ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.
ClickArr: a novel, high-throughput assay for evaluating β-arrestin isoform recruitment.
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DOI：
10.3389/fphar.2023.1295518
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发表时间：
2023
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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背景：定量G蛋白偶联受体（GPCR）信号偏差的现代方法依赖于使用单一β-抑制蛋白亚型。然而，越来越多的人认识到，两种β-抑制蛋白亚型具有独特的作用，需要评估β-抑制蛋白亚型偏好的能力。因此，需要有效筛选两种β-抑制蛋白同种型在细胞中竞争靶GPCR时的募集的方法。 研究方法：我们利用分子克隆技术将δ-阿片受体（δOR）、β-arrestin 1和β-arrestin 2融合到甲虫绿色和甲虫红色激酶的片段中。在该测定体系结构中，β-抑制蛋白1或2向δOR的募集产生光谱上不同的生物发光信号，使我们能够在激动剂激发前将所有三种构建体共转染到细胞中。 结果如下：我们证明了我们的新测定法，名为“ClickArr”，是一种活细胞测定法，它同时报告了两种β-抑制蛋白亚型的募集，因为它们竞争与δOR的相互作用。我们进一步发现，当将募集标准化为参考激动剂亮脑啡肽时，部分δOR激动剂TAN 67对β-抑制蛋白2相对于β-抑制蛋白1具有显著的功效偏倚。我们证实，ClickArr在作为高通量终点或高通量动力学测定运行时报告了该偏倚，并使用PathHunter测定（一种用于报告β-抑制蛋白募集至δOR的正交商业测定）交叉验证了该结果。 结论：我们的研究结果表明，激动剂：GPCR复合物可能具有相对的β-抑制蛋白亚型偏好，这是一种新的信号传导偏好，可能为药物开发开辟新的方向。
Background: Modern methods for quantifying signaling bias at G protein–coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both β-arrestin isoforms as they compete for a target GPCR in cells. Methods: We used molecular cloning to develop fusion proteins of the δ-opioid receptor (δOR), β-arrestin 1, and β-arrestin 2 to fragments of click beetle green and click beetle red luciferases. In this assay architecture, recruitment of either β-arrestin 1 or 2 to the δOR generates a spectrally distinct bioluminescent signal, allowing us to co-transfect all three constructs into cells prior to agonist challenge. Results: We demonstrate that our new assay, named “ClickArr,” is a live-cell assay that simultaneously reports the recruitment of both β-arrestin isoforms as they compete for interaction with the δOR. We further find that the partial δOR agonist TAN67 has a significant efficacy bias for β-arrestin 2 over β-arrestin 1 when recruitment is normalized to the reference agonist leu-enkephalin. We confirm that ClickArr reports this bias when run either as a high-throughput endpoint or high-throughput kinetic assay, and cross-validate this result using the PathHunter assay, an orthogonal commercial assay for reporting β-arrestin recruitment to the δOR. Conclusion: Our results suggest that agonist:GPCR complexes can have relative β-arrestin isoform bias, a novel signaling bias that may potentially open up a new dimension for drug development.
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