Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated, Ca2+ signaling
Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated, Ca2+ signaling
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DOI：
10.1002/jcp.20136
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发表时间：
2005-02-01
影响因子：
5.6
通讯作者：
Hirata, M
中科院分区：
文献类型：
--
作者：
Harada, K;Takeuchi, H;Hirata, M
PRIP-1是一种新型的1，4，5-三磷酸肌醇[Ins（1，4，5）P-3]结合蛋白，其结构域类似于磷脂酶C-delta 1（PLC-delta 1），但缺乏酶活性。进一步的研究表明PRIP-1的pleckstrin同源（PH）结构域是负责结合Ins（1，4，5）P-3的区域。在本研究中，我们旨在阐明生理浓度的PRIP-1在Ins（1，4，5）P-3介导的Ca 2+信号传导中的作用，因为我们先前使用过表达PRIP-1的COS-1细胞（Takeuchi等人，2000，Biochem J 349：357-368）。为此目的，我们使用先前产生的PRIP-1敲除（PRIP-1（-/-））小鼠（Kanematsu等人，2002，EMBO J 21：1004-1011）。从PRIP-1（-/-）小鼠制备的原代培养皮层神经元中，响应于嘌呤能受体刺激的游离Ca 2+浓度的增加低于野生型小鼠。在用[H-3]肌醇标记的神经元中测量的[H-3]Ins（1，4，5）P-3的相对量在来自PRIP-1（-/-）小鼠的细胞中也较低。相反，来自PRIP-1（-/-）小鼠的脑皮质样品中的PLC活性与野生型小鼠中的那些没有差异，表明来自PRIP-1（-/-）小鼠的细胞中Ins（1，4，5）P-3的水解增强。体外分析表明，1型肌醇多磷酸5-磷酸酶与PRIP-1的PH结构域（PRIP-1 PH）的物理相互作用，其酶活性被PRIP-1 PH抑制。然而，与这两种蛋白的物理相互作用似乎不是抑制酶活性的原因，表明Ins（1，4，5）P-3与PH结构域的结合阻止了其水解。总之，这些结果表明PRIP-1通过与Ins（1，4，5）P3结合调节1型肌醇多磷酸5-磷酸酶活性，在调节Ins（1，4，5）P3介导的Ca 2+信号传导中起重要作用。(C)2004 Wiley-Liss，Inc.
PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P-3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P-3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1(-/-)) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1(-/-) mice than in those from wild type mice. The relative amounts of [H-3]Ins(1,4,5)P-3 measured in neurons labeled with [H-3]inositol was also lower in cells from PRIP-1(-/-) mice. In contrast, PLC activities in brain cortex samples from PRIP-1(-/-) mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P-3 is enhanced in cells from PRIP-1(-/-) mice. In vitro analyses revealed that type 1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1 PH) and its enzyme activity was inhibited by PRIP-1 PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P-3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P-3-mediated Ca2+ signaling by modulating type 1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3. (C) 2004 Wiley-Liss, Inc.