Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model
Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model
复制标题
纳米荧光素酶报告基因对细胞膜受体内化的定量测量：以G蛋白偶联受体RXFP3为模型
DOI：
10.1016/j.bbamem.2014.11.026
复制
发表时间：
2015-02-01
影响因子：
3.4
通讯作者：
Guo, Zhan-Yun
中科院分区：
文献类型：
--
作者：
Liu, Yu;Song, Ge;Guo, Zhan-Yun
纳米荧光素酶（NanoLuc）是一种新开发的荧光素酶报告分子，具有迄今为止最亮的生物发光。在目前的工作中，我们开发了NanoLuc作为一个敏感的生物发光报告，以定量测量细胞膜受体的内化，基于报告活性的pH依赖性。G蛋白偶联受体RXFP3作为松弛素-3/INSL7的同源受体作为模型受体。我们首先生成稳定的HEK293T细胞，诱导共表达c端nanoluc标记的人RXFP3和c端增强的绿色荧光蛋白（EGFP）标记的人RXFP3。c端EGFP-tag和NanoLuc-tag对RXFP3的配体结合能力和细胞内转运没有不利影响。基于标记的EGFP报告基因的荧光，在荧光显微镜下直接观察配体诱导的RXFP3内化过程。基于标记的NanoLuc报告基因的生物发光特性，采用便捷的生物发光法定量测定配体诱导的RXFP3内化。egfp标记的失活[E141R]RXFP3的共表达对nanolac标记的野生型RXFP3的配体结合能力和配体诱导内化没有不利影响，这表明突变型RXFP3和野生型RXFP3是独立工作的。本生物发光内化实验可推广到其他G蛋白偶联受体和其他细胞膜受体，以研究配体-受体和受体-受体相互作用。(C) 2014 Elsevier B.V.版权所有
Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions. (C) 2014 Elsevier B.V. All rights reserved.