β-arrestin Signaling from the Cannabinoid Receptor 1

Background: G protein-coupled receptors (GPCRs) signal through G-proteins or β-arrestins. While G-protein pathways are well characterized, the mechanism controlling and the cascades mediated by β-arrestin are not well described. β-arrestin signaling initiates when receptors and β-arrestins associate in endocytic pits. Because GPCRs and β-arrestin interact during this process, we hypothesize that ligands control the time GPCRs and β-arrestin are bound at the cell surface into endocytic pits, dwell time, as a mechanism to control β-arrestin signaling. Our objective is to determine the mechanisms regulating β-arrestin signaling from the cannabinoid receptor 1 (CB1R), one of the most abundant receptors in the central nervous system (CNS), and test if this signaling can be manipulated. Methods: Endocytosis at the single pit level was investigated in human embryonic kidney (HEK)293 cells transfected with the CB1R tagged with the pH sensitive super-ecliptic phluorin (SEP) to increase the fluorescence from receptors located at the plasma membrane. Endocytosis was initiated by bath application of ligands and signaling was assessed by Western-blots before and after chemical or genetic modulation of the endocytic process. Results: We identified ligand-specific endocytic dwell times. 2-AG elicited prolonged dwell times and strong β-arrestin signaling, whereas WIN elicited short ones and no β-arrestin signaling. Furthermore, chemical inhibition of endocytosis significantly increased β-arrestin signaling. In addition, we have identified S426A/S430A in the receptor as key sited for ligand-specific dwell times. Conclusions: Ligand-specific dwell times could be a universal process for GPCRs and a novel therapeutic target to control β-arrestin signaling.