Differential beta-arrestin2 requirements for constitutive and agonist-induced internalization of the CB cannabinoid receptor

Abstract

CB1 cannabinoid receptor (CB1R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of beta-arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB1R binds beta-arrestin2 (beta-arr2), but not beta-arrestin1. Furthermore, both the expression of dominant-negative beta-arr2 (beta-arr2-V54D) and siRNA-mediated knock-down of beta-arr2 impaired the agonist-induced internalization of CB1R. In contrast, neither beta-arr2-V54D nor beta-arr2-specific siRNA had a significant effect on the constitutive internalization of CB1R. However, both constitutive and agonist-induced internalization of CB1R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB1R, beta-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB1R are different.