RT Journal Article SR Electronic T1 Sequence-Specific Regulation of Endocytic Lifetimes Modulates Arrestin-Mediated Signaling at the µ Opioid Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 416 OP 427 DO 10.1124/mol.116.106633 VO 91 IS 4 A1 Zara Y. Weinberg A1 Amanda S. Zajac A1 Tiffany Phan A1 Daniel J. Shiwarski A1 Manojkumar A. Puthenveedu YR 2017 UL http://molpharm.aspetjournals.org/content/91/4/416.abstract AB Functional selectivity at the µ opioid receptor (µR), a prototypical G-protein–coupled receptor that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. Functional selectivity is a cumulative effect of the magnitudes of individual signaling pathways, e.g., the Gαi-mediated and the arrestin-mediated pathways for µR. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface control the magnitude of arrestin signaling, and therefore functional selectivity, at µR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for µR, lengthens surface lifetimes of receptor-arrestin clusters significantly compared with morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail of µR. Mutation of these leucines to alanines decreased the magnitude of arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetimes by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on µR to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.