Reactive oxygen species are required for β2 adrenergic receptor-β-arrestin interactions and signaling to ERK1/2

The beta 2-adrenergic receptor (beta 2AR) is the prototypical member of the heptahelical G protein-coupled receptor (GPCR) superfamily and is well-known to elicit biological effects through both G protein-dependent and G protein-independent signaling cascades. Agonism of beta 2AR has been described to promote phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) via a G-protein/PKA pathway that transpires rapidly upon receptor agonism, as well as by a distinct beta-arrestin-mediated pathway that occurs at later time points. We have previously shown that beta 2AR agonism promotes generation of intracellular reactive oxygen species (ROS) and that beta 2AR-associated G protein signaling is dependent on ROS formation. It has also been suggested that beta 2AR-mediated ROS generation occurs via recruitment of beta-arrestins. In this study, we confirm the effects of beta-arrestin on beta 2AR-induced ROS generation, and investigate the ROS-dependency of beta-arrestin-linked beta 2AR signaling. In HEK293 cells, both coimmunoprecipitation and BRET studies reveal that ROS are vital for the physical interaction of beta 2AR with beta-arrestin partner proteins. Using phosphorylation of ERK1/2 as a functional endpoint to assess the role of ROS in beta 2AR-beta-arrestin signaling, our results show that inhibition of intracellular ROS abrogates both the beta-arrestin and G protein-mediated phosphorylation of ERK1/2 upon agonism of beta 2AR. Importantly, both the G protein and beta-arrestin components were reversed upon exogenous administration of ROS, suggesting a critical role for oxidants in stabilization of beta 2AR. Taken together, our data signify that ROS serve purposeful roles in stabilizing both G protein- and beta-arrestin-mediated beta 2AR signaling in HEK293 cells. (c) 2012 Elsevier Inc. All rights reserved.