Reactive oxygen species are required for β2 adrenergic receptor-β-arrestin interactions and signaling to ERK1/2.
The β2-adrenergic receptor (β2AR) 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 β2AR 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 β-arrestin-mediated pathway that occurs at later time points. We have previously shown that β2AR agonism promotes generation of intracellular reactive oxygen species (ROS) and that β2AR-associated G protein signaling is dependent on ROS formation. It has also been suggested that β2AR-mediated ROS generation occurs via recruitment of β-arrestins. In this study, we confirm the effects of β-arrestin on β2AR-induced ROS generation, and investigate the ROS-dependency of β-arrestin-linked β2AR signaling. In HEK293 cells, both coimmunoprecipitation and BRET studies reveal that ROS are vital for the physical interaction of β2AR with β-arrestin partner proteins. Using phosphorylation of ERK1/2 as a functional endpoint to assess the role of ROS in β2AR-β-arrestin signaling, our results show that inhibition of intracellular ROS abrogates both the β-arrestin and G protein-mediated phosphorylation of ERK1/2 upon agonism of β2AR. Importantly, both the G protein and β-arrestin components were reversed upon exogenous administration of ROS, suggesting a critical role for oxidants in stabilization of β2AR. Taken together, our data signify that ROS serve purposeful roles in stabilizing both G protein- and β-arrestin-mediated β2AR signaling in HEK293 cells.
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, USA.
Monalisa
M
Singh
Nader H
NH
Moniri
D019390:Arrestins / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004735:Energy Transfer; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
D019390:Arrestins; D017382:Reactive Oxygen Species; D018343:Receptors, Adrenergic, beta-2; D000071557:beta-Arrestins; D006861:Hydrogen Peroxide