Syntaxin-1a和granuphilin在胰岛素释放中的作用

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.37,pp.32244–32250,September16,2011©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedintheU.S.A.

LossofGranuphilinandLossofSyntaxin-1ACause

DifferentialEffectsonInsulinGranuleDockingandFusion*

Receivedforpublication,June5,2011,andinrevisedform,July3,2011Published,JBCPapersinPress,July18,2011,DOI10.1074/jbc.M111.268631

HaoWang ,RayIshizaki ,EriKobayashi ,TomonoriFujiwara§,KimioAkagawa§,andTetsuroIzumi 1

Fromthe DepartmentofMolecularMedicine,InstituteforMolecularandCellularRegulation,GunmaUniversity,Maebashi,Gunma371-8512andthe§DepartmentofCellPhysiology,KyorinUniversitySchoolofMedicine,Mitaka,Tokyo181-8611,Japan

TheRab27effectorgranuphilin/Slp4isessentialforthestableattachment(docking)ofsecretorygranulestotheplasmamem-brane,anditalsoinhibitssubsequentfusion.GranuphilinisthoughttomediatetheseprocessesthroughinteractionswithRab27onthegranulemembraneandwithsyntaxin-1aontheplasmamembraneanditsbindingpartnerMunc18-1.Consis-tentwiththishypothesis,bothsyntaxin-1a-andMunc18-1-de-ficientsecretorycells,aswellasgranuphilinnullcells,havebeenobservedtohaveadeficitofdockedgranules.However,todatetherehasbeennodirectcomparativeanalysisofthedockingdefectsinthosemutantcells.Inthisstudy,wemorphometricallycomparedgranule-dockingstatesbetweengranuphilinnullandsyntaxin-1anullpancreatic cellsderivedfrommicehavingthesamegeneticbackground.Wefoundthatlossofsyntaxin-1adoesnotcauseasignificantgranule-dockingdefect,incontrasttogranuphilindeficiency.Furthermore,wenewlygeneratedgranuphilin/syntaxin-1adoubleknock-outmice,characterizedtheirphenotypes,andfoundthatthedoublemutantmicerep-resentaphenocopyofgranuphilinnullmiceanddonotrepre-sentphenotypesofsyntaxin-1anullmice,includingtheirgran-ule-dockingbehavior.Becausegranuphilinbindstosyntaxin-2andsyntaxin-3aswellassyntaxin-1a,itlikelymediatesgranuledockingthroughinteractionswiththosemultiplesyntaxinsontheplasmamembrane.

Intheconstitutivesecretorypathwaythatoperatesinallcells,transportvesiclesleavethetransGolginetworkinasteadystream,andtheamountoftheproteinreleasedlargelydependsontheirsynthesisrate.Bycontrast,specializedsecretorycellshaveanothersecretorypathway,inwhichsolubleproteinsareinitiallystoredinsecretoryvesiclesandreleasedonlyinthepresenceofanappropriatesecretagogue.Inthisregulatedsecretorypathway,therate-limitingstepliesinoneoftheexo-cyticstepsfromtheintracellulartransporttothefinalfusionofthesecretoryvesiclemembranetotheplasmamembrane,whichallowsarapidsecretoryresponsetoextracellularstimu-lation.Althoughsecretoryvesiclesmusteventuallybetrans-portedclosetotheplasmamembraneforrelease,somevesiclesarealreadyattachedtotheplasmamembrane.Thosestably

*Thisworkwassupportedbygrantsinaidforscientificresearch(toT.I.and

R.I.),aGlobalCOEprogramgrantfromMinistryofEducation,Culture,Sports,ScienceandTechnologyofJapan,andinpartbygrantsfromtheMitsubishiFoundationandaNovoNordiskinsulinstudyaward(toT.I.).1

Towhomcorrespondenceshouldbeaddressed:3-39-15,Showa-machi,Maebashi,Gunma371-8512,Japan.Tel.:81-27-220-8856;Fax:81-27-220-8860;E-mail:tizumi@showa.gunma-u.ac.jp.

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dockedvesiclesarethoughttoconstituteareadilyreleasablepool,becausevesiclessuchassynapticvesiclesmustfusewithin1msafterstimulation.However,exocytosisofsecretorygran-ulesoccursmuchslower,atabout1–10s(1),andthegranulesthatfusedfirstmaynotnecessarilybederivedfromthedockedgranules.Infact,livingcellshavebeenobservedtopermitfusionwithoutpriorpausingattheplasmamembraneinanearlysecretoryphase(2,3).

Itisimportanttoelucidatethemolecularmechanismforthestabledockingofsecretoryvesiclestotheplasmamembranebecausethisphenomenonisauniquehallmarkofregulatedexocytosis.Inallintracellularpathwaysotherthanregulatedexocytosis,theattachmentofthevesicularmembranetothetargetingmembraneinstantlyleadstoconstitutivefusion,andtherefore,vesiclesstablydockedtothetargetmembranecanbarelybediscernedinstaticelectronmicrographs.Althoughdockedvesiclesinregulatedexocytosisappeartobepoisedforreleaseuponsecretagoguesensing,theymayresultfromtheinhibitorynatureofthissecretorypathway,suchthatincomingvesicleswouldnotfusespontaneously(4,5).WepreviouslydemonstratedthattheRab27effector,granuphilin/Slp4,isessentialforthestabledockingofsecretorygranulesinpancre-atic cells(6)andpituitaryendocrinecells(7).Granuphilin-deficientcellsshowaspecificlossofthegranulesdirectlyattachedtotheplasmamembraneundertheelectronmicro-scope.Nevertheless,theyexhibitelevatedsecretioninboththebasalandstimulatedstates(3,6).Granuphilinspecificallyinter-actswithaclosedformofsyntaxin-1a,asolubleN-ethylma-leimide-sensitivefactorattachmentproteinreceptors,ontheplasmamembrane(8).Thisformofsyntaxin-1acaninteractwithMunc18-1butnotwithothersolubleN-ethylmaleimide-sensitivefactorattachmentproteinreceptorstoexecuteafusionreaction(9).Thus,granuphilinstabilizesthefusion-in-competentsyntaxin-1a Munc18-1complexandinhibitssubse-quentfusionofdockedgranules.Theabsenceofgranuphilinandofstablydockedgranulesmayallowundockedgranulestogiveeasieraccesstofusion-competentsyntaxin-1aortofreeplasmamembrane,whichcouldincreasetheefficiencyoffusionfromundockedgranules.ConsistentwiththemodelthatgranuphilinmediatesthedockingprocessbylinkingRab27a/bonthegranulemembrane(10,11)andthesyntaxin-1a Munc18-1complexontheplasmamembrane(8,12),lossofMunc18-1leadstoadockingdefectinthesecretorygranulesinchromaffincells(13).Furthermore,syntaxin-1anull cellshaverecentlybeenreportedtoexhibitagranule-dockingdefect(14).However,directquantitativecomparisonofgranule-VOLUME286 NUMBER37 SEPTEMBER16,2011

RolesofGranuphilinandSyntaxinsinGranuleDocking

dockingdefectsamongthosemutantcellshasneverbeenperformed.

Inthisstudy,wemorphometricallycomparedintracellularinsulingranuledistributionsbetweengranuphilinnullandsyn-taxin-1anull cellsderivedfrommiceofidenticalgeneticbackground.Furthermore,togeneticallydeterminethefunc-tionalrelationshipbe …… 此处隐藏：6117字，全部文档内容请下载后查看。喜欢就下载吧 ……