摘要
目的:探讨β-抑制蛋白1分子在过度表达胰岛素样生长因子-1受体(IGF-1R)的肿瘤细胞中IGF-1R膜受体内化和泛素化降解的作用。方法:采用免疫印迹法(WB)、免疫沉淀技术(IP)和FACS技术对鼠胚胎成纤维瘤3种细胞株[P6、β-抑制蛋白1阳性(WT1)和β-抑制蛋白1阴性KO细胞]进行IGF-1R内化检测。结果:在IGF-1R过度表达的P6细胞随配体刺激时间的增加内化IGF-1R明显增加,但非内化IGF-1R的水平明显降低;在β-抑制蛋白1阴性细胞株随配体刺激时间的增加非内化IGF-1R的水平保持不变,而无IGF-1R内化产生,但阳性对照可以看到条带清晰的IGF-1R;使用FACS分析也证实了两个细胞株的WB结果,同时检测在细胞通路中IGF-1R内化的作用在β-抑制蛋白1阴性细胞检测不到磷酸化的pERK。结论:在β-抑制蛋白1缺乏的情况下IGF-1R内化不能发生,β-抑制蛋白1可作为一个调节IGF-1R内化的工具,也表明IGF-1R内化对细胞通路之一的MAPK是必需的。
Objective:To investigate whether the internalization of insulin-like growth factor-1 receptor (IGF-1R) is required for other functions apart from down-regulation of IGF-1R, and to determine the role of β-arrestin in the internalization of IGF-1R. Methods: IGF-1R internalization of three cell lines as P6, β-arrestin1 positive (WT1), and β-arrestin1 negative cell lines, were examined by Western blot (WB), immunoprecipitation, and FACS analysis. Results: In P6 cells, an obvious decrease in the level of un-internalized IGF-1R (surface-bound IGF-1R) could be seen as ligand stimulation time increased; but a clear and distinct increase in internalization of the IGF-1R could be seen. In β-arrestin1 negative cells, the IGF-1R bands could not be seen, indicating the absence of internalized IGF-1R, however, the positive control band could be seen. Un-internalized IGF-1R bands could be seen and its level remained constant. WB technique confirmed the FACS results both in P6 and β-arrestin1 negative cells. In the absence of β-arrestin1, the level of phosphorylation of pERK was not detected when the ligand stimulated the IGF-1R. Conclusion: IGF-1R internalization could not occur in the absence of β-arrestin1. By the modulation of β-arrestin1, IGF-1R internalization is required for the activation of the MAPK pathway.
出处
《武汉大学学报(医学版)》
CAS
北大核心
2010年第2期149-153,共5页
Medical Journal of Wuhan University
基金
湖北省卫生厅资助重点项目(NO:WJ301140502)