Molecular Basis of Taste Cell Signaling

描述（由申请人提供）：最近的研究已经确定了G蛋白偶联受体（gpcr）对鲜味，苦味和甜味刺激做出反应。通过生物化学和遗传分析的强大组合，这些gpcr的下游信号通路开始被理解。随着技术的进步，生理/行为分析与分子研究之间出现了显著的差异，特别是对鲜味的研究。酸味的机制尚不清楚，许多候选的换能器通道仍是候选通道。味觉细胞如何处理味觉信号并将信息传递给感觉传入纤维实际上是未知的。生理证据表明味觉细胞对多种味觉质量有反应，而分子证据表明味觉细胞似乎只对一种味觉质量表达gpcr，这两者之间存在着关键的差异。目前的应用程序解决了这些关键的开放性问题，使用新开发的方法来检查功能定义的味觉细胞的基因表达谱。我们假设来自受体细胞的信号汇聚到味蕾内的一类单独的输出细胞；只有输出细胞与感觉传入纤维形成突触。对这一假设的关键测试可能会解决目前关于味觉细胞调节宽度的争议。对于鲜味和酸味，我们将使用从切片制备的详细研究中得出的标准，对分离的味觉细胞进行功能成像。这些功能明确的味觉细胞随后将进行单细胞RT-PCR和/或差异文库筛选，以鉴定与功能表型相关的分子。为了在输出细胞上验证我们的假设，我们将使用小鼠，其中表达PLCb2的细胞(a)或合成生物胺的细胞(b)的功能细胞系被转基因标记为绿色荧光蛋白（GFP）或b-半乳糖苷酶。这些小鼠的味觉细胞的功能原位成像将使我们能够测试是否存在一种独立的味蕾输出细胞，类似于视网膜上的神经节细胞。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.