β2‐adrenergic receptor mediated generation of reactive oxygen species is a component required for signal transduction, desensitization, and homodimerization

Abstract

The β2‐adrenergic receptor (β2‐AR) has previously been shown to stimulate the formation of reactive oxygen species (ROS). The aim of the current study was to assess the importance of ROS on β2‐AR signaling cascades. Using HEK‐293 cells, which endogenously express β2‐AR, along with pharmacological inhibitors of various stages of ROS‐generating machinery, we examined the role of ROS generation on β2‐AR signaling, desensitization and homodimerization. Our results demonstrate that treatment of cells with the antioxidant NAC, the flavin‐containing oxidase inhibitor DPI, or the Rac1 inhibitor NSC23766, decreases β2‐AR‐mediated ROS generation, and importantly, that ROS inhibition abrogates β2‐AR‐mediated cAMP formation and PKA activation, as well as receptor phosphorylation and internalization. Additionally, our data demonstrate that β2‐AR‐β‐arrestin interactions, as well as β2‐AR‐β2‐AR homodimerization are decreased upon treatment of cells with ROS inhibitors. Taken together, these results suggest that intracellular ROS, which are generated upon agonist‐stimulation of β2‐AR, serve to stabilize receptor conformations that facilitate partner‐protein interactions which regulate downstream signaling, desensitization and homodimerization. Experiments to elucidate mechanisms of β2‐AR‐mediated ROS generation are currently underway in our laboratory.