Molecular Basis of Taste Cell Signaling

描述（由申请人提供）：最近的研究已经鉴定了响应鲜味、苦味和甜味刺激的G蛋白偶联受体（GPCR）。这些GPCR的下游信号通路开始通过生物化学和遗传分析的强大组合来理解。随着技术的进步，在生理/行为分析和分子研究之间出现了显着的差异，特别是对于鲜味。酸味的潜在机制远未被理解，许多候选换能器通道仍然是候选者。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。一个关键的差异存在于味觉细胞对多种味觉品质做出反应的生理学证据和味觉细胞似乎只表达一种品质的GPCR的分子证据之间。本申请使用新开发的方法解决了这些关键的开放问题，以检查功能上限定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个单独的输出细胞类别;只有输出细胞与感觉传入纤维形成突触。这一假设的关键测试可能会解决目前的争议的广度调整的味觉细胞。对于鲜味和酸味，我们将对分离的味觉细胞进行功能成像，使用来自切片制备中详细研究的标准。然后对这种功能上限定的味觉细胞进行单细胞RT-PCR和/或差异文库筛选以鉴定与功能表型相关的分子。为了测试我们关于输出细胞的假设，我们将使用小鼠，其中用绿色荧光蛋白（GFP）或β-半乳糖苷酶转基因标记表达PLCb 2的细胞（a）或合成生物胺的细胞（B）的功能性细胞谱系。这些小鼠味觉细胞的功能性原位成像将使我们能够测试是否存在单独类别的味蕾输出细胞，类似于视网膜中的神经节细胞。差异文库筛选将使我们开始确定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.