Characterization of reactive oxygen species as signal transducers in beta2 -Adrenergic receptor signaling and evaluation of their role in receptor-beta-arrestin interactions

Reactive oxygen species (ROS) seem to have dual roles in cellular patho-physiology. At higher concentrations, ROS induce cellular damage whereas, at lower concentrations, they can act as signal transducers. To date, very little is known about the role of ROS in G protein coupled receptor (GPCR) signal transduction, and specifically in G protein independent, beta-arrestin dependent signaling cascades. In this study we explored the role of ROS in beta-arrestin mediated signaling of the beta2-adrenergic receptor (beta2AR). Using (betaAR mediated ERK1/2 phosphorylation as a functional end point, our results demonstrated that agonist mediated ERK112 phosphorylation was inhibited by flavin oxidase inhibition and intracellular ROS depletion, an effect which was rescued upon exogenous ROS administration. Furthermore, using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays to assess the role of ROS in the direct physical interactions of the beta2AR with beta-arrestin, it was found that beta2AR-43-arrestin interactions were abolished upon ROS inhibition. Taken together, our results show that ROS are required for the direct association of beta2AR with beta-arrestin and are also required for G protein mediated as well as beta-arrestin mediated phosphorylation of ERKI/2 upon beta2AR agonism. These results suggest that ROS are vital for stabilizing beta2AR in signaling capable conformations and that both G protein dependent and 13-arrestin dependent beta2AR signaling rely on some degree of basal ROS for the efficient downstream signal transduction.