The membrane-target mechanism of taste receptors

我们的目标是了解分子机制，以积累味觉受体细胞的专门细胞膜上的味觉受体（存在于味觉孔）。已知味觉受体包括苦味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，在异源细胞中，属于相同化学受体家族的气味受体也不能充分靶向细胞表面。然而，最近的研究表明，与RTP和REEP家族的共表达促进了气味受体的功能性表面表达。我们推测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，我们将揭示这种受体靶向系统在味觉受体细胞。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BB 2）并被神经肽B激活 关于我们 通过IP 3途径诱导分泌素分泌。此外，Wu等证明STC-1细胞表达T2 R家族成员并对苦味物质应答。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细胞是否可以响应苦味信号以外的四种基本味觉刺激。STC-1细胞用五种碱性促味剂的几种化合物刺激后，我们使用Fluo 4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的味觉信号，这些信号可以被舌头上味蕾的味觉受体细胞检测到。(2)To为了检查STC-1细胞将味觉受体靶向细胞表面的能力，在HEK 293和STC-1细胞中表达mT 2 R8和毒蕈碱乙酰胆碱M1的N-末端标记的受体。HEK 293细胞可将M1受体靶向细胞表面，但不靶向mT 2 R8。在STC-1细胞中，M1和mT 2 R8的两种受体都靶向细胞表面。结果表明，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细胞中分离RNA，并从作为对照的NIH 3 T3细胞中分离RNA。RTP家族的表达水平无差异，但REEP家族的表达存在新的差异。观察到REEP 1、REEP 2、REEP 4和REEP 6的高水平表达。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