@article {Burns914, author = {Rebecca N. Burns and Nader H. Moniri}, title = {Agonist- and Hydrogen Peroxide-Mediated Oxidation of the β2 Adrenergic Receptor: Evidence of Receptor S-Sulfenation as Detected by a Modified Biotin-Switch Assay}, volume = {339}, number = {3}, pages = {914--921}, year = {2011}, doi = {10.1124/jpet.111.185975}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), have recently been shown to be generated upon agonism of several members of the G protein-coupled receptor (GPCR) superfamily, including β2-adrenergic receptors (β2ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates β2AR signaling, suggesting that β2AR-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 β2AR. 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 β2AR in human embryonic kidney 293 cells. It is noteworthy that stimulation of cells with the β-agonist isoproterenol produces both dose- and time-dependent S-sulfenation of β2AR, an effect that is receptor-dependent, and demonstrates that receptor-generated ROS are also capable of oxidizing the β2AR. Receptor-dependent S-sulfenation was inhibited 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 endogenously express physiologically relevant levels of β2AR, because treatment of human alveolar epithelial A549 cells with either H2O2 or the β2-selective agonist formoterol promoted receptor S-sulfenation. These findings provide the first evidence, 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 β2AR function.}, issn = {0022-3565}, URL = {https://jpet.aspetjournals.org/content/339/3/914}, eprint = {https://jpet.aspetjournals.org/content/339/3/914.full.pdf}, journal = {Journal of Pharmacology and Experimental Therapeutics} }