三通道荧光共振能量转移分析法的优化及其对活细胞内受体复合物组成的分析

OPTIMIZATION OF THREE-CUBE FRET ASSAY AND APPLICATION IN THE ANALYSIS OF SUBUNIT ASSEMBLING OF iGluR RECEPTORS IN LIVING CELLS

目的在活细胞内建立并优化三通道FRET检测方法,使之更好地适用于膜蛋白复合物亚单位的相互作用。方法利用在HEK293细胞中转染pCFP-YFP作为阳性对照,共转pCFP和pYFP作为阴性对照,用不同的公式如FRETN、NFRET、FR及NetFRET/Df等进行FRET定量计算,并通过改变供体与受体比例来观察所测得的FRET值与FRET转移效率的关系。在HEK293细胞内转染CFP或YFP标记的不同亲离子型谷氨酸受体(iGluR)亚单位,分析它们之间的相互作用。结果FRETN、NFRET、FR及NetFRET/Df等公式均可以适用于本研究所建的三通道FRET检测系统;对于不同的公式,供体与受体蛋白分子表达比例的变化会对测量结果产生不同的影响。用所建FRET技术,发现CFP-GluRl与YFP-GluRl之间,以及CFP-NRl与YFP-NRl具有确定FRET信号,而CFP-GluRl与YFP-NRl则没有FRET发生。结论在对不同iGluR亚单位之间相互作用的研究中证明同一受体亚型中的亚单位之间可以形成同聚体,而不同受体亚型的亚单位之间则不会形成复合物。

Objective To establish and optimize three-cube FRET assay in living cells and analyze subunit assembling of iGluR receptors. Methods Taking HEK293 cells cotransfed with pECFP and pEYFP as negative control, and those transfected with pECFP-YFP as positive control, different calculation methods using fluorescence microscopy were compared. Results These calculation methods were all suitable for FRET measurement in the system, but the measurement results were affected by the ratio of Donor/Acceptor （D/A） in some degree, and different calculation methods have different optimized conditions. FRET measurement using FR value showed subunit specific assembly of iGluR subtypes. Conclusion There are different optimized conditions for these different calculation methods in the three-cube FRET measurement system, and a further evidence is provided for subunit specific assembling of iGluR subtypes from the FRET assay.