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

我们分离了一种新的1,4,5-三磷酸肌醇（Ins(1,4,5)P3）结合蛋白，分子量为130kDa (p130或PRIP1)，后来发现该蛋白与磷脂酶C的δ同工酶相似，但没有催化活性。最近，我们建立了一个稳定过表达p130的细胞系（COS-1p130），以评估其生理功能。用缓激素（BK）或表皮生长因子（EGF）刺激负载fura -2的COS-1p130，监测游离Ca2+浓度的变化。与对照细胞相比，BK和EGF对游离Ca2+变化的响应减弱，而Ins(1,4,5)P3的产生水平没有变化，这表明p130对Ca2+响应的减弱可能是PLC激活产生的细胞Ins(1,4,5)P3结合的结果。p130可能是细胞中Ca2+信号通路的负调节因子。我们进一步尝试鉴定相互作用的分子，以帮助阐明p130的功能。通过酵母双杂交筛选分离到两个与p130相互作用的克隆，测序结果显示，一个是蛋白磷酸酶1 （PP1）催化亚基，另一个是GABARAP （GABAA受体相关蛋白）。然后，我们研究了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-hand基序相关。为了阐明p130对GABAA受体信号转导的作用，我们利用p130-/-小鼠急性解离海马CA1神经元，通过全细胞贴片记录研究了锌离子对IGABA的影响。锌离子对IGABA的抑制作用在p130-/-小鼠中显著降低。这些结果表明，p130不仅通过Ins(1,4,5)P3-Ca2+系统参与信号通路，还通过与PP1和GABARAP相关的GABAA受体信号通路参与信号通路。

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.