Internalizationofreceptorismeasuredasthereductionofreceptorsontheplasmamembranesurface.Surfacereceptorsarelabeledwithfluoresentantibodieswhichisinturnmeasuredusingaflowcytometer.TheantibodiesrecognizeanepitopeengineeredontotheN-terminusoftheGPCR.

ThefollowingprotocolisoptimizedforinternalizationofFLAG-taggedbeta2-adrenergicreceptor(B2AR)stablytransfectedintoHEK293cells.

Protocol

1)Growupreceptorsin37degCincubatoruntilconfluentonto6-cmdishes.

2)Filldisheswith2mLgrowthmediawith30mMHEPES,pH7.4.Setasideadishof293/B2ARat4degC.Youwillusethisdishtoestimatethetotalnumberofreceptorsonthesurfaceofasample(i.e.beforeagoNISTtreatment).HEPESreducesexposureofthecellstoextremepHvaluesoutsideoftheincubator.

3)Addagonisttodishesandplaceinincubator.

4)Attheappropriatetimepoints,removedishesfromincubatorandchillcellsonice.NOTE:Inordertopreventadditionalendocytosisofreceptors,youmustperformallofthefollowingproceduresonabedoficeorinthecoldroom.

5)Rinsealldisheswithice-coldPBS,includingthe293/B2ARsampleleftonice.

6)Harvestcells-rinsebrieflywithice-coldPBSCa-free,Mg-free,0.04%EDTA-add2.5mLice-coldCDBtoliftcells.

CDB-CellDissociationBuffer,Enzyme-free,PBS-based(GibcoBRLLifeTechnologies)Ourcampusflowcytometryfacilitymanagerrecommendedthisbufferforliftingcellsandreducingcellaggregates.However,theusercanprobablyalsosubstitutePBSCa-free,Mg-freeplus0.04%EDTAtoliftcells.

7)Pelletcellsincentrifugesetat4degCandrinse1xwithmedia.NOTE:Sincetheanti-FLAGM1antibody(Sigma,Covance)requiresCaionstobindtheFLAGepitope,youmustrinsethepelletwithmediatogetridofanyresidualchelatingagents.

8)Labelreceptorswithfluorescentantibody-resUSPendpelletin500µLofmediacontaining10mg/mLFITC-M1(M1whichhasbeencovalentlyboundtotheFITCfluorophore(Molecularprobes)usingastandardprocedure)-incubate45"onicewithoccasionalagitationtoensureevendistributionofantibody.

9)rightbeforeanalyzingsamplesontheflowcytometer,add1µLofpropidiumiodide(PI)(MolecularProbes)toeachsampletostaindeadcells.

10)Useflowcytometrytomeasurethefluorescenceofeachsample.Theusershouldconsulttheirlocalflowcytometryfacilityforgeneralinstructiononflowcytometryandadviceonspecificproceduresforcollectionandanalysisofsamples.WetypicallyuseaBectonDickinsonFACScan,gateoutPI-stainedcells,andusethemeanfluorescenceasameasureoffluorescentantibodyassociatedwiththecells.References:Chuetal.(1997)

Chuetal.(1997)JBiolChem.272(43):27124-30(figure2)(PubMed|FullText)

PatriciaTsaoandMarkvonZastrow,"Type-specificSortingofGProtein-coupledReceptorsafterEndocytosis."JBiolChem.April14,2000;275(15):11130-11140.[PubMed|FullText]