逮捕
内体
G蛋白偶联受体
细胞生物学
受体
旁观者效应
化学
内吞循环
信号转导
生物
内吞作用
生物化学
免疫学
作者
Yubo Cao,Yoon Namkung,Stéphane A. Laporte
出处
期刊:Methods in molecular biology
日期:2019-01-01
卷期号:: 59-68
被引量:10
标识
DOI:10.1007/978-1-4939-9158-7_3
摘要
β-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of β-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/β-arrestin complexes to endosomes. Trafficking of receptor/β-arrestin complexes can be assessed by BRET through tagging β-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of β-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring β-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and β2-adrenergic receptor (β2AR) at the PM and the trafficking of receptor/β-arrestin complexes into endosomes using EbBRET-based biosensors.
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