Clathrin regulates the β-arrestin pathway regardless of ligand bias

μ-opioid receptors (MOP) are thought to activate the G protein-mediated analgesic pathway and β-arrestin 2-mediated side effect pathway; however, ligands that recruit β-arrestin 2 only minimally to MOP may also cause opioid side effects. Such side effects are also induced in mutant mice lacking β-arrestin 2 or expressing phosphorylation-deficient MOP that do not recruit β-arrestin 2. These findings critically questioned whether β-arrestin 2 recruitment to MOP triggers side effects. Here, we show that β-arrestin 1 partially compensates for the lack of β-arrestin 2 in a neuronal cell line and thus might be involved in triggering such side effects in β-arrestin 2-null mice. Moreover, the magnitude of β-arrestin-mediated signals is not correlated with β-arrestin recruitment to MOP via phosphorylation of the carboxyl-terminal of MOP, which has long been used to evaluate β-arrestin bias of a ligand. Instead, β-arrestin activates downstream signals by binding with the clathrin heavy chain in the process of clathrin-coated pit formation. Our findings provide not only a novel insight into G protein-coupled receptor-mediated signalling to overcome opioid side effects but also an unexpected concept that the accumulation of molecules required for endocytosis is a key for activating the intracellular signalling.