Burström Viktor, Xu Kuiying, Garro-Martínez Emilio, Mach Robert H, Sahlholm Kristoffer, Betari Nibal
Department of Medical and Translational Biology, Wallenberg Centre for Molecular Medicine, Umeå University, 901 87, Umeå, Sweden.
Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104-6323, United States.
Biochem Biophys Rep. 2025 Apr 18;42:102019. doi: 10.1016/j.bbrep.2025.102019. eCollection 2025 Jun.
Luciferase complementation assays have emerged as a simple means of monitoring receptor-effector interactions in living cells in a time-resolved manner. Here, we describe a nanoluciferase complementation assay capable of reporting on β-arrestin2 recruitment to the human dopamine D receptor (DR) upon its activation in intact HEK293T cells. Using this assay in time-resolved experiments, we detect differences in arrestin response termination rates between the endogenous agonist dopamine and the synthetic DR agonist FAUC-73. We also investigate the influence of exogenous GRK2 on β-arrestin2 recruitment to the DR. We find that, in contrast to the DR and DR, the potency of dopamine to induce arrestin recruitment to DR is not significantly influenced by GRK2 overexpression. In further agreement with a lack of GRK2 regulation of DR signalling and again contrary to the DR and DR, we do not observe dopamine-induced recruitment of GRK2 to DR. Conversely, dopamine concentration-dependently decreases the interaction between GRK2 and DR. Additionally, we examine both the Ser-9 and Gly-9 variants of the human DR, which, according to some earlier reports, differ in terms of dopamine affinity and functional potency. However, we find no difference in the concentration-response relationships between these two variants, neither when arrestin recruitment nor GRK2 interactions are studied. In summary, the present report demonstrates the utility of nanoluciferase complementation for studying DR pharmacology in living cells.
荧光素酶互补分析已成为一种以时间分辨的方式监测活细胞中受体 - 效应器相互作用的简单方法。在此，我们描述了一种纳米荧光素酶互补分析，它能够报告在完整的HEK293T细胞中，人多巴胺D受体（DR）激活后β - 抑制蛋白2的募集情况。在时间分辨实验中使用该分析方法，我们检测到内源性激动剂多巴胺与合成DR激动剂FAUC - 73之间在抑制蛋白反应终止速率上的差异。我们还研究了外源性GRK2对β - 抑制蛋白2募集到DR的影响。我们发现，与DR和DR不同，多巴胺诱导抑制蛋白募集到DR的效力不受GRK2过表达的显著影响。与缺乏GRK2对DR信号传导的调节一致，并且再次与DR和DR相反，我们没有观察到多巴胺诱导GRK2募集到DR。相反，多巴胺浓度依赖性地降低了GRK2与DR之间的相互作用。此外，我们研究了人DR的Ser - 9和Gly - 9变体，根据一些早期报告，它们在多巴胺亲和力和功能效力方面存在差异。然而，当研究抑制蛋白募集或GRK2相互作用时，我们发现这两种变体之间在浓度 - 反应关系上没有差异。总之，本报告证明了纳米荧光素酶互补在研究活细胞中DR药理学方面的实用性。
