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The membrane-target mechanism of taste receptors

味觉受体的膜靶机制

基本信息

批准号：
18570138
负责人：
SAITOH Osamu
金额：
$ 2.63万
依托单位：
Nagahama Institute of Bio-Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570138/
关键词：
taste receptor plasma membrane G-protein IRTP REEP

项目摘要

Our goal is to understand the molecular mechanism to accumulate taste receptors on the specialized cell membrane of taste receptor cells (present in the taste pore). It is known that taste receptors including bitter taste receptors show only marginal cell surface expression in heterologous expression system. On the other hand, odorant receptors belonging to the same chemoreceptor family also insufficiently target to cell surface in heterologous cells. However, it was recently demonstrated that coexpression with RTP and REEP family promotes functional surface expression of odorant receptors. We hypothesized that taste receptor cells have the cell surface targeting system of taste receptors. In this study, we will unveil this receptor targeting system in taste receptor cells. STC-1 cells have been established as an enteroendocrine cell line. As cells of the upper small intestine, STC-1 cells express gastrin-releasing peptide receptors (GRP-R or BB2) and are activated by a neuropeptide, b … More ombesin, through the IP3 pathway to elicit secretin secretion. Furthermore, Wu, et al. demonstrated that STC-1 cells express T2R family members and respond to bitter taste substances. Then, we recently characterized bitter taste responses of STC-1 cells. Thus, STC-1 cells are considered to have the cell surface targeting system of taste receptors, and therefore we used STC-1 cells as culture cell model.(1) We first investigated whether intestinal STC-1 cells can respond four basic taste stimuli other than bitter signals. After STC-1 cells were stimulated with several compounds of five basic tastants, we monitored responses in the intracellular calcium using the Fluo4 calcium-indicator dye. We found that STC-1 cells can recognize all of five basic taste signals, which are detected by taste receptor cells of taste buds present on the tongue.(2)To examine the ability of STC-1 cells to target taste receptors to the cell surface, N-terminal tagged receptors of mT2R8 and muscarinic acetylcholine Ml were expressed in HEK293 and STC-1 cells. HEK293 cells could target M1 receptor to the cell surface, but mT2R8 was not targeted. In STC-1 cells, both receptors of M1 and mT2R8 were targeted to the cell surface. Results strongly demonstrated that the cell surface targeting system of taste receptors is present in STC-1 cells.(3) To investigate expression of members of RTP and REEP gene families in STC-1 cells, we performed RT-PCR analysis. RNA was isolated from STC-1 cells, and from NIH3T3 cells as control. No difference in expression level was observed for RTP family, but novel differences were found for expression of REEP family. High level expression was observed for REEP1, REEP2, REEP4 and REEP6. Less

我们的目标是了解味觉受体细胞（存在于味孔中）的特殊细胞膜上积累味觉受体的分子机制。已知包括苦味受体在内的味觉受体在异源表达系统中仅显示边缘细胞表面表达。另一方面，属于同一化学感受器家族的气味受体也不足以靶向异源细胞的细胞表面。然而，最近证明，与 RTP 和 REEP 家族共表达可促进气味受体的功能性表面表达。我们假设味觉受体细胞具有味觉受体的细胞表面靶向系统。在这项研究中，我们将揭示味觉受体细胞中的这种受体靶向系统。 STC-1 细胞已被确立为肠内分泌细胞系。作为小肠上部的细胞，STC-1 细胞表达胃泌素释放肽受体（GRP-R 或 BB2），并被神经肽 b … More ombesin 通过 IP3 途径激活，从而引发促胰液素分泌。此外，吴等人。证明 STC-1 细胞表达 T2R 家族成员并对苦味物质做出反应。然后，我们最近表征了 STC-1 细胞的苦味反应。因此，STC-1细胞被认为具有味觉受体的细胞表面靶向系统，因此我们使用STC-1细胞作为培养细胞模型。(1)我们首先研究了肠道STC-1细胞是否能够响应除苦味信号之外的四种基本味觉刺激。用五种碱性促味剂的几种化合物刺激 STC-1 细胞后，我们使用 Fluo4 钙指示剂染料监测细胞内钙的反应。我们发现STC-1细胞可以识别所有五种基本味觉信号，这些信号是由舌头上味蕾的味觉受体细胞检测到的。(2)为了检查STC-1细胞将味觉受体靶向细胞表面的能力，在HEK293和STC-1细胞中表达mT2R8和毒蕈碱乙酰胆碱Ml的N端标记受体。 HEK293细胞可以将M1受体靶向细胞表面，但mT2R8没有被靶向。在 STC-1 细胞中，M1 和 mT2R8 受体均靶向细胞表面。结果有力地证明了STC-1细胞中存在味觉受体的细胞表面靶向系统。(3)为了研究RTP和REEP基因家族成员在STC-1细胞中的表达，我们进行了RT-PCR分析。从 STC-1 细胞和作为对照的 NIH3T3 细胞中分离 RNA。 RTP家族的表达水平没有观察到差异，但REEP家族的表达发现了新的差异。观察到 REEP1、REEP2、REEP4 和 REEP6 的高水平表达。较少的