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Studies on the functions of a novel inositol 1, 4, 5-trisphosphate binding protein

新型肌醇1,4,5-三磷酸结合蛋白的功能研究

基本信息

批准号：
11694288
负责人：
HIRATA Masato
金额：
$ 4.1万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694288/
关键词：
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

项目摘要

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.

我们分离到一个新的1，4，5-三磷酸肌醇（Ins（1，4，5）P3）结合蛋白，分子量为130 kDa（p130或PRIP 1），后来发现它与磷脂酶C的δ-同工酶相似，但没有催化活性。最近，我们建立了一个细胞系，稳定过表达p130（COS-1 p130），以评估生理功能。用缓激肽（BK）或表皮生长因子（EGF）刺激载有Fura-2的COS-1 p130，并监测游离Ca ~（2+）浓度的变化。与对照组相比，BK和EGF对细胞内游离Ca ~（2+）变化的反应减弱，而对细胞内Ins（1，4，5）P_3产生的反应无明显变化，表明p130对Ca ~（2+）反应的减弱可能是PLC激活产生的Ins（1，4，5）P_3结合的结果。p130可能是细胞内Ca ~（2+）信号通路的负调控因子。我们进一步尝试识别相互作用的分子，以帮助阐明p130的功能。通过酵母双杂交筛选，获得了两个与p130相互作用的克隆，测序结果表明，其中一个为蛋白磷酸酶1（PP 1）催化亚基，另一个为GABAAA受体相关蛋白（GABARAP）。然后我们研究了负责它们相互作用的区域，发现PP-1和GABARAP分别与p130的pleckstrin同源结构域（PH结构域）和EF-手基序相关。为了阐明p130在GABAA受体信号转导中的作用，采用急性分离的p130-/-小鼠海马CA 1区神经元，采用全细胞膜片钳技术研究了锌离子对IGABA的影响。锌离子对p130-/-小鼠IGABA的抑制作用明显减弱。这些结果表明，p130不仅通过Ins（1，4，5）P3-Ca ~（2+）系统参与信号通路，而且还通过与PP 1和GABARAP相关的GABAA受体信号通路参与信号通路。