| Co-Investigator(Kenkyū-buntansha) |
MATILDA Katan 連合王国癌研究所, チェスタービーティーラボラトリー, 主任研究員
TAKEUCHI Hiroshi KYUSHU UNIVERSITY Faculty of Dental Science Assistant Prof., 歯学研究院, 助手 (70304813)
KANEMATSU Takashi KYUSHU UNIVERSITY Faculty of Dental Science Associate Prof., 歯学研究院, 助教授 (10264053)
KATAN Matilda Chester-Beatty Lab, (UK) Principle Investigator
八木澤 仁 姫路工業大学, 理学部, 助教授 (40192380)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Keywords | calcium ion / inositol 1, 4, 5-trisphospahte / gamma-aminobutyric acid / central nervous system / anti-anxiety drug / protein phosphatase / イノシトール1,4,5-三リン酸 / ホスホリパーゼC / カルシウムイオン / GABA受容体 / Ins(1,4,5)P_3 / 細胞機能 |
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| Research Abstract |
We isolated a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding proteins with a molecular mass of 130kDa (p130 or PRIP1), which was later found to be a similar protein to delta-isozyme of phospholipase C, but with no catalytic activity. Recently, we established a cell-line, stably over-expressing p130 (COS-1p130) in order to assess the physiological function. Fura-2-loaded COS-1p130 were stimulated with either bradykinin (BK) or epidermal growth factor (EGF) and the changes in free Ca2+ concentration was monitored. Compared to the control cells, the responses of free Ca2+ change were diminished without the changes of levels of Ins(1,4,5)P3 production in response to BK and EGF, indicating that the diminution of Ca2+ response by p130 could be a result of binding of cellular Ins(1,4,5)P3 produced by PLC activation. The p130 might be a negative regulator for Ca2+ signaling pathways in cells. We further attempted to identify interacting molecules to help elucidate the function of p130. We isolated two clones interacting with p130 by yeast two-hybrid screening, the sequencing of which revealed that one was protein phosphatase 1 (PP1) catalytic subunit and the other was GABARAP (GABAA receptor associated protein). We then investigated the region responsible for their interactions and found that PP-1 and GABARAP associated with the pleckstrin homology domain (PH domain) and EF-hand motifs of p130, respectively. To elucidate the function of p130 on GABAA receptor signaling, the effect of zinc ion on IGABA was investigated by whole cell patch recording using acutely dissociated hippocampal CA1 neurons from p130-/- mice. The inhibitory action of zinc ion on IGABA decreased significantly in p130-/-mice. These results suggest that p130 is involved in signaling pathway via not only an Ins(1,4,5)P3-Ca2+ system but also a GABAA receptor signaling concerning with PP1 and GABARAP.
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