清道夫受体介导的脂质蓄积的分子机制课件

IntracellularAccumulationofLipidsviaScavengerReceptorsQiChenNanjingMedicalUniversity2005.8IntracellularAccumulationof1FormationoffoamcellAthersclerosisisachronicinflammatoryreactioncausedbymacrophagesandlymphocytesthatuptakethepathogenicitylipidsinvadingthevessel.FormationoffoamcellAthe2SR-AandCD36aretheprincipalreceptorsresponsibleforfoam-cellformationMacrophagecelltakesuplipidbySR-ASR-AandCD36aretheprincipa3StructureofSR-A

SixdomainsThreetypesTrimericmoleculeStructureofSR-ASixdoma4

FoamcellformationMφadhesionHostdefenseandinnateimmunityAdaptiveresponsesDisordersofthenervoussystemPhagocytosisofapoptoticcellsFunctionsofSR-A

FoamcellformationFunctions5Thecytoplasmicdomainisthekeypartofthereceptor

Thecytoplasmicdomainisthe6

mediatetheinternalizationofligandssuchasox-LDL

participatetheregulationofcelladhesionandmigrationaccommodatetheexpressionandtransportofreceptorlink-coupledwithdownstreamofsignalingpathwayFunctionsofSR-Acytoplasmicdomain:mediatetheinternalizationo7

Cytoplasmicdomain：

Murine:MTKEMTENQRLCPHEREDADCSSESVKFDARSMTASLPHSTKNGPSVQEKLKSFK

(55)Rabbit:MAQWDSFTDQQEDTDSCSESVKFDARSNTALLPPNPKGPPLQEKLKSFK

(50)Bovine:MAQWDDFPDQQEDTDSCTESVKFDARSVTALLPPHPKNGPTLQERMKSYK(50)Human:MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK

(50)

Cytoplasmicdomain：8MSR-AAcLDLHSP90,HSP70,andGAPDHDirectlyInteractwiththeCytoplasmicDomainofMSRNakamuraT.etal.BBRC,290,858-864(2002)FunctionalandpossiblephysicalassociationofscavengerreceptorwithcytoplasmictyrosinekinaseLyninmonocyticTHP-l-derivedMacrophagesMikiS.etal.FEBSLetters,399,241-244(1996)HSP90HSP70GAPDHLynMSR-AAcLDLHSP90,HSP70,andGA9LigandBindingtoMacrophageScavengerReceptor-AInducesUrokinase-typePlasminogenActivatorExpressionbyaProteinKinase-dependentSignalingPathwayHsien-YehHsuetal.JBiolChem,273,1240-1246(1998)MSR-AAcLDLFucoidanPTKPLC-γPKCuPALigandBindingtoMacrophageS10MSR-AFucoidanHsp90,pp60SrcRac1PAKJNKp38PI3KproIL-1IL-1LigandsofMacrophageScavengerReceptorInduceCytokineExpressionviaDifferentialModulationofProteinKinaseSignalingPathwaysHsuHY.etal.JBiolChem,276,28719-28730(2001)MSR-AFucoidanHsp90,pp60SrcRac111MSR-AAcLDLinternalization,adhesionGi/oRegulationofacetylatedlowdensitylipoproteinuptakeinmacrophagesbypertussistoxin-sensitiveGproteinsStewartCetal.JLipidRes,41,807-813(2000)ClassAscavengerreceptorsmediatecelladhesionviaactivationofGi/oandformationoffocaladhesioncomplexes

PostSR.etal.JLipidRes,43,1829-1836(2002)MSR-AAcLDLinternalization,adh12phosphorylationofcytoplasmicdomain

signalsequenceincytoplasmicdomaininteractorswithcytoplasmicdomainFocusoncytoplasmicdomain：phosphorylationofcytoplasm13BovineModelHumanModelMurineModelRabbitModelBovineModelHumanModelMurine14Ser21（rat）increasesexpressionofreceptorpromotesmetabolismoflipidFongLGetal.JBiolChem,274,36088-36816(1999)phosphorylationofcytoplasmicdomainSer21（rat）phosphorylationofc15phosphorylationofcytoplasmicdomainSer49（rat）reducesuptakeoflipidFongLGetal.JBiolChem,274,36088-36816(1999)phosphorylationofcytoplasmic16FDARS

internalizationofreceptor

FongLGetal.JBiolChem,274,36088-36816(1999)signalsequenceofcytoplasmicdomainFDARSsignalsequenceofcyto17KLKSFKcelladhesionKosswigNetal.JBiolChem,278,34219-34225(2003)signalsequenceofcytoplasmicdomainKLKSFKsignalsequenceofcyto18VXFD(rabbit)internalizationofreceptor,transferencetocellsurfaceMorimotoKetal.BiolPharmBull,22,1022-1026(1999)signalsequenceofcytoplasmicdomainVXFD(rabbit)signalsequenceo19Ser48(human）expressionandinternalization

ofreceptor,cellmigration

HeiderHetal.FEBSLetters,505,185-190(2001)

phosphorylationofcytoplasmicdomainSer48(human）phosphorylationo20Di-leucinemotif?signalsequenceofcytoplasmicdomainDi-leucinemotif?signalseque21

1.TheroleofDi-leucinemotifinthecytoplasmicdomainofSRA1.TheroleofDi-leucinemot22(1)SR-Amutants/EGFPconstructs:WildtypeN3132LMEGFPSRASRA(1)SR-Amutants/EGFPconstruc23(2)ExpressionpatternsofSRAmutant:*P<0.05,n=3EGFPSR-AN3132LMEGFPSR-AN3132LM*02004006008001000controlEGFPSR-AN3132LM(2)ExpressionpatternsofSRA24(3)TheinternalizationofDiI-AcLDLviaSRAmutant:DiI-acLDLSR-Aexpression**DiI-acLDL*P<0.05,n=3**P<0.05,n=3010203040EGFPWildtypeN3132LM*EGFPWildtypeN3132LM0.020.040.060.080.100.12(3)TheinternalizationofDiI25(4)CelladhesionviaSRAmutant:00.10.20.30.40.50.60.70.80.9EGFPSR-AN3132LMratio%**P<0.05,n=3*(4)CelladhesionviaSRAmuta26EGFPSR-AN3132LMImmunofluorescence:TheCHOcellsweretransfectedwithSRA/EGFPorSRAMutant/EGFP,(5)Di-leucinemotifandClathrin-coatedpit:GFPAnti-clathrin

overlay

EGFPSR-AN3132LMImmunofluoresce27180KD97KDabca:EGFP-transfectedcellb:SR-A-transfectedcellc:N3132LMmutant-transfectedcellClathrinIP:180KD97KDab28NΔ1-27NΔ1-27LM***P<0.05,n=3SR-ANΔ1-27NΔ1-27LM97KDABC(6)ExpressingoftheSRAmutantsinCHOcellEGFPSR-A4006008001000NΔ1-27NΔ1-27LM200NΔ1-27NΔ1-27LM***P<0.05,n=3SR-29(7)TheinternalizationofDiI-AcLDLviaSRAmutant:*P<0.05,n=3*P<0.05,n=3****DiI-acLDLDiI-acLDLSR-Aexpression10203040EGFPSR-ANΔ1-27NΔ1-27LM0.120.100.080.060.040.020.000EGFPSR-ANΔ1-27NΔ1-27LM(7)TheinternalizationofDiI30*Di-leucinesignalmotifinthecytoplasmicdomainisrequiredforMSR-A-mediatedinternalization.

*Di-leucinesignalmotif-relatedendocytosisofAc-LDLmaydependontheclathrin-coatpit.*TheremightbemultiplesignalmotivesincytoplasmicdomainofMSR,whichadaptsthemultiplefunctionsofMSR.Di-LeucinemotifmaybeandistinctmotiffromtheVXFDinSR-Amediatedinternalizationandadhesion.*Di-leucinesignalmotifint312.IdentificationofpeptideligandsforthecytoplasmicdomainofSRA2.Identificationofpeptidel32(1)GST-cSRAfusionproteinexpressionandpurification:GSTfusionproteinwereseparatedona12%SDS.1,purifiedGSTfusionprotein;2,E.colisupernatantcontainingGSTfusionprotein;3and6,E.colisupernatant;4,molecularweightstandards;5,E.colisupernatantcontainingGST.123456Western-blotofthefusionproteinwithanti-GSTantibody.1,GST-cytoplasmicdomainofSRAfusionprotein;2,GST;3,E.colisupernatant;4,molecularweightstandards.1234(1)GST-cSRAfusionproteine33(2)Screeningthephagepeptidelibrarywiththefusionprotein:Biopanningrounds

Inputphages（pfu*）

Outputphages（pfu）Relativeoutputratio**

14.4×1011

5.9×103

1.34×10-824.25×1011

3.45×1048.11×10-632.75×1011

6.4×1052.33×10-642.5×1011

3.25×1061.3×10-5Thepoolofphagebecomesenrichedinfavourofsequencesthatbindtothetargetafterfourroundsofbio-panning*phageformunit**outputphages/inputphages(2)Screeningthephagepeptid34BindingspecificityofthepositivephageclonesanalysedbyELISAusinganti-M13antibody.Theblack,bindingwiththefusionprotein(P);Thewhite,bindingwithGST(N).Thepositiveclones:P/N>3.(3)IsolationofbindingphageclonesbyPhageELISA:II4II5II6II7II9II10II11II12M1300.10.20.30.40.50.60.70.8二4二5二6二7二9二10二11二12M13Bindingspecificityofthepos35(4)Sequencingtheselectedpositiveclones:*H11:TDRLFMNSIWPG(4,6,7,10,11,12)*H5:TFWDDGWLLLPR(5)*H9:YGDRWFMPVMRS(9