GPCR-G蛋白融合传感器检测GPCR与G蛋白相互作用的灵敏度

Sensitivity of GPCR-G protein fusion sensor to detect the interaction between GPCR and G protein

目的以GPR56-G蛋白融合传感器为例,建立一种检测GPCR与G蛋白相互作用的高灵敏方法。方法将带有NanoLuc荧光素酶(Nluc)的G蛋白α亚基的N端融合到GPCR的C端,构建GPCR和G蛋白α亚基两者的融合蛋白为实验组,受体和G蛋白共表达作为对照组。在受体表达水平相同的条件下,利用生物发光共振能量转移(BRET)方法检测GPR56-Gα_(12)亚基融合蛋白、GPR56和Gα_(12)亚基共表达的组成性活力。利用BRET方法检测GPR56激动剂(P19)对GPR56-Gα_(12)亚基融合蛋白相互作用的影响。结果BRET比率结果显示,与对照组GPR56和Gα_(12)亚基共表达相比,GPR56-Gα_(12)亚基融合蛋白具有更强的组成性活力(F=424.7,P<0.001);与对照组比较,P19激活实验组的半数有效浓度(EC_(50))下降,差异有统计学意义(t=13.36,P<0.001),GPR56-Gα_(12)亚基融合蛋白对激动剂(P19)的感知更灵敏,亲和力更强。结论GPCR-Gα亚基融合蛋白系统是一种检测GPCR与G蛋白相互作用更灵敏的方法。

Objective GPR56-G protein fusion sensor was used as a prototype to establish a highly sensitive method to detect the interaction between GPCR and G protein.Methods The N-terminus of the G proteinαsubunit with NanoLuc luciferase(Nluc)was fused to the C-terminus of the GPCR.The fusion protein of both GPCR and G proteinαsubunit was constructed as the experimental group,and the receptor and G protein were co-expressed as a control group.The constitutive activity of GPR56-Gα_(12)subunit fusion protein,GPR56 and Gα_(12)subunit co-expression were compared with bioluminescence resonance energy transfer(BRET)at the same level of receptor expression.The effect of agonist(P19)on the interaction of GPR56-Gα_(12)subunit fusion protein was detected with BRET.Results The BRET ratio results showed that the GPR56-Gα_(12)subunit fusion protein had stronger constitutive activity than the control group co-expressed with GPR56 and Gα_(12)subunits(F=424.7,P<0.001).Compared with the control group,the experimental group had decreased half effective concentration(EC_(50))of P19 activation(t=13.36,P<0.001).The GPR56-Gα_(12)subunit fusion protein was more sensitive to P19 and had a stronger affinity.Conclusion The GPCR-Gαsubunit fusion protein system is a sensitive method to detect the interaction between GPCRs and G protein.