TheN-TerminalHSDCIFMotifIsRequiredforCellSurfaceTraffickingandDimerizationofFamilyBGProteinCoupledReceptorPAC1
RongjieYu1*,XiaolingGuo1,JiapingZhong1,MeiLi1,ZhixingZeng1,HuahuaZhang21CellBiologyInstitute,theDepartmentofCellBiology,JinanUniversity,Guangzhou,China,2LaboratoryofMedicalGeneticsofGuangdongMedicalCollege,Dongguan,Guangdong,China
Abstract
PAC1isPACAP(pituitaryadenylatecyclase-activatingpolypeptide)preferringreceptorbelongingtoclassBGproteincoupledreceptor(GPCR)mediatingthemosteffectsofPACAP.TheimportantroleofGproteincoupledreceptorhomo/heteromerizationinreceptorfolding,maturation,trafficking,andcellsurfaceexpressionhasbecomeincreasinglyevident.Thebimolecularfluorescencecomplementation(BiFC)andbioluminescenceresonanceenergytransfer(BRET)assaywereusedinthisresearchtoconfirmthedimerizationofPAC1forthefirsttime.Thestructure-activityrelationshipfocusedontheN-terminalHSDCIFmotif,whichlocatesbehindthesignalsequenceandhashighhomologywithPACAP(1–6),wasassayedusingareceptormutantwiththedeletionoftheHSDCIFmotif.ThefluorescenceconfocalmicroscopeobservationshowedthatthedeletionoftheHSDCIFmotifimpairedthecelldeliveryofPAC1.TheresultsofBiFC,BRETandwesternblotindicatedthatthedeletionofHSDCIFmotifandthereplacementoftheCysresiduewithAlainHSDCIFmotifresultedinthedisruptionofreceptordimerization.AndtheexogenouschemicallysynthesizedoligopeptideHSDCIF(100nmol/L)notonlydown-regulatedthedimerizationofPAC1,inducedtheinternalizationofPAC1,butalsoinhibitedtheproliferationofCHOcellsexpressingPAC1stablyanddecreasedtheactivityofPACAPonthecellviability.AllthesedatasuggestedthattheN-terminalHSDCIFmotifplayedkeyroleinthetraffickingandthedimerizationofPAC1,andtheexogenousoligopeptideHSDCIFhadeffectsonthecellsignaling,traffickingandthedimerizationofPAC1.
Citation:YuR,GuoX,ZhongJ,LiM,ZengZ,etal.(2012)TheN-TerminalHSDCIFMotifIsRequiredforCellSurfaceTraffickingandDimerizationofFamilyBGProteinCoupledReceptorPAC1.PLoSONE7(12):e51811.doi:10.1371/journal.pone.0051811Editor:HubertVaudry,UniversityofRouen,France
ReceivedJune27,2012;AcceptedNovember6,2012;PublishedDecember21,2012
Copyright:?2012Yuetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(No.31100545),theScienceandTechnologyBureauofGuangdongProvince(2011B090400024)andtheNaturalScienceFoundationofGuangdongProvince(S2011010002931).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:rongjie_yu1123@163.com
Introduction
Theneuropeptidepituitaryadenylatecyclaseactivatingpoly-peptide(PACAP)isamemberofthevasoactiveintestinalpolypeptide(VIP)/secretingrowthhormone/eleasinghormone/glucagonsuperfamily,whichalsoincludessecretin,VIP,glucagon-likepeptide1(GLP-1)andglucose-dependentinsulinotropicpeptide(GIP)[1].PACAPelicitsvariousbiologicalactionsviathreetypesofG-protein–linkedreceptors,aPACAP-preferring(PAC1)andVIP-shared(VPAC1andVPAC2)receptorstermedaccordingtotheirrelativeaffinityforPACAPandVIP[1,2].PAC1receptorhasamuchhigheraffinityforPACAPthanforVIP,whereasVPACreceptorsrecognizeallpeptideswithsimilarhighaffinity[2].
PAC1andVPACsallbelongtoclassBGprotein-coupledreceptors(GPCR)family,whichareendogenouslyactivatedbylargepeptidehormones,includingmembersofthesecretin/glucagon/GHRHsuperfamily,parathyroidhormone(PTH),corticotropin-releasingfactor(CRF)andmembersofthecalcitoninfamily[3,4,5].ClassBisasmallfamilyofGPCRswithlowsequenceandstructuralhomologieswithothermembersoftheGPCRssuperfamily.IncommonwithothermembersofclassBGPCRs,PAC1showsaspecificstructuralfeature;alarge
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extracellularN-terminaldomainthatcontainsseveralconservedaminoacids,suchassixcysteines,ahydrophobicN-terminalsignalpeptideandseveralpotentialN-glycosylationsites,bothofwhichareconsideredcrucialforreceptorconformationandligandinteraction[3](showninFig.1A,B).Recently,ithasbeenshownthatthedimerizationoroligomerizationofGPCRmayaffectthephysiologicalandpharmacologicalprofilesofGPCR,suchastraffickingofnewlysynthesizedreceptorstothecellsurface,allostericmodulationofligandbinding,signalingspecificity,co-internalization,orcross-inhibitionofGPCRs.[6,7,8,9].Further-morethesecretinreceptor,VPAC1andVPAC2,whichalsoaremembersofclassBGPCRandhavecloserelationshipwithPAC1,havebeenreportedtoformoligomerization(homo-orhetero-)[10,11,12].WehypothesizedthatPAC1canformdimeroroligomer,buttillnow,thereisnodirectproofonthedimerizationortheoligomerizationofPAC1.Inthisresearch,weusedthetechniques:bioluminescenceresonanceenergytransfer(BRET)andbimolecularfluorescencecomplementation(BiFC),whichhavebeenacceptedinthedetectionofoligomerizationofGPCR[13],toverifythedimerizationofPAC1.
OurpreviousreporthasindicatedthattheN-terminalextracellulardomain,alsocalledthefirstextracellular(EC1)domainofPAC1,hasamotifconsistofsixaminoacidresidueHis-December2012|Volume7|Issue12|e51811
EffectsofHSDCIFonGPCRPAC1
Figure1.ThestructuresketchmapofPAC1(A,B).TheHSDCIFmotifisindicatedasred.ThehomologyanalysisbetweenPACAPandPAC1-EC1(C).TheredregionshowedtheHSDCIFmotifanditshighhomologuePACAP(1–6).TheconstructionofD-PAC1(D).ThegenecodingD-PAC1withthedeletionoftheHSDCIFmotifwasamplifiedusingover-lapPCR(ThearrowsrepresentedtheprimersusedtoamplifythegenescodingD-PAC1andPAC1.).
doi:10.1371/journal.pone.0051811.g001
Ser-Asp-Cys-Ile-Phe(HSDCIF)(indicatedinredinFig.1A,B),whichlocatesjustbehindtheputativesignalsequence,hasaveryhighhomologywithPACAP(1–6)(HSDGIF)[14].Thereisonlyoneresiduedifferent(Cysvs.Gly)betweentheHSDCIFmotifofPAC1andPACAP(1–6)(HSDGIF),whichisresponsiblefortheactivationofPAC1(Fig.1C)[15,16].AndthisCysresidueisnotbeincludedintheconservedsixcysteinesoftheextracellularN-terminaldomainofclassBGPCR(asshowninFig.1A,B)andisnotreportedtoformtheknowndisulfidebond.Thestructure-activityrelationshipfocusedonthisHSDCIFmotifattractedourgreatinterest.Inthisresearch,amutantPAC1withthedeletionoftheHSDCIFmotif(namedD-PAC1)andamutantPAC1withthereplacementofCysintheHSDCIFmotifwithAla(namedM-PAC1)wereconstructedandexpressedinChinesehamsterovary(CHO)cells.Bythisway,wewantedtodetecttheroleofthismotifanditseffectsonthepropertiesofPAC1,suchascellsurfacedeliveryanddimerization.
ComparedwiththeintactPAC1,thedeletionmutantofPAC1(D-PAC1)notonlylosttheabilityoftraffickingtothecellsurface,butalsolosttheabilitytoformdimer.TheexogenouschemicallysynthesizedoligopeptideHSDCIFinducedtheinternalizationofintactPAC1,disruptedthedimerizationofintactPAC1andinhibitedtheviabilityoftheCHOcellsexpressingPAC1stably.ItwasalsofoundthatM-PAC1wastraffickedtothecellmembrane
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normallybutfailedtoformdimer,whichindicatedthattheCysintheHSDCIFmotifplayedakeyroleinthedimerizationofPAC1.
MaterialsandMethodsMaterials
AllmaterialsforcellcultureandtransfectionreagentswerefromInvitrogen(Carlsbad,CA).ReagentsformolecularbiologicaltechniqueswereobtainedfromTakara(Dalian,China)andQIAGEN(Valencia,CA).TheoligopeptideHSDCIFandPA-CAP27weresynthesizedbyQiangraoBiologicalCompany(Shanghai,China).cDNAencodingmousePAC1(Normal/Hop)isoform(splicevariantwithnodeletioninEC1andwithahopinsertioninthethirdintracellularcytoplasmic(IC3)loop[17])wasfromGeneCopoeiaagentedbytheFunengGeneCompany(Guangzhou,China).TheeukaryoticexpressionvectorspEYFP-N(containingthegeneencodingyellowfluorescentprotein(YFP)),pRluc-N(containingthegeneencodingRenillaluciferase(Rlu))werepurchasedfromYingrunBiologicalCompany(Changsha,China).
PlasmidsandMutagenesis
ThegeneencodingthedeletionmutantPAC1withthedeletionoftheHSDCIFmotifnamedD-PAC1wasamplifiedbythree-step
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EffectsofHSDCIFonGPCRPAC1
PCRusingtheoverlapprimersasshowninFig.1DandTab.1.ThegeneencodingCysmutantPAC1(namedM-PAC1)withthereplacementoftheCysinHSDCIFmotifwithAlawasalsoamplifiedbytwo-stepPCRusingtheoverlapprimersasshowninTab.1.ForBRETstudies,theintactPAC1gene,theD-PAC1geneandtheM-PAC1genewereclonedintothevectorspEYFP-NandpRluc-NrespectivelytoconstructtherecombinantexpressionvectorsPAC-YFP,D-PAC-YFP,M-PAC-YFP,PAC-Rlu,D-PAC-RluandM-PAC-RluwhichmadethereceptorstaggedatthecarboxylterminuswitheitherYFP(usingasdonor)orRlu(usingasacceptor).ForBiFCstudies,thesequenceencodingtheN-terminal172aminoacidresiduesofYFPwasamplifyandaddedwiththeTAAterminationcodonbyPCR,andwassubclonedintotherecombinantexpressionvectorsPAC-YFP,D-PAC-YFPandM-PAC-YFPreplacingtheintactYFPgenetoproduceexpressionvectorsPAC-Y/N,D-PAC-Y/NandM-PAC-Y/N,whichallowedthereceptorstaggedatthecarboxylterminuswiththeN-terminal172aminoacidresiduesofYFP.AndtherecombinantvectorsPAC-Y/C,D-PAC-Y/CandM-PAC-Y/C,whichmadethereceptorstaggedatthecarboxylterminuswiththeC-terminal67aminoacidresiduesofYFPwereconstructedinthesameway.TheprimersforthealltheconstructionswereinTab.1.
FluorescenceConfocalMicroscopy
CellulartraffickingofreceptorconstructswereevaluatedbyvisualizingfluorescenceinCHOcellsexpressingYFP-taggedreceptorconstructs.ThetransfectedcellsgrownonPetridishweremountedonmicroscopicslides.YFPfluorescencewasacquiredusingappropriatespectralsettings(excitation,488nmargonlaser;emission,545nmfilter;pinholediameter2.3airyunits)oftheconfocalmicroscope(LSM510META;Zeiss,Thornwood,NY)equippedwithaPlan-Apochromat636/1.4numericalapertureoilobjective.ForthedetectiontheeffectsoftheoligopeptideHSDCIFonthetraffickingofPAC1,livecellstransfectedwithPAC-YFPinthedishweremountedonthemicroscopicslides,andfluorescentimageswerecollectedevery16sfortotal1120sstartingfromthetimeoftheadditionoftheoligopeptideHSDCIF(toafinalconcentrationof100nmol/L)on.
Immunofluorescence
TheexpressionofreceptorstaggedwiththeN-terminalfragmentofYFP(Y/N)ortheC-terminalfragmentofYFP(Y/C)wasdeterminedbyimmunofluorescence.Cellsexpressingthereceptorfusionproteinwerefixedwith4%(w/v)paraformalde-hyde(PFA)inPBSforamaximumof5minutesatroomtemperaturebeforebeingwashedtwicewithPBSandincubatedin3%(w/v)BSAinPBS(blockingsolution)foronehouratroomtemperature.Forcellsthatneededpermeabilization(toallowentryofanantibodythatrecognizesanintracellularepitope,e.g.BXP-21),a5minuteincubationin0.05%(v/v)TritonX-100inPBSwasincludedbeforetheblockingstep.Followingblocking,cellswerethenincubatedwithrabbitpolyclonalantibodyraisedagainstaminoacids61–115mappingwithinanN-terminalextracellulardomainofmousePAC1(1:100;SantaCruzBiotechnology,USA)for1hatroomtemperatureandthenwashedtwicewithPBS.Cellswerethenincubatedforafurther1hwithanAlexa594-conjugatedanti-rabbitantiserum(1:400).AfterwashingwithPBS,cellswereviewedusingtheOLYMPUSconvertedfluorescencemicroscopeIX71(Japan)withexcitation520630nmandemission595630nm.
CellCultureandTransfection
Chinesehamsterovary(CHO)celllineCHO-K1,whichhasbeenshowntoexpressneitherPACAPnorPAC1[18],wasusedforthetransientandstableexpressionofreceptorconstructs.CellsweregrowninDulbecco’smodifiedEagle’smedium(DMEM)supplementedwith10ttalcalfseruminahumidifiedatmosphereof95%air,5%CO2at37uC.ThecellsweretransfectedwithintactPAC1ordeletionmutantD-PAC1orCysmutantM-PAC1constructsusinglipofectamineLTXandOpti-MEMmedium(Invitrogen,CA)followingtheinstructionbook.Fortransientexpression,3ugoftotalplasmidDNAwastypicallyaddedintoeach10-cm2Petridish,cellswerestudied48to72haftertransfectiontoallowmaximallevelsofexpressionofrecombinantproteins.Forstableexpression,cellstransfectedwithplasmidswereselectedbasedonG418(0.8–1mg/mL)insensitiv-ity,thenwereclonedbysuccessivecyclesoflimitingdilutionandscreenedbythefluorescentsignal.CellsweremaintainedinDMEMmediumsupplementedwith10?Sand0.8mg/mLG418withanatmosphereof95%air,5%CO2at37uC.
BimolecularFluorescenceComplementation(BiFC)
TheBiFCassayisbasedonthereconstitutionofafluorescentproteinmoleculeuponre-associationofitstwononfluorescentfragments[19].IfYFPwascutintoN-terminal(173aminoacidresidues)andC-terminal(67aminoacidresidues)segments,neitherofthemdisplayedfluorescentpropertywhenexpressed
Table1.Oligonucleotideprimersemployed.
PrimerDPAC-YF-F1DPAC-YF-F2MPAC-YF-F1PAC/-YF-FPAC/-YF-RPAC/-Rlu-FPAC/-Rlu-RYF/N-FYF/N-RYF/C-FYF/C-R
Sequence59–39
CTGCTGCCTATGGCTATTGCTATGAAGAAGGAGCAAGCCATGTGCCTGGAGCTGCAGCTCTCCCTGACTGCTCTCCTCCTGCTGCCTATGGCTATTGCTATGTGACTGCTCTCCTCCTGCTGCCTATGGCTATTGCTATGCACTCTGACGCGATCTTCAAG
GAATTC_ATGGCCAGAACCCTGCAGCTCTCCCTGACTGCTCTCCTCCTGCTGCCGCGGGGTGGCCAAGTTGTCGGCCGGGAGGCT
CTCGAGATGGCCAGAACCCTGCAGCTCTCCCTGACTGCTCTCAAGCTTGGGTGGCCAAGTTGTCGGCCGGGAGGCTCCGCGGATGGTGAGCAAGGGCGAGGAGCTGTTC
GCGGCCGCTTACTCGATGTTGTGGCGGATCTTGAAGTTCCGCGGGACGGCAGCGTGCAGCTCGCCGACCACGCGGCCGCTTACTTGTACAGCTCGTCCATGCCGAG
Purpose
AmplificationofthegeneencodingD-PAC1byoverlapPCR(asshowninFig.1C)
AmplificationofthegeneencodingM-PAC1byoverlapPCR(GCGindicatedreplacingCyswithAla)
GenerationofPAC1,D-PAC1andM-PAC1taggedC-terminallywithcompleteYFP.(Restrictionsites,EcoRIandSacII)
GenerationofPAC1,D-PAC1andM-PAC1taggedC-terminallywithRlu.(Restrictionsites,XhoIandHindIII)
AmplificationoftheYFPN-terminal172AAandtaggingPAC1,D-PAC1andM-PAC1C-terminally.(Restrictionsites,SacIIandNotI)AmplificationoftheYFPC-terminal67AAandtaggingPAC1,D-PAC1andM-PAC1C-terminally.(Restrictionsites,SacIIandNotI)
doi:10.1371/journal.pone.0051811.t001
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EffectsofHSDCIFonGPCRPAC1
alone.Co-expressionofthesegmentslinkedtointeractingproteinsallowedthepartialreformationofYFPwiththeconcomitantappearanceofthefluorescentsignal.Cellswereco-transfectedwith3mgofreceptorDNAper10-cm2Petridish,dividedequallyamongtworeceptorconstructsforeachplasmidpair.And48hourssubsequently,cellswereimagedunderconvertedfluores-cencemicroscopeusingtheexcitation(480630nm)andemission(535625)filter.
Forstatisticanalysis,assayswereperformedforaliquotsofapproximately25,000cellsperwellin96-wellwhiteOptiplates.Inbrief,theCHOcellswereinnoculatedequablyin96-wellplateandtransfectedwiththeequalamountplasmids(1.0ugofDNA/56105dividedequallyintotworeceptorconstructsforeachplasmidpair).Thefluorescentsignalsfromthecells48hoursaftertransfectionweredetectedinVictor31420multi-labelcounter(PerkinElmer,Wellesley,MA)usingtheexcitation(480630nm)andemission(535640nm)filter.ThecellstransfectedwithD-PAC-YFPorM-PAC-YFPwereusedaspositivecontrol,andthecellwithouttransfectionasnegativecontrol.Theassaysforeachtransfectionwererepeatedatleastsixtimes.ForexaminationoftheeffectsoftheoligopeptideHSDCIFonthedimerization,thetransfectedCHOcellswereincubatedwitholigopeptideHSDCIF(100nmol/L)for2hat4uCbeforecollectingtheBiFCsignals.Allexperimentswererunwithatleastfourreplicatesinparallelandrepeatedsixtimes.
signalswerecollected.Allexperimentswererunwithatleastfourreplicatesinparallelandrepeatedsixtimes.
WesternBlotting
InordernottodestroythedimerofPAC1,SDS-PAGEwasconductedinnon-reducingcondition.Aftermixingthesampleswithloadingbufferwithoutreducingagentandboiling,bothcellmembranesamplesandcelllysatesweresubjectedtoSDS-PAGEanalysisusing4to12%Bis-Trisgels(NuPAGE;Invitrogen)andMOPSbuffer.Afterelectrophoresis,proteinsweretransferredontonitrocellulosemembranesthatwereincubatedin5%nonfatmilkand0.1%Tween20/PBSsolutionatroomtemperatureonarotatingshakerfor2htoblocknonspecificbindingsites.Themembranewasincubatedovernightwitharabbitpolyclonalantibodyraisedagainstaminoacids61–115mappingwithinanN-terminalextracellulardomainofmousePAC1(SantaCruzBiotechnology,USA)anddetectedusingahorseradishperoxi-dase-linkedanti-rabbitIgGsecondaryantiserum(GEHealthcare,LittleChalfont,Buckinghamshire,UK).Immunoblotsweredevelopedbytheapplicationofenhancedchemiluminescencesolution(PierceChemical,Rockford,IL).
CellProliferationAssay
ToinvestigatetheeffectsoftheoligopeptideHSDCIFandPACAPonthegrowthofCHOcellsexpressingPAC1stablynamedPAC1-CHOconstructedaspreviouslydescribed[21],thecells(26105cells/well)wereplatedin96-wellplatesovernightat37uCinDMEMwith0.5?S.ThenextdaythecellswereincubatedwithorwithoutgradientconcentrationsofHSDCIForPACAPintheabsenceofFBSfor24h.TheviabilityofthecellswasdeterminedusingacolorimetricMTTassay(Methylthiazo-letetrazoliumbromide,Sigma).ThisassayisbasedonthereductionofMTTintoablueformazandyebyviablemitochondria.Attheendofthe24htreatment,themediumwasdiscardedfromtheplates,andthecellsweresubsequentlywashedtwicewithPBS.ThecellswerethenincubatedwithPBScontaining0.5mg/mLMTTfor4hat37uCinanatmosphereof5%CO2.Thesolutionwasremovedcarefully,and1mLofdimethylsulfoxidewasaddedtodissolvetheblue-coloredformazanparticles.Thesamplesweretransferredtoa96-wellplate,andtheabsorbancewasmeasuredusinganELISAreader(Bio-Radmicroplatereader,Bio-Rad)at570nm,representingthevaluesinarbitraryunits(AU).Allexperimentswererunwithatleastfourreplicatesinparallelandrepeatedsixtimes,andtheresultsareexpressedasapercentageofthecontrol(treatmentwithoutanypeptides).InordertotesttheeffectsofHSDCIFontheactivityofPACAP,thecellsviabilitywasevaluatedinthepresenceofonedoseofHSDCIFpeptide(100nM)withPACAPingradientquantities(10to1000nM).Moreover,reverseexperimentwasperformedinthepresenceofonedoseofPACAP(100nM)withvariousdosesofHSDCIFpeptide(10to1000nM).
BioluminescenceResonanceEnergyTransferAssays(BRET)
TheCHOcellswereinnoculatedequablyin96-wellwhiteOptiPlateandsubmittedtoco-transfectionwithRluc-taggedandYFP-taggedreceptorconstructs.TheBRETassaywasinitiated48hoursaftertransfectionbyaddingthecell-permeantRlu-specificsubstratecoelenterazinehtothecellsuspensiontoyieldafinalconcentrationof5uMina96-wellwhiteOptiPlate.Lumines-cenceandYFPfluorescenceintensitieswerequantifiedinrepresentativealiquotsofthesamepopulationsofcellsutilizedineachsetofBRETstudies.TheBRETsignalwascollectedusingtheVictor31420multi-labelcounter(PerkinElmer,Wellesley,MA)withemissionfiltersetsforluminescence(460nm,bandwidth25nm)andfluorescence(535nm,bandwidth25nm).TheratiooffluorescencetoluminescenceemissionfromthecellstransfectedwithRlu-taggedreceptorconstructalone(1.0ugofDNA/56105)wasconsideredasbackgroundandusedforthedeterminationofcorrectfactor(Cf=Em535/Em460),whichdefinedtheamountofsignalintheacceptorportionthatwasattributabletodonorbioluminescence.TheBRETratiowascalculatedbasedontheratiooffluorescencetoluminescenceemissionusingthefollowingformula:(Em535–(Em4606Cf))/Em460.
BRETtitration(saturation)experimentswereperformedtovalidatetheobservationsinthestaticBRETstudiesimitatingthemethoddecribedbyHarikumarKG[20].Forthis,CHOcellsweretransfectedbothwithaconstantamountofdonorconstruct(Rlu-taggedreceptorconstructataconcentrationof1.0ugofDNA/56105cells)andwithincreasingamountsofacceptorconstruct(YFP-taggedreceptorconstructatconcentrationsof0.3to6.0ugofDNA/56105cells).TheBRETratioswereplottedagainsttheacceptor-to-donorratios.Curveswerefittothesedataandwereevaluatedforquality-of-fitbasedonR2valuesusingPrism3.0.Whenasinglephaseexponentialcurvewasfoundtorepresentasignificantlybetterfitthanthelinearfunction(Ftestdeterminationwithpvalue,0.05),itwasutilizedtocalculatetheBRETmaxandBRET50values.ForexaminationoftheeffectsoftheoligopeptideHSDCIF,thetransfectedCHOcellswereincubatedwith100nmol/Lfor2hat4uCbeforetheBRET
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StatisticalAnalysis
StatisticalanalysiswasperformedwithGraphPadPrism,usingtheunpairedttest.Differenceswithp,0.05wereconsideredtobestatisticallysignificant.
Results
ExpressionDifferencebetweenPAC1andD-PAC1inCHOCells
Theresultsoffluorescentconfocalimaging(Fig.2)showedthatPAC1wasnormallytraffickedtothecellsurfacewhenitwasexpressedtransientlyorstablyinCHOcells.Onthecontrary,D-December2012|Volume7|Issue12|e51811
