Agonist‐dependent oxidation of the β2 adrenergic receptor: Selective cysteine sulfenic acid formation as detected by a modified biotin‐switch assay

While traditional signaling mechanisms have been extensively investigated for the β2 adrenergic receptor (β2AR), our laboratory has recently found that reactive oxygen species (ROS) are key mediators of β2AR signaling. Previously, we showed that agonism of the β2AR generates ROS, and that these oxidants are indispensible for receptor function. Based on our previous data, we hypothesized that ROS, which are generated upon β2AR agonism, can feedback to oxidize the receptor, and that this modification allows β2AR to maintain functionally competent conformations. As ROS frequently oxidize cysteine residues, we developed a modified biotin‐switch assay to examine if β2AR agonism leads to selective oxidation of receptor cysteine sulfhydryl groups to sulfenic acids (Cys‐SOH). Our results show that agonism leads to oxidation of β2AR in a time and dose dependent manner. These effects are mimicked by treatment with exogenous oxidants and blocked by the β‐receptor antagonist propranolol as well as antioxidants. Potential molecular mechanisms and site(s) of sulfenic acid‐modification are also presented. Taken together, our results suggest that agonist‐mediated oxidation of β2AR cysteine residues to cysteine‐sulfenic acids can stabilize receptor conformations which allow for downstream signaling.