1、Intracellular Accumulation of Lipids via Scavenger Receptors Qi ChenNanjing Medical University2005.8Formation of foam cellMacrophage cell takes up lipid by SR-AStructure of SR-A Foam cell formation M adhesion Host defense and innate immunity Adaptive responses Disorders of the nervous system Phagocy
2、tosis of apoptotic cellsFunctions of SR-A The cytoplasmic domain is the key part of the receptor Functions of SR-A cytoplasmic domain:Cytoplasmic domain：MSR-AAcLDLHSP90,HSP70,and GAPDH Directly Interact with the Cytoplasmic Domain of MSRNakamura T.et al.BBRC,290,858-864(2002)Functional and possible
3、physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-l-derived MacrophagesMiki S.et al.FEBS Letters,399,241-244(1996)HSP90 HSP70 GAPDHLynLigand Binding to Macrophage Scavenger Receptor-A Induces Urokinase-type Plasminogen Activator Expression by a Protein
4、Kinase-dependent Signaling PathwayHsien-Yeh Hsu et al.J Biol Chem,273,1240-1246(1998)MSR-AAcLDLFucoidanPTKPLC-PKCuPAMSR-AFucoidanHsp90,pp60SrcRac1PAKJNKp38PI3KproIL-1IL-1Ligands of Macrophage Scavenger Receptor Induce Cytokine Expression via Differential Modulation of Protein Kinase Signaling Pathwa
5、ysHsu HY.et al.J Biol Chem,276,28719-28730(2001)MSR-AAcLDLinternalization,adhesionGi/oRegulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteinsStewart C et al.J Lipid Res,41,807-813(2000)Class A scavenger receptors mediate cell adhesionvia activat
6、ion of Gi/o and formation of focal adhesion complexes Post SR.et al.J Lipid Res,43,1829-1836(2002)Focus on cytoplasmic domain：Bovine ModelHuman ModelMurine ModelRabbit ModelSer21（rat）increases expression of receptor promotes metabolism of lipid Fong LG et al.J Biol Chem,274,36088-36816(1999)phosphor
7、ylation of cytoplasmic domainphosphorylation of cytoplasmic domainSer49（rat）reduces uptake of lipidFong LG et al.J Biol Chem,274,36088-36816(1999)FDARS internalization of receptor Fong LG et al.J Biol Chem,274,36088-36816(1999)signal sequence of cytoplasmic domainKLKSFK cell adhesionKosswig N et al.
8、J Biol Chem,278,34219-34225(2003)signal sequence of cytoplasmic domainVXFD(rabbit)internalization of receptor,transference to cell surfaceMorimoto K et al.Biol Pharm Bull,22,1022-1026(1999)signal sequence of cytoplasmic domainSer48(human）expression and internalization of receptor,cell migration Heid
9、er H et al.FEBS Letters,505,185-190(2001)phosphorylation of cytoplasmic domainDi-leucine motif?signal sequence of cytoplasmic domain 1.The role of Di-leucine motif in the cytoplasmic domain of SRA(1)SR-A mutants/EGFP constructs:Wild typeN3132LMEGFPSRASRA(2)Expression patterns of SRA mutant:*P0.05,n=
10、3EGFPSR-A N3132LMEGFPSR-AN3132LM*0 020020040040060060080080010001000controlcontrolEGFPEGFPSR-ASR-AN3132LMN3132LM(3)The internalization of DiI-AcLDL via SRA mutant:DiI-acLDLSR-A expression*DiI-acLDL*P0.05,n=3*P0.05,n=30 01010202030304040EGFPEGFPWild typeWild typeN3132LMN3132LM*EGFPEGFPWild typeWild t
11、ypeN3132LMN3132LM0.020.020.040.040.060.060.080.080.100.100.120.12(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 N3132LMN3132LMratioratio%*P0.05,n=3*EGFPSR-AN3132LMImmunofluorescence:The CHO cells were transfected with SRA/EGFP or SRA Mutant
12、/EGFP,(5)Di-leucine motif and Clathrin-coated pit:GFPAnti-clathrinoverlay180KD97KD a b ca:EGFP-transfected cell b:SR-A-transfected cell c:N3132LM mutant-transfected cellClathrin IP:N1-27N1-27LM*P0.05,n=3SR-A N1-27 N1-27LM97KD(6)Expressing of the SRA mutants in CHO cellEGFPEGFPSR-ASR-A400400600600800
13、80010001000N1-27N1-27LM200200(7)The internalization of DiI-AcLDL via SRA mutant:*P0.05,n=3*P3.(3)Isolation of binding phage clones by Phage ELISA:II4 II5 II6 II7 II9 II10 II11 II12 M1300.10.20.30.40.50.60.70.8二4二5二6二7二9二10二11二12M13(4)Sequencing the selected positive clones:(5)Searching the PIR-PSD d
14、atabase:(6)Construction of expression plasmidsPEGFP-N1-H5PEGFP-N1-H9PEGFP-N1-H11(7)Subcellular localization of the peptide/EGFP in CHO cells expressing SRA(8)DiI-AcLDL uptake assay0102030405060EGFP119fluoresenceEGFP-H11 inhibits SRA-mediated DiI-AcLDL association.CHO cells stably expressing SRA were transfected with EGFP-N1,EGFP-N1-H11,EGFP-N1-H9,then incubated with DiI AcLDL(5ug/ml)for 2 hours.*p=0.03710.05AcknowledgementThanks
