DescriptionCatalogueNumberHTS004MBrandFamilyChemicon®TradeNameChemiScreenChemiconDescriptionChemiSCREEN™CXCR4MembranePreparationOverviewFull-lengthhumanCXCR4CDNABackgroundInformationThechemokineSDF-1aanditsGPCRreceptorCXCR4haveaone-to-onespecificitythatisuniqueamongchemokinesandtheirreceptors.SDF-1abindstoCXCR4expressedonhematopoieticandlymphopoieticcells,anddirectstheirtraffickingtoandretentioninhemato-andlymphatopoieticorgansandsitesofinflammation(Kuciaetal.,2004).CXCR4isexpressedonseveraltumorcelllineages,andmightberesponsIBLeformetastasistositesofSDF-1aexpression,suchasboneandlymphnodes(Mulleretal.,2001).Inaddition,CXCR4isacoreceptorfortheHIVenvelopeglycoproteingp120(Fengetal.,1996).SmallmoleculeantagoNISTsofCXCR4havebeendevelopedandshowntoinhibitinfectivityofT-tropicHIVstrainsandtoimpairgrowthofbraintumors(Arakakietal.,1999;Rubinetal.,2003).Chemicon"sCXCR4membranepreparationsarecrudemembranepreparationsmadefromourproprietarystablerecombinantcelllinestoensurehigh-levelofGPCRsurfaceexpression;thus,theyareidealtoolsforscreeningforantagonistsofinteractionsbetweenCXCR4andSDF-1a.RADIolabeledSDF-1abindstotheCXCR4membraneswithaKdof0.9nM.With0.04-0.13nM125I-labeledSDF-1a,anapproximately3-foldwindowofspecificbindingisobtained.ProductInformationFormatMembranesPresentationPackagingbuffer:50mMTrispH7.4,10%glyceroland1%BSAPackagingmethod:Membranesproteinwereadjustedto0.5mg/mlin1mlpackagingbuffer,rapidlyfrozen,andstoredat-80°C.StorageandShippingInformationStorageConditionsMaintainfrozenat-70°Cforupto2years.Donotfreezeandthaw.ApplicationsApplicationHumanCXCR4GPCRmembranepreparationforRadioligandbindingAssays>PgammaSbinding.KeyApplicationsRadioligandbindingassayGTPγSbindingApplicationNotesRadioligandbindingassayandGTPgammaSbinding.BIOLOGicalInformationConcentration2mL,0.5mg/mLEntrezGeneNumberNM_001008540.1NM_003467.2EntrezGeneSummaryThisgeneencodesaCXCchemokinereceptorspecificforstromalcell-derivedfactor-1.Theproteinhas7transmembraneregionsandislocatedonthecellsurface.ItactswiththeCD4proteintosupportHIVentryintocellsandisalsohighlyexpressedinbreastcancercells.MutationsinthisgenehavebeenassociatedwithWHIM(warts,hypogammaglobulinemia,infections,andmyelokathexis)syndrome.Alternatetranscriptionalsplicevariants,encodingdifferentisoforms,havebeencharacterized.GeneSymbolCXCR4WHIMNPY3RNPYY3RFB22LAP3FusinCXCR-4LCR1NPYRHM89fusinHSY3RRCXC-R4NPYY3NPYRLD2S201ECD184LESTRGPCRClassAHostCellsChem-1ProteinTargetCXCR4TargetSub-FamilyChemokineUniProtNumberP61073UniProtSummaryFUNCTION:SwissProt:P61073#ReceptorfortheC-X-CchemokineCXCL12/SDF-1.Transducesasignalbyincreasingtheintracellularcalciumionslevel.Involvedinhaematopoiesisandincardiacventricularseptumformation.Playsalsoanessentialroleinvascularizationofthegastrointestinaltract,probablybyregulatingvascularbranchingand/orremodelingprocessesinendothelialcells.Couldbeinvolvedincerebellardevelopment.IntheCNS,couldmediatehippocampal-neuronsurvival.Actsasacoreceptor(CD4beingtheprimaryreceptor)forHIV-1X4isolatesandasaprimaryreceptorforsomeHIV-2isolates.PromotesEnv-mediatedfusionofthevirus.SIZE:352aminoacids;39746DaSUBUNIT:InteractswithHIV-1surfaceproteingp120andTat.SUBCELLULARLOCATION:Cellmembrane;Multi-passmembraneprotein.TISSUESPECIFICITY:Expressedinnumeroustissues,suchasperipheralbloodleukocytes,spleen,thymus,spinalcord,heart,placenta,lung,liver,skeletalmuscle,kidney,pancreas,cerebellum,cerebralcortexandmedulla(inmicrogliaaswellasinastrocytes),brainmicrovascular,coronaryarteryandumbilicalcordendothelialcells.Isoform1ispredominantinalltissuestested.DOMAIN:SwissProt:P61073Theamino-terminusiscriticalforligandbinding.ResiduesinallfourextracellularregionscontributetoHIV-1coreceptoractivity.PTM:Sulfated.DISEASE:SwissProt:P61073#DefectsinCXCR4areacauseofWHIMsyndrome[MIM:193670];alsocalledwarts,hypogammaglobulinemia,infections,andmyelokathexis.WHIMsyndromeisanimmunodeficiencydiseasecharacterizedbyneutropenia,hypogammaglobulinemiaandextensivehumanpapillomavirus(HPV)infection.Despitetheperipheralneutropenia,bonemarrowaspiratesfromaffectedindividualscontainabundantmaturemyeloidcells,aconditiontermedmyelokathexis.SIMILARITY:BelongstotheG-proteincoupledreceptor1family.IncubationConditionsRECOMMENDEDASSAYCONDITIONS:Membranesaremixedwithradioactiveligandandunlabeledcompetitor(seeFigures1and2forconcentrationstested)inbindingbufferinanonbinding96-wellplate,andincubatedfor1-2h.Priortofiltration,aGF/C96-wellfilterplateiscoatedwith0.33%polyethyleneiminefor30min,thenwashedwith50mMHEPES,pH7.4,0.5%BSA.Bindingreactionistransferredtothefilterplate,andwashed3times(1mLperwellperwash)withWashBuffer.Theplateisdriedandcounted.Bindingbuffer:50mMHepes,pH7.4,5mMMgCl2,1mMCaCl2,0.2%BSA,filteredandstoredat4°CRadioligand:[125I]SDF-1alpha(Perkinelmer#:NEX346)WashBuffer:50mMHepes,pH7.4,500mMNaCl,0.1%BSA,filteredandstoredat4°C.Onevialcontainsenoughmembranesforatleast200assays(units),whereanunitistheamountofmembranethatwillyieldgreaterthan4-foldsignal:backgroundwith125I-labeledSDF-1alphaat0.13nM.PhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation