全内反射荧光显微术在G蛋白偶联受体研究中的应用

全内反射荧光显微术在G蛋白偶联受体研究中的应用

方三华，杨 丹，刘 丽，肖桂凤

（浙江大学医学院公共技术平台，浙江杭州310058）

摘  要 G蛋白偶联受体是最大的细胞膜受体超家族，是许多临床治疗药物的靶点。放射性配体结合、酶联免疫吸附和免疫共沉淀是研究它们结构和功能的主要技术，但这些属于群体平均(ensemble average)研究，无法得到单个分子图像和动态变化。全内反射荧光显微镜利用隐逝波激发临界面下200 nm范围内荧光物质成像，具有信噪比高、Z轴分辨率高、速度快和对生物样本损伤小等优点，是常用的单分子荧光成像技术。本文在介绍全内反射荧光显微镜发展历史、原理、组成和成像优势的基础上，详细阐述全内反射荧光显微镜在研究G蛋白偶联受体构象变化、寡聚化、内吞和再循环以及信号转导等方面的应用，并提出未来展望。

关键词  全内反射原理；荧光显微镜；G蛋白偶联受体；应用

中图分类号：R966；Q336  文献标识码：A  doi:10.3969/j.issn.1000-6281.2023.03.013

Application of total internal reflection fluorescence microscopy in the study on G protein coupled receptors

FANG San-hua，YANG Dan，LIU Li

(Core Facilities, Zhejiang University School of Medicine, Hangzhou Zhejiang 310058, China)

Abstract G protein coupled receptors are the largest superfamily of cell membrane receptors and the targets of many clinical therapeutic drugs. Their structures and functions are mainly studied by radioligand binding, enzyme-linked immunosorbent assay and immunocoprecipitation. However, these techniques are group average and could not obtain single molecular images and dynamic changes. Total internal reflection fluorescence microscope uses an evanescent wave to excite fluorescent materials in the range of 200 nm under the critical interface. It has the advantages of high signal-to-noise ratio, high Z-axis resolution, fast speed and little damage to biological samples. It is a commonly used single molecule fluorescence imaging technology. Based on the introduction of the development history, principle, composition and imaging advantages of total internal reflection fluorescence microscopy, this paper describes in detail the application of total internal reflection fluorescence microscopy in studying the conformational changes, oligomerization, endocytosis and recycling of G protein coupled receptors, and signal transduction.

Keywords   principle of total internal reflection; fluorescence microscopy; G protein coupled receptors; application

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