Reyes-Alcaraz Arfaxad, Lee Yoo-Na, Yun Seongsik, Hwang Jong-Ik, Seong Jae Young
Graduate School of Medicine, Korea University;
Graduate School of Medicine, Korea University.
J Vis Exp. 2019 Jun 28(148). doi: 10.3791/59994.
Interactions between G-protein coupled receptors (GPCRs) and β-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with β-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-β-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-β-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-β-arrestin complex.
G蛋白偶联受体（GPCRs）与β-抑制蛋白之间的相互作用是具有极其重要生理意义的关键过程。目前，在GPCR药物研发领域，尤其是在GPCR偏向性激动作用的研究中，鉴定新型药物与β-抑制蛋白及其他胞质蛋白之间的相互作用极具价值。在此，我们展示了一种新型结构互补检测方法的应用，该方法能够在实时活体系统中准确监测受体与β-抑制蛋白的相互作用。此方法简单、准确，可轻松扩展至任何感兴趣的GPCR，并且具有克服因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的优势。这种结构互补检测方法提供了关键特性，能够准确、精确地监测受体与β-抑制蛋白的相互作用，使其适用于任何GPCR系统的偏向性激动作用研究，以及由不同GPCR激酶（GRKs）编写的GPCR C末端“磷酸化密码”和稳定或破坏受体 - β-抑制蛋白复合物的抑制蛋白的翻译后修饰研究。
