Abstract
β2-adrenergic receptor (β2AR) agonists (β2-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β2ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β2ARs as a measure of resensitization. Despite significant accumulation of β2ARs in the endosomes of asthmatic HASMCs, endosomal β2ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β2AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β2AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.
Highlights
Introduction β-adrenergic receptor is a proto-typical member of a large family of seven transmembrane cell surface receptors termed G protein-coupled receptors (GPCR) [1,2] [3]. βAR consists of β1, β2 and β3 subtypes, of which β2-adrenergic receptor (β2AR) is widely distributed in the respiratory tract and the most well studied in asthma [4,5,6,7,8]. β2AR-agonist (β2-agonist) binding evokes coupling of β2ARs to G-protein releasing Gs and Gβγ subunits
Dissociated Gβγ subunits recruit G-protein coupled receptor kinase 2 (GRK2) that phosphorylates β2ARs resulting in β-arrestin binding [11,12,13] desensitizing the receptors. β-arrestin targets β2ARs to undergo internalization but dissociates from β2AR complex prior to internalization [14]. β2ARs are resensitized by dephosphorylation through protein phosphatase 2A (PP2A) in the early endosomes before recycling to the plasma membrane as agonist ready receptors [15]
To test whether asthmatic airway smooth muscle (ASM) is characterized by β2AR dysfunction, non-asthmatic and asthmatic human airway smooth muscle cells (HASMCs) were challenged with albuterol (β-agonist) acutely (0, 5, 10 and 20 minutes) and cAMP generation assessed as a measure of βAR function
Summary
Introduction β-adrenergic receptor (βAR) is a proto-typical member of a large family of seven transmembrane cell surface receptors termed G protein-coupled receptors (GPCR) [1,2] [3]. βAR consists of β1, β2 and β3 subtypes, of which β2AR is widely distributed in the respiratory tract and the most well studied in asthma [4,5,6,7,8]. β2AR-agonist (β2-agonist) binding evokes coupling of β2ARs to G-protein releasing Gs and Gβγ subunits. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects.
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