Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA.
Integr Biol (Camb). 2013 Oct;5(10):1253-61. doi: 10.1039/c3ib40112j.
The canonical model of G protein-coupled receptor (GPCR) signalling states that it is solely initiated at the cell surface. In recent years, a handful of evidence has started emerging from high-resolution molecular assays that the internalized receptors can mediate the third wave of signalling, besides G protein- and β-arrestin-mediated signalling both initiating at the cell surface. However, little is known about the functional consequences of distinct waves of GPCR signalling, in particular, at the whole cell system level. We here report the development of label-free biosensor antagonist reverse assays and their use to differentiate the signalling waves of an endogenous β2-adrenergic receptor (β2-AR) in A431 cells. Results showed that the persistent agonist treatment activated the β2-ARs, leading to a long-term sustained dynamic mass redistribution (DMR) signal, a whole cell phenotypic response. Under the persistent treatment scheme in microplates, a panel of known β-blockers all dose-dependently and completely reversed the DMR signal of epinephrine at a relatively low dose (10 nM), except for sotalol which partially reversed the DMR. Under the perfusion conditions with microfluidics, the subsequent perfusion with sotalol only reversed the DMR induced by epinephrine or isoproterenol at 10 nM, but not at 10 μM. Furthermore, the degree of the DMR reversion by sotalol was found to be in an opposite relation with the duration of the initial agonist treatment. Together, these results suggest that the hydrophilic antagonist sotalol is constrained outside the cells throughout the assays, and the early signalling wave initiated at the cell surface dominates the DMR induced by epinephrine or isoproterenol at relatively low doses, while a secondary and late signalling wave is initiated once the receptors are internalized and contributes partially to the long-term sustainability of the DMR of epinephrine or isoproterenol at high doses.
G 蛋白偶联受体 (GPCR) 信号的经典模型表明，它仅在细胞表面起始。近年来，一些高分辨率分子分析的证据开始出现，表明除了细胞表面起始的 G 蛋白和β-arrestin 介导的信号外，内化的受体也可以介导第三波信号。然而，对于不同的 GPCR 信号波的功能后果，特别是在整个细胞系统水平上，人们知之甚少。我们在这里报告了无标记生物传感器拮抗剂反向测定法的开发及其在 A431 细胞中区分内源性β2-肾上腺素能受体 (β2-AR) 信号波的用途。结果表明，持续激动剂处理激活了β2-AR，导致长期持续的动态质量重分布 (DMR) 信号，这是一种全细胞表型反应。在微孔板中的持续处理方案下，一系列已知的β阻断剂均以剂量依赖性且完全的方式在相对较低的剂量（10 nM）下逆转肾上腺素的 DMR 信号，除了索他洛尔部分逆转了 DMR。在微流体灌注条件下，随后用索他洛尔灌注仅在 10 nM 时逆转肾上腺素或异丙肾上腺素诱导的 DMR，但在 10 μM 时则不能。此外，发现索他洛尔逆转 DMR 的程度与初始激动剂处理的持续时间呈相反关系。总之，这些结果表明，亲水性拮抗剂索他洛尔在整个测定过程中都被约束在细胞外，在相对较低的剂量下，起始于细胞表面的早期信号波主导肾上腺素或异丙肾上腺素诱导的 DMR，而一旦受体内化，就会启动二次和晚期信号波，部分导致肾上腺素或异丙肾上腺素的 DMR 长期持续。
