蛋白激酶C抑制剂Staurosporine对A类清道夫受体胞浆域结构去除后功能的影响  

The Role of Staurosporine in Mediating The Function of Cytoplasmic Domain-truncated Scavenger Receptor

作　　者：管晓翔[1] 陈琪[1] 范乐明[1] 

机构地区：[1]南京医科大学动脉硬化研究中心

出　　处：《生物化学与生物物理进展》2004年第8期693-698,共6页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展规划项目 ( 973 ) (TG2 0 0 0 5 6910 );江苏省科委应用基础资助项目 (BK2 0 0 1113 )~~

摘　　要：A类清道夫受体 (scavengerreceptor,SR A)是一种主要位于巨噬细胞膜表面的同源三聚体糖蛋白 ,能够结合和摄取多种配基并介导内移 .在清道夫受体胞浆域有几个潜在的磷酸化位点 ,有关这些磷酸化位点与受体功能之间的确切关系目前尚所知甚少 .为深入探讨A类清道夫受体胞浆域与磷酸化之间的关系 ,以及受体胞浆域磷酸化对受体功能的影响 ,实验以含有SR AcDNA质粒为模板 ,采用PCR方法扩增不含胞浆域序列的清道夫受体 ,同时扩增全长清道夫受体作为对照 .PCR产物经纯化酶切后 ,进一步亚克隆到PcDNA3 1/HisB中 ,测序结果表明 ,重组产物能够编码正确的氨基酸序列 .重组产物经脂质体Lipofectamine (LF2 0 0 0 )介导转化入CHO细胞中 ,在含G4 18选择性培养液中培养筛选 14天后 ,分离阳性克隆 ,继续培养 .采用流式细胞计数仪 (FACS)鉴定转化筛选后细胞能否表达具有功能的清道夫受体 .结果发现 ,转化的CHO细胞可以稳定表达SR A的蛋白质 ,但受体胞浆域去除后 ,摄取配基的能力明显弱于全长组 (1∶1 337) .用荧光DiI标记乙酰化低密度脂蛋白(DiI AcLDL) ,37℃孵育转化细胞 5h后 ,激光共聚焦显微镜下观察到 :全长受体转化组细胞荧光散在分布于细胞膜和细胞器 ,而去除胞浆域组荧光只局限于细胞膜 ,说明SRThe class A scavenger receptor (SR-A) is a glycoprotein expressed on the cell surface of macrophages that mediates internalization of chemically modified lipoprotein. It was reported that the receptor internalization required the presence of internalization signal motif and the rate of receptor internalization was governed by the pattern of receptor phosphorylation induced by the ligands. However, the role of the cytoplasmic domain played in the receptor-mediated endocytosis is not fully characterized. Here the changes in internalization process of the receptor were reported when the whole cytoplasmic domain sequence ( 150 base pairs) was truncated. Both the full length and truncated were recombinated into PeDNA3. 1/HisB vector and were then transfected to CHO cells separately. Measurement of uptake of DiI-AcLDL by transfected cells with FACS showed that the bind and uptake of the ligand in full length SR-A was higher than that of truncated receptor (1.3 fold increase). After incubated with Dil-acetyl-LDL (Dil-AcLDL), the full length SR-A-transfected cells showed a diffuse distribution of the Dil-AcLDL in cytoplasm as well as in cell membrane when monitored under laser confocal microscopy. But in the truncated SR-A-transfected CHO cells, Dil-AcLDL mostly distributes at the cell surface only. In order to elucidate the role of phosphorylation played in mediating the function of cytoplasmic domain of SR-A, transfected CHO cells were preincubated with Staurosporine for 1 h at the concentration of 0.4 mumol/L. Then the cells were refed with medium containing Dil-AcLDL at the concentration of 10 mg/L at 37degreesC for 2 h, the DiI specially associated to cells was measured by spectrofluoremeter. The result indicated that staurosporine did not changed Dil-AcLDL bound and untaken by truncated receptor, which was different from the full length SR-A that increased obviously. The research here demonstrated that cytoplasmic domain regulate the receptor activity of SR-A, in which the phosphorlation or dephosphorlation of the

关 键 词：A类清道夫受体 胞浆域 磷酸化 蛋白激酶C抑制剂 巨噬细胞 

分 类 号：Q26[生物学—细胞生物学] Q78