一种快速荧光 GPCR-β-arrestin 相互作用检测法。
A rapid fluorogenic GPCR-β-arrestin interaction assay.
机构信息
Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, California.
Cardiovascular Research Institute, University of California-San Francisco, San Francisco, California.
出版信息
Protein Sci. 2018 Apr;27(4):874-879. doi: 10.1002/pro.3385. Epub 2018 Feb 23.
Abstract
Detection of protein-protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein-coupled receptor (GPCR)-β-arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal-to-noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease-activated receptor-1 activation developed in minutes and required specific serine-threonine residues in the receptor carboxyl tail, consistent with a classical G protein-coupled receptor kinase dependent β-arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.
摘要
检测活细胞和生物体内信号转导涉及的蛋白质-蛋白质相互作用具有多种重要的应用。我们报告了一种荧光测定法，用于检测 G 蛋白偶联受体（GPCR）-β-arrestin 相互作用，该方法是遗传编码的，可推广到多种 GPCR，并具有高信噪比，因为荧光在 GPCR 激活时其成分相互作用之前是不存在的。蛋白酶激活受体-1 激活后几分钟内出现荧光，需要受体羧基末端的特定丝氨酸-苏氨酸残基，与经典的 G 蛋白偶联受体激酶依赖的β-arrestin 募集机制一致。该测定法为其他体内 GPCR 激活检测提供了有用的补充。
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