Differential regulation of RhoA by TAS2R signaling in airway smooth muscle relaxation

Abstract

RATIONALE: Airway smooth muscle (ASM) hypercontraction and hyperresponsiveness are hallmark features of bronchoconstrictive disorders, which affect over 300 million people worldwide. Furthermore, the current standard of care, β-(adrenergic) agonists, lose their effectiveness for over 50% of patients that use them. Previous work in our lab resulted in the discovery of the expression of type 2 taste receptors (TAS2Rs) in human ASM. Interestingly, TAS2R agonists were found to elicit relaxation in a phospholipase C beta (PLCβ)-dependent manner. Furthermore, recent phosphoproteomics data from our group suggest that RhoA, an important contractile-related effector of PLCβ, is differentially regulated by TAS2R and β-agonists. Therefore, the goal of this study was to validate these findings and elucidate differences in the effects of multiple GPCR pathways on RhoA activity and ASM relaxation. METHODS: We determined the level of RhoA activity in human ASM cells (hASM) using the Rhotekin protein construct, which contains a Rho-binding domain with a high affinity for active RhoA. Additionally, we utilized lentiviral particles containing a single chain fluorescence resonance energy transfer (FRET) biosensor construct (RhoA2G) with a Rho-binding domain specific for active RhoA. Healthy hASMs were transduced with lentivirus and RhoA activity was monitored using fluorescent confocal microscopy. Cells were treated with TAS2R (chloroquine or flufenamic acid) or β (isoproterenol) agonists alone or in conjunction with the Gq agonist histamine to study the effects of these relaxant ligands on histamine-mediated RhoA activation. RESULTS: Our RhoA pulldown data using the Rhotekin construct showed that isoproterenol partially ablated histamine-mediated RhoA activation compared to the complete ablation of the signal by chloroquine (300 μM). Additionally, we observed complete ablation of histamine-mediated RhoA2G activity by isoproterenol while chloroquine and flufenamic acid evoked a reduction of histamine-mediated activity to below the observed baseline. This further validates our phosphoproteomics findings which suggest that histamine-mediated phosphorylation of ARHGEF12, a highly expressed RhoA activator, is partially attenuated by isoproterenol but fully attenuated by chloroquine (300 μM). Further, the RhoA FRET biosensor was useful in assessing the activation kinetics of RhoA in hASMs in real time. CONCLUSION: RhoA is an important contractile-related effector that is regulated differentially by isoproterenol and TAS2R agonists to evoke ASM relaxation. The studies in this abstract are funded by the NHLBI Diversity Supplement for the NIH grant R01-HL137030 (D.A. Deshpande) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.