A beta-arrestin green fluorescent protein biosensor for detecting G protein-coupled receptor activation
A beta-arrestin green fluorescent protein biosensor for detecting G protein-coupled receptor activation
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DOI：
10.1074/jbc.272.44.27497
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Caron, MG
中科院分区：
文献类型：
--
作者：
Barak, LS;Ferguson, SSG;Caron, MG
G蛋白偶联受体（GPCR）代表了医学治疗中最重要的药物靶点，来自基因组测序和基因组数据库的信息大大加速了它们的发现。缺乏系统的方法来鉴定新的GPCR的功能或将其与同源配体相关联，增加了孤儿受体的数量。在这项工作中，我们提供了一种新的方法来解决这个问题，使用β-arrestin 2/绿色荧光蛋白缀合物（β arr 2-GFP）。它提供了一种实时和基于单细胞的测定来监测GPCR活化和GPCR-Gr蛋白偶联受体激酶或GPCR-抑制蛋白相互作用。共聚焦显微镜证实了β arr 2-GFP易位到超过15种不同的配体活化的GPCR。这些数据清楚地支持了活化受体的β-抑制蛋白结合是GPCR信号传导的会聚步骤的共同假设，已知与β-抑制蛋白相互作用的GPCR数量增加5倍，证明细胞溶质是生物活性β-抑制蛋白的主要储存库，并首次直接证明G蛋白偶联受体激酶磷酸化对活细胞中β-抑制蛋白活性和GPCR信号转导的生物调节的关键重要性，使用β arr 2-GFP作为生物传感器来识别不同GPCR的激活，应该加速孤儿受体的鉴定，并允许对普通生物化学方法难以处理的信号转导生物学进行光学研究。
G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a beta-arrestin2/green fluorescent protein conjugate (beta arr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-Gr protein-coupled receptor kinase or GPCR-arrestin interactions, Confocal microscopy demonstrates the translocation of beta arr2-GFP to more than 15 different ligand-activated GPCRs, These data clearly support the common hypothesis that the beta-arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with beta-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active beta-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of beta-arrestin activity and GPCR signal transduction in living cells, The use of beta arr2-GFP as a biosensor to recog- nize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.