Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Int J Mol Sci. 2019 Jun 17;20(12):2958. doi: 10.3390/ijms20122958.
G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR-GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein-protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR-GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR-GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.
G 蛋白偶联受体（GPCRs）有形成同源和异源二聚体的倾向。这些二聚体的功能障碍与多种疾病有关，例如先兆子痫、精神分裂症和抑郁症等。在过去的二十年中，人们为开发用于研究活细胞中这些 GPCR 二聚体复合物的筛选测定方法做出了相当大的努力。作为第一步，在过表达系统中建立稳健的体外测定方法对于鉴定和表征特定的 GPCR-GPCR 相互作用至关重要，然后是在内源性水平和最终体内证明关联的方法。本综述重点介绍了用于研究 GPCR 寡聚化的蛋白互补测定法（PCAs）。这些方法通常基于荧光和发光，使得鉴定和定位蛋白-蛋白相互作用成为可能。感兴趣的 GPCR 与互补的荧光或发光片段融合，一旦 GPCR 二聚化或寡聚化，就可能重新组成功能性报告子，其活性可以测量。各种蛋白互补测定法都有一个缺点，即重新组装的分裂片段之间的相互作用是不可逆的，这可能导致假阳性读数。可逆系统具有几个优点，因为它们不仅允许跟踪 GPCR-GPCR 相互作用的动力学，还允许评估配体（激动剂或拮抗剂）对受体复合物的调制。蛋白互补测定法也可用于高通量筛选，这是非常相关的，因为越来越多的人有兴趣和努力来识别潜在的针对疾病相关二聚体的小分子药物。除了概述 PCAs 如何使我们更好地了解 GPCR-GPCR 相互作用之外，本综述还旨在提供关于如何进行基于 PCA 的测定的实用指南。
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