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Ligand Binding to the β2‐Adrenergic Receptor is Dependent Upon its Oxidation State

Inhaled β2‐adrenergic receptor (β2AR) agonists are the gold standard treatment of an acute asthma attack and function by eliciting rapid bronchial dilation. Asthmatic tissue is characterized by increased expression of NADPH Oxidase (NOX) and subsequent increased levels of reactive oxygen species (ROS) compared to healthy tissue. We and others have previously showed that agonism of β2AR with ‐isoproterenol (ISO) leads to activation of NOX, and generation of intracellular ROS. We have also previously shown that these ROS are capable of oxidizing cysteine residues on the β2AR itself, a process known as cysteine‐S‐sulfenation. Emerging evidence has established cysteine‐S‐sulfenation as a functionally significant post translational protein modification, which thus far has not been evaluated for its effects on β2AR ligand binding. Utilizing isolated plasma membrane and whole cell radioligand binding, the current study evaluates oxidation state‐dependent alterations to ligand binding to the β2AR in HEK293 cells over‐expressing β2AR and in immortalized human lung epithelial cells. Specific ligand binding is significantly increased in the oxidized receptor state in both cell lines and in both membrane and whole cell assays. This effect is reversed by receptor labeling with the sulfenic acid selective small molecule dimedone, indicating increased ligand binding is specific to the presence of cysteine sulfenic acids on the β2AR. Furthermore, isoproterenol can fully displace radioligand binding in all redox states to the same extent in a monophasic manner, indicating redox state either directly or indirectly modifies the β2AR's orthosteric binding pocket. Given the oxidative environment native to asthmatic tissue, the findings presented here have implications for asthma treatment with β2AR agonists, and present a need to better understand how β2AR may interact differently with ligands in asthmatic tissue compared to healthy models.