FRET技术研究Apelin受体与G蛋白的相互作用  

Using fluorescence resonance energy transfer technology to study the interaction between G protein and Apelin receptor

作　　者：姜云璐 田艳君[1] 王正文[1] 吕志瞳 白波[1] 陈京[1] JIANG Yunlu TIAN Yanjun WANG Zhengwen L V Zhitong BAI Bo CHEN Jing(Neurobiology institute, Jining Medical University, Jining 272067, Chin)

机构地区：[1]济宁医学院神经生物研究所,济宁272067

出　　处：《济宁医学院学报》2017年第1期9-15,共7页Journal of Jining Medical University

基　　金：国家自然基金项目(3097108;816712276;31271243;81070961);山东省高校科技计划项目(ZR2014HL404;JY2015KJ004;JY2015KJ002;2015-57-6;2015-57-62;J15LE19)

摘　　要：目的探讨Apelin受体与G蛋白之间的相互作用。方法在HEK293T细胞中建立Apelin受体与各亚型G蛋白的过表达体系,利用荧光共振能量转移技术(fluorescence resonance energy transfer technology,FRET)实时、动态、连续的监测Apelin受体与G蛋白偶联的机制。结果加入激动剂Apelin-13刺激后,Apelin受体与Gαi1亚基的FRET值没有发生明显的变化(P>0.05),Apelin受体与Gαi2、Gαi3亚基的FRET信号显著增加(P<0.05),而Apelin受体与Gαo、Gαq亚基的FRET信号相对于刺激前显著降低(P<0.05)。结论 Apelin受体在其激动剂Apelin-13作用下,能够激活G蛋白的Gαi2、Gαi3、Gαo、Gαq亚基,并引起Gαi2、Gαi3亚基构象重排进而与Gγ亚基相互靠近,Gαo、Gαq亚基远离Gγ2亚基,而不与Gαi1亚基发生相互作用。Objective To explore the interaction between Apelin receptor and G protein. Methods To mo- nitor Apelin receptor mediated activation of G proteins in living ceils in real-time and dynamic, and the estab- lished cell lines that expressed APJ or some one of G protein using fluorescence resonance energy transfer technology. Results Upon superfusion with Apelin-13 and recording of whole-cell uorescence, there was no significant change in FRET ratio （P 〉 0. 05 ）. There was an agonist-induced increase in FRET in in HEK293T cells which transfected with Gαi2/Gαi3, Gα1 Gα2-CFP and Apelin receptor （P 〈 0.05 ）. And there was an ag- onist-induced decrease in FRET in Gαo/Gαq with APJ （ P 〈 0.05 ）. Conclusion These results demonstrate that activation of APJ with Apelin-13 also leads to the activation of Gαi, Gαo, Gαq proteins, and Apelin recep- tor activation causes Gαi2 and Gαi3 subtmits activation through a unique rearrangement process instead of subunit dissociation,and leads to Gαo and Gαq disassociation with Gα1α2 and Gαi1 is not activated follow- ing APJ activity or APJ only cause a very small portion of the Gail activation.

关 键 词：Apelin受体 荧光共振能量转移技术 信号转导 G蛋白偶联受体 

分 类 号：R346[医药卫生—基础医学]