Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Biochem Pharmacol. 2018 Feb;148:298-307. doi: 10.1016/j.bcp.2018.01.008. Epub 2018 Jan 5.
Besides classical G protein coupling, G protein-coupled receptors (GPCRs) are nowadays well known to show significant signalling via other adaptor proteins, such as β-arrestin2 (βarr2). The elucidation of the molecular mechanism of the GPCR-βarr2 interaction is a prerequisite for the structure-activity based design of biased ligands, which introduces a new chapter in drug discovery. The general mechanism of the interaction is believed to rely on phosphorylation sites, exposed upon agonist binding. However, it is not known whether this mechanism is universal throughout the GPCR family or if GPCR-specific patterns are involved. In recent years, promising orally active agonists for the human A adenosine receptor (AAR), a GPCR highly expressed in inflammatory and cancer cells, have been evaluated in clinical trials for the treatment of rheumatoid arthritis, psoriasis, and hepatocellular carcinoma. In this study, the effect of cytoplasmic modifications of the AAR on βarr2 recruitment was evaluated in transiently transfected HEK293T cells, using a live-cell split-reporter system (NanoBit®, Promega), based on the structural complementation of NanoLuc luciferase, allowing real-time βarr2 monitoring. The AAR-selective reference agonist 2-Cl-IB-MECA yielded a robust, concentration dependent (5 nM-1 µM) recruitment of βarr2 (logEC50: -7.798 ± 0.076). The role of putative phosphorylation sites, located in the C-terminal part and cytoplasmic loops, and the role of the 'DRY' motif was evaluated. It was shown that the AAR C-terminus was dispensable for βarr2 recruitment. This contrasts with studies in the past for the rat AAR, which pointed at crucial C-terminal phosphorylation sites. When combining truncation of the AAR with modification of the 'DRY' motif to 'AAY', the βarr2 recruitment was drastically reduced. Recruitment could be partly rescued by back-mutation to 'NQY', or by extending the C-terminus again. In conclusion, other parts of the human AAR, either cytosolic or exposed upon receptor activation, rather than the C-terminus alone, are responsible for βarr2 recruitment in a complementary or synergistic way.
除了经典的 G 蛋白偶联，G 蛋白偶联受体（GPCR）现在也被广泛认为通过其他衔接蛋白（如β-arrestin2，βarr2）显示出显著的信号传导。阐明 GPCR-βarr2 相互作用的分子机制是基于结构活性的偏倚配体设计的前提，这为药物发现开辟了新的篇章。这种相互作用的一般机制被认为依赖于激动剂结合后暴露的磷酸化位点。然而，目前尚不清楚这种机制是否在整个 GPCR 家族中普遍存在，或者是否涉及 GPCR 特异性模式。近年来，已在临床试验中评估了具有口服活性的人类 A 腺苷受体（AAR）激动剂，该受体在炎症和癌细胞中高度表达，用于治疗类风湿关节炎、银屑病和肝细胞癌。在这项研究中，通过瞬时转染 HEK293T 细胞中的活细胞分割报告系统（NanoBit®，Promega），使用基于 NanoLuc 荧光素酶结构互补的方法，评估了 AAR 细胞质修饰对βarr2 募集的影响，从而可以实时监测βarr2。AAR 选择性参考激动剂 2-Cl-IB-MECA 产生了一种强大的、浓度依赖性的（5 nM-1 μM）βarr2 募集（logEC50：-7.798±0.076）。评估了位于 C 末端和细胞质环中的假定磷酸化位点以及“DRY”基序的作用。结果表明，AAR 的 C 末端对于βarr2 的募集是可有可无的。这与过去对大鼠 AAR 的研究形成了对比，后者指出了关键的 C 末端磷酸化位点。当 AAR 的截断与“DRY”基序的修饰相结合为“AAY”时，βarr2 的募集大大减少。通过回突变为“NQY”或再次延长 C 末端，可以部分挽救募集。总之，人类 AAR 的其他部分，无论是细胞质的还是受体激活后暴露的，而不仅仅是 C 末端，以互补或协同的方式负责βarr2 的募集。
