Abstract 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. One Sentence summary Agonist-specific differences in β-arrestin recruitment are mainly determined by the ligand dissociation rate and G protein activation at the endosomes.

Receptor endocytosis orchestrates the spatiotemporal bias of β-arrestin signaling