The membrane-target mechanism of taste receptors

我们的目标是了解味觉感受器细胞(存在于味觉孔中)的特殊细胞膜上积累味觉感受器的分子机制。已知在异源表达系统中，包括苦味受体在内的味觉受体仅表现为边缘细胞表面表达。另一方面，属于同一化学感受器家族的气味受体在异种细胞中也不足以靶向细胞表面。然而，最近的研究表明，与RTP和REEP家族共表达可以促进气味受体的功能性表面表达。我们假设味觉感受器细胞具有味觉感受器的细胞表面靶向系统。在这项研究中，我们将在味觉感受器细胞中揭示这种受体靶向系统。STC-1细胞已被建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-…激活。更多的奥贝菌素，通过IP3途径诱导分泌素的分泌。此外，Wu等人也提出了自己的观点。证实STC-1细胞表达T2R家族成员，并对苦味物质产生反应。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，因此我们选择STC-1细胞作为培养细胞模型。(1)我们首先研究了肠道STC-1细胞是否能够响应四种基本的味觉刺激，而不是苦味信号。在STC-1细胞被五种基本味精的几种化合物刺激后，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细胞可以识别舌上存在的味蕾的味觉感受器细胞检测到的所有五种基本味觉信号。(2)为了检测STC-1细胞将味觉受体靶向细胞表面的能力，在HEK293和STC-1细胞中表达了标记的mT2R8和M受体。HEK293细胞可靶向M1受体，而mT2R8不能靶向。在STC-1细胞中，M1和mT2R8受体均定位于细胞表面。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NIH3T3细胞中提取RNA作为对照。RTP家族的表达水平没有差异，而REEP家族的表达存在新的差异。REEP1、REEP2、REEP4和REEP6均有高水平表达。较少

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