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The Journal of pharmacology and experimental therapeutics

Agonist- and hydrogen peroxide-mediated oxidation of the β2 adrenergic receptor: evidence of receptor s-sulfenation as detected by a modified biotin-switch assay.


PMID 21917560

Abstract

Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), have recently been shown to be generated upon agonism of several members of the G protein-coupled receptor (GPCR) superfamily, including β(2)-adrenergic receptors (β(2)ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates β(2)AR signaling, suggesting that β(2)AR-mediated ROS generation is capable of feeding back to regulate receptor function. Given that ROS, specifically H(2)O(2), 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 β(2)AR. Using a modified biotin-switch assay that is selective for cysteine-sulfenic acids, our results demonstrate for the first time that H(2)O(2) treatment facilitates S-sulfenation of transiently overexpressed β(2)AR 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 β(2)AR, an effect that is receptor-dependent, and demonstrates that receptor-generated ROS are also capable of oxidizing the β(2)AR. 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 β(2)AR, because treatment of human alveolar epithelial A549 cells with either H(2)O(2) or the β(2)-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 β(2)AR function.