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

我们分离到了一个新的结合蛋白1，4，5-三磷酸(Ins(1，4，5)P3)，其分子量为130 kDa(p130或PRIP1)，后来被发现与磷脂酶C的Delta-同工酶相似，但没有催化活性。最近，我们建立了稳定高表达p130(COS-1p130)的细胞系，以评价其生理功能。用缓激肽(BK)或表皮生长因子(EGF)刺激Fura-2负载的COS-1p130，观察细胞内游离钙浓度的变化。与对照组相比，BK和EGF对细胞内游离钙变化的反应减弱，INS(1，4，5)P3的产生水平没有变化，提示p130抑制细胞内钙离子的反应可能是PLC激活细胞产生的INS(1，4，5)P3结合的结果。P130可能是细胞内钙信号通路的负调节因子。我们进一步试图确定相互作用的分子，以帮助阐明p130的功能。通过酵母双杂交筛选获得了两个与p130相互作用的克隆，测序表明其中一个为蛋白磷酸酶1催化亚基，另一个为GABARAP(GABAA受体相关蛋白)。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-Hand基序相关。为阐明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CA1区神经元全细胞膜片记录技术，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+)系统参与信号转导，而且还通过与PP1和GABARAP有关的GABAA受体参与信号转导。

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.