U50,488H-induced Internalization of the Human κ Opioid Receptor Involves a β-Arrestin- and Dynamin-dependent Mechanism

Abstract

Agonist-promoted internalization ofsome G protein-cou- pled receptors has been shown to mediate receptor desen- sitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internal- ization of the human and rat ? opioid receptors stably ex- pressed in Chinese hamster ovary cells, the potential mech- anisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Ex- posure of the human ? receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etor- phine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat ? opioid receptor. U50,488H-induced human ? receptor internalization was time- and concentration-dependent, with 30–40% of the receptors internalized following a 30- minexposure to 1?MU50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50,488H- induced internalization without affecting internaliza- tion itself, while pretreatment with pertussis toxin had no effect on U50,488H-induced internalization. In con- trast, incubation with sucrose (0.4–0.8 M) significantly reduced U50,488H-induced internalization of the ? recep- tor. While co-expression ofthe wild type GRK2,␤-arrestin, or dynamin I had no effect on ? receptor internalization, co-expression of the dominant negative mutants GRK2- K220R, ␤-arrestin (319–418), or dynamin I-K44A signifi- cantly inhibited receptor internalization. Whether recep- tor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant nega- tive mutants of ␤-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etor- phine, which did not promote human ?receptor internal- ization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat ? receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internal- ization of the human ? opioid receptor in Chinese ham- ster ovary cells occurs via a GRK-, ␤-arrestin-, and dy- namin I-dependent process that likely involves clathrin coated pits. In addition, internalization of the ? receptor is not required for activation of MAP kinase.