GPCR-G蛋白融合传感器检测GPCR与G蛋白相互作用的灵敏度

doi:10.6040/j.issn.1671-7554.0.2022.0368

摘要/Abstract

摘要： 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-Gα₁₂亚基融合蛋白、GPR56 和 Gα₁₂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-Gα₁₂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 Gα₁₂亚基共表达相比,GPR56-Gα₁₂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(EC₅₀)下降,差异有统计学意义(t=13.36,P<0.001),GPR56-Gα₁₂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。

关键词: GPCR-Gα 亚基融合蛋白, GPR56, Gα₁₂, 组成性活力, P19

Abstract: 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 protein α subunit with NanoLuc luciferase(Nluc)was fused to the C-terminus of the GPCR. The fusion protein of both GPCR and G protein α subunit was constructed as the experimental group, and the receptor and G protein were co-expressed as a control group. The constitutive activity of GPR56-Gα₁₂ subunit fusion protein, GPR56 and Gα₁₂ 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-Gα₁₂ subunit fusion protein was detected with BRET. Results The BRET ratio results showed that the GPR56-Gα₁₂ subunit fusion protein had stronger constitutive activity than the control group co-expressed with GPR56 and Gα₁₂ subunits(F=424.7, P<0.001). Compared with the control group, the experimental group had decreased half effective concentration(EC₅₀)of P19 activation (t=13.36, P<0.001). The GPR56-Gα₁₂ subunit fusion protein was more sensitive to P19 and had a stronger affinity. Conclusion The GPCR-Gα subunit fusion protein system is a sensitive method to detect the interaction between GPCRs and G protein.

Key words: GPCR-Gα subunit fusion protein, GPR56, Gα₁₂, Constitutive activity, P19

中图分类号:

Q5-33

马传顺,林慧,孙金鹏,张道来. GPCR-G蛋白融合传感器检测GPCR与G蛋白相互作用的灵敏度[J]. 山东大学学报 (医学版), 2022, 60(9): 85-90.

MA Chuanshun, LIN Hui, SUN Jinpeng, ZHANG Daolai. Sensitivity of GPCR-G protein fusion sensor to detect the interaction between GPCR and G protein[J]. Journal of Shandong University (Health Sciences), 2022, 60(9): 85-90.

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