Development and validation of novel optical methods for direct screening of taste receptor activation

项目总结 味觉家族1受体(T1Rs，形成甜味和鲜味受体)和味觉家族2受体 (T2Rs，形成苦味受体)是一种重要的G蛋白偶联受体(GPCRs)，表达于白纹伊蚊的味蕾中 舌头。T1Rs和T2Rs在全身组织中也发挥着重要的化学感觉作用，包括 在呼吸道上皮细胞和免疫细胞中。我们和其他人将T1R和T2R描述为 先天免疫力。他们检测细菌代谢物以刺激快速的先天免疫反应。 虽然目前的筛选方法揭示了许多关于激活特定T1R的特定激动剂的信息 和T2Rs，了解复杂混合物(例如植物提取物或有条件的植物提取物)对T1R和T2R的激活 来自细菌的介质)目前非常困难。现有的筛选方法主要集中在钙信号转导AS 一次读出。许多化合物激活培养细胞中不依赖味觉感受器的钙信号，并且 HEK293细胞通常表达内源性T2Rs。以钙为重点的筛查方法也有 可能遗漏的“偏向激动剂”，即不激活T2RG蛋白信号但激活的激动剂 Arrestin信号。许多GPCR已经报道了这一点，但还没有人筛选有偏见的激动剂。 味觉感受器。这是T1R和T2R生物学研究中的一个关键差距，这里将讨论这一问题。 为了克服基于钙的分析的局限性并揭示对T2R-arrestin信号的新见解， 我们将采用一种基于荧光的三方GFP分析(称为TRIO分析)，直接 通过arrestin与激活的受体结合，可视化异源表达的受体激活。这 与传统方法相比，分析方法不受复杂混合物或单一化合物偏离目标效应的限制。 目前的方法。它还适用于高通量平板阅读器仪器。这种化验速度更快(1-2 小时)，而不是其他依赖于转录作为读出的GPCR分析(12-24小时)。我们已经利用了这种化验方法 在以往的研究中，研究蛋白酶激活和肾上腺素能受体的激活和抑制。初步 数据表明，这种检测方法对T2Rs很有效。我们假设T1R和T2R三重分析将揭示 新的配体-T1R/T2R相互作用，并可能有助于使剩余的孤儿T2R亚型去孤儿。 在这项提案中，目标1将专注于使用已知的苦味和甜味来验证这种光学分析 化合物。Aim 2将使用这一方法来筛选针对常见致病菌和 真菌，以确定哪些受体被哪些病原体激活。新的临床相关数据将是 本研究中验证的表达结构将成为味觉受体的有用工具 研究。所有表达载体将在首次发表后通过Addgene广泛提供。这 研究对免疫研究以及分子感官营养和味道都具有重要的实用价值。 研究。

PROJECT SUMMARY Taste family 1 receptors (T1Rs, forming sweet and umami receptors) and taste family 2 receptors (T2Rs, forming bitter receptors) are important G-protein couple receptors (GPCRs) expressed in taste buds of the tongue. T1Rs and T2Rs also play important chemosensory roles in tissues all over the body, including the airway epithelium and in immune cells. We and other have characterized T1Rs and T2Rs as novel players in innate immunity. They detect bacterial metabolites to stimulate rapid innate immune responses. While current screening methods have revealed a lot about specific agonists that activate specific T1Rs and T2Rs, understanding T1R and T2R activation by complex mixtures (e.g., plant extracts or conditioned media from bacteria) is currently very difficult. Existing screening methods are focused on calcium signaling as a read-out. Many compounds activate calcium signals independently of taste receptors in cultured cells, and the HEK293 cells commonly used express endogenous T2Rs. Calcium-focused screening methods have also likely missed “biased agonists,” that is, agonists which do not activate T2R G protein signaling but activate arrestin signaling. This has been reported for many GPCRs, but no one has yet screened for biased agonists of taste receptors. This is a critical gap in the studies of T1R and T2R biology that will be addressed here. To overcome limitations of calcium-based assays and reveal new insights into T2R-arrestin signaling, we will adapt a fluorescence-based tripartite GFP-based assay (known as the TRIO assay) that directly visualizes heterologously-expressed receptor activation through arrestin binding to the activated receptor. This assay is much less limited by off-target effects of complex mixtures or single compounds compared with current methods. It is also adaptable to high throughput plate-reader instrumentation. This assay is faster (1-2 hrs) than other GPCR assays which rely on transcription as a readout (12-24 hrs). We have utilized this assay to study activation and inhibition of protease-activated and adrenergic receptors in prior studies. Preliminary data suggest this assay works well with T2Rs. We hypothesize that T1R and T2R TRIO assays will reveal novel ligand-T1R/T2R interactions and may be useful to de-orphanize the remaining orphan T2R isoforms. In this proposal, Aim 1 will focus on validation of this optical assay using known bitter and sweet compounds. Aim 2 will use this assay to screen taste receptors against common pathogenic bacteria and fungi to characterize which receptors are activated by which pathogens. New clinically relevant data will be revealed, and the expression constructs validated in this study will become useful tools for taste receptor research. All expression vectors will be made widely available via Addgene after initial publication. This research has important utility for both immune research as well as molecular sensory nutrition and taste research.