浙师大《细胞生物学》cha.ppt

Chapter11 Cell to CellSignaling A OverviewofExtracellularSignalingB SignalingviaHydrophobicMoleculesC SignalingviaIonChannelsD SignalingviaG Protein CoupledReceptorsE SignalingviaReceptorTyrosineKinasesF MAPKinasePathwaysG InteractionandRegulationofSignalingPathways A OverviewofExtracellularSignaling Communicationbyextracellularsignalsusuallyinvolvessixsteps 1 synthesisand 2 releaseofthesignalingmoleculebythesignalingcell 3 transportofthesignaltothetargetcell 4 detectionofthesignalbyaspecificreceptorprotein 5 achangeincellularmetabolism function ordevelopmenttriggeredbythereceptor signalcomplex and 6 removalofthesignal whichoftenterminatesthecellularresponse 1 Generalschemesofintercellularsignalinginanimals Inanimals signalingbyextracellular secretedmoleculescanbeclassifiedintothreetypesbasedonthedistanceoverwhichthesignalacts Inaddition certainmembrane boundproteinsononecellcandirectlysignalanadjacentcell Inendocrinesignaling signalingmolecules calledhormones actontargetcellsdistantfromtheirsiteofsynthesisbycellsofendocrineorgans Inparacrinesignaling thesignalingmoleculesreleasedbyacellonlyaffecttargetcellsincloseproximitytoit Inautocrinesignaling cellsrespondtosubstancesthattheythemselvesrelease Manygrowthfactorsactinthisfashion andculturedcellsoftensecretegrowthfactorsthatstimulatetheirowngrowthandproliferation a c Cell to cellsignalingbyextracellularchemicalsoccursoverdistancesfromafewmicrometersinautocrineandparacrinesignalingtoseveralmetersinendocrinesignaling d Proteinsattachedtotheplasmamembraneofonecellcaninteractdirectlywithreceptorsonanadjacentcell 2 ReceptorProteinsExhibitLigand BindingandEffectorSpecificity Thesignalingmolecule hormone pheromone orneurotransmitter actsasaligand whichbindsto or fits asiteonthereceptor intracellularreceptors andcell surfacereceptors Bindingofaligandtoitsreceptorcausesaconformationalchangeinthereceptorthatinitiatesasequenceofreactionsleadingtoaspecificcellularresponse 3 HormonesCanBeClassifiedBasedonTheirSolubilityandReceptorLocation Mosthormonesfallintothreebroadcategories 1 smalllipophilicmoleculesthatdiffuseacrosstheplasmamembraneandinteractwithintracellularreceptors and 2 hydrophilicor 3 lipophilicmoleculesthatbindtocell surfacereceptors Recently nitricoxide agas hasbeenshowntobeakeyregulatorcontrollingmanycellularresponses Somehormonesbindtointracellularreceptors others tocell surfacereceptors a Steroidhormones thyroxine andretinoids beinglipophilic aretransportedbycarrierproteinsintheblood Afterdissociationfromthesecarriers suchhormonesdiffuseacrossthecellmembraneandbindtospecificreceptorsinthecytosolornucleus Thereceptor hormonecomplexthenactsonnuclearDNAtoaltertranscriptionofspecificgenes b Polypeptidehormonesandcatecholamines e g epinephrine whicharewatersoluble andprostaglandins whicharelipophilic allbindtocell surfacereceptors Thisbindingtriggersanincreaseordecreaseinthecytosolicconcentrationofsecondmessengers e g cAMP Ca2 activationofaproteinkinase orachangeinthemembranepotential HydrophobicmoleculesIonchannelsG protein coupledreceptorsEnzymes e g RTKs Inmanycases thesignalcontinuestopropagatewithinthecellandoftenreachesnuclearDNAtoexpressproteins 4 Theexternalsignalmayenteracellviafourmajorpathways Majorpathwaysforsignalstoenteracell Cell SurfaceReceptorsBelongtoFourMajorClasses SectionB SignalingviaHydrophobicMolecules Hydrophobicmoleculescanmoveinandoutofcellsbypassingthroughlipidbilayers Nitricoxide arachidonicacidandsteroidshavebeenshowntoplayimportantrolesincellsignaling SignalingwithNOorarachidonicacid Unlikemostsignalingcascadeswhichoccurwithinthesamecell newlygeneratedNOorarachidonicaciddiffusestoactontargetmoleculesinneighboringcells Nitricoxide NO isproducedfromthefollowingreaction whereNOSrepresentsNOsynthase AfterNOisgeneratedinacell itdiffusestoactontargetmoleculesinneighboringcells NOmaystimulatesolubleguanylylcyclasetoproducecGMPwhichregulatesseveralenzymesandionchannels Insmoothmuscle animportantactionofcGMPistoinducemusclerelaxation Normally cGMPwillsoonbeconvertedintoGMPbyphosphodiesterase Thewellknowndrugforimpotence Viagra sildenafilcitrate inhibitsphosphodiesterase Nitricoxide Arachidonicacidisgeneratedfromphospholipidhydrolysiscatalyzedbyphospholipase Afterdiffusingtotargetcells arachidonicacidmayactivateproteinkinase resultinginphosphorylationoftargetmolecules Manyofitstargetmoleculesareinvolvedinlearningandotherneuronalactivities Arachidonicacid GenerationofarachidonicacidfromphospholipidbyphospholipaseA2 PLA2 SteroidsThemajorroleofsteroidsistoregulatetranscription sincemanysteroidreceptorsaretranscriptionfactors SectionC SignalingviaIonChannels Ionchannelsaremembraneproteinsthatallowionstopassthrough Intermsofionselectivity theyareclassifiedascalciumchannels sodiumchannels potassiumchannels etc Intermsofgating theymaybeclassifiedasvoltage gatedchannels ligand gatedchannels etc istoregulatemembranepotential However thecalciumionalsoplaysimportantrolesinothercellularfunctions sincemanyenzymesarecalcium dependent Throughvoltage activatedCa2 channelswhoseopeningprobabilitydependsonthemembranepotential ThroughIP3 sensitiveCa2 channelswhoseopeningprobabilityisregulatedbyIP3 inositoltrisphosphate TheopeningofCa2 storesintheendoplasmicreticulummayresultincalciumwaves Themajorroleofsodiumandpotassiumions ThemajorroleofG protein coupledreceptorsistotransmitsignalsintothecell Theyarecharacterizedbyseventransmembranesegments Thisclassofmembraneproteinscanrespondtoawiderangeofagonists includingphoton amines hormones neurotransmittersandproteins Someagonistsbindtotheextracellularloopsofthereceptor othersmaypenetrateintothetransmembraneregion SectionD SignalingviaG protein CoupledReceptors 1 G Protein CoupledReceptors2 GProteins3 Effectors www gpcr orgG Protein CoupledReceptorDatabase 1 G protein CoupledReceptors ThemajorroleofG protein coupledreceptorsistotransmitsignalsintothecell Thefullnameof Gprotein isGTP bindingproteinbecauseintheactivestateitbindstoGTP guanosinetriphosphate 2 GProteins TherearetwotypesofGproteins heterotrimericGproteinsandmonomericGproteins orsmallGproteins RasandRanaresmallGproteins G protein coupledreceptorsarecoupledtoheterotrimericGproteins ThestructureofheterotrimericGprotein consistingofthreesubunits a bandg Intheinactivestate GDP Basedonthedifferencesintheirgenes 20a 6band12gsubunitshavebeenidentified Theirmolecularweightsareinthefollowingranges asubunit 39 46kDbsubunit 35 39kDgsubunit 8kD CyclingofGproteinbetweenactiveandinactivestates InteractionbetweenGaandtheagonist stimulatedreceptorcausesthereleaseofGDP GTPthenbindstotheemptysitebecauseitsconcentrationinthecellishigherthanGDP 3 GProteinEffectors EffectorsarethetargetmoleculesofGproteinaorbgsubunit Thetableliststhemajoreffectorsoftheasubunit means activate and means inhibit Uponbindingofthecholeratoxin theGa boundGTPcannotbeconvertedintoGDPsothattheGproteinremainsintheactivestate Bycontrast pertussistoxinpreventsGDPreleasefromtheasubunitsothattheGproteinislockedintheinactivestate a BeforeagonistbindingtotheG protein coupledreceptor thethreesubunitsofGproteinareboundtogether b TheagonistbindingcausesinteractionbetweentheG protein coupledreceptorandtheGprotein c TheirinteractionresultsinthedissociationbetweenaandbgsubunitsoftheGprotein Theseparatedaand orbgsubunitsmaytheninteractwitheffectors SignalingviaG protein CoupledReceptors Thebgsubunitsmayactonadenylatecyclase phospholipaseA2 phospholipaseC ionchannels calciumATPaseetc AdenylatecyclaseAdenylatecylasecatalyzestheconversionofATPtocAMP whichisanimportant secondmessenger Secondmessengersarethesignalingmoleculesgeneratedbythestimulationofcell surfacereceptors Forexample cAMP arachidonicacid DAGandIP3generatedbytheactivationofG protein coupledreceptorsaresecondmessengers TheagonistswhichactivateG protein coupledreceptorsarefirstmessengers SignalingwithcAMP AcAMP PKAsignalingpathway PKAconsistsoftwocatalyticsubunitsandtworegulatorysubunits cAMPbindstotwositesoneachregulatorysubunit BindingoffourcAMPmoleculescausesthereleaseoffreeandactivecatalyticsubunits whichmayphosphorylateserineandthreonineresiduesontargetproteins Inthisfigure theactivesubunitphosphorylatesaCREBprotein resultingintranscription PhospholipaseC PLC alsocleavesphospholipids generatingdiacylglycerol DAG andIP3 inositol1 4 5 trisphosphate IP3canactivateanIP3 sensitiveCa2 channelinendoplasmicreticulum SignalingwithDAGandIP3 RTKsarethesecondmajortypeofcell surfacereceptors TheligandsforRTKsaresolubleormembrane boundpeptide proteinhormonesincludingnervegrowthfactor NGF platelet derivedgrowthfactor PDGF fibroblastgrowthfactor FGF epidermalgrowthfactor EGF andinsulin Bindingofaligandtothistypeofreceptorstimulatesthereceptor sintrinsicprotein tyrosinekinaseactivity whichsubsequentlystimulatesasignal transductioncascadeleadingtochangesincellularphysiologyand orpatternsofgeneexpression RTKsignalingpathwayshaveawidespectrumoffunctionsincludingregulationofcellproliferationanddifferentiation promotionofcellsurvival andmodulationofcellularmetabolism SectionE SignalingviaReceptorTyrosineKinases RTKs ActivationofRTKstransmitahormonesignaltoRas 1 LigandBindingLeadstoAutophosphorylationofRTKs 2 RasBelongstotheGTPaseSuperfamilyofIntracellularSwitchProteins RasisaGTP bindingswitchproteinthat liketheGasubunitsindifferentGproteins alternatesbetweenanactiveonstatewithaboundGTPandaninactiveoffstatewithaboundGDP Rasactivationisacceleratedbyaproteincalledguaninenucleotideexchangefactor GEF whichbindstotheRas GDPcomplex 3 AnAdapterProteinandGEFLinkMostActivatedRTKstoRas ActivationofRasfollowingbindingofahormone e g EGF toanRTK TheadapterproteinGRB2bindstoaspecificphosphotyrosineontheactivatedRTKandtoSos whichinturninteractswiththeinactiveRas GDP Theguaninenucleotideexchangefactor GEF activityofSosthenpromotesformationoftheactiveRas GTP Thephosphategroupofphosphotyrosine phosphorylatedtyrosine canformseveralhydrogenbondswiththeSH2 Srchomology2 domainofsomeproteinsinvolvedinthesignalingviatyrosinekinases ReceptorTyrosineKinases DomainstructureofsomeSH2 containingproteins Theepidermalgrowthfactor EGF peptideinducescellularproliferationthroughtheEGFreceptor whichhasatyrosinekinasecytoplasmicdomain asingletransmembranedomainandanextracellulardomaininvolvedinEGFbindingandreceptordimerization InhibitorsoftheEGFreceptorarebeingpursuedaspotentialcancertherapiesandEGFmaystimulatewoundhealing MutationoftheEGFreceptorhasbeenassociatedwithcancerinhumans TheproliferativeeffectsofEGFaresignaledthroughseveralpathways BindingofEGFresultsinEGFreceptordimerization autophosphorylationofthereceptor andtyrosinephosphorylationofotherproteins TheEGFreceptoractivatesrasandtheMAPkinasepathway ultimatelycausingphosphorylationoftranscriptionfactorssuchasc FostocreateAP 1andELK 1thatcontributetoproliferation ActivationofSTAT 1andSTAT 3transcriptionfactorsbyJAKkinasesinresponsetoEGFcontributestoproliferativesignaling PhosphatidylinositolsignalingandcalciumreleaseinducedbyEGFactivateproteinkinaseC anothercomponentofEGFsignaling CrosstalkofEGFsignalingwithotherpathwaysmaketheEGFreceptorajunctionpointbetweensignalingsystems PlateletDerivedGrowthFactor PDGF playsacriticalroleincellularproliferationanddevelopment ThebiologicallyactiveformisadimerformedfromtheAandBchains PDGFisactivetoadifferingdegreedependingonwhichdimerisformed AA AB orBB ThePDGFReceptor PDGFR isalsoadimerandcanformfromthecombinationofthealphaandbetachainsinanyorder alpha alpha alpha beta beta beta ThePDGFRdimerisonlyformedafterligandbindingsothealpha betacompositionofthereceptorcanbeinfluencedbytheformofPDGFthatispresent UponbindingofligandthePDGFRistyrosinephosphorylatedandleadstothephosphorylationofseveralothercellularproteins Receptortyrosinekinases RTKs whichbindtopeptide proteinhormones mayexistasdimersordimerizeduringbindingtoligands Ligandbindingleadstoactivationofthekinaseactivityofthereceptorandautophosphorylationoftyrosineresiduesinitscytosolicdomain Theactivatedreceptoralsocanphosphorylateotherproteinsubstrates RasisanintracellularGTPaseswitchproteinthatactsdownstreamfrommostRTKs LikeGsa RascyclesbetweenaninactiveGDP boundformandactiveGTP boundform Rascyclingrequirestheassistanceoftwoproteins GEFandGAP whereasGsacyclingdoesnot SUMMARYofRTKs UnlikeGPCRs whichinteractdirectlywithanassociatedGprotein RTKsarelinkedindirectlytoRasviatwoproteins GRB2andSos TheSH2domaininGRB2 anadapterprotein bindstospecificphosphotyrosinesinactivatedRTKs ThetwoSH3domainsinGRB2thenbindSos aguaninenucleotideexchangefactor therebybringingSosclosetomembrane boundRas GDPandactivatingitsexchangefunction BindingofSostoinactiveRascausesalargeconformationalchangethatpermitsreleaseofGDPandbindingofGTP Normally RasactivationandthesubsequentcellularresponseisinducedbyligandbindingtoanRTK However incellsthatcontainaconstitutivelyactiveRas thecellularresponseoccursintheabsenceofligandbinding AllRas linkedRTKsinmammaliancellsappeartoutilizeahighlyconservedsignal transductionpathwayinwhichthesignalinducedbyligandbindingiscarriedviaGRB2andSostoRas leadingtoitsactivation ActivatedRastheninducesakinasecascadethatculminatesinactivationofMAPkinase Thisserine threoninekinase whichcantranslocateintothenucleus phosphorylatesmanydifferentproteinsincludingtranscriptionfactorsthatregulateexpressionofimportantcell cycleanddifferentiation specificproteins ActivationofMAPkinaseintwodifferentcellscanleadtosimilarordifferentcellularresponses ascanactivationinthesamecellbystimulationofdifferentRTKs SectionF MAP Mitogen activatedprotein KinasePathways 1 SignalsPassfromActivatedRastoaCascadeofProteinKinases AremarkableconvergenceofbiochemicalandgeneticstudieshasrevealedahighlyconservedcascadeofproteinkinasesthatoperateinsequentialfashiondownstreamfromactivatedRas 1 ActivatedRasbindstotheN terminaldomainofRaf aserine threoninekinase 2 RafbindstoandphosphorylatesMEK adual specificityproteinkinasethatphosphorylatesbothtyrosineandserineresidues 3 MEKphosphorylatesandactivatesMAPkinase anotherserine threoninekinase 4 MAPkinasephosphorylatesmanydifferentproteins includingnucleartranscriptionfactors thatmediatecellularresponses Inunstimulatedcells mostRasisintheinactiveformwithboundGDP bindingofagrowthfactortoitsRTKleadstoformationoftheactiveRas GTP AsignalingcomplexthenisassembleddownstreamofRas leadingtoactivationofMAPkinasebyphosphorylationofthreonineandtyrosineresiduesseparatedbyasingleaminoacid Biochemicalstudiesshowedthesignalingpathwayappearstobealinearone activatedRTK Ras Raf MEK MAPkinase ActivationofRasinducesakinasecascade 2 KsrMayFunctionasaScaffoldfortheMAPKinaseCascadeLinkedtoRas Twoadditionalproteins 14 3 3andKsr participateintheMAPkinasecascadedownstreamfromRas Althoughtheprecisefunctionsoftheseproteinsarenotyetknown theybothappeartoplayimportantrolesinformingproteincomplexesnecessaryforsignalingfromRaftoMAPkinase Inarestingcellpriortostimulation Rafispresentinthecytosolinaninactiveconformationstabilizedbyadimerof14 3 3 Each14 3 3monomerbindstoaphosphoserineresidueinRaf andthedimerappearstohaveamoregeneralfunctioninlinkingtogethersignalingcomponentsthroughphosphoserineresidues ActivationofRafalsorequiresKsr whichcontainsbindingsitesforRaf 14 3 3 MEK andMAPkinase Ksrmayfunctionasanadapterprotein providingascaffoldforformationofalargesignalingcomplexthatcontinuestooperateafterRafdissociatesfromRas GDP AlthoughKsrhasakinasedomain geneticstudiesinDrosophilahaveshownthatthisdomainhasanegativeregulatoryfunction perhapsactingaspartofaswitchforregulatingtheturnoverofcomponentswithinthecomplex FuctionofKsr 3 PhosphorylationofaTyrosineandaThreonineActivatesMAPKinase StructuresofMAPkinaseinitsinactiveandactiveform BoththephosphorylatedtyrosineandthreonineresiduesinMAPkinaseinteractwithadditionalaminoacids therebyconferringanalteredconformationtothelipregion whichinturnpermitsbindingofATPtothecatalyticsite ThephosphotyrosineresidueplaysakeyroleinformingthebindingsiteforspecificsubstrateproteinsonthesurfaceofMAPkinase PhosphorylationpromotesnotonlythecatalyticactivityofMAPkinasebutalsoitsdimerization ThedimericformofMAPkinasecanbetranslocatedtothenucleuswhereitregulatestheactivityofanumberofnuclearlocalizedtranscriptionfactors 4 VariousTypesofReceptorsTransmitSignalstoMAPKinase Althoughyeastsandothersingle celledeukaryoteslackRTKs theyhavebeenfoundtopossessMAPkinasepathways Andindifferentcelltypesofhighereukaryotes stimulationofreceptorsotherthanRTKsalsocaninitiatesignalingpathwaysleadingtoactivationofMAPkinase ThematingpathwayinS cerevisiaeisawell studiedexampleofaMAPkinasecascadethatislinkedtoGproteincoupledreceptors inthiscasefortwosecretedpeptidepheromones theaandafactors 5 MultipleMAPKinasePathwaysAreFoundinEukaryoticCells Bothyeastsandhighereukaryoticcellscontainotherfunctionallyequivalentproteins includingtheJunN terminalkinases JNKs andp38kinasesinmammaliancellsandsixyeastproteins Alltheseproteinsareserine threoninekinasesthatareactivatedinthecytosolinresponsetospecificextracellularsignalsandcanbetranslocatedtothenucleus Thusinalleukaryoticcells bindingofawidevarietyofextracellularsignalingmoleculestriggershighlyconservedkinasecascadesculminatinginactivationofaparticularMAPkinase ThedifferentMAPkinasesmediatespecificcellularresponses includingmorphogenesis celldeath andstressresponses OverviewoffiveMAPkinasepathwaysinS cerevisiae EachpathwayistriggeredbyaspecificextracellularsignalandleadstoactivationofasingleMAPkinase whichmediatescharacteristiccellularresponses eachpathwayactivatesonlyoneMAPkinase Fus3inthematingpathway Kss1inthefilamentationpathway andHog1intheosmoregulationpathway Aswe vejustexplained Ste11isacomponentoftheyeastmating filamentation andosmoregulatoryMAPkinasepathways Acomplexsetofregulatoryinteractionsappearstolimitintracellularsignalinginresponsetoaparticularextracellularsignaltotheappropriatepathway HerewedescriberecentfindingsabouttwomechanismsthatcandeterminethespecificityofMAPkinasepathways 6 SpecificityofMAPKinasePathwaysDependsonSeveralMechanisms ToillustrateonewaymultipleMAPkinasesaresegregated weconsiderhowachangeinosmolarityactivatesSte11butdoesnotleadtoactivationofdownstreamcomponentsinth