LRP6受体胞内区赖氨酸位点突变和经典Wnt信号通路相关性分析  

Correlation analysis between mutations of LRP6 cytosolic lysine residues and classic Wnt signaling pathway

作　　者：林翠鸿[1] 柯蒙[1] 黄品芳[1] 王长连[1] 

机构地区：[1]福建医科大学附属第一医院药学部,福建福州350005

出　　处：《中国医院药学杂志》2015年第16期1461-1464,共4页Chinese Journal of Hospital Pharmacy

基　　金：福建省战略性新兴产业技术应用基础研究项目(编号:2013J01368);福建省卫生系统中青年骨干人才培养项目资助计划重点项目(编号:2014-ZQN-ZD-15)

摘　　要：目的:探讨LRP6受体胞内区赖氨酸位点突变对经典Wnt信号通路影响。方法:构建胞内区3个赖氨酸位点(K8R、K48R及K82R)同时突变的突变型LRP6受体,瞬时转染HEK293细胞,运用荧光素酶报告基因技术分析其对β-catenin/TCF转录活性影响;同时运用谷胱甘肽转移酶-E-钙粘蛋白(GST-E-cadherin)结合法提取胞浆游离β-连环蛋白(β-catenin),Western blot法测定总β-连环蛋白、胞浆游离β-连环蛋白、低密度脂蛋白相关受体-6(LRP6)及磷酸化LRP6(p-LRP6)蛋白水平。结果 :和转染空白质粒PcDNA3比较,转染LRP6增强TOPflash活性593倍(P<0.05),提高胞浆游离β-catenin水平126.3%;而转染赖氨酸突变的LRP6将进一步增强TOPflash活性,达2 989倍(P<0.05),胞浆游离β-catenin水平提高580.5%;和转染未突变LRP6相比,转染胞内区赖氨酸突变LRP6受体激活Wnt信号通路作用更强。结论:LRP6胞内区赖氨酸位点在LRP6参与Wnt信号通路活化过程中可能具有重要作用。OBJECTIVE To investigate correlation between mutations of LRP6 cytosolic lysine residues and classic Wnt signaling pathway. METHOBS PcDNA3 constructed with LRP6 or LRP6 receptor with KSR, K48R and K82R mutations （LRP6 M） were transfeeted transiently into HEK293 cells. Luciferase reporter gene assay was performed to detect changes of transcriptional activity of 13-catenin/TCF in HEK293 cells. Free 13-catenin in cytoplasm of HEK293 cells were harvested by GST-Ecadherin binding method. Western blot analysis was performed to detect changes of total β-catenin, free β-catenin, LRP6 and p- LRP6 levels. RESULTS Compared with HEK293 cells transfected with PcDNA3, TOPflash activity and free β-catenin levels of cells transfected with wild type LRP6 increased by 593 times and 126. 3%, respectively, while those transfected with mutated type LRP6 increased by 2 989 times and 580. 5% , respectively. These resialts showed that LRP6 M had stronger Wnt activation effects compared with wild type LRP6. CONCLUSION LRP6 cytosolic lysine residues may play an important role in activation of classic Wnt signaling pathway.

关 键 词：赖氨酸残基 低密度脂蛋白相关受体-6 经典Wnt信号通路 突变 

分 类 号：R927.2[医药卫生—药学]