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MethodsinCell Biology GProtein-CoupledReceptors: Signaling,Traffickingand Regulation Volume132 SeriesEditors LeslieWilson
DepartmentofMolecular,CellularandDevelopmentalBiology
UniversityofCalifornia
SantaBarbara,California
PhongTran
UniversityofPennsylvania
Philadelphia,USA&
InstitutCurie,Paris,France
MethodsinCell Biology GProtein-CoupledReceptors: Signaling,Traffickingand Regulation Volume132 Editedby
ArunK.Shukla
DepartmentofBiologicalSciencesandBioengineering, IndianInstituteofTechnology,Kanpur,India
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Contributors
AgnesM.AcevedoCanabal
InstituteofNeurobiology,UniversityofPuertoRicoMedicalSciencesCampus, SanJuan,PR,USA;DepartmentofAnatomyandNeurobiology,Schoolof Medicine,UniversityofPuertoRico,SanJuan,PR,USA
D.Agranovich
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
StefanAmisten
DiabetesResearchGroup,King’sCollegeLondon,London,UK
GabrielaAntunes
LaboratoryofNeuralSystems(SisNE),DepartmentofPhysics,Faculdadede FilosofiaCie ˆ nciaseLetrasdeRibeira ˜ oPreto,UniversidadedeSa ˜ oPaulo, Ribeira ˜ oPreto,Brazil
ChaitanyaA.Athale DivisionofBiology,IISERPune,Pune,India
NicolasAudet
DepartmentofPharmacologyandTherapeutics,McGillUniversity,Montreal,QC, Canada
MohammedAkliAyoub
BiologieetBioinformatiquedesSyste ` mesdeSignalisation,InstitutNationaldela RechercheAgronomique,UMR85,Unite ´ PhysiologiedelaReproductionetdes Comportements;CNRS,UMR7247,Nouzilly,France;LESTUDIUM LoireValley InstituteforAdvancedStudies,Orle ´ ans,France
R.Bar-Shavit
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
DamianBartuzi
DepartmentofSynthesisandChemicalTechnologyofPharmaceutical SubstanceswithComputerModellingLab,FacultyofPharmacywithDivisionof MedicalAnalytics,MedicalUniversityofLublin,Lublin,Poland
MaikBehrens
DepartmentofMolecularGenetics,GermanInstituteofHumanNutrition Potsdam-Rehbruecke,Nuthetal,Germany
NicolasF.Berbari
DepartmentofBiology,IndianaUniversity-PurdueUniversityIndianapolis, Indianapolis,IN,USA
He ´ le ` neBonin
DepartmentofBiochemistryandMolecularMedicine,InstituteforResearchin ImmunologyandCancer,Universite ´ deMontre ´ al,Montreal,QC,Canada
MichelBouvier
DepartmentofBiochemistryandMolecularMedicine,InstituteforResearchin ImmunologyandCancer,Universite ´ deMontre ´ al,Montreal,QC,Canada
AmitabhaChattopadhyay
CSIR-CenterofCellularandMolecularBiology,Hyderabad,India
LinjieChen
InstituteofBiochemistry,CollegeofLifeSciences,ZijingangCampus,Zhejiang University,Hangzhou,Zhejiang,China
SantiagoCuevas
DivisionofRenalDiseases&Hypertension,DepartmentofMedicine,TheGeorge WashingtonUniversitySchoolofMedicineandHealthSciences,WA,USA
FrancheskaDelgado-Peraza
InstituteofNeurobiology,UniversityofPuertoRicoMedicalSciencesCampus, SanJuan,PR,USA;DepartmentofAnatomyandNeurobiology,Schoolof Medicine,UniversityofPuertoRico,SanJuan,PR,USA
DominicDevost
DepartmentofPharmacologyandTherapeutics,McGillUniversity,Montre ´ al,QC, Canada
AntonellaDiPizio
InstituteofBiochemistry,FoodScienceandNutrition,TheRobertH.Smith FacultyofAgriculture,FoodandEnvironment,TheHebrewUniversity,Rehovot, Israel
ShaliniDogra
DivisionofPharmacology,CSIR-CentralDrugResearchInstitute,Lucknow,Uttar Pradesh,India
ZyanyaP.Espinosa-Riquer
DepartamentodeFarmacobiologı´a,CentrodeInvestigacio ´ nydeEstudios
AvanzadosdelIPN,Me ´ xicoD.F.,Mexico
TimothyN.Feinstein
DepartmentofDevelopmentalBiology,UniversityofPittsburghSchoolof Medicine,Pittsburgh,PA,USA
ColleenA.Flanagan
SchoolofPhysiologyandMedicalResearchCouncilReceptorBiologyResearch Unit,FacultyofHealthSciences,UniversityoftheWitwatersrand,WitsParktown, Johannesburg,SouthAfrica
AlexandreGidon
MolecularMechanismsofMycobacterialInfection,CenterforMolecular InflammationResearch,NorwegianUniversityofScienceandTechnology, Trondheim,Norway
ClaudiaGonza ´ lez-Espinosa
DepartamentodeFarmacobiologı´a,CentrodeInvestigacio ´ nydeEstudios
AvanzadosdelIPN,Me ´ xicoD.F.,Mexico
S.Grisaru-Granovsky
DepartmentofObstetricsandGynecology,ShaareZedek,Jerusalem,Israel
AylinC.Hanyaloglu
InstituteofReproductiveandDevelopmentalBiology,ImperialCollegeLondon, London,UK
TerenceE.He ´ bert
DepartmentofPharmacologyandTherapeutics,McGillUniversity,Montre ´ al,QC, Canada
MellisaM.Hege
DepartmentofBiology,IndianaUniversity-PurdueUniversityIndianapolis, Indianapolis,IN,USA
IlpoHuhtaniemi
InstituteofReproductiveandDevelopmentalBiology,ImperialCollegeLondon, London,UK
M.Jaber
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
KimC.Jonas
InstituteofReproductiveandDevelopmentalBiology,ImperialCollegeLondon, London,UK;InstituteofMedicalandBiomedicalEducation,StGeorge’s UniversityofLondon,London,UK
PedroA.Jose
DivisionofRenalDiseases&Hypertension,DepartmentofMedicine,TheGeorge WashingtonUniversitySchoolofMedicineandHealthSciences,WA,USA
ManaliJoshi
SavitribaiPhulePuneUniversity,Pune,India
AgnieszkaA.Kaczor
DepartmentofSynthesisandChemicalTechnologyofPharmaceutical SubstanceswithComputerModellingLab,FacultyofPharmacywithDivisionof MedicalAnalytics,MedicalUniversityofLublin,Lublin,Poland;Schoolof Pharmacy,UniversityofEasternFinland,Kuopio,Finland
A.Kancharla
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
RafikKaraman
BioorganicChemistryDepartment,FacultyofPharmacy,Al-QudsUniversity, Jerusalem,Israel
HiroyukiKobayashi
DepartmentofBiochemistryandMolecularMedicine,InstituteforResearchin ImmunologyandCancer,Universite ´ deMontre ´ al,Montreal,QC,Canada
AjeetKumar
DivisionofPharmacology,CSIR-CentralDrugResearchInstitute,Lucknow,Uttar Pradesh,India
ChristianLeGouill
DepartmentofBiochemistryandMolecularMedicine,InstituteforResearchin ImmunologyandCancer,Universite ´ deMontre ´ al,Montreal,QC,Canada
AnatLevit
DepartmentofPharmaceuticalChemistry,UniversityofCalifornia San Francisco,SanFrancisco,CA,USA
BinLu
InstituteofBiochemistry,CollegeofLifeSciences,ZijingangCampus,Zhejiang University,Hangzhou,Zhejiang,China
ViktoryaLukashova
DepartmentofBiochemistryandMolecularMedicine,InstituteforResearchin ImmunologyandCancer,Universite ´ deMontre ´ al,Montreal,QC,Canada
MarinaMacı´as-Silva
DepartamentodeBiologı´aCelularyDesarrollo,InstitutodeFisiologı´aCelular, UniversidadNacionalAuto ´ nomadeMe ´ xico,Me ´ xicoD.F.,Mexico
M.Maoz
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
DariuszMatosiuk
DepartmentofSynthesisandChemicalTechnologyofPharmaceutical SubstanceswithComputerModellingLab,FacultyofPharmacywithDivisionof MedicalAnalytics,MedicalUniversityofLublin,Lublin,Poland
JeremyC.McIntyre
DepartmentofNeuroscience,UniversityofFlorida,Gainesville,FL,USA;Center forSmellandTaste,UniversityofFlorida,Gainesville,FL,USA
MashaY.Niv
InstituteofBiochemistry,FoodScienceandNutrition,TheRobertH.Smith FacultyofAgriculture,FoodandEnvironment,TheHebrewUniversity,Rehovot, Israel;FritzHaberCenterforMolecularDynamics,TheHebrewUniversity, Jerusalem,Israel
CarlosNogueras-Ortiz
InstituteofNeurobiology,UniversityofPuertoRicoMedicalSciencesCampus, SanJuan,PR,USA
MelaniePhilipp
InstituteforBiochemistryandMolecularBiology,UlmUniversity,Ulm,Germany
CristinaRoman-Vendrell
InstituteofNeurobiology,UniversityofPuertoRicoMedicalSciencesCampus, SanJuan,PR,USA;DepartmentofPhysiology,SchoolofMedicine,Universityof PuertoRico,SanJuan,PR,USA
EwelinaRutkowska
DepartmentofBiopharmacy,FacultyofPharmacywithDivisionofMedical Analytics,MedicalUniversityofLublin,Lublin,Poland
JanaSelent
ResearchProgrammeonBiomedicalInformatics(GRIB),UniversitatPompeu Fabra,IMIM(HospitaldelMarMedicalResearchInstitute),Barcelona,Spain
DurbaSengupta
CSIR-NationalChemicalLaboratory,Pune,India
YingShi
InstituteofBiochemistry,CollegeofLifeSciences,ZijingangCampus,Zhejiang University,Hangzhou,Zhejiang,China
FabioMarquesSimoesdeSouza
CenterforMathematics,ComputationandCognition,FederalUniversityofABC, SaoBernardodoCampo,Brazil
MichalSlutzki
InstituteofBiochemistry,FoodScienceandNutrition,TheRobertH.Smith FacultyofAgriculture,FoodandEnvironment,TheHebrewUniversity,Rehovot, Israel
ChandanSona
DivisionofPharmacology,CSIR-CentralDrugResearchInstitute,Lucknow,Uttar Pradesh,India
KatarzynaM.Targowska-Duda
DepartmentofBiopharmacy,FacultyofPharmacywithDivisionofMedical Analytics,MedicalUniversityofLublin,Lublin,Poland
TeresaCasarTena
InstituteforBiochemistryandMolecularBiology,UlmUniversity,Ulm,Germany
B.Uziely
SharettInstituteofOncology,Hadassah-HebrewUniversityMedicalCenter, Jerusalem,Israel
GenaroVa ´ zquez-Victorio
DepartamentodeBiologı´aCelularyDesarrollo,InstitutodeFisiologı´aCelular, UniversidadNacionalAuto ´ nomadeMe ´ xico,Me ´ xicoD.F.,Mexico
Jean-PierreVilardaga
LaboratoryforGPCRBiology,DepartmentofPharmacology&ChemicalBiology, UniversityofPittsburghSchoolofMedicine,Pittsburgh,PA,USA
VanAnthonyM.Villar
DivisionofRenalDiseases&Hypertension,DepartmentofMedicine,TheGeorge WashingtonUniversitySchoolofMedicineandHealthSciences,WA,USA
RichardWargachuk
DepartmentofPharmacologyandTherapeutics,McGillUniversity,Montre ´ al,QC, Canada
KunhongXiao
LaboratoryforGPCRBiology,DepartmentofPharmacology&ChemicalBiology, UniversityofPittsburghSchoolofMedicine,Pittsburgh,PA,USA
PremN.Yadav
DivisionofPharmacology,CSIR-CentralDrugResearchInstitute,Lucknow,Uttar Pradesh,India
GuillermoA.Yudowski
InstituteofNeurobiology,UniversityofPuertoRicoMedicalSciencesCampus, SanJuan,PR,USA;DepartmentofAnatomyandNeurobiology,Schoolof Medicine,UniversityofPuertoRico,SanJuan,PR,USA
YapingZhang
InstituteofBiochemistry,CollegeofLifeSciences,ZijingangCampus,Zhejiang University,Hangzhou,Zhejiang,China
XiaoxuZheng
DivisionofRenalDiseases&Hypertension,DepartmentofMedicine,TheGeorge WashingtonUniversitySchoolofMedicineandHealthSciences,WA,USA
CynthiaZhou
DepartmentofPharmacologyandTherapeutics,McGillUniversity,Montre ´ al,QC, Canada
NaimingZhou
InstituteofBiochemistry,CollegeofLifeSciences,ZijingangCampus,Zhejiang University,Hangzhou,Zhejiang,China
Preface Gprotein coupledreceptors(GPCRs)alsoreferredasseventransmembrane receptors(7TMRs)lieattheheartofalmosteveryphysiologicalandpathophysiologicalprocessinourbody.Thesereceptorsbindtoandgetactivatedbyawide rangeofligandsrangingfromsmallmolecules,hormones,peptides,proteinsto lipids.TheoverallactivationandsignaltransductionmechanismsofGPCRsare highlyconservedwherebindingofanagonistresultsinaconformationalchange inthereceptorfollowedbyactivationofheterotrimericGproteinsandsubsequent generationofsecondmessengersanddownstreamsignaling.Downregulationof GPCRsisalsoprimarilyaconservedprocesswhereactivatedreceptorsare phosphorylatedbyGRKs(GPCRkinases)followedbybindingofbetaarrestins whichleadstoreceptordesensitizationandinternalization.GPCRsaretargetedby aboutone-thirdofthecurrentlyprescribeddrugswhichincludeangiotensinblockers forhypertension,beta-blockersforheartfailure,antihistaminesforallergymanagement,andopioidagonistsasanalgesicmedication.
InthisvolumeofMethodsinCellBiolo gy,wecovermultipleaspectsofGPCR signaling,trafficking,regulation,andcellularassaysinaformofeitheranovervieworasstep-by-stepprotocol.Thisisanefforttobringtogetherdifferent domainsofGPCRpharmacologyandsignalingontoacommonplatformandhighlighttheincrediblyversatilenatureanddiversefunctionalmanifestationof GPCRs.SectionIincludeschaptersonGPCRtraffickinginlipidraftsandcilia, imagingendogenousreceptorinneurons,singlemoleculeimagingofGPCRs, andacomprehensiveanalysisofGPCRsinadiposetissue.InSectionII,wecover topicsrangingfromGPCRsignalingfromendosomes,olfactoryreceptorsignal transduction,studiesofaspecializedGPCRsmoothenedinzebrafishmodel, andtheoutcomeofGPCRsignalingincytoskeletaldynamics.Inrecentyears,a keyfocusareainGPCRbiologyhasbeenthedevelopmentofnovelandmoresensitivecellularassaystoinvestigateGPCRexpression,signaling,anddownregulation.SectionIIIofthisvolumeisfocusedonGPCRassayswhichincludeclassical radioligandbinding,label-free,biosensorandfluorescence basedapproachesto studyGPCRtraffickingandsignaling,andTANGOassayformeasuringGPCRbeta-arrestininteraction.Finally,SectionIVconsistsofchaptersonstructural andcomputationalaspectsofprotease-activatedreceptors,bittertastereceptors, andGPCRdimerization.
Iwouldliketothankalltheauthorswhohavecontributedtothisfocusedvolume despitetheirbusyschedule.Ialsoexpressmysinceregratitudetothejournaleditorialstaffandproductionteamforawonderfuljobinputtingthisvolumetogetherina timelyfashion.Withthisbriefbackground,onbehalfoftheentireMethodsinCell
BiologyTeam,Ipresenttoyouthisvolumeentitled“GProtein CoupledReceptors: Signaling,Trafficking,andRegulation.”Isincerelyhopethatyouenjoythetopics coveredinthisissueandpleasefeelfreetoshareyourfeedbackwithus.
ArunK.Shukla IndianInstituteofTechnology,Kanpur,India
Localizationandsignaling ofGPCRsinlipidrafts 1 VanAnthonyM.Villar1,SantiagoCuevas,XiaoxuZheng,PedroA.Jose1
DivisionofRenalDiseases&Hypertension,DepartmentofMedicine,TheGeorgeWashington UniversitySchoolofMedicineandHealthSciences,WA,USA
1Correspondingauthors:E-mail:vvillar@gwu.edu;pjose@mfa.gwu.edu
CHAPTEROUTLINE 1.LocalizationofGPCRsinLipidRafts........................................................................6
1.1IsolationofLipidRafts............................................................................7
1.1.1Detergent-freemethod.......................................................................... 7
1.1.2Detergent-basedmethod...................................................................... 9
1.1.3Immunoblottinganddatainterpretation............................................... 10
1.2LocalizationofGPCRsinLipidRafts.......................................................11
1.2.1Cellsinsuspension.............................................................................
1.2.2Adherentcells....................................................................................
2.GPCRSignalinginLipidRafts...............................................................................15
2.1PerturbationofRaftStability..................................................................15
2.2ChangingtheCholesterolContent...........................................................16
2.3FluorescenceImaging............................................................................16
Abstract
Theunderstandingofhowbiologicalmembranesareorganizedandhowtheyfunction hasevolved.Insteadofjustservingasamediuminwhichcertainproteinsarefound, portionsofthelipidbilayerhavebeendemonstratedtoformspecializedplatformsthat fostertheassemblyofsignalingcomplexesbyprovidingamicroenvironmentthatis conduciveforeffectiveprotein proteininteractions.Gprotein-coupledreceptors (GPCRs)andrelevantsignalingmolecules,includingtheheterotrimericGproteins,key enzymessuchaskinasesandphosphatases,traffickingproteins,andsecondarymessengers,preferentiallypartitiontothesehighlyorganizedcellmembranemicrodomains, calledlipidrafts.Assuch,lipidraftsarecrucialforthetraffickingandsignalingof GPCRs.ThestudyofGPCRbiologyinthecontextoflipidraftsinvolvesthelocalization oftheGPCRofinterestinlipidrafts,atthebasalstateanduponreceptoragonism,and
MethodsinCellBiology,Volume132,ISSN0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.11.008
2016ElsevierInc.Allrightsreserved.
theevaluationofthebiologicalfunctionsoftheGPCRinappropriatecelllines.Thelack ofstandardizedmethodologytostudylipidrafts,ingeneral,andoftheworkingsof GPCRsinlipidrafts,inparticular,andtheinherentdrawbacksofcurrentmethodshave hamperedthecompleteunderstandingoftheunderlyingmolecularmechanisms.Newer methodologiesthatallowthestudyofGPCRsintheirnativeformareneeded.Theuseof complementaryapproachesthatproducemutuallysupportiveresultsappeartobethebest wayfordrawingconclusionswithregardstothedistributionandactivityofGPCRsin lipidrafts.
INTRODUCTION LipidRaftMicrodomains.Theplasmamembraneisasemipermeable,biological membranethatdemarcatestheintracellularmilieufromtheextracellularenvironment.Amphipathiclipids,suchasphospholipidsandsphingolipids,arethebuilding blocksofthesebilipidmembranesbecauseoftheiraggregativeproperties,i.e.,their hydrophobictailsassociatetogether,whiletheirhydrophilicheadsinteractwithboth extra-andintracellularaqueousenvironments(Sonnino&Prinetti,2013).The fluidityofthefattyacylgroupsofphospholipidsat37 Cenablesthemembranes toactasamediuminwhichdissolvedmembraneproteinsareaffordedamplelateral mobility,especiallyinresponsetoenvironmentalcues.Sincethefirstdescriptionof an“organizationofthelipidcomponentsofmembranesintodomains”(Karnovsky etal.,1982)andtheelaborationofthe“lipidrafthypothesis”bySimonsandvan Meer(vanMeer&Simons,1988;Simons&Ikonen,1997;Simons&vanMeer 1998),theexistenceoflipidraftsisnowestablished.
Lipidraftsaretightlypacked,highlyorganizedplasmamembranemicrodomains thatareenrichedinphospholipids,glycosphingolipids,andcholesterolandserveas aplatformfortheorganizationanddynamicinteractionofbiomoleculesinvolvedin variousbiologicalprocesses(Figure1).Thecholesterolbestowsasemblanceof rigidityandorderbyintertwiningintothehydrophobicgapsbetweenthephospholipidacylchains.Certainstructuralproteinsaboundinlipidraftstoserveasscaffold oranchorforotherproteins,includingcaveolins(Head,Patel,&Insel,2014;Quest, Leyton,&Pa ´ rraga,2004;Yu,Villar,&Jose,2013;Yuetal.,2004),flotillins (Rajendran,LeLay,&Illges,2007;Yuetal.,2004)andtetraspanins(Hemler, 2005),andglycosylphosphatidylinositol-linked(GPI-linked)proteins.Thespatial concentrationandorganizationofspecificsetsofmembraneproteinsallowgreater efficiencyandspecificityofsignaltransductionbyfacilitatingprotein protein interactionsandbypreventingcrosstalkbetweencompetingpathways.The nonhomogeneouslateraldistributionofmembranecomponentshelpsexplainthe differencesincompositionbetweenapicalandbasolateralmembranedomainsof polarizedepithelialcells(Sonnino&Prinetti,2013).
Thebestcharacterizedlipidraftmicrodomainsarethecaveolae,whichwerefirst describedbyPaladeandYamadainthe1950s(Palade,1953;Yamada,1955).These aresmall(60 80nm)invaginationsoftheplasmamembraneformedbythe polymerizationofcaveolinswithcholesterol(Parton&delPozo,2013).Caveolae
FIGURE1ALipidRaftMembraneMicrodomain.
Lipidraftsarehighlyorganizedplasmamembranemicrodomainsenrichedinphospholipids, glycosphingolipids,andcholesterol,andserveasmatrixforreceptors,suchasGproteincoupledreceptors(GPCRs),andothersignalingmolecules.(Seecolorplate) VanAnthonyM.Villar,MD,PhD.
havebeenimplicatedinavarietyofcellularprocesses,includingsignaltransduction,endocytosis,transcytosis,andcholesteroltrafficking(Barnett-Norris,Lynch,& Reggio,2005).Lipidraftsaccumulateintheapicalplasmamembraneinpolarized epithelialcellsandinaxonalmembranesinneurons.Basolateralanddendritic membranescontainlipidraftsbutinmorelimitedquantities(Simons&Ikonen, 1997).Interestingly,caveolaearefoundmostlyatthebasolateralmembranethat facesthebloodsupplyandismoreactiveduringsignaltransduction(Simons& Toomre,2000).Lipidraftsaremostlyfoundattheplasmamembrane;however, theymayalsobefoundinintracellularmembranesinvolvedinthebiosynthetic andendocyticpathways.Lipidraftmicrodomainsplayacrucialroleincellularprocessessuchasmembranesorting,receptortrafficking,signaltransduction,andcell adhesion.
GPCRSignalingandTrafficking.Gprotein-coupledreceptors(GPCRs) constitutethelargestsuperfamilyofseventransmembraneproteinsthatrespond toamyriadofenvironmentalstimulithataretransducedintracellularlyasmeaningfulsignalsthroughsecondarymessengers.AgoniststimulationofaGPCRleadstoa conformationalchangethatpromotestheexchangeofGDPforGTPontheGa subunitoftheGprotein,resultingintheuncouplingoftheGproteinfromtheGPCRand thedissociationofGa andGbg subunits.TheGa subuniteitheractivatesorinhibits intracellularsignalingpathwaysdependingonthereceptorsubtype,whiletheGbg subunitrecruitsGprotein-coupledreceptorkinaseswhichselectivelyphosphorylate serineandthreonineresidueslocalizedwithinthethirdintracellularloopand carboxyl-terminaltaildomainsofthereceptortopromotethebindingofcytosolic cofactorproteinscalledarrestins(Lefkowitz,1998).The b-arrestinsplayapivotal roleintheuncouplingprocessandinthesequestrationandinternalizationofGPCRs
throughadynamin-dependent,clathrin-mediatedendocytosis.Onceinternalized, theGPCRs,invesiclestermedasearlyendosomes,aresortedbysortingnexins andfollowdivergentpathways(Worby&Dixon,2002).Thereceptorsaresorted intorecyclingendosomesfortheirreturntothecellmembrane(recyclingand resensitization),accumulateinlateendosomeswhichtargetthelysosomesfortheir subsequentdegradation,ortransportedinitiallytotheperinuclearendosomes(transGolginetwork)andthentothelateendosomesforeventuallysosomaldegradation. Additionalproteolyticmechanisms,suchasproteasomesorcell-associatedendopeptidases,arealsoimplicatedinmediatingthedownregulationofcertainGPCRs (vonZastrow,2003).
ThesignaltransductionthatfollowsligandoccupationoftheGPCRishighly regulatedtoensurethespecificityofthecellularresponse,bothtemporallyand spatially.Thesignaltransductioncanbeattenuatedwithrelativelyfastkinetics throughaprocesscalleddesensitizationorthroughamuchslowerprocessofdownregulationfollowingprolongedorrepeatedexposuretoanagonist.Desensitization, orthewaningofareceptor’sresponsivenesstoagonistwithtime,isaninherent molecular“feedback”mechanismthatpreventsreceptoroverstimulationandhelps increatinganintegratedandmeaningfulsignalbyfilteringoutinformationfrom weakerGPCR-mediatedsignals(Ferguson,2001).
Itisaccomplishedthroughtwocomplementarymechanisms,i.e.,thefunctional uncouplingofGPCRsfromtheircognateGproteins,whichoccurswithoutany detectablechangeinthenumberofcellsurfacereceptors,andGPCRphosphorylation,sequestration,andinternalization/endocytosis.GPCRresensitizationprotects thecellsfromprolongeddesensitizationandiscarriedoutviadephosphorylation byphosphatasesastheGPCRtrafficsthroughtheendosomalpathway.GPCRactivityisthenetresultofacoordinatedbalancebetweenreceptordesensitizationand resensitization.
ItisnowestablishedthatlipidraftsserveasdynamicplatformsforGPCRsand pertinentsignalingmoleculessuchasGproteins,enzymes,andadaptors(BarnettNorrisetal.,2005;Lingwood&Simons,2010).However,understandingthe molecularmechanismsinvolvedhasbeenhamperedbythelackofstandardized methodologytostudylipidrafts,ingeneral,andoftheworkingsofGPCRsinlipid rafts,inparticular.Moreover,theminutesizeoflipidraftshasmadelipidrafts difficulttoresolvebystandardlightmicroscopy,unlessthelipidraftcomponents arecross-linkedwithantibodiesorlectins(Simons&Toomre,2000).Studying howGPCRworksinlipidraftsmaybeaccomplishedbydeterminingifthe GPCRofinterestlocalizestothelipidraftsandbyevaluatingifGPCRsignaling andactivityarelostwhenlipidraftsaredisrupted.
1. LOCALIZATIONOFGPCRsINLIPIDRAFTS SeveraltechniquesareavailableforthedetectionandlocalizationofGPCRsinlipid raftmicrodomainsincells.Themostcommonlyemployedapproachutilizescell
fractionationproceduresthatbreakthecellsapartanddestroycellmorphology beforeGPCRanalysisusingbiochemicalorimmunologicalassays.AcomplementarybiophysicalapproachinvolvesthevisualizationofGPCRsinintactcell membranes.
1.1 ISOLATIONOFLIPIDRAFTS Lipidraftsarecharacterizedbytheirrelativeinsolubilityinnonionicdetergentsat
4 Candlightbuoyantdensityonsucrosegradient(Schnitzer,McIntosh,Dvorak, Liu,&Oh,1995).Theisolationoflipidraftscanbeperformedusingeither detergent-basedordetergent-freemethods(Yuetal.,2013),withthelattergenerating agreaterfractionofinnerleafletmembraneraftsandproducingmorereplicable results(Pike,2004). Schnitzeretal.(1995) employedadetergent-freemethodto isolatelipidraftsusingcationiccolloidalsilicaparticles,whichisappropriatefor non-cellculturestudies.Lipidraftsmaybeextractedfromtotalcellmembranes (Songetal.,1996)orjustfromsurfaceplasmamembranes(Smart,Ying,Mineo,& Anderson,1995).Detergentinsolubilityresultsfromthesegregationofmembraneassociatedproteinsintothelipidrafts,whichareabundantincholesteroland glycosphingolipids.Nonionicdetergents,suchasTritonX-100, b-octylglucoside, CHAPS,deoxycholate,LubrolWX,LubrolPX,Brij58,Brij96,andBrij98,have beenusedtopreparelipidraftfractions(Macdonald&Pike,2005),resultingin varyingyieldsofproteins.Samplesobtainedbydetergent-basedmethodsaretermed detergent-resistantmembranesordetergent-insolublefractions.Differentdetergents mayyielddifferentlipidraftcomponentsbecauseofthevaryingdegreesofresistancebytheproteinstoextractionusingdifferentreagents.Themethodsdetailed belowarebasedon Yuetal.(2013)
1.1.1 Detergent-freemethod Materials
2-N-morpholinoethanesulfonicacid(Mes),250mM,pH ¼ 6.8
Mes-bufferedsolution(MBS),25mMMes þ 150mMNaCl
Sodiumcitrate,500mM,pH w 11(addproteaseinhibitors)
Sucrose,5%,35%,and80%inMBSsolution(addproteaseinhibitors)
Methyl-b-cyclodextrin(b-MCD),2%dissolvedincellculturemedia
Cholesterol þ b-MCD(Sigmacatalog#C4951),dissolvedincellculture media
1XPBS,forwashing
1. Cellcultureandcellpelletcollection.Toobtainsufficientamountsoflipidraft fraction,cellsshouldbegrownin150-mmdishesuntilalmostconfluentusing theappropriatemediaat37 Cwith95%airand5%CO2.Separatedishesof cellsshouldalsobetreatedforcholesteroldepletionandrepletionasexperimentalcontrols(Figure2).Cholesteroldepletiontodisruptthelipidraftsis commonlyperformedbypretreatmentwith b-MCDfor1hat37 C.
FIGURE2ComparisongroupsforGPCRlocalizationinlipidrafts.
Methyl-a-cyclodextrin(a-MCD)maybeusedascontrolfor b-MCD(Vial& Evans,2005).Cholesterolrepletionisperformedbypretreatingwithcholesterol/b-MCDsolutionfor1hat37 C.Cholestane-3,5,6-triol,aninactive analogofcholesterol,maybeusedascontrolfortheuseofexogenous cholesterol(Murtazina,Kovbasnjuk,Donowitz,&Li,2006).Todeterminethe effectofagonistorantagonisttreatment,cellsshouldbeserum-starvedforat least1hpriortotreatmenttoachieve“basal”conditionspriortotreatment. Additionalcontrols,suchastheuseofthedrugvehicle,shouldbeconcomitantlyperformed.
1.1 WashcellswithcoldPBSandscrapethecellsusingarubber-tippedcell scraper.
1.2 Transfercellsuspensioninto15-mLtubeandspinat2000 g for5min.
1.3 Decantthesupernatanttoobtainthecellpellet.
2. Cellhomogenatepreparation. Allstepsarecarriedoutat4 C.
2.1 Tothecellpellet,add1.5mL500mMsodiumcarbonateandvortex.
2.2 Homogenizethecellsuspensionbysonicationusingfive20-sburstsonice.
2.3 Add1.5mLof80%sucroseandmixbyvortexandsonication(three20-s bursts)onice.Proteinconcentrationmaybedeterminedatthistimeusinga BCAkit.
3. Sucrosegradientultracentrifugation.Prepare5%,35%,and80%sucrose solutionsinMBSsolution.TheuseofMBSsolutionwithpHcloseto7.0may beadvantageousformostproteins.
3.1 Place3mLofcellhomogenatesintothebottomofprecooled12-mL ultracentrifugetubes.
3.2 Overlaysequentially4.5mLof35%sucroseand4.5mLof5%sucroseto eachtube.
3.3 WiththetubessecurelybalancedinanSW-41bucket,spinat180,000 g (38,000rpm)for16hat4 CinaBeckmanSW-41centrifuge.
4. Lipidraftfractionpreparation.Alight-scatteringbandthatisenrichedwith caveolae/lipidraftscanbeobservedbetweenthe5%and35%sucrosegradients andcorrespondstothefourthfraction.
4.1 Carefullyaspirate121-mLfractionsfromthetopofthetubeandtransfer intoprelabeled1.5microcentrifugetubes.
4.2 Prepare0.5mLofeachfractionbyadding0.1mL6Xsamplebuffer,vortex, andboilfor5minbeforeuseforimmunoblotting.Thesesamplescanbe storedat 20 C,whiletherestofthefractionswithoutthe6Xsample buffercanbestoredat 80 C.
1.1.2 Detergent-basedmethod Materials
50%OptiprepStocksolution(45mLof60%Optiprep þ 9mLofOptiprep diluent)
MBSTSbuffer(MBS þ 0.5%TritonX-100 þ proteaseinhibitorsin10%sucrose)
Sucrosesolutions(Table1):
Table1 PreparationofOptiprepGradientSolutions
1. Cellcultureandcellpelletpreparation.Thesameaswiththedetergent-free method.
2. Cellextractpreparation.
2.1 Add0.3mLice-coldMBSTStocellpelletandpushthrougha25G needle10 .
2.2 Adjustcellextract(w0.4mL;cellpelletvolumeis w0.1mL)to40% Optiprepbyadding0.8mLofcold60%Optiprepandvortex.Determine proteinconcentrationusingaBCAkit.
3. Optiprepgradientultracentrifugation.
3.1 Place1mLofthecellextractintothebottomofprecooled5-mLultracentrifugetubes.
3.2 Overlaywith1mLeachof30%,25%,20%,and0%Optiprepsolutionsin MBSTSbuffer.
3.3 SecureeachtubeinaBeckmanSW50.1bucketandspinat175,000 g (42,000rpm)at4 Cfor4h.Otherrotorsmaybeused,suchastheSW55 (170,000 g for4h)orTLS55(250,000 g for2.5h).
4. Lipidraftfractionpreparation.
4.1 Carefullyaspirateten0.5-mLfractionsfromthetopofthetubeandtransfer intoprelabeled1.5microcentrifugetubes.
4.2 Prepare0.25mLofeachfractionbyadding0.5mL6Xsamplebuffer, vortex,andboilfor5minbeforeuseforimmunoblotting.Thesesamples canbestoredat 20 C,whiletherestofthefractionswithoutthe6X samplebuffercanbestoredat 80 C.
1.1.3 Immunoblottinganddatainterpretation Westernblotisthemostcommonlyusedmethodtodeterminethelipidraftdistributionofproteins,suchasGPCRs.Antibodyspecificityiscrucialfortheidentification oftheGPCRofinterest.Thelipidraftproteinsarefoundinthemorebuoyant fractions(top5 6fractions);however,theirdistributionamongthesefractionsis notuniform.Immunoblottingforlipidraftmarkersmayhelpindeterminingthe fractionswherethelipidraftsaremostabundant.Caveolin-1isthemostcommonly usedproteinmarkerforlipidrafts,specificallyforcaveolae(Inseletal.,2005; Lingwood&Simons,2010).Thereareseveralothermarkersforlipidrafts,such asflotillin-1,CD55,alkalinephosphatase,andpore-formingtoxins,suchascholera toxinsubunitB(CTxB),equinatoxinII,perfringolysin(Foster,DeHoog,&Mann, 2003;Salzer&Prohaska,2001;Skocajetal.,2013).Flotillin-1hasbeenusedasa lipidraftmarkerproteinincellsthatdonotcontaincaveolae,i.e.,bloodcells (Salzer&Prohaska,2001),neuralcells(Huangetal.,2007),andratrenalproximal tubulecells(Breton,Lisanti,Tyszkowski,McLaughlin,&Brown,1998;Riquier,Lee, &McDonough,2009)andhumanembryonickidney(HEK)-293cells(Yuetal., 2004).Thereisspeciesspecificitybecausehumanrenalproximaltubulecells expresscaveolin-1(Gildeaetal.,2009),whileHEK-293cellsexpresscaveolin-2. Thesemarkersmayalsobeusedtoindicatetheintegrityoflipidraftsincholesterol depletionorrepletionexperiments.Ingeneral,thesemarkersshouldbedistributedin themorebuoyantfractionsandshouldredistributeintothelessbuoyantfractions (fractions7 12)aftercholesteroldepletionwith b-MCD(Figure3).Cholesterol repletionreconstitutesthelipidraftsandthus,thesemarkersshouldbeobservedin themorebuoyantfractions.
FIGURE3LipidRaftDistributionofCaveolin-1andD1R.
Lipidraftandnon-lipidraftfractionsfromhumanrenalproximaltubulecellstreatedwith b-MCD,acholesterol-depletingandlipidraft-disruptingagent,werepreparedbydetergentfreemethodandsucrosegradientultracentrifugation.Thedistributionofcaveolin-1,alipid raftmarker,andthedopamineD1 receptor(D1R),aGPCR,isshownintheimmunoblots. ImagesarecourtesyofPeiyingYu,MD.
1.2 LOCALIZATIONOFGPCRsINLIPIDRAFTS AnotherwaytodemonstratethedistributionofGPCRsinlipidraftsisbyvisualizing theminintactcells,livingorfixed,andtissues.TherearenowcommerciallyavailablekitsthathavebeendevelopedforlabelingthelipidraftsusingtheCTxBthatis taggedwithfluorophores(Figure4).CTxBbindstothepentasaccharidechainof gangliosideGM1,whichselectivelypartitionsintolipidrafts.Forvisualizinglipid rafts,cellsarelabeledwithCTxBtaggedwithAlexaFluor 488,AlexaFluor
FIGURE4ColocalizationoftheD1 dopaminereceptor(D1R)inLipidRaftsofHumanRenal ProximalTubuleCells.
Humanrenalproximaltubulecellsweregrownonapoly-L-Lysine-coatedcoverslipto50% confluenceandserum-starvedfor1htodeterminethebasaldistributionofD1Rpriorto fixationwith4%paraformaldehydeandpermeabilizationwith0.5%TritonX-100.Thelipid raftswerelabeledusingcholeratoxinsubunitB(CTxB)taggedwithAlexaFluor 555 (MolecularProbes),whiletheendogenousD1Rwasimmunostainedusingaproprietary rabbit-anti-D1Rantibodyandadonkeyanti-rabbitsecondaryantibodytaggedwithAlexa Fluor 488(MolecularProbes).DAPIwasusedtovisualizethenucleus.Atthebasalstate, mostoftheD1Rwerefoundintracellularly,justbelowtheinnerleafletoftheplasma membrane,althoughsomecolocalizedwiththelipidrafts(yellowareaspointedatbyarrows). Therawimageswerecapturedvialaserscanningconfocalmicroscopeusingseparate channelsandthecompositeimagewasobtainedusingZen2011software.630X magnification,scalebar ¼ 10 mm.(Seecolorplate)
VanAnthonyM.Villar,MD,PhD.
555,orAlexaFluor 647beforecross-linkingwithananti-CTxBtomaintainthe insitu proteindistribution.TodemonstratethelipidraftdistributionofGPCRs,colocalizationexperimentsmaybeperformedvialaserscanningconfocalmicroscopyby labelingthelipidraftsusingCTxBandimmunostainingtheGPCRofinterestusing specificantibodiesonthesamecell.CTxBlabelingmayalsobeusedtodemonstrate lipidraftendocytosisuponagoniststimulationinlivecells(Qi,Mullen,Baker,& Holl,2010)andculturedexplants(Hansenetal.,2005).Thec-subunitofcytolethal distendingtoxin(cdt)mayalsobeutilizedforlipidraftcolocalizationexperiments (theprotocolisdetailedin Boesze-Battaglia,2006).Otherpore-formingtoxins,besidesCTxB,usedtovisualizelipidraftsincludeequinatoxinIIwhichbindsdispersed sphingomyelin,lyseninwhichbindsclusteredsphingomyelin,perfringolysinOwhich bindstocholesterol,andostreolysinwhichbindstothecombinationofsphingomyelin andcholesterol(Makinoetal.,2015;Skocajetal.,2013).
AnalternativetousingCTxB,cdt,andotherpore-formingtoxinsistouse antibodiesthatspecificallytargetthelipidraftproteinmarkers,suchas caveolin-1,caveolin-3,andflotillin-1.Conversely,transferrinreceptors,CD71, andgeranylatedproteinsarenon-lipidraftmarkers( Boesze-Battaglia,2006; Magee,Adler,&Parmryd,2005 ).ThegangliosideGM1 maybelabeledwithsingle quantumdotstomeasurethelateralmo bilityandextentofmovementofthelipid rafts(Chang&Rosenthal,2012 ).Recently,GPI-anchoredproteinsthatsegregate intolipidraftshavebeenvisualizedusin ganovelmethodcalledenzyme-mediated activationofradicalsources( Miyagawa-Yamaguchi,Kotani,&Honke,2015 ). Probesthattargetthelipidcontentoflipidraftshavealsobeenusedtovisualize thesemembranemicrodomains.Laurdan(6-dodecanoyl-2-(dimethylamino)naphthalene)andC-laurdan(6-dodecanoyl-2-[N-methyl-N-(carboxymethyl) amino]-naphthalene),whicharemembra neprobesthataresensitivetomembrane polarity,allowtheobservationoflipidraftsviatwo-photonmicroscopy(Gaus, Zech,&Harder,2006;Kimetal.,2007,2008 ).Afluorophore-taggeddomain D4ofperfringolysinO,acholesterol-bindingcytolysinproducedby Clostridium perfringens ,hasbeenusedasprobetostudymembranecholesterol(OhnoIwashitaetal.,2004).
Asidefromconfocalmicroscopy,othe rbiophysicalapproachesmayalsobe employedtostudylabeledGPCRsand/orli pidrafts.Singlefluorophoretracking microscopy( Schu ¨ tz,Kada,Pastushenko,&Schindler,2000 )andfluorescence recoveryafterphotobleaching(Kenworthy,2007 )maybeusedtomonitorlateral diffusionoflipidraft-anchoredGPCRs,w hilefluorescence lifetimeimaging microscopy fluorescenceresonanceenergytransfer(FLIM-FRET)(Kenworthy, Petranova&Edidin,2000;Thaa,Herrmann,&Veit,2010 )maybeusedtodeterminetheproximityofGPCRswithotherproteinsofinterest,oroflipidraftsizes dependingonmembranecomposition( deAlmeida,Loura,Fedorov,&Prieto, 2005).Atomicforcemicroscopymaybeused tovisualizetheeffectsofdetergent solubilizationofmembranesduringlipidraftstudies(Garner,Smith,&Hooper, 2008).Lipidraftscannowbevisualizedusingsuperresolutionimagingbelow the200nmlimitofconventionalmicroscopes,e.g.,includingstructured
illuminationmicroscopy,stimulatedemissiondepletion(STED)microscopy,nearfieldscanningopticalmicroscopy,pho toactivatedlocalizationmicroscopy (PALM),andstochasticopticalreconstructionmicroscopy(dSTORM)(Owen& Gaus,2013;Tobinetal.,2014;Wuetal.,2013 ).
Materials
Vybrant LipidRaftLabelingKits(Catalog#V-34403,V-34404,orV-34405) preparefreshworkingsolutionsaccordingtomanufacturer’sinstructions
PrimaryantibodyagainsttheGPCRofinterest
Secondaryantibodyagainstthehostoftheprimaryantibody
10%bovineserumalbumin(BSA)solution
4%ParaformaldehydeinPBS
Mountingmedium(EMScatalog#17985)without40 ,6-diamidino-2phenylindole(DAPI)
DAPI,anuclearstain,10mMstocksolution
TritonX-100,20%stocksolutionindeionizedwater 1XPBSforwashing
1.2.1 Cellsinsuspension ColocalizationofGPCRswithlipidraftscannowbeaccomplishedwiththeconcomitantuseofCTxBandanantibodyagainsttheGPCRofinterestoncells.Thecellscan belabeledinsuspensionandthenmountedonglassslidesforimaging,orthecellscan begrownandlabeledoncoverslipsorinTranswells cellcultureinsertswhencell polarityisimportanttodistinguishbetweenapical versus basolateralmembranes.
1. Fluorescentlabelingofcells.
1.1 Spincellsat2000 g for5minanddecantthemedium.
1.2 Resuspendthecellsincoldmedium,spin,anddecantthemedium.
1.3 Resuspendthecellsin2mLofCTxB AlexaFluor workingsolutionat 4 Cfor10min.TheprimaryantibodyagainsttheGPCRofinterestmay beaddedtothisworkingsolutionat1:100dilution.Theprimaryantibody againsttheGPCRshouldberaisedinmouse,goat,rat,orchickenbutnotin rabbitwhenusingtheVybrant LipidRaftLabelingKits.Alternatively, theprimaryantibodyagainsttheGPCR(especiallyifonlyarabbitantibodyisavailable)maybeprelabeledwithaFluorotherthantheoneused forCTxB.Directlylabelingtheprimaryantibodyprecludestheuseofa secondaryantibody(instep1.5).
1.4 Gentlywashcells3 withcoldPBS.Spincellsanddecantwashbuffer.
1.5 Resuspendin2mLoftherabbitCTxBantibodyworkingsolutionat4 C for30min.TherabbitCTxBantibodycross-linksittothelipidraftdomains.Thesecondaryantibodyagainsttheprimaryantibodymaybeadded tothisworkingsolutionat1:100dilution.Thesecondaryantibodyshould betaggedwithaFluorotherthantheoneusedtolabeltheCTxB. Ascounterstain,300nMDAPImayalsobeaddedtothisworkingsolution.
1.6 Gentlywashcells3 withcoldPBS.Spincellsanddecantwashbuffer.
2. Mountingandimaging.
2.1 (Optional)Fixcellswith4%paraformaldehydeatroomtemperaturefor 15min.Paraformaldehydeisacross-linkerfixativethatpreservesthe architectureofthecellbutmayreducetheantigenicityofsomecell componentsandthus,requiresanadditionalpermeabilizationstepif additionalintracellularproteinsareneededtobevisualized.Fixationmay alsobeachievedusingorganicsolvents,suchasalcoholsandacetone,but theseremovelipidsandprecipitatetheproteinsandoftendisruptthecell structure.
2.2 MountlivecellsincoldPBSorfixedcellsinmountingmediumonglass slideandcoverwithcoverslip.
2.3 Imagethecellsusingalaserscanningconfocalmicroscope.Theappropriate filtersshouldbeuseddependingontheAlexaFluor dyethatwasusedand whetherDAPIwasusedasanuclearstainornot(Table2).
1.2.2 Adherentcells
1. Cellcultureoncoverslips.
1.1 Growcellson12-mmcoverslipsplacedina24-welltissuecultureplateto w50%confluenceusingcompletecellculturemediumat37 Cin95%air and5%CO2.Coverslipscoatedwithlysine,laminin,orcollagenmay improvecellattachmentforcellsthateasilydetach,suchasHEK-293cells. Todeterminetheeffectofagonist/antagonisttreatmentonGPCR trafficking,cellsshouldbeserum-starvedforatleast1hpriortotreatment toachieve“basal”conditionspriortotreatment.Additionalcontrols,such asvehicletreatment,shouldbeperformed.
1.2 DrawoffthemediumandwashcellswithcoldPBS.Placethecellculture plateonicetostopfurtherreceptorendocytosisandendosomaltrafficking.
2. Fluorescentlabeling,fixation,andpermeabilization.
2.1 Add0.3mLofCTxB AlexaFluor workingsolutionat4 Cfor10min.
2.2 DrawoffthesolutionandwashcellswithcoldPBS.
2.3 Fixcellswith0.3mLof4%paraformaldehydeatroomtemperaturefor 15min.
2.4 WashcellswithPBS.Subsequentstepscanbeperformedatroom temperature.
Table2 FluorescenceSpectraofCTxBConjugates
AlexaFluor 488(V-34403)495/519 AlexaFluor 555(V-34404)555/565 AlexaFluor 594(V-34405)590/617
ThemaximumabsorptionandemissionforDAPIare358/461nm.
2.5 Permeabilizethecellswith0.3mLof0.5%TritonX-100indeionizedwater for10min.Permeabilizationprovidesaccesstointracellularantigens. TritonX-100caneffectivelysolvatecellularmembraneswithoutdisturbingprotein proteininteractions.Otherdetergentssuchassaponin, Tween-20,orsodiumdodecylsulfatemayalsobeused.
2.6 WashcellswithPBS.
3. Immunostaining.
3.1 Add0.3mLoftheprimaryantibodyagainsttheGPCRofinterestdissolved in10%BSA(1:100 200dilution)for30 60min.
3.2 Washcells3XwithPBS.
3.3 Add0.3mLofthesecondaryantibody(againstthehostoftheprimary antibodyusedinstep3.1)in10%BSA.Thesecondaryantibodyshouldbe taggedwithaFluorotherthantheoneusedtolabeltheCTxB.Ascounterstain,300nMDAPImayalsobeaddedtothisworkingsolution.
3.4 Wash2XwithPBSandoncewithdeionizedwater.Theuseofdeionized waterwashesawaytheresidualNaClcrystalsfromPBS.
3.5 Mountcoverslipsusingamountingmediumonglassslide.Gentlyremove excessmountingmediumbyaspiration.Allowthemountingmediumto hardencompletely.
3.6 Imagethecellsusingalaserscanningconfocalmicroscope.Theappropriate filtersshouldbeuseddependingontheAlexaFluor dyethatwasusedand whetherDAPIwasusedasanuclearstain.
2. GPCRSIGNALINGINLIPIDRAFTS TherearemanyestablishedprotocolsavailablethatallowthestudyofGPCRactivity perse usingcommerciallyavailablekitsor,lesscommonly,proprietarymaterials. StudyingtheactivityofGPCRsinthecontextoftheirresidencyinlipidraftsoften requiresadditionalstepsthatwoulddisrupttheintegrityofthelipidraftmicrodomainordissociatetheproteinofinterestfromtherafts.Mostofthecurrentstrategies todisruptlipidraftinvolveseitherperturbationoftheraftstabilityormodifyingthe cholesterolcontentofthelipidrafts.Mostofthesetreatmentsareperformedoncells priortoagonist/antagonisttreatmentandfunctionalassays,suchascAMPproduction,sodiumtransport,andNADPHoxidaseactivity(Gildeaetal.,2009;Hanetal., 2008;Yuetal.,2004,2014).
2.1 PERTURBATIONOFRAFTSTABILITY Lipidraftsaredynamicassembliesofphospholipidsandglycosphingolipidsthat containmostlysaturatedhydrocarbonchainswhichallowcholesteroltointercalate betweenthefattyacylchains.Thesurroundingmembranehasgreaterfluidity becauseofthepreponderanceofphospholipidswithunsaturatedacylgroups.The additionofexogenousgangliosides(Webb,Hermida-Matsumoto,&Resh,2000)
