Beta adrenergic receptor resensitization in asthma

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Abstract Our proposal investigates mechanisms of β2-adrenergic receptor (β2AR) dysfunction that underlies airway hyper-responsiveness in asthma accounting for paradoxical loss of asthma control observed with frequent use of β-agonist. Asthma pathology is associated with elevation of nitric oxide (NO) and indeed, patients with higher fraction of exhaled NO (FENO) have greater hyper-reactivity and airflow obstruction with paradoxical loss to β-agonist challenge and re-challenge (Bonini et al., AJRCCM, 2013). β-agonist activation of βAR causes airway smooth muscle relaxation via cAMP in parallel leading to βAR phosphorylation, desensitization and internalization. βAR desensitization is well understood but mechanistically, little is known about β2AR resensitization to recover active β2ARs on cell surface. We recently uncovered mechanisms regulating β2AR reactivation pathway wherein, dephosphorylation/reactivation of the β2AR by protein phosphatase 2A (PP2A) is blocked by phosphoinositide 3-kinase γ (PI3Kγ), which serves to inhibit β2AR resensitization limiting βAR response in the cell to β-agonist [PI3Kγ-PP2A axis] (Vasudeven et al., Mol Cell, 2011). It is not known whether desensitization and/or resensitization mechanisms underlie the loss in β- agonist response with elevated NO in asthma. Contrary to NO mediating β2AR desensitization via S- nitrosylation of proteins, our preliminary data in primary murine airway smooth muscle cells (ASMCs) reveal that NO causes β2AR dysfunction not via desensitization rather, via resensitization pathways. NO activates PI3Kγ by S-nitrosylation of PI3Kγ blocking PP2A activity thereby, inhibiting β2AR resensitization. Further, PP2A activity is determined by its methylation and we find that nitrosylated PI3Kγ inhibits PP2A activity in part by de-methylation. Consistently, primary ASMCs from asthmatic patients have markedly more intracellular phosphorylated-β2ARs, less cAMP response to β-agonist, greater PI3Kγ activity and lesser PP2A activity compared to non-asthmatics at plasma membrane and endosome. Based on these findings, we hypothesize that NO inhibits β2AR resensitization by nitrosylation/activation of PI3Kγ which inhibits PP2A and limits β2AR reactivation in asthmatic airways with high NO. The result is cumulative loss of functional cell surface β2ARs, pre-disposing cells to greater sensitivity for broncho-constrictors, i.e. airway hyper-reactivity (AHR). In strong support of our hypotheses, PI3Kγ null (PI3Kγ-/-) mice have minimal AHR, and AHR returns in the PI3Kγ-/- upon PP2A inhibition. To address our hypothesis, we propose to comprehensively determine the effects of elevated NO on β2AR resensitization (Aim1) and its role in airway hyper-responsiveness (Aim 2), and to identify mechanisms of NO-mediated non-canonical activation of PI3Kγ that inhibits PP2A and β2AR resensitization in asthma (Aim 3).