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1、Intracellular Accumulation of Lipids via Scavenger Receptors Qi ChenNanjing Medical University2005.8Formation of foam cell AthersclerosisAthersclerosisisachronicinflammatoryreactioncausedbymacrophagesisachronicinflammatoryreactioncausedbymacrophagesandlymphocytesthatuptaketheandlymphocytesthatuptake
2、thepathogenicitypathogenicitylipidsinvadingthevessel.lipidsinvadingthevessel.SR-AandCD36aretheSR-AandCD36aretheprincipalreceptorsprincipalreceptorsresponsibleforfoam-responsibleforfoam-cellformationcellformationMacrophage cell takes up lipid by SR-AStructure of SR-A SixdomainsSixdomains ThreetypesTh
3、reetypes TrimericTrimericmoleculemolecule FoamcellformationMadhesionHostdefenseandinnateimmunityAdaptiveresponsesDisordersofthenervoussystemPhagocytosisofapoptoticcellsFunctions of SR-A The cytoplasmic domain is the key part of the receptor mediatethemediatetheinternalizationinternalizationofofligan
4、dsligandssuchasox-LDLsuchasox-LDL participatetheregulationofcelladhesionandmigrationparticipatetheregulationofcelladhesionandmigration accommodatetheexpressionandtransportofreceptoraccommodatetheexpressionandtransportofreceptor link-coupledwithdownstreamofsignalingpathwaylink-coupledwithdownstreamof
5、signalingpathwayFunctions of SR-A cytoplasmic domain:Cytoplasmic domain：MurineMurine:MTKEMTENQRLCPHEREDADCSSES:MTKEMTENQRLCPHEREDADCSSESVKFDARSVKFDARSMTAMTASLSLPPHSTKNGPSVQEHSTKNGPSVQEKLKSFKKLKSFK(55)(55)Rabbit:MAQWDSFTDQQEDTDSCSESRabbit:MAQWDSFTDQQEDTDSCSESVKFDARSVKFDARSNTANTALLLLPPNPKPPNPKGPPLQEGP
6、PLQEKLKSFKKLKSFK(5050)Bovine:MAQWDDFPDQQEDTDSCTESBovine:MAQWDDFPDQQEDTDSCTESVKFDARSVKFDARSVTAVTALLLLPPHPKNPPHPKNGPTLQERMKSYKGPTLQERMKSYK(50)(50)Human:MEQWDHFHNQQEDTDSCSESHuman:MEQWDHFHNQQEDTDSCSESVKFDARSVKFDARSMTAMTALLLLPPNPKPPNPKNSPSLQENSPSLQEKLKSFKKLKSFK(50)(50)MSR-AAcLDLHSP90,HSP70,andGAPDHDirect
7、lyInteractwiththeCytoplasmicDomainofMSRNakamuraT.etal.BBRC,290,858-864(2002)FunctionalandpossiblephysicalassociationofscavengerreceptorwithcytoplasmictyrosinekinaseLyninmonocyticTHP-l-derivedMacrophagesMikiS.etal.FEBSLetters,399,241-244(1996)HSP90 HSP70 GAPDHLynLigandBindingtoMacrophageScavengerRece
8、ptor-AInducesUrokinase-typePlasminogenActivatorExpressionbyaProteinKinase-dependentSignalingPathwayHsien-YehHsuetal.JBiolChem,273,1240-1246(1998)MSR-AAcLDLFucoidanPTKPLC-PKCuPAMSR-AFucoidanHsp90,pp60SrcRac1PAKJNKp38PI3KproIL-1IL-1LigandsofMacrophageScavengerReceptorInduceCytokineExpressionviaDiffere
9、ntialModulationofProteinKinaseSignalingPathwaysHsuHY.etal.JBiolChem,276,28719-28730(2001)MSR-AAcLDLinternalization,adhesionGi/oRegulationofacetylatedlowdensitylipoproteinuptakeinmacrophagesbypertussistoxin-sensitiveGproteinsStewartCetal.JLipidRes,41,807-813(2000)ClassAscavengerreceptorsmediatecellad
10、hesionviaactivationofGi/oandformationoffocaladhesioncomplexes PostSR.etal.JLipidRes,43,1829-1836(2002)phosphorylationphosphorylationofcytoplasmicdomainofcytoplasmicdomain signalsequenceinsignalsequenceincytoplasmiccytoplasmicdomaindomain interactorsinteractorswithcytoplasmicdomainwithcytoplasmicdoma
11、inFocus on cytoplasmic domain：BovineModelHumanModelMurineModelRabbitModelSer21（rat）increasesexpressionofreceptorpromotesmetabolismoflipidFongLGetal.JBiolChem,274,36088-36816(1999)phosphorylationofcytoplasmicdomainphosphorylationofcytoplasmicdomainSer49（rat）reducesuptakeoflipidFongLGetal.JBiolChem,27
12、4,36088-36816(1999)FDARS internalizationofreceptor FongLGetal.JBiolChem,274,36088-36816(1999)signalsequenceofcytoplasmicdomainKLKSFK celladhesionKosswigNetal.JBiolChem,278,34219-34225(2003)signalsequenceofcytoplasmicdomainVXFD(rabbit)internalizationofreceptor,transferencetocellsurfaceMorimotoKetal.B
13、iolPharmBull,22,1022-1026(1999)signalsequenceofcytoplasmicdomainSer48(human）expressionandinternalization ofreceptor,cellmigration HeiderHetal.FEBSLetters,505,185-190(2001)phosphorylationofcytoplasmicdomainDi-leucine motif?signalsequenceofcytoplasmicdomain 1.The role of Di-leucine motif in the cytopl
14、asmic domain of SRA(1)SR-A mutants/EGFP constructs:Wild typeN3132LMEGFPSRASRA(2)Expression patterns of SRA mutant:*P0.05,n=3EGFPSR-AN3132LMEGFPSR-AN3132LM*0 020020040040060060080080010001000controlcontrolEGFPEGFPSR-ASR-AN3132LMN3132LM(3)The internalization of DiI-AcLDL via SRA mutant:DiI-acLDLSR-Aex
15、pression*DiI-acLDL*P0.05,n=3*P0.05,n=30 01010202030304040EGFPEGFPWild typeWild typeN3132LMN3132LM*EGFPEGFPWild typeWild typeN3132LMN3132LM0.020.020.040.040.060.060.080.080.100.100.120.12(4)Cell adhesion via SRA mutant:0 00.10.10.20.20.30.30.40.40.50.50.60.60.70.70.80.80.90.9EGFPEGFPSR-A SR-A N3132LM
16、N3132LMratioratio%*P0.05,n=3*EGFPSR-AN3132LMImmunofluorescence:TheCHOcellsweretransfectedwithSRA/EGFPorSRAMutant/EGFP,(5)Di-leucine motif and Clathrin-coated pit:GFPAnti-clathrin overlay 180KD97KDabca:EGFP-transfectedcellb:SR-A-transfectedcellc:N3132LMmutant-transfectedcellClathrin IP:N1-27N1-27LM*P
17、0.05,n=3SR-AN1-27N1-27LM97KDA AB BC C(6)Expressing of the SRA mutants in CHO cellEGFPEGFPSR-ASR-A40040060060080080010001000N1-27N1-27LM200200(7)The internalization of DiI-AcLDL via SRA mutant:*P0.05,n=3*P3.(3)Isolation of binding phage clones by Phage ELISA:II4II5II6II7II9II10II11II12M1300.10.20.30.
18、40.50.60.70.8二4二5二6二7二9二10二11二12M13(4)Sequencing the selected positive clones:*H11:TDRLFMNSIWPG(4,6,7,10,11,12)*H11:TDRLFMNSIWPG(4,6,7,10,11,12)*H5:TFWDDGWLLLPR(5)*H5:TFWDDGWLLLPR(5)*H9:YGDRWFMPVMRS(9)*H9:YGDRWFMPVMRS(9)(5)Searching the PIR-PSD database:H5:CaseinkinaseIIphosphorylationsite(H5:Casein
19、kinaseIIphosphorylationsite(TFWDTFWD)H11:ProteinkinaseCphosphorylationsite(H11:ProteinkinaseCphosphorylationsite(TDRTDR)(6)Construction of expression plasmidsPEGFP-N1-H5PEGFP-N1-H9PEGFP-N1-H11EGFPEGFPPeptide/EGFPPeptide/EGFP(7)Subcellular localization of the peptide/EGFP in CHO cells expressing SRA(
20、8)DiI-AcLDL uptake assayEGFP-H11inhibitsSRA-mediatedDiI-AcLDLassociation.CHOcellsstablyexpressingSRAweretransfectedwithEGFP-N1,EGFP-N1-H11,EGFP-N1-H9,thenincubatedwithDiIAcLDL(5ug/ml)for2hours.*p=0.03710.05*Acknowledgement YaoyuYaoyuChenChenXiaohuaXiaohuaWangWang JingjingJingjingBenBenHuiBaiHuiBai LemingLemingFanFan YongJiYongJiXiuyingChenXiuyingChenThanksThanks