Elucidating the Bitter Taste Signaling Paradox in Airway Smooth Muscle Relaxation

Airway smooth muscle (ASM) hypersensitivity and hypercontractility play a pivotal role in the pathophysiology of obstructive airway diseases such as asthma. Furthermore, over half of affected individuals are unable to reliably control these disorders using the current standard of care, β-(adrenergic) agonists. Previously, our group discovered the expression of a novel group of G protein-coupled receptors (GPCRs), type 2 taste receptors (TAS2Rs), in the human airway which evoke ASM relaxation. Previous work in our lab suggests that TAS2R agonists evoke relaxation in a G protein beta-gamma subunit-, phospholipase C beta- and calcium-dependent manner. We hypothesized that the downstream molecular targets of contractile-related signaling components are differentially regulated by TAS2Rs compared to other GPCRs. The goal of this study was to establish the molecular mechanism by which TAS2Rs induce ASM relaxation.We employed a phosphoproteomic approach, involving dual-mass spectrometry, to determine differences in the phosphorylation status of contractile-related proteins in ASM following the stimulation of TAS2Rs and other GPCRs. Healthy human ASM cells were treated with the TAS2R agonist chloroquine or β-agonist isoproterenol alone or in combination with the bronchoconstrictive agonist histamine to study the ability of each relaxant compound to attenuate the histamine-mediated phosphorylation profile of contractile-related proteins. These phosphoproteomic profiles were compared and a select set of targets were validated using western blot analysis.Our phosphoproteomic data demonstrated that histamine stimulation results in increased phosphorylation of Ras homolog family member A (RhoA) guanine nucleotide exchange factor (ARHGEF) 12 and A kinase anchoring proteins (AKAP) 2 and 12, as well as decreased phosphorylation of RhoA GTPase-activating protein (ARHGAP) 6. Furthermore, chloroquine treatment attenuated histamine-induced phosphorylation of ARHGEF12 but not AKAP2 or AKAP12. Interestingly, isoproterenol attenuated histamine-induced phosphorylation of AKAP2 and AKAP12 but not ARHGEF12. Validation studies using western blot analysis revealed chloroquine mediated inhibition of histamine-induced RhoA activation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation at the T853 residue, which was not observed with isoproterenol.Our findings were able to show that contractile-related proteins are phosphorylated differentially by chloroquine and isoproterenol to elicit ASM relaxation. Furthermore, our studies strongly suggest the importance of RhoA and the T853 residue on MYPT1 as points of mechanistic divergence between TAS2R and other GPCR pathways. These findings provide further insight into the mechanism by which TAS2R signaling reduces airway contractility and its potential as a novel therapy for obstructive airway disease.