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.
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