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.
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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, USA.
authors.forename: Rebecca N
authors.initials: RN
authors.lastname: Burns
authors.forename: Nader H
authors.initials: NH
authors.lastname: Moniri
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D000478:Alkylation; D001681:Biological Assay; D001710:Biotin / Q000378:metabolism; D003512:Cyclohexanones / Q000494:pharmacology; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects; D004983:Ethanolamines / Q000494:pharmacology; D000068759:Formoterol Fumarate; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011650:Pulmonary Alveoli; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D003512:Cyclohexanones; D004983:Ethanolamines; D017382:Reactive Oxygen Species; D018343:Receptors, Adrenergic, beta-2; D013434:Sulfenic Acids; D001710:Biotin; C004774:dimedone; D006861:Hydrogen Peroxide; C100870:cysteinesulfenic acid; D003545:Cysteine; D007545:Isoproterenol; D000068759:Formoterol Fumarate