Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University Health Sciences Center, Mercer University, Atlanta, GA30341, United States.
Sci Rep. 2020 Feb 19;10(1):2934. doi: 10.1038/s41598-020-59983-4.
Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. β2-adrenergic receptor (β2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that β2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes β2AR cysteine thiols to Cys-S-sulfenic acids (Cys-S-OH). Furthermore, highly oxidative environments can irreversibly oxidize Cys-S-OH to Cys-S-sulfinic (Cys-SOH) or S-sulfonic (Cys-SOH) acids, which are incapable of further participating in homeostatic redox reactions (i.e., redox-deficient). The aim of this study was to examine the vitality of β2AR-ROS interplay and the resultant functional consequences of β2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that β2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing β2AR, we show that receptor redox state profoundly influences β2AR orthosteric ligand binding and downstream function. Specifically, homeostatic β2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to β-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient β2AR states exhibit decreased ability to signal via either Gαs or β-arrestin. Together, our results demonstrate a β2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.
支气管收缩性气道疾病，如哮喘，其特征是炎症和活性氧（ROS）增加，从而产生高度氧化的环境。β2-肾上腺素能受体（β2AR）激动剂是哮喘临床治疗的主要方法，吸入后可使支气管松弛。我们之前已经表明，β2AR 激动剂会产生细胞内 ROS，这种作用是受体功能所必需的，并且会使β2AR 半胱氨酸巯基氧化为 Cys-S-磺酸（Cys-S-OH）。此外，高度氧化的环境会不可逆地将 Cys-S-OH 氧化为 Cys-S-亚磺酸（Cys-SOH）或 S-磺酸（Cys-SOH），这些物质无法进一步参与体内稳态的氧化还原反应（即氧化还原缺陷）。本研究的目的是研究β2AR-ROS 相互作用的活力以及β2AR Cys 氧化还原在受体天然、氧化和氧化还原缺陷状态下的功能后果。在这里，我们首次表明β2AR 可以在原位被氧化为 Cys-S-OH，此外，我们使用克隆细胞和内源性表达β2AR 的人气道上皮细胞系表明，受体氧化还原状态对β2AR 正位配体结合和下游功能有深远影响。具体来说，维持性β2AR 氧化还原状态对于激动剂诱导的 cAMP 形成以及随后的 CREB 和 G 蛋白依赖性 ERK1/2 磷酸化、β-抑制蛋白 2 募集以及下游抑制蛋白依赖的 ERK1/2 磷酸化和内化至关重要。相反，氧化还原缺陷的β2AR 状态表现出降低的通过 Gαs 或β-抑制蛋白 2 信号转导的能力。总之，我们的结果表明存在一个β2AR-ROS 氧化还原轴，如果该轴受到干扰，会干扰受体的正常功能。
