Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, USA.
FASEB J. 2024 Jul 31;38(14):e23842. doi: 10.1096/fj.202400452R.
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 β-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.
G 蛋白偶联受体（GPCRs）属于 2 型味觉受体（TAS2Rs）家族，主要存在于味觉细胞中，以感知苦味化合物。TAS2Rs 也已被证明在人呼吸道平滑肌（ASM）中表达，并且 TAS2R 激动剂可松弛 ASM 细胞并扩张气道，尽管细胞内钙升高。这种钙“悖论”（钙通过促收缩的 Gq 偶联 GPCR 介导收缩）以及 TAS2R 激动剂松弛 ASM 的机制仍知之甚少。为了深入了解 TAS2Rs 产生的促松弛机制，我们采用了一种无偏见的磷酸蛋白质组学方法，涉及双质谱法，以确定 TAS2R 激动剂、组胺（Gq 偶联 H1 组胺受体激动剂）或异丙肾上腺素（Gs 偶联 β-肾上腺素受体激动剂）单独或联合刺激细胞后，ASM 中与收缩相关的蛋白质磷酸化的差异。我们的研究确定了调节收缩的蛋白质的差异磷酸化，包括 A-激酶锚定蛋白（AKAP）2、AKAP12 和 RhoA 鸟嘌呤核苷酸交换因子（ARHGEF）12。随后的信号分析显示，RhoA 和肌球蛋白轻链磷酸酶（MYPT）1 上的 T853 残基是 TAS2R 和 Gs 偶联 GPCR 途径之间机制分歧的关键点。与 Gs 偶联受体信号不同，后者通过蛋白激酶 A（PKA）依赖性抑制细胞内钙动员来抑制组胺诱导的肌球蛋白轻链（MLC）20 磷酸化，而 HSP20 和 ERK1/2 活性，TAS2Rs 被证明通过抑制 RhoA 活性和 MYPT1 上的 T853 残基磷酸化来抑制组胺诱导的 pMLC20。这些发现通过定义调节收缩的 pMLC20 抑制以松弛收缩的 ASM 的独特信号机制，深入了解了 TAS2R 在 ASM 中的信号转导。
