Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
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DOI：
10.3791/59994
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发表时间：
2019-06-01
影响因子：
1.2
通讯作者：
Seong, Jae Young
中科院分区：
文献类型：
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作者：
Reyes-Alcaraz, Arfaxad;Lee, Yoo-Na;Seong, Jae Young
G蛋白偶联受体(GPCRs)和β-抑制素之间的相互作用是具有重要生理意义的重要过程。目前，新药与β-抑制素和其他胞浆蛋白相互作用的表征在GPCR类药物发现领域，特别是在GPCR型偏向激动剂的研究中具有极其重要的价值。在这里，我们展示了一种新的结构互补分析的应用，以准确监测受体-β-arrestin在实时生命系统中的相互作用。这种方法简单、准确，可以很容易地扩展到任何感兴趣的GPCR，而且它的优点是克服了由于每个载体系统中存在低表达启动子而导致的非特异性相互作用。这种结构互补分析提供了关键特征，允许准确和精确地监测受体-β-arrestin的相互作用，使其适用于任何GPCR系统的偏向激动性的研究，以及由不同GPCRK(GRKs)编写的GPCRc末端‘磷酸化密码’的研究，以及稳定或破坏受体-β-arrestin复合体的抑制素的翻译后修饰。
Interactions between G-protein coupled receptors (GPCRs) and beta-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with beta-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-beta-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-beta-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus 'phosphorylation codes' written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-beta-arrestin complex.