| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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
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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
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