浙师大《细胞生物学》cha.ppt

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1、Chapter11:Cell-to-CellSignalingA:OverviewofExtracellularSignalingB:SignalingviaHydrophobicMoleculesC:SignalingviaIonChannelsD:SignalingviaG-Protein-CoupledReceptorsE:SignalingviaReceptorTyrosineKinasesF:MAPKinasePathwaysG:InteractionandRegulationofSignalingPathwaysA:OverviewofExtracellularSignalingC

2、ommunicationbyextracellularsignalsusuallyinvolvessixsteps:(1)synthesisand(2)releaseofthesignalingmoleculebythesignalingcell;(3)transportofthesignaltothetargetcell;(4)detectionofthesignalbyaspecificreceptorprotein;(5)achangeincellularmetabolism,function,ordevelopmenttriggeredbythereceptor-signalcompl

3、ex;and(6)removalofthesignal,whichoftenterminatesthecellularresponse.1.Generalschemesofintercellularsignalinginanimals.Inanimals,signalingbyextracellular,secretedmoleculescanbeclassifiedintothreetypesbasedonthedistanceoverwhichthesignalacts.Inaddition,certainmembrane-boundproteinsononecellcandirectly

4、signalanadjacentcell.(1)Inendocrinesignaling,signalingmolecules,calledhormones,actontargetcellsdistantfromtheirsiteofsynthesisbycellsofendocrineorgans.(2)Inparacrinesignaling,thesignalingmoleculesreleasedbyacellonlyaffecttargetcellsincloseproximitytoit.(3)Inautocrinesignaling,cellsrespondtosubstance

5、sthattheythemselvesrelease.Manygrowthfactorsactinthisfashion,andculturedcellsoftensecretegrowthfactorsthatstimulatetheirowngrowthandproliferation.(a-c)Cell-to-cellsignalingbyextracellularchemicalsoccursoverdistancesfromafewmicrometersinautocrineandparacrinesignalingtoseveralmetersinendocrinesignalin

6、g.(d)Proteinsattachedtotheplasmamembraneofonecellcaninteractdirectlywithreceptorsonanadjacentcell.2.ReceptorProteinsExhibitLigand-BindingandEffectorSpecificityThesignalingmolecule(hormone,pheromone,orneurotransmitter)actsasaligand,whichbindsto,orfits,asiteonthereceptor(intracellularreceptors;andcell

7、-surfacereceptors).Bindingofaligandtoitsreceptorcausesaconformationalchangeinthereceptorthatinitiatesasequenceofreactionsleadingtoaspecificcellularresponse.3.HormonesCanBeClassifiedBasedonTheirSolubilityandReceptorLocationMosthormonesfallintothreebroadcategories:(1)smalllipophilicmoleculesthatdiffus

8、eacrosstheplasmamembraneandinteractwithintracellularreceptors;and(2)hydrophilicor(3)lipophilicmoleculesthatbindtocell-surfacereceptors.Recently,nitricoxide,agas,hasbeenshowntobeakeyregulatorcontrollingmanycellularresponses.Somehormonesbindtointracellularreceptors;others,tocell-surfacereceptors.(a)St

9、eroidhormones,thyroxine,andretinoids,beinglipophilic,aretransportedbycarrierproteinsintheblood.Afterdissociationfromthesecarriers,suchhormonesdiffuseacrossthecellmembraneandbindtospecificreceptorsinthecytosolornucleus.Thereceptor-hormonecomplexthenactsonnuclearDNAtoaltertranscriptionofspecificgenes.

10、(b)Polypeptidehormonesandcatecholamines(e.g.,epinephrine),whicharewatersoluble,andprostaglandins,whicharelipophilic,allbindtocell-surfacereceptors.Thisbindingtriggersanincreaseordecreaseinthecytosolicconcentrationofsecondmessengers(e.g.,cAMP,Ca2+),activationofaproteinkinase,orachangeinthemembranepot

11、ential.HydrophobicmoleculesIonchannelsG-protein-coupledreceptorsEnzymes(e.g.RTKs).Inmanycases,thesignalcontinuestopropagatewithinthecellandoftenreachesnuclearDNAtoexpressproteins.4.Theexternalsignalmayenteracellviafourmajorpathways:Majorpathwaysforsignalstoenteracell.5.Cell-SurfaceReceptorsBelongtoF

12、ourMajorClassesSectionB:SignalingviaHydrophobicMoleculesHydrophobicmoleculescanmoveinandoutofcellsbypassingthroughlipidbilayers.Nitricoxide,arachidonicacidandsteroidshavebeenshowntoplayimportantrolesincellsignaling.SignalingwithNOorarachidonicacid.Unlikemostsignalingcascadeswhichoccurwithinthesamece

13、ll,newlygeneratedNOorarachidonicaciddiffusestoactontargetmoleculesinneighboringcells.Nitricoxide(NO)isproducedfromthefollowingreaction:whereNOSrepresentsNOsynthase.AfterNOisgeneratedinacell,itdiffusestoactontargetmoleculesinneighboringcells.NOmaystimulatesolubleguanylylcyclasetoproducecGMPwhichregul

14、atesseveralenzymesandionchannels.Insmoothmuscle,animportantactionofcGMPistoinducemusclerelaxation.Normally,cGMPwillsoonbeconvertedintoGMPbyphosphodiesterase.Thewellknowndrugforimpotence,Viagra(sildenafilcitrate),inhibitsphosphodiesterase.NitricoxideArachidonicacidisgeneratedfromphospholipidhydrolysi

15、scatalyzedbyphospholipase.Afterdiffusingtotargetcells,arachidonicacidmayactivateproteinkinase,resultinginphosphorylationoftargetmolecules.Manyofitstargetmoleculesareinvolvedinlearningandotherneuronalactivities.ArachidonicacidGenerationofarachidonicacidfromphospholipidbyphospholipaseA2(PLA2).Steroids

16、Themajorroleofsteroidsistoregulatetranscription,sincemanysteroidreceptorsaretranscriptionfactors.Atranscriptionalactivationmechanismbysteroidreceptor(SR).Steroid-boundSRmayrecruitSRC(steroidreceptorcoactivator)andhistoneacetyltransferasestostimulatetranscriptionofthetargetgene.SectionC:SignalingviaI

17、onChannelsIonchannelsaremembraneproteinsthatallowionstopassthrough.Intermsofionselectivity,theyareclassifiedascalciumchannels,sodiumchannels,potassiumchannels,etc.Intermsofgating,theymaybeclassifiedasvoltage-gatedchannels,ligand-gatedchannels,etc.istoregulatemembranepotential.However,thecalciumional

18、soplaysimportantrolesinothercellularfunctions,sincemanyenzymesarecalcium-dependent.1.Throughvoltage-activatedCa2+channelswhoseopeningprobabilitydependsonthemembranepotential.2.ThroughIP3-sensitiveCa2+channelswhoseopeningprobabilityisregulatedbyIP3(inositoltrisphosphate).TheopeningofCa2+storesintheen

19、doplasmicreticulummayresultincalciumwaves.ThemajorroleofsodiumandpotassiumionsThemajorroleofG-protein-coupledreceptorsistotransmitsignalsintothecell.Theyarecharacterizedbyseventransmembranesegments.Thisclassofmembraneproteinscanrespondtoawiderangeofagonists,includingphoton,amines,hormones,neurotrans

20、mittersandproteins.Someagonistsbindtotheextracellularloopsofthereceptor,othersmaypenetrateintothetransmembraneregion.SectionD:SignalingviaG-protein-CoupledReceptors1.G-Protein-CoupledReceptors2.GProteins3.EffectorsG-Protein-CoupledReceptorDatabase1.G-protein-CoupledReceptorsThe major role of G-prote

21、in-coupled receptors is to transmit signals into the cell.ThefullnameofGproteinisGTP-bindingproteinbecauseintheactivestateitbindstoGTP(guanosinetriphosphate).2.GProteinsTherearetwotypesofGproteins:heterotrimericGproteinsandmonomericGproteins(orsmallGproteins).RasandRanaresmallGproteins.G-protein-cou

22、pledreceptorsarecoupledtoheterotrimericGproteins.ThestructureofheterotrimericGprotein,consistingofthreesubunits:a,bandg.Intheinactivestate(GDP)Basedonthedifferencesintheirgenes,20a,6band12gsubunitshavebeenidentified.Theirmolecularweightsareinthefollowingranges:asubunit:39-46kDb subunit:35-39kDg subu

23、nit:8kDCycling of G protein between active and inactive statesInteractionbetweenGaandtheagonist-stimulatedreceptorcausesthereleaseofGDP.GTPthenbindstotheemptysitebecauseitsconcentrationinthecellishigherthanGDP.3.GProteinEffectorsEffectorsarethetargetmoleculesofGproteinaorbgsubunit.Thetableliststhema

24、joreffectorsoftheasubunit.(+)meansactivateand(-)meansinhibit.Uponbindingofthecholeratoxin,theGa-boundGTPcannotbeconvertedintoGDPsothattheGproteinremainsintheactivestate.Bycontrast,pertussistoxinpreventsGDPreleasefromtheasubunitsothattheGproteinislockedintheinactivestate.ClassGene VariantEffectorToxi

25、n SensitivityGsa as(s)a as(L)(+)Adenylyl cyclase(+)Ca2+channel(-)Na+channelCholeraa aolf(+)Adenylyl cyclaseCholeraGia ai1a ai2a ai3(-)Adenylyl cyclase(-)Ca2+channel(+)K+channelPertussisa aoa a aob(-)Ca2+channel(+)Phospholipase C(+)Phospholipase A2Pertussisa at1 a at2(+)cGMP phosphodiesteraseCholera

26、and Pertussisa ag(+)Phospholipase CPertussisa a2(-)Adenylyl cyclaseGqa aq a a11 a a14a a15a a16(+)Phospholipase CG12a a12 a a13?(a)BeforeagonistbindingtotheG-protein-coupledreceptor,thethreesubunitsofGproteinareboundtogether.(b)TheagonistbindingcausesinteractionbetweentheG-protein-coupledreceptorand

27、theGprotein.(c)TheirinteractionresultsinthedissociationbetweenaandbgsubunitsoftheGprotein.Theseparatedaand/orbgsubunitsmaytheninteractwitheffectors.Signaling via G-protein-Coupled ReceptorsThebgsubunitsmayactonadenylatecyclase,phospholipaseA2,phospholipaseC,ionchannels,calciumATPaseetc.Adenylatecycl

28、aseAdenylatecylasecatalyzestheconversionofATPtocAMP,whichisanimportantsecondmessenger.Second messengersarethesignalingmoleculesgeneratedbythestimulationofcell-surfacereceptors.Forexample,cAMP,arachidonicacid,DAGandIP3generatedbytheactivationofG-protein-coupledreceptorsaresecondmessengers.Theagonists

29、whichactivateG-protein-coupledreceptorsarefirst messengers.SignalingwithcAMPAcAMP/PKAsignalingpathway.PKAconsistsoftwocatalyticsubunitsandtworegulatorysubunits.cAMPbindstotwositesoneachregulatorysubunit.BindingoffourcAMPmoleculescausesthereleaseoffreeandactivecatalyticsubunits,whichmayphosphorylates

30、erineandthreonineresiduesontargetproteins.Inthisfigure,theactivesubunitphosphorylatesaCREBprotein,resultingintranscription.PhospholipaseC(PLC)alsocleavesphospholipids,generatingdiacylglycerol(DAG)andIP3(inositol1,4,5-trisphosphate).IP3canactivateanIP3-sensitiveCa2+channelinendoplasmicreticulum.Signa

31、lingwithDAGandIP3RTKsarethesecondmajortypeofcell-surfacereceptors.TheligandsforRTKsaresolubleormembrane-boundpeptide/proteinhormonesincludingnervegrowthfactor(NGF),platelet-derivedgrowthfactor(PDGF),fibroblastgrowthfactor(FGF),epidermalgrowthfactor(EGF),andinsulin.Bindingofaligandtothistypeofrecepto

32、rstimulatesthereceptorsintrinsicprotein-tyrosinekinaseactivity,whichsubsequentlystimulatesasignal-transductioncascadeleadingtochangesincellularphysiologyand/orpatternsofgeneexpression.RTKsignalingpathwayshaveawidespectrumoffunctionsincludingregulationofcellproliferationanddifferentiation,promotionof

33、cellsurvival,andmodulationofcellularmetabolism.SectionE:SignalingviaReceptorTyrosineKinases(RTKs)ActivationofRTKstransmitahormonesignaltoRas.1.LigandBindingLeadstoAutophosphorylationofRTKs2.RasBelongstotheGTPaseSuperfamilyofIntracellularSwitchProteinsRasisaGTP-bindingswitchproteinthat,liketheGasubun

34、itsindifferentGproteins,alternatesbetweenanactiveonstatewithaboundGTPandaninactiveoffstatewithaboundGDP.Rasactivationisacceleratedbyaproteincalledguaninenucleotideexchangefactor(GEF),whichbindstotheRasGDPcomplex.3.AnAdapterProteinandGEFLinkMostActivatedRTKstoRasActivation of Ras following binding of

35、 a hormone(e.g.,EGF)to an RTK.TheadapterproteinGRB2bindstoaspecificphosphotyrosineontheactivatedRTKandtoSos,whichinturninteractswiththeinactiveRasGDP.Theguaninenucleotideexchangefactor(GEF)activityofSosthenpromotesformationoftheactiveRasGTP.Thephosphategroupofphosphotyrosine(phosphorylatedtyrosine)c

36、anformseveralhydrogenbondswiththeSH2(Srchomology2)domainofsomeproteinsinvolvedinthesignalingviatyrosinekinases.ReceptorTyrosineKinasesDomainstructureofsomeSH2-containingproteins.Theepidermalgrowthfactor(EGF)peptideinducescellularproliferationthroughtheEGFreceptor,whichhasatyrosinekinasecytoplasmicdo

37、main,asingletransmembranedomainandanextracellulardomaininvolvedinEGFbindingandreceptordimerization.InhibitorsoftheEGFreceptorarebeingpursuedaspotentialcancertherapiesandEGFmaystimulatewoundhealing.MutationoftheEGFreceptorhasbeenassociatedwithcancerinhumans.TheproliferativeeffectsofEGFaresignaledthro

38、ughseveralpathways.BindingofEGFresultsinEGFreceptordimerization,autophosphorylationofthereceptor,andtyrosinephosphorylationofotherproteins.TheEGFreceptoractivatesrasandtheMAPkinasepathway,ultimatelycausingphosphorylationoftranscriptionfactorssuchasc-FostocreateAP-1andELK-1thatcontributetoproliferati

39、on.ActivationofSTAT-1andSTAT-3transcriptionfactorsbyJAKkinasesinresponsetoEGFcontributestoproliferativesignaling.PhosphatidylinositolsignalingandcalciumreleaseinducedbyEGFactivateproteinkinaseC,anothercomponentofEGFsignaling.CrosstalkofEGFsignalingwithotherpathwaysmaketheEGFreceptorajunctionpointbet

40、weensignalingsystems.PlateletDerivedGrowthFactor(PDGF)playsacriticalroleincellularproliferationanddevelopment.ThebiologicallyactiveformisadimerformedfromtheAandBchains.PDGFisactivetoadifferingdegreedependingonwhichdimerisformed(AA,AB,orBB).ThePDGFReceptor(PDGFR)isalsoadimerandcanformfromthecombinati

41、onofthealphaandbetachainsinanyorder(alpha-alpha,alpha-beta,beta-beta).ThePDGFRdimerisonlyformedafterligandbindingsothealpha/betacompositionofthereceptorcanbeinfluencedbytheformofPDGFthatispresent.UponbindingofligandthePDGFRistyrosinephosphorylatedandleadstothephosphorylationofseveralothercellularpro

42、teins.1.Receptortyrosinekinases(RTKs),whichbindtopeptide/proteinhormones,mayexistasdimersordimerizeduringbindingtoligands.2.Ligandbindingleadstoactivationofthekinaseactivityofthereceptorandautophosphorylationoftyrosineresiduesinitscytosolicdomain.Theactivatedreceptoralsocanphosphorylateotherproteins

43、ubstrates.3.RasisanintracellularGTPaseswitchproteinthatactsdownstreamfrommostRTKs.LikeGsa,RascyclesbetweenaninactiveGDP-boundformandactiveGTP-boundform.Rascyclingrequirestheassistanceoftwoproteins,GEFandGAP,whereasGsacyclingdoesnot.SUMMARYofRTKs4.UnlikeGPCRs,whichinteractdirectlywithanassociatedGpro

44、tein,RTKsarelinkedindirectlytoRasviatwoproteins,GRB2andSos.5.TheSH2domaininGRB2,anadapterprotein,bindstospecificphosphotyrosinesinactivatedRTKs.ThetwoSH3domainsinGRB2thenbindSos,aguaninenucleotideexchangefactor,therebybringingSosclosetomembrane-boundRasGDPandactivatingitsexchangefunction.6.Bindingof

45、SostoinactiveRascausesalargeconformationalchangethatpermitsreleaseofGDPandbindingofGTP.7.Normally,RasactivationandthesubsequentcellularresponseisinducedbyligandbindingtoanRTK.However,incellsthatcontainaconstitutivelyactiveRas,thecellularresponseoccursintheabsenceofligandbinding.AllRas-linkedRTKsinma

46、mmaliancellsappeartoutilizeahighlyconservedsignal-transductionpathwayinwhichthesignalinducedbyligandbindingiscarriedviaGRB2andSostoRas,leadingtoitsactivation.ActivatedRastheninducesakinasecascadethatculminatesinactivationofMAPkinase.Thisserine/threoninekinase,whichcantranslocateintothenucleus,phosph

47、orylatesmanydifferentproteinsincludingtranscriptionfactorsthatregulateexpressionofimportantcell-cycleanddifferentiation-specificproteins.ActivationofMAPkinaseintwodifferentcellscanleadtosimilarordifferentcellularresponses,ascanactivationinthesamecellbystimulationofdifferentRTKs.SectionF:MAP(Mitogen-

48、activatedprotein)KinasePathways1.SignalsPassfromActivatedRastoaCascadeofProteinKinasesAremarkableconvergenceofbiochemicalandgeneticstudieshasrevealedahighlyconservedcascadeofproteinkinasesthatoperateinsequentialfashiondownstreamfromactivatedRas:1.ActivatedRasbindstotheN-terminaldomainofRaf,aserine/t

49、hreoninekinase.2.RafbindstoandphosphorylatesMEK,adual-specificityproteinkinasethatphosphorylatesbothtyrosineandserineresidues.3.MEKphosphorylatesandactivatesMAPkinase,anotherserine/threoninekinase.4.MAPkinasephosphorylatesmanydifferentproteins,includingnucleartranscriptionfactors,thatmediatecellular

50、responses.Inunstimulatedcells,mostRasisintheinactiveformwithboundGDP;bindingofagrowthfactortoitsRTKleadstoformationoftheactiveRasGTP.AsignalingcomplexthenisassembleddownstreamofRas,leadingtoactivationofMAPkinasebyphosphorylationofthreonineandtyrosineresiduesseparatedbyasingleaminoacid.Biochemicalstu