Agonist- and Hydrogen Peroxide-Mediated Oxidation of the 2 Adrenergic Receptor: Evidence of Receptor S-Sulfenation as Detected by a Modified Biotin-Switch Assay□S

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), have recently been shown to be generated upon ago-nism of several members of the G protein-coupled receptor (GPCR) superfamily, including 2-adrenergic receptors (2ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates 2AR signaling, suggest-ing that 2AR-mediated ROS generation is capable of feeding back to regulate receptor function. Given that ROS, specifically H2O2, are able to post-translationally oxidize protein cysteine sulfhydryls to cysteine-sulfenic acids, the goal of the current study was to assess whether ROS are capable of S-sulfenating 2AR. Using a modified biotin-switch assay that is selective for cysteine-sulfenic acids, our results demonstrate for the first time that H2O2 treatment facilitates S-sulfenation of transiently overexpressed 2AR in human embryonic kidney 293 cells. It is noteworthy that stimulation of cells with the -agonist isopro-terenol produces both dose- and time-dependent S-sulfenation of 2AR, an effect that is receptor-dependent, and demon-strates that receptor-generated ROS are also capable of oxi-dizing the 2AR. Receptor-dependent S-sulfenation was inhib-ited by the chemoselective sulfenic acid alkylator dimedone and the cysteine antioxidant N-acetyl-L-cysteine. Moreover, our results reveal that receptor oxidation occurs in cells that en-dogenously express physiologically relevant levels of 2AR, because treatment of human alveolar epithelial A549 cells with either H2O2 or the 2-selective agonist formoterol promoted receptor S-sulfenation. These findings provide the first evi-dence, to our knowledge, that a mammalian GPCR can be oxidized by S-sulfenation and signify an important first step toward shedding light on the overlooked role of ROS in the regulation of 2AR function