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Cysteine redox state regulates human ?2-adrenergic receptor binding and function.

ABSTRACT: Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. ?2-adrenergic receptor (?2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that ?2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes ?2AR 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-SO2H) or S-sulfonic (Cys-SO3H) 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 ?2AR-ROS interplay and the resultant functional consequences of ?2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that ?2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing ?2AR, we show that receptor redox state profoundly influences ?2AR orthosteric ligand binding and downstream function. Specifically, homeostatic ?2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to ?-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient ?2AR states exhibit decreased ability to signal via either G?s or ?-arrestin. Together, our results demonstrate a ?2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.

SUBMITTER: Rambacher KM

PROVIDER: S-EPMC7031529 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Cysteine redox state regulates human β2-adrenergic receptor binding and function.

Rambacher Kalyn M KM Moniri Nader H NH

Scientific reports 20200219 1

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 Cy ...[more]

PMID: 32076070

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Project description:RationaleStimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects.ObjectiveWe tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation.Methods and resultsCardiomyocytes expressing wild-type (WT) PKG1α (PKG1αWT) or cysteine-42 to serine mutation redox-dead (PKG1αCS/CS) were exposed to ET-1 (endothelin 1). Cells expressing PKG1αWT exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1αCS/CS expression. Mice with global knock-in of PKG1αCS/CS subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1αWT mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1αCS/CS more than PKG1αWT myocardium following PO. TSC2S1365A/S1365A (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1αCS/CS mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1αCS/CS mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1αWT and PKG1αCS/CS after PO and blocked ET-1 stimulated mTORC1 in TSC2S1365A-expressing myocytes.ConclusionsOxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.

Dataset Information

Cysteine redox state regulates human ?2-adrenergic receptor binding and function.

Publications

Cysteine redox state regulates human β2-adrenergic receptor binding and function.

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