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.
摘要:
属于2型味觉受体(TAS2R)家族的G-蛋白偶联受体(GPCR)主要存在于味觉细胞中以允许感知苦味化合物。TAS2R也被证明在人气道平滑肌(ASM)中表达,TAS2R激动剂放松ASM细胞和支气管扩张气道,尽管细胞内钙升高。这种钙“悖论”(钙通过促收缩Gq偶联的GPCRs介导收缩)和TAS2R激动剂放松ASM的机制仍然知之甚少。为了深入了解TAS2R影响的促松弛机制,我们采用了一种无偏的磷酸蛋白质组学方法,涉及双质谱,以确定在用TAS2R激动剂刺激细胞后,ASM中收缩相关蛋白的磷酸化差异。单独或组合使用的组胺(Gq偶联的H1组胺受体的激动剂)或异丙肾上腺素(Gs偶联的β2-肾上腺素受体的激动剂)。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(ARHBEF)12。随后的信号分析显示,肌球蛋白轻链磷酸酶(MYPT)1上的RhoA和T853残基是TAS2R和Gs偶联的GPCR途径之间的机制差异点。与Gs偶联受体信号传导不同,通过蛋白激酶A(PKA)依赖性抑制细胞内钙动员,抑制组胺诱导的肌球蛋白轻链(MLC)20磷酸化,HSP20和ERK1/2活性,TAS2R显示通过抑制RhoA活性和T853残基处的MYPT1磷酸化来抑制组胺诱导的pMLC20。这些发现通过定义调节pMLC20抑制以放松收缩的ASM的独特信号机制,提供了对ASM中TAS2R信号传导的洞察。