Molecular Basis of Taste Cell Signaling

描述(申请人提供)：最近的研究已经确定G蛋白偶联受体(GPCRs)对鲜味、苦味和甜味刺激做出反应。通过生化和遗传分析的强大组合，人们开始了解这些GPCR的下游信号通路。随着这些进步，在生理/行为分析和分子研究之间出现了显著的差异，尤其是对鲜味的研究。人们对酸味背后的机制知之甚少，许多候选的转导通道仍然是候选通道。味觉细胞如何处理味觉信号并将信息传递到感觉传入纤维几乎是未知的。在味觉细胞对多种味觉品质作出反应的生理证据和味觉细胞似乎只表达一种味觉品质的GPCRs的分子证据之间存在着关键的差异。本申请使用新开发的方法来研究功能定义的味觉细胞的基因表达谱，以解决这些关键的开放问题。我们假设，来自感受器细胞的信号汇聚到味蕾内的一类单独的输出细胞上；只有输出细胞与感觉传入纤维形成突触。对这一假设的批判性测试可能会解决目前关于味觉细胞调谐广度的争议。对于鲜味和酸味，我们将使用切片准备过程中详细研究得出的标准，对分离的味觉细胞进行功能成像。然后，这些功能定义的味觉细胞将接受单细胞RT-PCR和/或差异文库筛选，以确定与功能表型相关的分子。为了验证我们对输出细胞的假设，我们将使用小鼠，在小鼠中，表达PLCb2或(B)合成生物胺的细胞的功能细胞谱系被转基因标记绿色荧光蛋白(GFP)或b-半乳糖苷酶。对这些小鼠的味觉细胞进行功能性原位成像将使我们能够测试是否存在一种单独的味蕾输出细胞，类似于视网膜中的神经节细胞。然后，差异文库筛选将允许我们开始定义受体和输出细胞之间的功能关系。

DESCRIPTION (provided by applicant): Recent studies have identified G protein coupled receptors (GPCRs) that respond to umami, bitter and sweet taste stimuli. Downstream signaling pathways for these GPCRs are beginning to be understood through powerful combinations of biochemical and genetic analyses. With the advances, have come significant discrepancies between physiological/behavioral analyses and molecular studies, especially for umami taste. The mechanisms underlying sour taste are far less understood, and many candidate transducer channels remain as candidates. How taste cells process taste signals and transmit information to sensory afferent fibers is virtually unknown. A critical discrepancy exists between physiological evidence that taste cells respond to multiple taste qualities, and molecular evidence that taste cells appear to express GPCRs for only one quality. The present application addresses these key open questions using newly developed methods to examine the gene expression profile of functionally defined taste cells. We hypothesize that signals from receptor cells converge onto a separate class of output cells within taste buds; only output cells form synapses with sensory afferent fibers. Critical tests of this hypothesis may resolve the current controversy on the breadth of tuning of taste cells. For umami and acid tastes, we will carry out functional imaging on isolated taste cells, using criteria derived from detailed studies in the slice preparation. Such functionally defined taste cells will then be subjected to single-cell RT-PCR and/or differential library screening to identify molecules associated with the functional phenotype. To test our hypothesis on output cells, We will employ mice in which functional cell lineages for cells (a) that express PLCb2 or (b) that synthesize biogenic amines are transgenically labeled with Green Fluorescent Protein (GFP) or b-galactosidase. Functional in situ imaging of taste cells from these mice will allow us to test whether there is a separate category of taste bud output cells, akin to ganglion cells in the retina. Differential library screening will then allow us to begin defining the functional relationship between receptor and output cells.