestin2wasquantifiedbydensitometry.Valuesshownarepercentagesofthelevelof β‐arrestinsincontrolsiRNA‐transfectedcells.ERK1/2phosphorylationwasquantifiedbydensitometry andexpressedasapercentageofthebasalphosphorylationofERK1/2obtained5minafterghrelin additiontocontrolsiRNAtransfectedcells(mean±SE).Immunoblotsarerepresentativeofthree independentexperiments.*P<0.05. A B
Chapter1 72 Figure4.A.Ghrelin‐inducedERK1/2phosphorylationintheabsenceorpresenceofPTX(100ng/ml,12 h).Serum‐starvedcellswerestimulatedwithghrelin(100nM)forthetimeperiodsindicated.ERK1/2 phosphorylationwasquantifiedbydensitometryandexpressedasapercentageofthemaximal phosphorylationofERK1/2(mean±SE).Dottedcurvepredictsthetime‐coursefortheGi/o‐dependent activationofERK1/2.B.EffectsofBAPTAandwortmanninonERK1/2phosphorylation.Serum‐starved cellswerepretreatedwithBAPTA(30μM,30min)orwortmannin(1μM,30min)beforeghrelin stimulation(100nM,5min).ERK1/2phosphorylationwasquantifiedbydensitometryandexpressedas apercentageofthebasalphosphorylationofERK1/2obtainedincontrolcells(mean±SE).Immunoblots arerepresentativeofthreeindependentexperiments.*P<0.05. A B
Chapter1 73 NextweexploredtheroleofPKC,usingthePKCinhibitorsGö6976andcalphostinC.Ghrelin‐ inducedERK1/2activationwasreduced41%bypretreatmentwithGö6976(100nM,30min), aselectiveinhibitorofCa+2dependentPKCαandPKCβIisoenzymes,and67%bypretreatment withcalphostinC(10μM,30min),anon‐selectivePKCinhibitor(Figure5A).Todeterminethe temporalcontributionofPKCα/βonERK1/2activation,cellsweretreatedwithGö6976prior tostimulationwithghrelin.AsshowninFigure5B,Gö6976partiallyreducedtheearlytime componentofghrelin‐inducedERK1/2activation(30%,5–10min).Unexpectedly,thegreatest effectonthetime‐coursewasobservedonthesustainedERK1/2activationat20–60minutes afterghrelinstimulation.Subtractionofthetime‐courseobtainedfromtheGö6976treatment curve,tothecontrolcurve,predictsthetime‐coursefortheGq/11/PKCα/β‐dependent activationofERK1/2(Figure5B,dottedcurve).Takentogetherwiththepotassiumdepletion mediumandPTXdataabove,theseresultssuggestthatβ‐arrestin‐mediatedcomponentof ERK1/2activationisdependentof‘‘interrelated’’componentsactivatedbyGi/0‐andGq/11‐ proteins. Importantrolesofnon‐receptortyrosinekinases(RTK),forexamplemembersofthecSrc family,havebeenreportedduringGPCR‐mediatedERK1/2activationinvariouscelltypes. Ghrelin‐inducedERK1/2activationwasstronglyinhibitedbygenistein(2μM,30min)andPP2 (5μM,30min),aselectivecSrcinhibitor(Figure6A).Thisinhibitionwasspecific,since pretreatmentwithPP3(5μM,30min),anegativecontrolforPP2,hadnoeffectontheghrelin‐ inducedERK1/2phosphorylation(Figure6A),supportingaroleforcSrcproteininghrelin signaling.ToinvestigatetheactivationofcSrc,wecomparedthephosphorylationofbothcSrc regulatorytyrosines,namelyTyr527andTyr416.PhosphorylationofTyr416displayedan earlyincreaseat5minutesofghrelinstimulationandremainedgreaterthanovertwofold basalforatleast60minutesafterstimulation.Conversely,thephosphorylationofinhibitory Tyr527showedadecreaseconcurrentwiththedynamicofphosphorylationofTyr416(Figure 6B).Furthermore,PP2attenuatedthestimulatoryeffectofghrelinonphosphorylationofcSrc atTyr416(datanotshown).
Chapter1 80 Figure10.MechanismofPKCεactivationbyghrelin.Serum‐starvedcellswerepretreatedwith(A)PTX (100ng/ml,12h)or(B)PI3Kinhibitorwortmannin(1μM,30min)andthenstimulatedwithghrelin(100 nM,10min).PKCεphosphorylationwasquantifiedbydensitometryandexpressedaspercentagesof thebasalphosphorylationobtainedincontrolcells(mean±SE).Inmunblotsarerepresentativeofthree independentexperiments.*P<0.05. A B
Chapter1 81 Afterdeterminethatβ‐arrestinsandcSrcplayaroleintheGHSR‐1a‐mediatedERK1/2 activation,wenextinvestigatedwhetherghrelininducesformationofacomplexincludingβ‐ arrestins,theinternalizatedreceptorandotherkinases.Toaddressthisquestion immunoprecipitationexperimentsweremade.Theresultsobtainedshowedthat immunoprecipitationofghrelin‐stimulatedcellswithantibodiestophospho‐ERK1/2co‐ precipitatescSrc,Raf‐1andGHSR‐1a.Similarly,immunoprecipitationwithantibodiestoβ‐ arrestin1orβ‐arrestin2co‐precipitatephospho‐ERK1/2andGHSR‐1a(Figure11). Withthepreviousdataobtained,wewantedtotestthehypothesisofcSrcbindingtoβ‐ arrestins,whichthenbindstoreceptor,toformthecomplexpreviouslydescribed.Tothis purpose,theghrelin‐inducedcSrcactivationwasevaluatedafterreducingcellularlevelsofβ‐ arrestin1orβ‐arrestin2bytransfectingwithsiRNA.Theresultsshowedthatinthepresence ofβ‐arrestin1siRNA(47%reductioninβ‐arrestin1expression);activationofcSrcbyghrelin (100nM)wasreducedby45%.Inthepresenceofβ‐arrestin2siRNA(57%reductioninβ‐ arrestin2expression)activationofcSrcbyghrelin(100nM)wasreducedby60%(Figure12).
Chapter1 82 Figure11.EffectofghrelinontheassemblyofcomplexescontainingGHSR‐1a,β‐arrestins1and2, cSrc,Raf1,andphospho‐ERK1/2.Serum‐starvedcellswerestimulatedwithghrelin(100nM)forthe timeperiodsindicated,thenlysedandimmunoprecipitated(IP)withantibodiestophospho‐ERK1/2 (upperpanel)orβ‐arrestin1orβ‐arrestin2(lowerpanel),thenanalyzedbyimmunoblottingwith antibodiestocSrc,Raf‐1,GHSR‐1aandphospho‐ERK1/2.Immunoblotsarerepresentativeofthree independentexperiments.
Chapter1 83 Figure12.ActivationofcSrcbyβ‐arrestins.Cellstransfectedwithβ‐arrestin1orβ‐arrestin2siRNA wereserum‐starvedandthenstimulatedwithghrelin(100nM,5min).Afterstimulation,cellextracts werepreparedandequalamountsofproteinineachsamplewereusedtovisualizeexpressionofβ‐ arrestins1and2(A1CTantibody)(A)andphospho‐cSrc(Tyr416)proteinband(B)byimmunoblotting analysis.Expressionofβ‐arrestinswasdeterminedbydensitometry.Valuesshownarepercentagesof thelevelofthecorrespondingβ‐arrestinsincontrolsiRNAtransfectedcells.Phospho‐cSrc(Tyr416) wasquantifiedbydensitometryandexpressedasthepercentageofthebasalphosphorylation obtainedincontrolsiRNA‐transfectedcells(mean±SE).Immunoblotsarerepresentativeofthree independentexperiments.*P<0.05. A B
CHAPTER2
Chapter2 87 cSrcregulatesAktsignalinginresponsetoghrelinviaβ‐arrestinsignaling‐ independentand‐dependentmechanisms. Firstofall,thetime‐courseofAktactivationwasdefinedafterthestimulationofHEK‐GHSR‐1a cellswithghrelin(100nM).AsFigure1shows,ghrelininducedmaximallevelsofAkt phosphorylationinboththeactivationloopwithinthekinasedomain[A‐loop(T308)]andthe hydrophobicmotifintheC‐terminalregion[HM(S473)]within20minuteskeepingthe maximumby60minutes. Figure1.Time‐courseoftheeffectofghrelinonAktHM(S473)andA‐loop(T308)phosphorylation. Serum‐starvedHEK‐GHSR‐1acellswerestimulatedwithghrelin(100nM)forthetimeperiodsindicated. CellswerelysedandanalyzedbyimmunoblottingusingspecificantibodiesagainstpAkt(S473)and (T308).Aktphosphorylationwasquantifiedbydensitometryandexpressedasapercentageofthe maximalphosphorylationobtainedforeachresidue(mean±SE).Immunoblotsarerepresentativeof threeindependentexperiments.
Chapter2 88 TheinvolvementofGi/o‐proteinswasevaluatedbymeansofpretreatmentwithPTX(100 ng/mL,12h).TheresultshowsthatPTXreducedtheghrelin‐inducedphosphorylationofthe AktA‐loop(T308).Surprisingly,theeffectofPTXontheotherresidue,(S473),wasincreased. Meanwhile,theeffectofPI3Kinhibitorwortmannin(1μM,30min)showedadecreaseof ghrelin‐inducedAktphosphorylationatbothresidues.Itwasalsocheckedtheinvolvementof theβγ‐subunitofG‐proteinsthroughtheuseoftheconstructβ‐ARK‐CT,whichalsodecreased theeffectofghrelinonAktphosphorylationatbothresidues(Figure2). AktphosphorylationwasstronglyinhibitedatbothresiduesbytheselectivecSrcinhibitorPP2 (5μM,30min)foralltimetested.Thisinhibitionwasspecific,sincepretreatmentwithPP3(5 μM,30min),anegativecontrolforPP2,hadnoeffectonghrelin‐inducedAktphosphorylation (datanotshown).ThesiRNAexperimentstargetingcSrcreduceditsexpressionby57±2%.cSrc siRNAdecreasedghrelin‐activatedAktphosphorylationwithrespecttosiRNAcontrolforall timetestedandforbothresidues[54±3%atHM(S473);and45±6%atA‐loop(T308)](Figure3).
Chapter2 89 Figure2.RoleofGi/o‐proteins,GβγdimmersandPI3KinAktphosphorylationinresponsetoghrelin. Ghrelin‐inducedAktphosphorylationintheabsenceorpresenceofPTX(100ng/mL,12h),PI3Kinhibitor wortmannin(1μM,30min)andβγsequesterβ‐ARK‐CT.Serum‐starvedHEK‐GHSR‐1acellswere stimulatedwithghrelin(100nM)forthetimeperiodsindicated.Cellswerelysedandanalyzedby immunoblottingusingspecificantibodies.Aktphosphorylationwasquantifiedbydensitometryand expressedasapercentageofthemaximalphosphorylationobtainedforeachresidue(mean±SE). Immunoblotsarerepresentativeofthreeindependentexperiments.
Chapter2 96 Thenextstepwasdilucidatetheroleofβ‐arrestinsinAktphosphorylation.ThesiRNA experimentstargetingβ‐arrestin1orβ‐arrestin2(45±2%and55±4%reductionrespectively) showedthatβ‐arrestin1andβ‐arrestin2siRNAledtorapidAktphosphorylation,which decreasedafter10minutesofghrelinstimulation(100nM)withrespecttosiRNAcontrol [41±2%and39±3%atHM(S473)forβ‐arrestin1andβ‐arrestin2siRNArespectively;and 47±5%and40±4%atA‐loop(T308)forβ‐arrestin1andβ‐arrestin2siRNArespectively].This inhibitoryeffectstayedonbothresiduesforatleast60minutes(Figure8). Immunoprecipitationofghrelin‐stimulatedcells(100nM,10min)withantibodiestoβ‐arrestin 1orβ‐arrestin2co‐precipitatedpAkt(T308,S473)whilefailedtoco‐precipitatedpPDK‐1 (S241),RictorandmTOR(Figure9).Theβ‐arrestin‐dependentAktphosphorylationwas dependentoncSrcasnoimmunoprecipitationofpAktwasobtainedwithantibodiestoβ‐ arrestin1orβ‐arrestin2inghrelin‐stimulatedcells(100nM,10min)afterpretreatmentwith PP2(5μM,30min).Thisinhibitionwasspecific,sincepretreatmentwithPP3(5μM,30min) hadnoeffectontheβ‐arrestin‐dependentAktphosphorylationinducedbyghrelin(datanot shown). Figure10showsthatin3T3‐L1cells,ghrelininducedAktphosphorylationatbothresiduesand atdifferenttimepoints,reachingmaximallevelswithin20–60minutesofghrelinstimulation (100nM).Thistime‐coursewassimilartothetime‐courseobservedinHEK‐GHSR‐1acells.Ina similarway,theeffectofPP2(5μM,30min)reducedAktphosphorylationatbothresidues, meanwhilePP3pretreatment(5μM,30min),anegativecontrolforPP2,showednoeffect (Figure10). Immunoprecipitationexperimentsperformedonghrelin‐stimulatedcells(100nM,10minutes) withantibodiestoβ‐arrestin1andβ‐arrestin2co‐precipitatedfull‐activatedAktshowingan increaseontyrosinephosphorylationcomparedtounstimulatedcells.Inaddition,the activatedformofcSrc,pcSrc(Tyr416)co‐immunoprecipitatedwithβ‐arrestin1andβ‐arrestin 2(Figure11).
Chapter2 97 Figure8.EffectofsiRNAdepletionofβ‐arrestin1orβ‐arrestin2onghrelin‐inducedAkt phosphorylation.HEK‐GHSR‐1acellstransfectedwithβ‐arrestin1orβ‐arrestin2siRNAwereserum starvedandthenstimulatedwithghrelin(100nM).Equalamountsofproteinineachsamplewereused toassesstheexpressionofβ‐arrestin1orβ‐arrestin2(upperpanel)andAktphosphorylation(lower panel)byimmunoblotting.Expressionofβ‐arrestin1orβ‐arrestin2wasquantifiedbydensitometry. Valuesshownarepercentagesofthelevelofβ‐arrestinsincontrolsiRNA‐transfectedcells(mean±SE). Aktphosphorylationwasquantifiedbydensitometryandexpressedasapercentageofthemaximal phosphorylationatHM(S473)andA‐loop(T308)afterghrelinadditiontocontrolsiRNA‐transfectedcells (mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments.(*,P<0.05).
Chapter2 98 Figure9.Effectofghrelinontheassemblyofcomplexescontainingβ‐arrestins1and2andpAkt. Serum‐starvedHEK‐GHSR‐1acellswerestimulatedwithghrelin(100nM,10min),lysedand immunoprecipitated(IP)withantibodiestoβ‐arrestin1(leftpanel)orβ‐arrestin2(rightpanel),and thenanalyzedbyimmunoblottingwithpAkt[HM(S473),A‐loop(T308),pPDK‐1(S241)],Rictor,mTOR, andβ‐arrestin(A1CT)antibodies.Immunoblotsarerepresentativeofthreeindependentexperiments.
Chapter2 99 Figure10.EffectofthecSrcinhibitorPP2andPP3,ontheghrelin‐inducedAktactivationin3T3‐L1 preadipocytecells.Serum‐starved3T3‐L1cellswerepretreatedwithPP2(5μM,30min)orPP3(5μM, 30min)beforeghrelinstimulation(100nM)fortheindicatedtimeperiodsat37°C.Then,cellswere lysedandanalyzedbyimmunoblottingwithpAktHM(S473)andA‐loop(T308).Aktphosphorylationwas quantifiedbydensitometryandexpressedasapercentageofthemaximalphosphorylationatHM (S473)andA‐loop(T308)(mean±SE).Immunoblotsarerepresentativeofthreeindependent experiments.
Chapter2 100 Figure11.Effectofghrelinontheassemblyofcomplexescontainingβ‐arrestins1and2andpAktin 3T3‐L1preadipocytecells.Serum‐starved3T3‐L1cellswerestimulatedwithghrelin(100nM,10min), lysedandimmunoprecipitated(IP)withantibodiestoβ‐arrestin1and2andthenanalyzedby immunoblottingwithpAktHM(S473),pAktA‐loop(T308),pYandpcSrc(Y416)antibodies.Immunoblots arerepresentativeofthreeindependentexperiments.
CHAPTER3
Chapter3 103 SHP‐1proteintyrosinephosphatasenegativelymodulatesAktsignalingin ghrelin/GHSR‐1asystem.Implicationsinwhiteadiposetissue. PhosphorylationoftheC‐terminalY536residueofSHP‐1[pSHP‐1(Y536)]afterghrelin stimulationinHEK‐GHSR‐1acellswasevaluated.AsFigure1shows,ghrelin(100nM)increased pSHP‐1(Y536),reachingmaximallevelswithin10minutes,keepingthisdegreebyatleast60 minutes. Figure1.Time‐courseoftheeffectofghrelinonSHP‐1C‐terminalY536residuephosphorylation [pSHP‐1(Y536)].Serum‐starvedHEK‐GHSR‐1acellswerestimulatedwithghrelin(100nM)forthetime periodsindicated.Cellswerelysedandanalyzedbyimmunoblottingusingspecificantibodiesagainst pSHP‐1andactin.SHP‐1phosphorylationwasquantifiedbydensitometryandexpressedasafoldof controlcells(mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments.
Chapter3 104 TheroleofSHP‐1onghrelin‐inducedphosphorylationoftheAktA‐loop(T308)andHM(S473) wasevaluatedbymeansofoverexpressionofadominant‐negativeSHP‐1mutant(SHP‐1dn). Ghrelin‐inducedAktactivitywasincreasedinSHP‐1dn‐transfectedcells,withhighereffecton AktA‐loop(T308)phosphorylationthanthatobservedforAktHM(S473)phosphorylation within20minutesofghrelinstimulation(100nM)(Figure2).Aktphosphorylationatboth residueswasnotalteredafter20minutesofghrelintreatmentintheSHP‐1dn‐transfectedcells (Figure2). PI3KactivationwasevaluatedbymeansofoverexpressionofSHP‐1dn.Forthispurpose, controlandSHP‐1dn‐transfectedcellswereimmunoprecipitatedwithp85andthetyrosine phosphorylation(Y),underghrelinstimulationwasevaluated.ResultsinFigure3showedthat thelevelofp85‐tyrosinephosphorylationinducedbyghrelin(100nM,5min)wasmarkedly higherinSHP‐1dn‐transfectedcellscomparedwithcontrolcells. cSrcactivationwasalsoevaluatedthroughoverexpressionofSHP‐1dn.Ghrelin‐stimulatedcSrc phosphorylation[pSrc(Y416)]wasmeasuredincontrolandSHP‐1dn‐transfectedcells.As showninFigure4,thelevelofpcSrc(Y416)ismarkedlyhigherincellsoverexpressingSHP‐1dn comparedwithcontrolcells.Inaddition,theeffectofSHP‐1dnonPDK‐1phosphorylationatits conservedserineresidue,S241,wasalsoevaluated.OverexpressionofSHP‐1dnincreased ghrelin‐inducedPDK‐1phosphorylation[pPDK‐1(S241)](Figure4). TheeffectofsiRNA‐mediatedsuppressionofβ‐arrestins1and2expressiononthekineticsof pSHP‐1followingghrelinstimulationwasexamined.siRNAexperimentstargetingβ‐arrestin1 orβ‐arrestin2reducedtheirexpressionby50±2%and65±4%,respectively(Figure5).Inthe presenceofanon‐targetingcontrolsiRNA,ghrelin‐activatedpSHP‐1(Y536)wasidenticalto thatobservedwithoutanytransfection(datanotshown).β‐arrestin1andβ‐arrestin2siRNA decreasedghrelin‐activatedpSHP‐1(Y536)withrespecttosiRNAcontrol,withmaximal inhibitoryeffectatthelatertimepoints[34±3%and60±9%at10minforβ‐arrestin1andβ‐ arrestin2siRNA,respectively(Figure5)].
Chapter3 105 Figure2.Effectofoverexpressionofdominant‐negativeSHP‐1mutant(SHP‐1dn)onghrelin‐mediated Aktactivation.Serum‐starvedHEK‐GHSR‐1acellswerestimulatedwithghrelin(100nM)forthetime periodsindicated.Cellswerelysedandanalyzedbyimmunoblottingusingspecificantibodiesagainst pAkt(S473)and(T308).Aktphosphorylationwasquantifiedbydensitometryandexpressedasafoldof controlcells(mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments. ImmunoblotanalysisofSHP‐1expressionincontrolandSHP‐1dn‐transfectedHEK‐GHSR‐1acellsis showninthecenterofthepanel.(*,P<0.05).
Chapter3 112 Figure8.A.ImmunoblotanalysisofSHP‐1inextractsfrom3T3‐L1cells,WAT,skeletalmuscleandliver ofcontrolmice.Sameamountofprotein(40μg)wasusedforthisanalysis.SHP‐1wasdetectedby immunoblottingandnormalizedforactin.Resultswereexpressedasafoldofrespectivecontrol (mean±SE;n=10).Immunoblotsarerepresentativeofthreeindependentexperiments.B. ImmunocytochemicaldetectionofSHP‐1in3T3‐L1preadipocyteandadipocytecells(objective magnification40x).SHP‐1immunostainingwashigherinpreadipocytethanadipocytecellsbeingmainly concentratedinperinuclearcompartmentsinboth3T3‐L1cells. A B
Chapter3 113 Figure9.Time‐courseoftheeffectofghrelinonpSHP‐1(Y536)in3T3‐L1preadipocyteandadipocyte cells.Serum‐starved3T3‐L1cellswerestimulatedwithghrelin(100nM)forthetimeperiodsindicated. CellswerelysedandanalyzedbyimmunoblottingusingspecificantibodiesagainstpSHP‐1andSHP‐1. SHP‐1(Y536)phosphorylationwasquantifiedbydensitometryandexpressedasafoldofcontrolcells (mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments.
Chapter3 114 Figure10.ImmunohistochemicaldetectionofSHP‐1inomental(upperpanel)andsubcutaneous(lower panel)WATobtainedfromcontrolandHFD‐treatedmice(objectivemagnification20x).DecreasedSHP‐ 1immunostainingwasevidentinomentalWATcomparedtocontrolmice.
Chapter3 115 Figure11.A.Effectofghrelin(100nM,1h)onpSHP‐1(Y536)inomentalandsubcutaneousWATexplants invitrofromcontrol(A)andHFD(B)mice.pSHP‐1(Y536)levelswereexpressedasafoldofcontrol (unstimulated)WAT(n=10pergroup).InA,subcutaneousSHP‐1levelwasexpressedasfoldofomental WAT.InB,SHP‐1levelwasexpressedasfoldofWATfromcontrolmice.(*,P<0.05). B A
Chapter3 116 TofurtherdefinetheroleforSHP‐1inmodulatingghrelinaction,GHSR‐1asignalingtoAktwas furtherexaminedinWATtissueexplantsinvitro.Ghrelin‐stimulatedAktactivity(100nM,1h) wasincreasedonbothA‐loop(T308)andHM(S473)regulatorysitesinomentalWATofHFD micecomparedtoomentalWATofcontrolmice(∼1.4‐and1.5‐foldhigher,respectively).In contrast,ghrelin‐inducedpAkt(T308)levelswere37%lessinsubcutaneousWATofHFD relativetosubcutaneousWATofcontrolmice(Figure12A).HowevertheextentofpAkt(S473) stimulationafterghrelintreatmentinsubcutaneousWATofHFDwasquitesimilartoits respectivecontrol(Figure12A).TotalAktlevelswerecomparableinallinstances(Figure12B). NochangesweredetectedinGHSR‐1aandp85levels(Figure13AandB,respectively)inall instances.cSrcexpressionwasmarkedlyenhancedinomentalandsubcutaneousWATofHFD comparedtoWATofcontrolmice(∼1.9‐and∼1.6‐fold,respectively;Figure14A).PDK‐1levels wereenhancedinsubcutaneousWATofHFDmicerelativetocontrol(∼0.9fold),although werenotalteredinomentalWATofHFDmice(Figure14B).Onthecontrary,mTORlevels wereincreasedinomentalWATofHFDmiceandwerediminishedinsubcutaneousWATof HFDmice(Figure14C).
Chapter3 117 Figure12.Effectofghrelin(100nM,1h)onpAkt(T308)andpAkt(S473)(A)andAkt(B)inWATexplants invitroinomentalandsubcutaneousWATobtainedfromcontrolandHFD‐treatedmice(n=10per group).Proteinexpressionwasnormalizedforactinandresultswereexpressedasafoldofrespective controlasmean±SE.(*,P<0.05). A B
Chapter3 118 Figure13.Effectofghrelin(100nM,1h)onGHSR‐1a(A)andp85(B)inWATexplantsinvitroinomental andsubcutaneousWATobtainedfromcontrolandHFD‐treatedmice(n=10pergroup).Protein expressionwasnormalizedforactinandresultswereexpressedasafoldofrespectivecontrol (mean±SE). A B
Chapter3 119 Figure14.Effectofghrelin(100nM,1h)oncSrc(A),PDK‐1(B)andmTOR(C)inWATexplantsinvitroin omentalandsubcutaneousWATobtainedfromcontrolandHFD‐treatedmice(n=10pergroup).Protein expressionwasnormalizedforactinandresultswereexpressedasafoldofrespectivecontrol (mean±SE).(*,P<0.05). B C A
CHAPTER4
Chapter4 128 Figure5.ExpressionlevelsofSSTRinHEK‐WTandHEK‐GHSR‐1acells.AbsolutemRNAcopynumberof SSTR1,SSTR2,SSTR3,SSTR4,SSTR5,SST5MD5andSST5MD4measuredbyqRT‐PCR.Valuesindicate meanmRNAcopynumber±SEofeachtranscriptadjustedbyβ‐actinmRNAcopynumber.
Chapter4 129 Figure6.EffectofsiRNAdepletionofSSTR2onCST‐17‐inducedERK1/2andAktHM(S473) phosphorylation.HEK‐GHSR‐1acellstransfectedwithSSTR2siRNAwereserum‐starvedandthen stimulatedwithCST‐17(200nM)forthetimeperiodsindicated.Equalamountsofproteinineach samplewereusedtoassesstheexpressionofSSTR2(upperpanel)orERK1/2andAkt(S473) phosphorylation(lowerpanel)byimmunoblottinganalysis.ExpressionofSSTR2wasquantifiedby densitometryandexpressedaspercentagesofthelevelofSSTR2incontrolsiRNA‐transfectedcells (mean±SE).Immunoblotsarerepresentativeofthreeindependentexperiments.(*,P<0.05).
Chapter4 130 Ghrelin‐induced[Ca2+]irisewasinhibitedafterpre‐stimulationwithCST‐17orCST‐14(200nM, 30sec)(Figure7A).Thisinhibitoryeffectwasdose‐dependent,exceeding50%atadoseof500 nMforbothCST‐17andCST‐14.HowevertheeffectdidnotsharewithSSTpretreatment(1.0 μM,30secbeforeghrelinstimulus)(Figure7A).HavingestablishedpreviouslythatCST‐17 exertsaninhibitoryroleinghrelinsignaling,theroleofSSTR2inthisinhibitoryeffectwas evaluated.Tothisend,wefirstinvestigatedthepossibleroleofGi/o‐proteinsbymeansof pretreatmentwithpertussistoxin(PTX,100ng/mL,12h).AsshowninFigure7B,PTX,which uncouplesGi/ofromreceptors,reversedtheinhibitoryeffectofCST‐17onghrelin‐induced calciumrise.TheinhibitoryeffectofCST‐17onghrelin‐inducedcalciummobilizationwasnot reversedafterapretreatmentwithPMA(1μmol/l,5minbeforeghrelinstimulus),a characteristicsharedwithghrelin‐activatedcalciummobilizationthroughGHSR‐1a(Figure7C). TheeffectofCST‐17onERK1/2phosphorylationcomparedtotheeffectofghrelin,wasalso evaluatedandisshowninFigure8.CST‐17pretreatment(200nM,30secbeforeghrelin stimulus)inhibitedghrelin‐inducedERK1/2phosphorylation(200nM,5min).However ghrelin‐inducedERK1/2phosphorylation(200nM,5min)wasnotmodifiedbySST(200nM, 30secbeforeghrelinstimulus).ItwasalsoshownthatinHEK‐GHSR‐1acells,SSTactivates phosphorylationofERK1/2(datanotshown).
Chapter4 131 Figure7.A.EffectofCST‐17,CST‐14orSSTonghrelin‐induced[Ca+2]IinHEK‐GHSR‐1acells.Resultsare expressedas%ofmaximalghrelinresponse(200nM;mean±SE;n=3).B.EffectofCST‐17(200nM)on ghrelin‐induced[Ca2+]i(200nM)intheabsenceorpresenceofPTX(100ng/mL,12h).C.EffectsofPMA (1μM,5minutesbeforestimulus)ontheinhibitoryeffectofCST‐17(200nM)onghrelin‐induced[Ca2+]I (200nM).ForA,BandCresultsareexpressedas%ofmaximalghrelinresponse(mean+SE;n=6). A CB
Chapter4 132 Figure8.Effectofghrelin,CST‐17andSSTonbasal‐andghrelin‐inducedERK1/2phosphorylation.HEK‐ GHSR‐1acellswereserumstarvedandthenstimulatedwithghrelinalone(200nM),CST‐17alone(200 nM),SSTalone(200nM),ghrelinplusCST‐17(200nM,30secondspriortoghrelinstimulation)or ghrelinplusSST(200nM,30secondspriortoghrelinstimulation)for5minutes.Cellswerelysedand analyzedbyimmunoblottingusingspecificantibodiesagainstpERK1/2.ERK1/2phosphorylationwas quantifiedbydensitometryandexpressedasthepercentageofthebasalphosphorylation(mean±SE). Immunoblotsarerepresentativeofthreeindependentexperiments.
5. DISCUSSION
Discussion 135 Sinceghrelindiscovery,morethanadecadeago,numerouspublicationshavebeenreported withthedifferentrolesofthisacylatedpeptidichormone,rangingfromregulationofappetite andfoodintaketothereleaseofdistincthormones,effectsonglucoseandlipidmetabolism, cardiovasculareffects,modulationofcellproliferationoreffectsonreproductivesystem.10 Onlyunderstandingindetailthebasicsmechanismsthatcontrolandregulatetheresponseof thishormonethroughitsreceptor,willallowusabetterknowledgetodevelopnewdrugs actingonGHSR‐1aorinitstargets. Ghrelin/MAPKpathway Withregardtotheroleofghrelinincellproliferation,theMAPKpathwayrepresentsthemain pathwayinvolvedinmitogenicprocesses. 10TheclassicalmechanismbywhichG‐protein‐ coupledreceptors(GPCR)activateMAPKisthroughheterotrimericG‐proteins.Thesereceptors stimulateG‐proteinsubunits,whichactivatetheRas‐dependentcascade,leadingtothe activationofRaf‐1andMAPkinasekinase1,aspecificactivatorofextracellularsignal‐ regulatedkinases1and2(ERK1/2). Forexample,in3T3‐L1preadipocytes,themitogeniceffectofghrelinismediatedviaactivation oftheMAPKandphosphoinositide3‐kinase(PI3K)pathwaysthroughapertussistoxin(PTX)‐ sensitiveG‐protein(Gi/o).84ThePI3K/Aktpathwayhasalsobeenimplicatedinthemitogenic effectsofghrelinonpancreaticadenocarcinomacells.121 Meanwhile,inhumanandratadrenal zoneglomerulosacells,themitogeniceffectofghrelininvolvesactivationofatyrosinekinase‐ dependentMAPKp42/43mechanismandseemstobeindependentofcAMP‐dependent proteinkinase(PKA)andproteinkinaseC(PKC).82,83Itwasalsoseenthatinhepatomacells, ghrelinmodulatesthedownstreamstagesofinsulinsignalingbystimulatingcellproliferation throughthetyrosinephosphorylationofinsulinreceptorsubstrate‐I(IRS‐I),leadingassociation ofIRS‐Iwiththeadaptormoleculegrowthfactorreceptor‐boundprotein2(GRB2)andthus activatingMAPKactivity.85 Thus,therearedifferentpaperswheretheghrelin‐inducedMAPKactivationhasbeen describedandtheintracellularsignalingmechanismshavebeenelucidated. RecentevidencehassuggestedthatERK1/2activationmayalsobeachievedviapathways involvingβ‐arrestins.Theβ‐arrestins,originallythoughtonlytobeinvolvedinreceptor 121DuxburyMS,WaseemT,ItoH,RobinsonMK,ZinnerML,AshleySW,WhangEE.Ghrelinpromotes pancreaticadenocarcinomacellularproliferationandinvasiveness.BiochemBiophysResCommun. 2003;309:464–68.
Discussion 136 uncouplingandinternalization,serveasmultifunctionaladaptor,scaffoldsandsignal transducersthatconnectactivatedreceptorswithdiversesignalingmoleculeswithinthecell.5 β‐arrestin‐mediatedactivationofERK1/2appearstobelinkedtothefunctionofβ‐arrestinsin mediatingendocytosisofanumberofGPCR,inwhichtheyactasscaffoldsforthecomponent kinasesthatactivateERK1/2.5,122Inpathwaysmediatedbytheclass‐BGPCR,theβ‐arrestin scaffoldedsignalingcomplexpersistsforprolongedperiodswhileinpathwaysmediatedby class‐AGPCR,thescaffoldismuchlesspersistent.5,122Thesedifferencesaredeterminedby thestabilityoftheβ‐arrestin/receptorcomplexes.100,123,124Thesescaffoldingcomplexescan determinethesubcellularlocationandspecificityofactivatedERK1/2,promoting phosphorylationofdiversecytosolicsubstratesandtherebyhavingdifferentphysiological consequences,liketheangiotensinIItype1areceptor(AT1AR)forexample.116Thisisowingto thefactthatGq‐protein‐mediatedactivationisrapidandtransientleadingtonuclear translocationoftheactivatedERK1/2withconsequentmitogenicactivity.Incontrast,β‐ arrestin‐mediatedactivationischaracterizedbyretentionoftheactivatedERK1/2inendocytic vesicles,sothereisnoinductionofmitogenesis,asitwasreportedforthetype2vasopressin receptor(V2R).125Thus,itmaybeconcludedthatthephysiologicalconsequenceswere differentsincetheactivatedERK1/2poolsgeneratedbythetwopathwayswouldhave differentspatialandtemporaldistributionsandsubcellularlocations.Anotherimportantthing tomentionisrelatedtotheactionofthesepathways.Itisnotcleartheinterplaybetweenthe β‐arrestin‐dependentandG‐protein‐dependentpathways.ThereareexampleswheretheG‐ proteinandβ‐arrestin‐dependentpathwaysactsequentially125andcaseswhereboth pathwayscanactinparallel,asdescribedforAT1AR116andβ‐2adrenergicreceptor(β2AR).126 Itiswell‐knownthatERK1/2activationviatheGHSR‐1areceptorhasbeenreportedtoinvolve Gi‐mediatedsignalingandPI3K,PKC,tyrosinekinasephosphorylations.However,itisunclear howthisreceptorcoupletoMAPKsignalingpathwayand,furthermore,whetherthereare subtype‐relatedregulationsinthissignalingpathway.Theresultspresentedhereallowusto 122ReiterE,LefkowitzRJ.GRKsandβ‐arrestins:Rolesinreceptorsilencing,traffickingandsignaling. TrendsEndocrinolMetab.2006;17:159–65. 123FergusonSS.EvolvingconceptsinG‐protein‐coupledreceptorendocytosis:Theroleinreceptor desensitizationandsignaling.PharmacolRev.2001;53:1–24. 124LuttrellLM,LefkowitzRJ.Theroleofβ‐arrestinsintheterminationandtransductionofG‐protein‐ coupledreceptorsignals.JCellSci.2002;115:455–65. 125RenXR,ReiterE,AhnS,KimJ,ChenW,LefkowitzRJ.DifferentG‐protein‐coupledreceptorkinases governG‐proteinandβ‐arrestin‐mediatedsignalingofV2vasopressinreceptor.ProcNatlAcadSciUSA. 2005;102:1448–53. 126ShenoySK,DrakeMT,NelsonCD,HoutzDA,XiaoK,MadabushiS,ReiterE,PremontRT,LichtargeO, LefkowitzRJ.β‐arrestin‐dependent,G‐protein‐independentERK1/2activationbytheβ2adrenergic receptor.JBiolChem.2006;281:1261–73.
Discussion 137 delineatethepathwayofMAPKactivationinducedbyghrelininresponsetobindingtoGHSR‐ 1ainHEK‐GHSR‐1acells.Thesignalingmechanismsthatunderliethephosphorylationand activationofERK1/2arecomplexandresultfromtheinterplayofthreedifferentsignaling pathwayswhichdependonbothclassicalG‐protein‐regulatedeffectorsandβ‐arrestins‐ dependentmechanisms.OnepathwayisGq/11‐dependentandinvolvesaCa+2‐dependentPKC (PKCα/β)andcSrc.AsecondpathwayisGi/o‐dependentandinvolvesPI3K,PKCε,andcSrc.The thirdpathwayismediatedbyβ‐arrestins1and2andrequirestheentryofthereceptorintoa multiproteincomplexwiththeβ‐arrestins,cSrc,Raf‐1,ERK1/2,andperhapsother componentsoftheMAPKcascade.Inaddition,ithasbeenshownthattheGq/11‐andtheGi/o‐ proteinsarecruciallyinvolvedandconvergeintheβ‐arrestin1and2‐mediatedERK1/2 activation.AnotherpointtohighlightisthecrucialroleofcSrcplayingasanodelinkingboth pathways.Thus,thesefindingssupporttheviewthatghrelinactivatesdifferentERK1/2pools thatdifferintheirtemporalandspatialdistributions,andthusprobablyhavedifferent physiologicalconsequences. StartingfromthepreviousdatareportedwhereERK1/2activationviatheGHSR‐1ainvolveda Gi‐protein‐mediatedmechanismwecontinuedtoexploretheinvolvementofotherG‐proteins whichmightbemediatingERK1/2activation.ThediscoveryoftheGq/11‐dependentpathway wasachievedaftertestingtheeffectofBAPTA,whichpartiallydecreasedtheERK1/2 activationafterghrelinstimulation,pointingtoaCa+2‐dependentsignalingpathway.Itiswell‐ knownthatGq/11‐proteinsactivatephosphatidylinositol‐specificphospholipaseC(PI‐PLC)which generatesIP3andDAGfromphosphatidylinositol4,5‐biphosphatewiththesubsequent liberationofCa+2fromIP3‐sensitivestoresandactivationofCa+2‐dependentPKCα/β.In addition,theuseofaselectiveinhibitoroftheCa+2‐andDAG‐dependentPKCαandPKCβ isoforms,Gö6976,causedasimilardecreaseintheghrelin‐inducedERK1/2activation.Besides, thedataobtainedfromPKCαandPKCβactivationinresponsetoghrelinparallelthetime‐ courseofghrelin‐inducedERK1/2activation.ItwasalsodeterminedtheroleofcSrc, confirmingitsparticipationinthisroutedownstreamthePKCα/β,sinceGö6976pretreatment hadasignificanteffectoncSrcactivation. TheotherpathwaydescribedbelongstotheGi/o‐dependentandwasindicatedbythe inhibitoryeffectonERK1/2activationachievedafterPTXpretreatment,whichdisablesGi/o‐ protein.ItalsohasbeenshownthatcSrc‐tyrosinephosphorylationwasreducedbythis pretreatment,involvingthisnon‐receptortyrosinekinaseinthesignalingpathway.Similarly, thePI3Kblockerwortmannin,inhibitedcSrcphosphorylation.Inaddition,thetime‐courseof PKCεwascloselysimilarwiththedynamicsofghrelin‐inducedERK1/2activationandacontrol
Discussion 144 domain,itwasproposedthatcSrcinteractswiththePXXPmotifofAkt176whichisconsistent withthis,thatis,AkttranslocatestotheplasmamembranethroughthebindingofitsPH domaintoIP3generatedbyPI3K.ThisallowstheinteractionofmembraneboundcSrcandAkt throughitsPXXPmotifintheC‐terminalregulatoryregionandtheSH3domainofcSrc.Then cSrcphosphorylatesAktattyrosineresidue(s),whichtriggersAktA‐loop(T308)andHM(S473) phosphorylationbyPDK‐1andmTORC2respectively.Altogetherthesedatasupporttheidea thatcSrcoperatesasa‘‘switch’’inconcertwithPI3KforactivationofAktinresponseto ghrelin. Ontheotherside,thedataobtainedshowaroleofβ‐arrestinsinghrelin‐inducedAkt activation,rolethatisrelatedtoreceptorendocytosis.Thisisclearsinceβ‐arrestindepletion usingsiRNAreducedthemagnitudeofAktactivationinresponsetoghrelin.Besides,theco‐ immunoprecipitationassaysshowedthatβ‐arrestins,actinglikeadaptorproteins,recruitAkt totheghrelin‐occupiedreceptor.AlthoughthemechanismofactivationofAkt‐associatedtoβ‐ arrestinsisnotclear,fromthedataobtaineditcanbeconcludedthatcSrcisessentialforAkt activationevenwhenisassociatedtoβ‐arrestins.Asitalreadywasdescribedpreviously,160it wouldbepossiblethatAktinteractswithcSrcassociatedtoGHSR‐1a‐β‐arrestincomplexas consequenceofcolocalizationatplasmamembranewhenPI3Kisactivatedinresponseto ghrelin.ThisAkt‐cSrcassociationispresumedtoinitiateAktphosphorylationbyallowing tyrosinephosphorylation.Furthermore,inlinewithitsfunction,β‐arrestin‐scaffoldedcomplex placesthedifferentcomponentsoftheAktcascadeincloseproximitytoeachother,ensuring substratespecificity.ThisfactissupportedbythedatashowingthatneitherPDK‐1nor mTORC2co‐immunoprecipitatedwithβ‐arrestins.Itisalsonoteworthythatbothβ‐arrestins seemtoberequiredtopromoteAktactivation.Thiscouldbeindicativeofaneedtoform heterodimers,asitwasdescribedforMAPKactivation.160However,thismechanismcannotbe generalizatedsinceinothersystemsβ‐arrestinsshowoppositeeffects.ThisisthecaseofD2‐ classreceptorswhereβ‐arrestin2facilitatesthedephosphorylationofAktbyphosphatase2A (PP2A)inresponsetodopamine.177,178OtherexamplesaretheprostaglandinE2,179IGF‐1,180 176JiangT,QiuY.InteractionbetweenSrcandaC‐terminalproline‐richmotifofAktisrequiredforAkt activation.JBiolChem.2003;278:15789–93. 177BeaulieuJM,MarionS,RodriguizRM,MedvedevIO,SotnikovaTD,GhisiV,WetselWC,LefkowitzRJ, GainetdinovRR,CaronMG.Aβ‐arrestin2signalingcomplexmediateslithiumactiononbehavior.Cell. 2008;132:125–36. 178BeaulieuJM,SotnikovaTD,MarionS,LefkowitzRJ,GainetdinovRR,CaronMG.AnAkt/β‐arrestin 2/PP2Asignalingcomplexmediatesdopaminergicneurotransmissionandbehavior.Cell.2005;122: 261–73. 179BuchananFG,GordenDL,MattaP,ShiQ,MatrisianLM,DuBoisRN.Roleofβ‐arrestin1inthe metastaticprogressionofcolorectalcancer.ProcNatlAcadSciUSA.2006;103:1492–97.
Discussion 145 α‐thrombin181andβ‐1adrenergicreceptors182,183whichactivateAktpathwaythroughβ‐ arrestin1dependentmechanisms.Thus,itseemsthateachreceptordeterminetheβ‐arrestin‐ associatedfunctionsonbasisofthereceptor‐associatedisoforms. Asitisreportedinthebibliography,ghrelinregulatesAktactivitythroughtheGHSR‐1ain differentcellularsystems.156‐159Particularly,themitogeniceffectofghrelinin3T3‐L1 preadipocytesismediatedbythePI3K/AktandMAPKpathwaysviaaGi‐protein.84Inlinewith thepreviousdataobtainedinHEK‐GHSR‐1acells,co‐immunoprecipitationassaysperformedin 3T3‐L1cellsindicatedthatβ‐arrestinsrecruitAkt,leadingtoitsactivation.Inaddition,cSrcacts upstreamofAkt,asPP2experimentsabolishedtheghrelin‐inducedAktactivation. Furthermore,theco‐immunoprecipitationassaysalsoshowthatcSrcinteractswithAktin responsetoghrelin,allowingthephosphorylationofAktattyrosineresidue(s). Insummary,theresultspresentedhereshowthatAkttranslocatestotheplasmamembrane throughthebindingofitsPHdomaintothesecondmessengerIP3generatedbyPI3Kwhichis activatedthroughGi/o‐protein‐dependentsignalingpathway.Aktisphosphorylatedattyrosine bythemembraneboundcSrcviatheinteractionbetweenitsC‐terminalproline‐richmotifand theSH3domainofcSrc.ThistyrosinephosphorylationisfollowedbyphosphorylationofAktA‐ loop(T308)andHM(S473)byPDK‐1andmTORC2respectively.Oncethereceptorisactivated, asecondsignalingpathwayismediatedbyβ‐arrestins1and2involvingtherecruitmentof GHSR‐1a,cSrcandAktintoaβ‐arrestin‐scaffoldedcomplex.ThuscSrcfunctionsasaswitch thatinitiatestheAktpathwayassociatedtoboththeGi/o‐protein‐dependentpathwayandβ‐ arrestin‐scaffoldedcomplex. 180PovsicTJ,KohoutTA,LefkowitzRJ.β‐arrestin1mediatesinsulin‐likegrowthfactor1(IGF‐1)activation ofphosphatidylinositol3‐kinase(PI3K)andanti‐apoptosis.JBiolChem.2003;278:51334–39. 181GoelR,Phillips‐MasonPJ,RabenDM,BaldassareJJ.α‐ThrombininducesrapidandsustainedAkt phosphorylationbyβ‐arrestin1‐dependentand‐independentmechanisms,andonlythesustainedAkt phosphorylationisessentialforG1phaseprogression.JBiolChem.2002;277:18640–48. 182NomaT,LemaireA,NagaPrasadSV,Barki‐HarringtonL,TilleyDG,ChenJ,LeCorvoisierP,ViolinJD, WeiH,LefkowitzRJ,RockmanHA.β‐arrestin‐mediatedβ1‐adrenergicreceptortransactivationofthe EGFRconferscardioprotection.JClinInvest.2007;117:2445–58. 183MoriscoC,MarroneC,GaleottiJ,ShaoD,VatnerDE,VatnerSF,SadoshimaJ.Endocytosismachinery isrequiredforβ1‐adrenergicreceptor‐inducedhypertrophyinneonatalratcardiacmyocytes. CardiovascRes.2008;78:36–44.
Discussion 146 Aktpathway/SHP‐1 Overthepastseveralyears,muchprogresshasbeenmadeinelucidatingintracellularsignaling eventsmediatingmetabolicactionsbytheghrelin/GHSR‐1asystem.However,themolecular processesthatmediatetheinactivationoftheseeventsremainlessdefined.Theresults obtainedhereofferthreemajorfindingsrelatedtotheregulationAktactivityinresponseto ghrelin.First,SHP‐1isoneofthenegativeregulatorsofGHSR‐1a‐mediatedAktactivation. Second,activationofSHP‐1iscSrc‐dependentandinvolvestheinterplayofG‐protein‐andβ‐ arrestins‐dependentsignalingpathways.Third,whiteadiposetissue(WAT)expressesrelatively highlevelsofSHP‐1andthisexpressionisdynamicallyregulatedinresponsetohighfat feeding.Thus,SHP‐1attenuatestheactionofghrelinbydephosphorylatingPI3KandcSrcand therebyinhibitingghrelinsignalingtotheAktpathway. TheproteintyrosinephosphataseSHP‐1playsavarietyofrolesinmultiplesignaltransduction events184bytyrosinedephosphorylation.185,186Thiseventscomprisesignalingofcytokine receptors,187,188GPCR119,189,andRTK.In theseandmanyothercases,SHP‐1regulates signalinginanegativemanner;inotherpathwaysSHP‐1exertsapositivefunction.190,191 TheresultspresentedhereareinfavoroftheroleofSHP‐1asanegativeregulatorof ghrelin/GHSR‐1asignaling.TheexpressionofcatalyticallyinactiveSHP‐1increasedghrelin‐ inducedAktphosphorylation,withgreaterrepercussiononAktA‐loop(T308)phosphorylation. 184NeelBG,TonksNK.Proteintyrosinephosphatasesinsignaltransduction.CurrOpinCellBiol.1997;9: 193‐204. 185NeelBG,GuH,PaoL.The'Shp'ingnews:SH2domain‐containingtyrosinephosphatasesincell signaling.TrendsBiochemSci.2003;28:284‐93. 186ZhangJ,SomaniAK,SiminovitchKA.RolesoftheSHP‐1tyrosinephosphataseinthenegative regulationofcellsignalling.SeminImmunol.2000;12:361‐78. 187KlingmüllerU,LorenzU,CantleyLC,NeelBG,LodishHF.SpecificrecruitmentofSH‐PTP1tothe erythropoietinreceptorcausesinactivationofJAK2andterminationofproliferativesignals.Cell.1995; 80:729‐38. 188KozlowskiM,LaroseL,LeeF,LeDM,RottapelR,SiminovitchKA.SHP‐1bindsandnegatively modulatesthec‐Kitreceptorbyinteractionwithtyrosine569inthec‐Kitjuxtamembranedomain.Mol CellBiol.1998;18:2089‐99. 189FengYH,SunY,DouglasJG.Gbetagamma‐independentconstitutiveassociationofGalphaswith SHP‐1andangiotensinIIreceptorAT2isessentialinAT2‐mediatedITIM‐independentactivationofSHP‐ 1.ProcNatlAcadSciUSA.2002;99:12049‐54. 190WishcamperCA,CoffinJD,LurieDI.LackoftheproteintyrosinephosphataseSHP‐1resultsin decreasednumbersofgliawithinthemotheaten(me/me)mousebrain.JCompNeurol.2001;441:118‐ 33. 191KrautwaldS,BüscherD,KummerV,BuderS,BaccariniM.Involvementoftheproteintyrosine phosphataseSHP‐1inRas‐mediatedactivationofthemitogen‐activatedproteinkinasepathway.Mol CellBiol.1996;16:5955‐63.
Discussion 147 WhilemuchofthecharacterizationofSHP‐1iscenteredonitsfunctionasaPTP,192the regulationofitsactivationisconfused.193,194Despitesuchpuzzlement,itwasdemonstrated thattyrosinephosphorylation(Y)oftheC‐terminalY536inSHP‐1increasesitsPTPactivity.193, 195Thisisconsistentwiththeseresults,whichshowedthatghrelinisabletophosphorylate SHP‐1atC‐terminusY536inintactcellsandWAT.Additionally,theresultsshowthatablating cSrcbysiRNA,impairedghrelin‐stimulatedC‐terminusY536phosphorylationofSHP‐1.This resultlocatestocSrcasanupstreamsignalthatregulateactivationofSHP‐1.Indeedaprevious reportshowedthatcSrcphosphorylatesSHP‐1onYinvitro,leadingtoanincreaseinthe activityofthephosphatase.195ThisisalsoconsistentwithrecentworksinwhichangiotensinII type2receptor(AT2R)regulatesSHP‐1activitythroughcSrcactivation.189,196Thisdatasupport theideathatcSrcoperatesasa“switch”inconcertwithPI3KformodulationofAktactivityin responsetoghrelin. AnalysisoftheupstreamtargetsinghrelinsignalingforAktactivationundertheexpressionof catalyticallyinactiveSHP‐1revealedanincreaseofghrelin‐inducedtyrosinephosphorylationof p85regulatorysubunitofPI3K.Underrestingconditions,p85actstobothstabilizeand inactivatethep110catalyticsubunitPI3KoftheN‐terminaldomain.197Ourdataconfirmthat tyrosine‐phosphorylatedp85isatargetofSHP‐1andcellslackingSHP‐1increasePI3Kactivity asitwaspreviouslydescribedforotherfactors.198,199TheinhibitoryeffectofSHP‐1onPI3K activityisconsequenceofitstyrosinedephosphorylation(Y688),200aresiduethat,when phosphorylated,interactswiththep85N‐terminalSH2domainreleasingtheinhibitoryactivity 192LorenzU,RavichandranKS,PeiD,WalshCT,BurakoffSJ,NeelBG.Lck‐dependenttyrosyl phosphorylationofthephosphotyrosinephosphataseSH‐PTP1inmurineTcells.MolCellBiol.1994;14: 1824‐34. 193UchidaT,MatozakiT,NoguchiT,YamaoT,HoritaK,SuzukiT,FujiokaY,SakamotoC,KasugaM. InsulinstimulatesthephosphorylationofTyr538andthecatalyticactivityofPTP1C,aproteintyrosine phosphatasewithSrchomology‐2domains.JBiolChem.1994;269:12220‐8. 194ZhangZ,ShenK,LuW,ColePA.TheroleofC‐terminaltyrosinephosphorylationintheregulationof SHP‐1exploredviaexpressedproteinligation.JBiolChem.2003;278(7):4668‐74. 195FrankC,BurkhardtC,ImhofD,RingelJ,ZschörnigO,WieligmannK,ZachariasM,BöhmerFD. EffectivedephosphorylationofSrcsubstratesbySHP‐1.JBiolChem.2004;279:11375‐83. 196AlvarezSE,SeguinLR,VillarrealRS,NahmiasC,CiuffoGM.Involvementofc‐Srctyrosinekinasein SHP‐1phosphataseactivationbyAngIIAT2receptorsinratfetaltissues.JCellBiochem.2008;105:703‐ 11. 197CuevasBD,LuY,MaoM,ZhangJ,LaPushinR,SiminovitchK,MillsGB.Tyrosinephosphorylationof p85relievesitsinhibitoryactivityonphosphatidylinositol3‐kinase.JBiolChem.2001;276:27455‐61. 198ImaniF,RagerKJ,CatipovicB,MarshDG.Interleukin‐4(IL‐4)inducesphosphatidylinositol3‐kinase (p85)dephosphorylation.ImplicationsfortheroleofSHP‐1intheIL‐4‐inducedsignalsinhumanBcells.J BiolChem.1997;272:7927‐31. 199YuZ,SuL,HoglingerO,JaramilloML,BanvilleD,ShenSH.SHP‐1associateswithbothplatelet‐derived growthfactorreceptorandthep85subunitofphosphatidylinositol3‐kinase.JBiolChem.1998;273: 3687‐94. 200CuevasB,LuY,WattS,KumarR,ZhangJ,SiminovitchKA,MillsGB.SHP‐1regulatesLck‐induced phosphatidylinositol3‐kinasephosphorylationandactivity.JBiolChem.1999;274:27583‐9.
Discussion 148 ofp85onthep110catalyticsubunit.198‐200Infact,activationofdownstreamPI3Ktargets,PDK‐ 1[pPDK‐1(S241)]andcSrc[pSrc(Y416)]areincreasedincellslackingSHP‐1underghrelin stimulation.Thisisalsoconsistentwiththeincreaseonghrelin‐stimulatedA‐loop(T308) phosphorylationofAktinthecontextofSHP‐1deficiency. Thepresenceofapathwayinvolvingreceptorendocytosisissupportedbythefactthatβ‐ arrestin1and2depletionusingspecificsiRNAreducedthemagnitudeofghrelin‐stimulatedC‐ terminusY536phosphorylationofSHP‐1.Co‐immunoprecipitationassaysrevealthatβ‐ arrestinsfunctionasadaptorsrecruitingSHP‐1totheghrelin‐occupiedreceptorthroughthe formationofβ‐arrestincomplexes.Thisisalsoconsistentwiththeincreaseonghrelin‐ stimulatedAktphosphorylationinthecontextoffunctionalSHP‐1deficiency.Themechanism ofactivationofSHP‐1‐associatedtoβ‐arrestinsisnotcompletelydelineated.Availabledata suggestthatcSrcisessentialforAktactivationevenwhenisassociatedtoβ‐arrestins.201Given thatcSrcisrecruitedtoGHSR‐1a‐β‐arrestincomplextoinitiateAktphosphorylationupon ghrelinstimulation,160itwouldbepossiblethatSHP‐1interactswithcSrcassociatedtothis complex,exertingamodulatoryroleoncSrcactivityasp85isnotassociatedtoGHSR‐1a‐β‐ arrestincomplex.160Interestingly,bothβ‐arrestin1andβ‐arrestin2arerequiredtopromote theactivationofSHP‐1.Therequirementofbothproteinsmightbeindicativeofaneedto formheterodimerstoactivatetheβ‐arrestin‐dependentsignalingpathwayforGHSR‐1a. PTPhaveemergedasmainregulatorsofkeymetabolicprocessessuchasinsulinsensitivity202, 203andglucosehomeostasis.204,205,206Fromthisgroup,themetabolicroleofSHP‐1has remainedunstudied,probablyasresultoftheobservationthatitismainlyexpressedin 201LodeiroM,TheodoropoulouM,PardoM,CasanuevaFF,CamiñaJP.c‐SrcregulatesAktsignalingin responsetoghrelinviaβ‐arrestinsignaling‐independentand‐dependentmechanisms.PLoSOne.2009; 4:e4686. 202ElcheblyM,PayetteP,MichaliszynE,CromlishW,CollinsS,LoyAL,NormandinD,ChengA,Himms‐ HagenJ,ChanCC,RamachandranC,GresserMJ,TremblayML,KennedyBP.Increasedinsulinsensitivity andobesityresistanceinmicelackingtheproteintyrosinephosphatase‐1Bgene.Science.1999;283: 1544‐8. 203KlamanLD,BossO,PeroniOD,KimJK,MartinoJL,ZabolotnyJM,MoghalN,LubkinM,KimYB,Sharpe AH,Stricker‐KrongradA,ShulmanGI,NeelBG,KahnBB.Increasedenergyexpenditure,decreased adiposity,andtissue‐specificinsulinsensitivityinprotein‐tyrosinephosphatase1B‐deficientmice.Mol CellBiol.2000;20:5479‐89. 204MaegawaH,HasegawaM,SugaiS,ObataT,UgiS,MorinoK,EgawaK,FujitaT,SakamotoT,NishioY, KojimaH,HanedaM,YasudaH,KikkawaR,KashiwagiA.ExpressionofadominantnegativeSHP‐2in transgenicmiceinducesinsulinresistance.JBiolChem.1999;274:30236‐43. 205GoldsteinBJ.Protein‐tyrosinephosphatases:emergingtargetsfortherapeuticinterventionintype2 diabetesandrelatedstatesofinsulinresistance.JClinEndocrinolMetab.2002;87:2474‐80. 206Asante‐AppiahE,KennedyBP.Proteintyrosinephosphatases:thequestfornegativeregulatorsof insulinaction.AmJPhysiolEndocrinolMetab.2003;284:E663‐70.
Discussion 149 hematopoieticlineages.207However,SHP‐1expressionisalsofoundinperipheraltissues includinginsulintargettissuessuchasliverandmuscle,208,209suggestingamorewidespread involvementofSHP‐1.Infact,SHP‐1regulatesglucosehomeostasisthroughmodulationof insulinsignalinginliverandmuscle.208Furthermore,SHP‐1expressionwasalsofoundinother celltypesandtissuessuchasRPEandperivascularcells(glialcells)intheretinaandinrenal podocytes,suggestingtheinvolvementofSHP‐1indiabeticpathology.210Asreportedhere, WATexpressesSHP‐1albeittoalesserextentthanliver.ThesubcellulardistributionofSHP‐1 isprimarilycytosolicandconcentratedinperinuclearcompartmentsinpreadipocyteand adipocyte3T3‐L1cells.AccordingtotheresultsinHEK‐GHSR‐1acells,ghrelinstimulatedC‐ terminusY536phosphorylationofSHP‐1inboth3T3‐L1cellsandWAT.AnalysisofSHP‐1levels incontrolmiceexhibitedhigherexpressioninomentalthansubcutaneousWAT.Furthermore, thispatternofexpressionisinvertedinHFD‐treatedmice,suggestingaroleofSHP‐1in controllingghrelinsensitivityinthisorgan.Indeed,deficiencyofSHP‐1wasassociatedhere withanincreaseinghrelin‐evokedpAkt(T308)inomentalWATaswellasadecreasein activationofT308phosphorylationunderabundanceofSHP‐1insubcutaneousWAT. Intriguingly,theinhibitoryeffectofSHP‐1onpAkt(S473)showedlowerrepercussionasitwas describedinHEK‐GHSR‐1acellmodel.TheSHP‐1‐regulatedtargetsassociatedtothe phosphorylationofA‐loop(T308)andHM(S473)mightexplainthisdifference.Indeed,SHP‐1 attenuatesPDK‐1activitythroughPI3KwithnoapparenteffectonmTORC2.Thisisfurther supportedbythefactthatbothA‐loop(T308)andHM(S473)phosphorylationsare independent.201AnalysisofinitialstepsinghrelinsignalingtoAkt revealedmodifiedlevelsfor cSrc,PDK‐1andmTORinWATfromHFD‐treatedmicerelativetocontrolmice,whileno changeinAktandGHSR‐1aexpressionsweredetected.Howeverthisdifferentialpatterndoes notappeartoexplainthedifferencesofAktactivitydetectedbetweenbothgroups.Infact, enhancedcSrcandPDK‐1expressioninsubcutaneousWATfromHFDmicedoesnot counteracttheattenuationofghrelin‐evokedpAkt(T308).Basedonthisinformation,itis possibletoproposethatghrelinactioninWATis,atleastinpart,underregulationofSHP‐1.As 207YiTL,ClevelandJL,IhleJN.ProteintyrosinephosphatasecontainingSH2domains:characterization, preferentialexpressioninhematopoieticcells,andlocalizationtohumanchromosome12p12‐p13.Mol CellBiol.1992;12:836‐46. 208DuboisMJ,BergeronS,KimHJ,DombrowskiL,PerreaultM,FournèsB,FaureR,OlivierM, BeaucheminN,ShulmanGI,SiminovitchKA,KimJK,MaretteA.TheSHP‐1proteintyrosinephosphatase negativelymodulatesglucosehomeostasis.NatMed.2006;12:549‐56. 209NorrisK,NorrisF,KonoDH,VestergaardH,PedersenO,TheofilopoulosAN,MøllerNP.Expressionof protein‐tyrosinephosphatasesinthemajorinsulintargettissues.FEBSLett.1997;415:243‐8. 210GeraldesP,Hiraoka‐YamamotoJ,MatsumotoM,ClermontA,LeitgesM,MaretteA,AielloLP,Kern TS,KingGL.ActivationofPKC‐deltaandSHP‐1byhyperglycemiacausesvascularcellapoptosisand diabeticretinopathy.NatMed.2009;15:1298‐306.
Discussion 150 isconsistentwithdatafromcellmodelstudiesofSHP‐1functions,deficiencyofSHP‐1is associatedwithenhancedactivationofPI3K,PDK‐1,cSrcandAkttherebyincreasingghrelin controlongrowthandmetabolisminWATandviceversa.211,212Thesedataareinlinewiththe attenuatingroleofSHP‐1onAktsignalinginliverandmuscle,whichdeterminesinsulin sensitivityandglucosemetabolism. Inconclusion,wehaveidentifiedtheSHP‐1tyrosinephosphataseasanegativeregulatorof ghrelinsignaling.Dataareconsistentwithamodelinwhichghrelin‐activatedAkttranslocates totheplasmamembranethroughthebindingofitsPHdomaintothesecondmessengerIP3 generatedbyPI3KwhichisactivatedthroughtheGi/o‐protein‐dependentsignalingpathway. AktisphosphorylatedattyrosinebythemembraneboundcSrc.Thistyrosinephosphorylation isfollowedbyphosphorylationofAktA‐loop(T308)andHM(S473)byPDK‐1andmTORC2 respectively.Oncethereceptorisactivated,asecondsignalingpathwayismediatedbyβ‐ arrestins1and2,involvingtherecruitmentofGHSR‐1a,cSrcandAktintoaβ‐arrestin‐ scaffoldedcomplex.InbothsignalingpathwayscSrcphosphorylatestheSHP‐1C‐terminus (Y536)whichexertsaninhitoryeffectontheactivityofPI3KandAkt.ConsequentlycSrc functionsasaswitchthatcontrolstheAktpathwaysassociatedtoboththeGi/o‐protein dependentpathwayandβ‐arrestin‐scaffoldedcomplex.Althoughfurtherstudiesarerequired, theresultsshowthatSHP‐1isexpressedinwhiteadiposetissue,oneoftheghrelintarget tissue,andthatitmodulatesghrelinsignalingtoAkt,determiningghrelinsensitivityinWAT, basedonadiposedepotdifferences. Ghrelin/CST‐17 ElucidationoftheintracellularsignalingeventsmediatingGHSR‐1aactionsconstitutesan importantpointtoabetterknowledgeaboutghrelin/GHSR‐1asystem.GHSR‐1ashowsabroad bindingcapacitythatallowstheinteractionofpeptidic/nonpeptidicGHS,notrelated structurallytoghrelin,andothernaturalligandssuchasadenosine59,60,CST62GHRH.76 CST‐17isahormoneoriginallydescribedintherat,mouse,andhumancerebralcortexthat displaysstructuralandfunctionalsimilaritiestoSST,bindingtoallfivesomatostatinreceptors 211DaviesJS,KotokorpiP,EcclesSR,BarnesSK,TokarczukPF,AllenSK,WhitworthHS,GuschinaIA,Evans BA,ModeA,ZigmanJM,WellsT.GhrelininducesabdominalobesityviaGHS‐R‐dependentlipid retention.MolEndocrinol.2009;23:914‐24. 212WellsT.Ghrelin‐Defenderoffat.ProgLipidRes.2009;48:257‐74.
Discussion 151 (SSTR).213,214Amongitsfunctions,CSTinhibitsGHandinsulinsecretioninphysiological conditionsandinacromegaly,66italsoinhibitsprolactin(PRL)secretionfromcultured prolactinomas65and,recently,aroleforCSTastherapeuticagenttoautoimmunediseaseshas beenproposed.67Becauseofthesedifferentbiologicalactivities,itwasproposedthe existenceofaCSTspecificreceptor.Infact,anorphanreceptorcalledMrgX2hasbeenshown tobindCSTwithselectiveaffinityoverSST.64,215However,MrgX2wasfoundtobindadditional peptidesasproadrenomedullinN‐terminalpeptide12(PAMP12)withsimilaraffinitytothat describedforCST.216 TheideathatCSTmightbealigandofGHSR‐1aemergesfrombindingstudiesthat demonstratesacrosscompetitionwithghrelinbindinginmembranesfromhuman hypothalamusandpituitarygland62,217suggestingapotentialfunctionalinteractionbetween CSTandghrelinsystem.Toconsideratrueligand‐receptorinteraction,tworequirementsmust befulfilled:specificbindingandbiologicaleffect.Inthepresentstudy,wehaveprovidedthat CST‐17sharedspecificbindingsitesinHEK‐GHSR‐1acells,bindingthatweredisplaceablewith unlabeledCST‐17andghrelin.Contrarily,WTcellsexhibitedanon‐specificbindingfor[125I]‐ CST‐17thatwasnotdisplacedbysaturatingdoseofCST‐17.Ontheotherside,[125I]‐ghrelin bindingtoHEK‐GHSR‐1acellswascompletelydisplacedinadose‐dependentmannerby ghrelinandtoalesserextentbyCST‐17.DespiteitspotentialbindingcapacitytoGHSR‐1a,CST‐ 17didnotshowghrelin‐associatedsignalingpathway.CST‐17failedtoinduceintracellular calciumreleaseforalldosetestedinHEK‐GHSR‐1acells.Howevertheghrelin‐induced[Ca2+]i risewasinhibitedwhencellswerepre‐stimulatedwithCST‐17,effectthatwasdose‐ dependentandnotsharedwithSSTpretreatment.Inaddition,CST‐17didactivateneitherERK 1/2norAktphosphorylationforanyofthetimeordosestestedinHEK‐WTcells.However,the effectinHEK‐GHSR‐1acellswasdifferent;sinceCST‐17activatedAktandERK1/2 phosphorylationreachingmaximallevelswithin20minutesofCST‐17stimulation.This activationwasdependentonβ‐arrestins,sincesiRNAtargetingbothβ‐arrestin1andβ‐arrestin 2abolishedcompletelythepatternofERK1/2andAktactivation.Besides,surprisingly,CST‐17 213VolanteM,RosasR,AllìaE,GranataR,BaragliA,MuccioliG,PapottiM.Somatostatin,cortistatinand theirreceptorsintumours.MolCellEndocrinol.2008;286:219‐29. 214BroglioF,PapottiM,MuccioliG,GhigoE.Brain‐gutcommunication:cortistatin,somatostatinand ghrelin.TrendsEndocrinolMetab.2007;18:246‐51. 215RobasN,MeadE,FidockM.MrgX2isahighpotencycortistatinreceptorexpressedindorsalroot ganglion.JBiolChem.2003;278:44400‐4. 216KamoharaM,MatsuoA,TakasakiJ,KohdaM,MatsumotoM,MatsumotoS,SogaT,HiyamaH,Kobori M,KatouM.IdentificationofMrgX2asahumanG‐protein‐coupledreceptorforproadrenomedullinN‐ terminalpeptides.BiochemBiophysResCommun.2005;330:1146‐52. 217MuccioliG,PapottiM,LocatelliV,GhigoE,DeghenghiR.Bindingof125I‐labeledghrelintomembranes fromhumanhypothalamusandpituitarygland.JEndocrinolInvest.2001;24:RC7‐9.
Discussion 152 pre‐stimulationbeforeghrelinstimulusinhibitedghrelin‐inducedERK1/2phosphorylation, actionthatwasnotmodifiedbySST,establishingthatCST‐17exertsaninhibitoryrolein ghrelinsignaling.Inaddition,theimmunoprecipitationassaysdemonstratedthatCST‐17 promotesGHSR‐1aphosphorylationatthesamelevelthatghrelin,andtoagreatextentwhen iscombinatedtoghrelin.ItwasalsoprovedthatCST‐17promotesβ‐arrestinrecruitment, demonstratingadirectinteractionoftheseproteinsafterGHSR‐1adesensitization. Therefore,presentdataseemtoindicatethatCST‐17inhibitsthemechanismsofsignaling stimulatedbyghrelin,bothinitsG‐protein‐dependentpathwayandintheβ‐arrestin‐ dependentone.ThismightbeconsideredthebiologicaleffectoftheCST‐17. Toexplaintheabovediscrepancy,wedecidedtoexploretheSSTRexpressiononourcell model.SSTRexpressioninHEK‐WTandHEK‐GHSR‐1ashowedthatSSTR2mRNAis significativelypresentinbothcelltypes.ExperimentsofsiRNAtargetingSSTR2inHEK‐GHSR‐1a cellsaffectedtotheERK1/2andAktdynamicsstimulatedbyCST‐17andarealiketheobserved onesforthecaseoftheghrelin.ThisfactpointstoaroleforSSTR2subtypeinCST‐17signaling, possiblythroughadimerizationprocessbetweenbothreceptors.Theseresultsseemto establishreasonstoproposeCST‐17asamodulatorofghrelinsignaling,inhibitingtheghrelin‐ mediatedsignaling.Onthebasisoftheobtainedresultsitseemsreasonabletoproposetwo hypothesestoexplicatetheseresults.First,CST‐17mightbeconsideredasatrueligandforthe GHSR‐1aactingthroughthemodulationoftheghrelinactivity.Second,CST‐17actsasaSSTR2 ligandpromotingtheformationofdimmerswiththeGHSR‐1aand,therefore,modifyingthe actionofthisone.Accordingtothefirsthypothesis,bindinganddisplacementassayswould supporttheCST‐17uniontotheGHSR‐1a,fulfillingthereforeoneoftwonecessary requirementstoconsideratrueligand‐receptorinteraction.Inaddition,theeffectonthe activationofghrelintargets,asERK1/2andAkt,inHEK‐GHSR‐1arespecttoWTwouldassure thesecondpointnecessaryoftheligand‐receptorinteraction.Furthermore,thefactthatCST‐ 17modifiestheghrelin‐inducedAktandERK1/2activationrevealsthat,somehow,itconcerns thesignalingpathwaysofthisone.Thesefacts,togetherwiththeresultsoftargetingofβ‐ arrestinsandGHSR‐1aphosphorylation,pointtoCST‐17actingthroughthemodificationor modulationofthesignalingpathwaysmediatedbyghrelinthroughGHSR‐1a. TheresultsprovingtheexpressionofSSTR2inHEK‐GHSR‐1acellswouldsupportthesecond hypothesis.ItisknownthatSSTR2signalsthroughGi‐dependentproteins,soitwouldbe possiblethatCST‐17wouldexerciseitsfunctionsonghrelintargetsthroughthismechanism, whichwouldalsojustifythedataobtained.Inaddition,theintracellularcalciummobilization assaysinpresenceofPTXreversedtheinhibitoryeffectofCST‐17onghrelin‐inducedcalcium
Discussion 153 rise(effectthatitwasnotreversedafterapretreatmentwithPMA).DataaboutGHSR‐1a phosphorylationandβ‐arrestinwouldalsoexplainthishypothesis,throughtheeffectthat SSTR2presencewouldcauseontheGHSR‐1adesensitization. Therefore,CST‐17mightbindtotheghrelinreceptor,actingasapotentialligandfortheGHSR‐ 1aasithasbeendescribedinothercasesasforexampleforGHRH,76theGABAβ2receptor218 orthenon–peptidylgrowthhormonesecretagogueL‐692,429.219Ontheotherhand,CST‐17 alsocouldbindtoSSTR2,formingadimmerwiththeGHSR‐1aandaffectinginthiswaytothe ghrelin‐mediatedsignalingasitwasdescribedforothercases.220 Insummary,CST‐17showstomodulateghrelinsignalingthroughSSTR2.Furthermore,CST‐17 showedthecapacitytoactivateGHSR‐1ainabsenceofSSTR2displayingapatternof intracellularsignalingdifferenttothatdescribedforghrelin,whichsuggestabiasedligandfor thisreceptor. 218BinetV,BrajonC,LeCorreL,AcherF,PinJP,PrézeauL.TheheptahelicaldomainofGABA(β2)is activateddirectlybyCGP7930,apositiveallostericmodulatoroftheGABA(B)receptor.JBiolChem. 2004;279:29085‐91. 219HolstB,BrandtE,BachA,HedingA,SchwartzTW.Nonpeptideandpeptidegrowthhormone secretagoguesactbothasghrelinreceptoragonistandaspositiveornegativeallostericmodulatorsof ghrelinsignaling.MolEndocrinol.2005;19:2400‐11. 220LeungPK,ChowKB,LauPN,ChuKM,ChanCB,ChengCH,WiseH.Thetruncatedghrelinreceptor polypeptide(GHS‐R1b)actsasadominant‐negativemutantoftheghrelinreceptor.CellSignal.2007;19: 1011‐22.