Molecular mechanism of bitter taste receptor agonist-mediated relaxation of airway smooth muscle

Abstract

G-protein-coupled receptors (GPCRs) belonging to the type 2 taste receptors (TAS2Rs) family are predominantly present in taste cells to allow the perception of bitter-tasting compounds. TAS2Rs have also been shown to be expressed in human airway smooth muscle (ASM), and TAS2R agonists relax ASM cells and bronchodilate airways despite elevating intracellular calcium. This calcium "paradox" (calcium mediates contraction by pro-contractile Gq-coupled GPCRs) and the mechanisms by which TAS2R agonists relax ASM remain poorly understood. To gain insight into pro-relaxant mechanisms effected by TAS2Rs, we employed an unbiased phosphoproteomic approach involving dual-mass spectrometry to determine differences in the phosphorylation of contractile-related proteins in ASM following the stimulation of cells with TAS2R agonists, histamine (an agonist of the Gq-coupled H1 histamine receptor) or isoproterenol (an agonist of the Gs-coupled β₂-adrenoceptor) alone or in combination. Our study identified differential phosphorylation of proteins regulating contraction, including A-kinase anchoring protein (AKAP)2, AKAP12, and RhoA guanine nucleotide exchange factor (ARHGEF)12. Subsequent signaling analyses revealed RhoA and the T853 residue on myosin light chain phosphatase (MYPT)1 as points of mechanistic divergence between TAS2R and Gs-coupled GPCR pathways. Unlike Gs-coupled receptor signaling, which inhibits histamine-induced myosin light chain (MLC)20 phosphorylation via protein kinase A (PKA)-dependent inhibition of intracellular calcium mobilization, HSP20 and ERK1/2 activity, TAS2Rs are shown to inhibit histamine-induced pMLC20 via inhibition of RhoA activity and MYPT1 phosphorylation at the T853 residue. These findings provide insight into the TAS2R signaling in ASM by defining a distinct signaling mechanism modulating inhibition of pMLC20 to relax contracted ASM.

Keywords: TAS2R; airway smooth muscle; asthma; chloroquine; rho a.

Figure 1.. Agonist-specific differences of phosphorylated proteins in multiple signaling pathways.

Phosphoproteomic analysis was performed on ASM cells to determine the profile for all proteins phosphorylated at serine, tyrosine and threonine residues within the cell proteome. A) Numerical values correspond to the percentage of proteins observed to be differentially phosphorylated in each respective signaling pathway following stimulation with 10 μM histamine (H) or dual agonist treatment (300 μM chloroquine: CQ+H and 1 μM isoproterenol: I+H). Pathways that showed no significant change in treatment versus control conditions are denoted as N/A (n=3). B) The degree of perturbance within each signaling pathway with respect to treatment condition as denoted by a negative log₁₀ transformation of the p-value. Pathways with transformed p-values greater than 1.3 (p < 0.05) were determined to be significantly perturbed. C) Quantification of the number of contractile-related proteins that were differentially regulated between treatment conditions. P < 0.05 versus control.

Figure 2.. Agonist-specific differences of individual phosphorylated proteins of interest.

Above are representative graphs of contractile-related proteins of interest compiled from our phosphoproteomics analysis: (A) AKAP2, (B) AKAP12, (C) ARHGAP6, (D) ARHGEF12, (E) MYPT1 and (F) MYLK. ASM cells were treated with 1 μM isoproterenol (I), 300 μM chloroquine (CQ), 10 μM histamine (H) or dual agonist treatment (I+H and CQ+H) prior to phosphoproteomic analysis. Fold change is relative to non-treated control samples and increases/decreases in phosphorylation correspond with positive and negative fold changes (n=3). * p < 0.05 versus basal and # p < 0.05 versus histamine alone. Data were analyzed using a 1-way ANOVA and are represented as mean±SEM.

Figure 3.. MLC20 is negatively regulated by TAS2R and β₂AR pathways.

The phosphorylation of MLC20 was measured in ASM cells treated with 1 μM isoproterenol (I), 300 μM or 1 mM chloroquine (CQ), 30 μM or 300 μM flufenamic acid (FFA), 10 μM histamine (H) or dual agonist treatment (I+H, CQ+H or FFA+H). A) Representative western blots of phosphorylated MLC20 in agonist-stimulated ASM cells. B) Graph depicting the level of MLC20 phosphorylation normalized to β-actin in each sample (n=5–8). * p < 0.05 versus basal and # p < 0.05 versus histamine alone. Data were analyzed using a 1-way ANOVA and are represented as mean±SEM.

Figure 4.. PKC may be differentially regulated by TAS2R and β₂AR agonists.

A) The phosphorylation of PKC substrates was measured in ASM cells treated with 1 μM isoproterenol (I), 300 μM or 1 mM chloroquine (CQ), 30 μM or 300 μM flufenamic acid (FFA), 10 μM histamine (H) or dual agonist treatment (I+H, CQ+H or FFA+H). A) Representative western blot of phosphorylated PKC substrates normalized to β-actin in each sample. B) Representative graph of the amount of a 66–70 kDa phosphorylated PKC substrate normalized to β-actin in agonist-stimulated ASM cells (n=3–5). * p < 0.05 versus basal. Data were analyzed using a 1-way ANOVA and are represented as mean±SEM.

Figure 5.. TAS2R and β₂AR differentially regulate RhoA activity.

A and B) RhoA activity was measured in ASM cells transduced with a lentiviral FRET construct containing RhoA (n=5–8). Cells were treated with isoproterenol (I), chloroquine (CQ), flufenamic acid (FFA), histamine (H) or dual agonist treatment (I+H, CQ+H or FFA+H). C) Representative images of ASM cells (4 minutes after treatment) with a pseudocolor lookup table. The arrows highlight areas of low or high RhoA activity as represented by fluorescent intensity. D) Representative graph and western blot depicting the level of active RhoA “pulled down” using a GST-tagged Rhotekin RhoA-binding domain conjugated to agarose beads normalized to total RhoA in each sample (n=3–5). E) Representative graph and western blot depicting phosphorylation of RhoA at residue S188 normalized to β-actin in each sample (n=5–6). * p < 0.05 versus basal and # p < 0.05 versus histamine alone. Data were analyzed using a 1-way ANOVA and are represented as mean±SEM.

Figure 6.. MYPT1 is differentially regulated by TAS2R and β₂AR agonists.

The phosphorylation of MYPT1 at inhibitory residues T696 and T853 was measured in ASM cells treated with 1 μM isoproterenol (I), 300 μM or 1 mM chloroquine (CQ), 30 μM or 300 μM flufenamic acid (FFA), 10 μM histamine (H) or dual agonist treatment (I+H, CQ+H or FFA+H). A) Representative western blot and graph B) of MYPT1 T696 phosphorylation normalized to β-actin in agonist-stimulated ASM cells (n=4–6). C) Representative western blot and graph D) of MYPT1 T853 phosphorylation normalized to β-actin in agonist-stimulated ASM cells (n=3–7). * p < 0.05 versus basal and # p < 0.05 versus histamine alone. Data were analyzed using a 1-way ANOVA and are represented as mean±SEM.

Figure 7.. Regulation of Gq-mediated contractile proteins by TAS2R and Gs signaling.

Binding of histamine to the H1 receptor leads to downstream activation of RhoA, attenuation of MYPT1 and activation of MLC20, leading to actin-myosin cross-bridge cycling and ASM contraction. Activation of the TAS2R leads to decreased RhoA activity, increased MYPT1 activity via prevention of T853 phosphorylation and attenuation of histamine-induced MLC20 phosphorylation. Conversely, β₂AR signaling partially reduces histamine-induced MLC20 phosphorylation via PKA-mediated signaling, which partially reduces RhoA activity by phosphorylating RhoA at serine 188. This phosphorylation increases RhoA affinity for Rho-GDI, which leads to the sequestration of active GTP-bound RhoA away from the plasma membrane into the cytosol.