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R7R8WTsevenlessR7receptorisrequiredforUVresponsesevBossStrategy:movingfromRTKdown:discoveringSOSRASGDPRASGTPPiGTPGDPGAPtargetproteinActivestateInactiveRTKSonofSevenless:hyperactivemutationRogerandBenejee,Cell1990ThestoryaboutRonRoger-+Sev/RTK(lf)SevenlessSev(lf)+SOS(gf)R7fineSevSOSR7fateLeadstoModel:A:yesB:maynot.Drosophila:F1screenvsF2screenX**X**F3homozygotesmutagenXTMTM**F1F2screenmutagenX**F1F1screenTMAconversationwithRubinMikeSimon:landmarkmodifyscreenSimonetal.1991CellSevRASSOSR7Rougheye,noR7Mostlywt,small%defectsRougheyeRougheyeHighTLowTLowTLowTTsalleleX**F1F1screenTM+/-+/-SOSencodesanexchangefactorforGTPaseSimonetal.1991CellRASGDPRASGTPPiGTPGDPSOSGAPtargetproteinActivestateInactiveRTKQuickTime?andaGIFdecompressorareneededtoseethispicture.Movingfromreceptordown:biochemistryRASGDPRASGTPPiGTPGDPGAPtargetproteinActivestateInactiveRTKSOSDiscoveryofGRB2EGFRYEGFRLigandactivationY-PScreenlGT11proteinexpressionlibrary9GRBproteinsWhichoneisonemediatedRasactivation?Y-PDiscoveryofGRB2:complementaryworksbybiochemistsandgeneticistsAcDNAcloneencodinganovel,widelyexpressedprotein(calledgrowthfactorreceptor-boundprotein2orGRB2)containingonesrchomology2(SH2)domainandtwoSH3domainswasisolated.ImmunoblottingexperimentsindicatethatGRB2associateswithtyrosine-phosphorylatedepidermalgrowthfactorreceptors(EGFRs)andplatelet-derivedgrowthfactorreceptors(PDGFRs)viaitsSH2domain.Interestingly,GRB2exhibitsstrikingstructuralandfunctionalhomologytotheC.elegansproteinsem-5.Ithasbeenshownthatsem-5andtwoothergenescalledlet-23(EGFRlike)andlet-60(raslike)liealongthesamesignaltransductionpathwaycontrollingC.elegansvulvalinduction.ToexaminewhetherGRB2isalsoacomponentofrassignalinginmammaliancells,microinjectionstudieswereperformed.WhileinjectionofGRB2orH-rasproteinsaloneintoquiescentratfibroblastsdidnothavemitogeniceffect,microinjectionofGRB2togetherwithH-rasproteinstimulatedDNAsynthesis.TheseresultssuggestthatGRB2/sem-5playsacrucialroleinahighlyconservedmechanismforgrowthfactorcontrolofrassignaling.Lowensteinetal.Cell.Aug.1992Thesem-5story-early1992SEM-5wasidentifiedasbyasuppressormutantscreenClarketal.NatureMar1992lin-15(-)Multivulvasem-5(-)Vulvalesslin-15(-);sem-5(-)VulvalessRasSEM-5LIN-15EGFRLIN-15RasVulvalinductionKnownSince1990SEM-5ishomologoustoGRB-2.Combinethedatafromthetwofield:RasGRB2/SEM-5EGFRras(gf)Mutivulvasem-5(-)Vulvalessras(gf);sem-5(-)MultivulvaTherestofthestoryisthereinarushandclash-mid1993OlivierJP,RaabeT,HenkemeyerM,DicksonB,MbamaluG,MargolisB,SchlessingerJ,HafenE,Pawson.ADrosophilaSH2-SH3adaptorproteinimplicatedincouplingthesevenlesstyrosinekinasetoanactivatorofRasguaninenucleotideexchange,Sos.Cell.1993Apr9;73:179-91.EganSE,GiddingsBW,BrooksMW,BudayL,SizelandAM,WeinbergRA.AssociationofSosRasexchangeproteinwithGrb2isimplicatedintyrosinekinasesignaltransductionandtransformation.Nature.1993May6;363:45-51.Rozakis-AdcockM,FernleyR,WadeJ,PawsonT,BowtellD.TheSH2andSH3domainsofmammalianGrb2coupletheEGFreceptortotheRasactivatormSos1.Nature.1993May6;363:83-5.BudayL,DownwardJ.Epidermalgrowthfactorregulatesp21rasthroughtheformationofacomplexofreceptor,Grb2adapterprotein,andSosnucleotideexchangefactor.Cell.1993May7;73:611-20.ChardinP,CamonisJH,GaleNW,vanAelstL,SchlessingerJ,WiglerMH,Bar-SagiD.HumanSos1:aguaninenucleotideexchangefactorforRasthatbindstoGRB2.Science.1993May28;260:1338-43.SkolnikEY,BatzerA,LiN,LeeCH,LowensteinE,MohammadiM,MargolisB,SchlessingerJ.ThefunctionofGRB2inlinkingtheinsulinreceptortoRassignalingpathways.Science.1993Jun25;260:1953-5.BaltenspergerK,KozmaLM,CherniackAD,KlarlundJK,ChawlaA,BanerjeeU,CzechMP.BindingoftheRasactivatorsonofsevenlesstoinsulinreceptorsubstrate-1signalingcomplexes.Science.1993Jun25;260:1950-2.SimonMA,DodsonGS,RubinGM.SH3-SH2-SH3proteinisrequiredforp21Ras1activationandbindstosevenlessandSosproteinsinvitro.Cell.1993Apr9;73:169-77.ManySmallGTPaseshaveaconservedC-terminusRasislipidmodifiedforitsmembranebindingRASGDPSH2SH3SH3SOSSH3SH3SH2GRB2EGFRYRASGDPSH2SH3SH3SOSEGFRY-PRASGTPHalfwaydoneRASGDPRASGTPPiGTPGDPGAPActivestateInactiveRTKSOSGRB2Summaryoftheoriginalscreenslin-15(-),Muvulvalin-15(-);suppressor;Vulvalessmutagenwormbiochembindingtophosphorylatedtyosineofgrowthfactor:Grb2DiscoveryofGAP.RTKGRB2SOSRaswormLET-23SEM-5SOS-1LET-60flySevenlessDRKSOSRas1sev(lf),sev(lf);suppressor/enhancer;flyR7fatereducedmutagenR7fatebetterorworseGAPGAP-1Gap1Theholygrail:whatistheeffectorofRas?TheRaseffector1.MustinteractwiththeRaseffectordomain2.TheinteractionmustberequiredforRasfunction3.MustberequiredtointeractwithactivatedRas(oncoproteins).A:Yes,B:noRASGDPRASGTPPiGTPGDPEffectorproteinActivestateInactiveTheRaseffectordomainisdefinedby1.MutationsintheregionaffectRasbiologicalfunction2.Themutationshavenoeffectonotherbiochemproperties3.Thedomainisproposedtointeractwiththeeffector/targetQuickTime?andaGIFdecompressorareneededtoseethispicture.QuickTime?andaGIFdecompressorareneededtoseethispicture.1259116effector185164GAP=RaseffectorNature.April1988GAP=RaseffectorScience.April1988KeyargumentA.Thebiochemicalandgeneticc