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

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 ß2-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.

Diacylglycerol kinase is a keystone regulator of signaling relevant to the pathophysiology of asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

BACKGROUND: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). METHODS: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. RESULTS: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. CONCLUSIONS: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.