Receptor endocytosis orchestrates the spatiotemporal bias of β-arrestin signaling

The varying efficacy of biased and balanced agonists is generally explained by the stabilization of different active receptor conformations. In this study, systematic profiling of transducer activation of AT ₁ angiotensin receptor agonists revealed that the extent and kinetics of β-arrestin binding exhibit substantial ligand-dependent differences, which however completely disappear upon the inhibition of receptor internalization. Even weak partial agonists for the β- arrestin pathway acted as full or near full agonists, if receptor endocytosis was prevented, indicating that receptor conformation is not an exclusive determinant of β-arrestin recruitment. The ligand-dependent variance in β-arrestin translocation at endosomes was much larger than it was at the plasma membrane, showing that ligand efficacy in the β-arrestin pathway is spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shape the effects of agonists on endosomal receptor–β-arrestin binding and thus determine the extent of bias. Among others, ligand dissociation rate and G protein activity have particularly strong impact on receptor–β-arrestin interaction, and their effects are integrated at endosomes. Our results highlight that endocytosis forms a key spatiotemporal platform for biased GPCR signaling and can aid the development of more efficacious functionally-selective compounds.