Studies on the molecular mechanisms underlying anxiety and convulsion with special reference to the roles for PRIP

焦虑和惊厥的分子机制研究，特别是 PRIP 的作用

• 批准号: 14370595
• 负责人: HIRATA Masato
• 金额: $ 9.6万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370595/
• 关键词: Ins(1,4,5)P_3; GABA_A receptor; GABARAP; knock-out mice; anti-anxiety drugs; hippocampus

The protein PRIP was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. In this study, we show that PRIP plays an important role in signaling by the type A receptor for γ-aminobutyric acid(GABA). Yeast two-hybrid screening identified GABARAP(GABA_A receptor-associated protein), which is proposed to contribute to the sorting, targeting, or clustering of GABA_A receptors, and PP1(protein phosphatase 1), as proteins that interact with PRIP. PRIP competitively inhibited the binding of the γ2 subunit of the GABA_A receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl^-current by Zn^<2+> or diazepam, both of which act at GABA_A receptors containing y subunits, is impaired in hippocampal neurons of PRIP knockout mice(PRIP^<-/->mice). Behavioral analysis revealed that, compared with wild-type animals, motor coordination was imp … More aired and the intraperitoneal injection of diazepam induced markedly reduced sedative and anti-anxiety effects in the mutant mice. These results indicate that PRIP is essential for the function of GABA_A receptors, especially in response to the agents acting on a γ2 subunit. Furthermore, we have examined the role for PRIP, which binds and inactivates PP1,in facilitating GABA_A receptor phospho-dependent regulation, using PRIP^<-/-> mice. In wild-type animals, robust phosphorylation and functional modulation of GABA_A receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP^<-/-> mice. PRIP^<-/-> mice exhibited enhanced PP1 activity compared to controls. Furthermore, PRIP was able to interact directly with GABA_A receptor p subunits, and moreover, these proteins were found to be PP1 substrates. Finally, phosphorylation of PRIP on threonine 94 facilitated the dissociation of PP1/PRIP complexes, providing a local mechanism for the activation of PP1. Together, these results suggest an essential role for PRIP in controlling GABAA receptor activity, via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors. Less

PRIP蛋白是从大鼠脑中分离的一种肌醇1，4，5-三磷酸结合蛋白，其结构域类似于磷脂酶C-δ1，但缺乏PLC活性。在这项研究中，我们发现PRIP在γ-氨基丁酸（GABA）A型受体的信号传导中起着重要作用。酵母双杂交筛选鉴定出GABARAP（GABA_A受体相关蛋白）和PP 1（蛋白磷酸酶1），GABARAP（GABA_A受体相关蛋白）被认为有助于GABA_A受体的分选、靶向或聚集，PP 1（蛋白磷酸酶1）是与PRIP相互作用的蛋白质。PRIP在体外竞争性抑制GABA_A受体γ2亚基与GABARAP的结合。电生理学分析显示，在PRIP基因敲除小鼠（PRIP^-/->小鼠）的海马神经元中，Zn^<2+>或地西泮对GABA诱导的Cl^-电流的调制受到损害，这两种物质都作用于含有γ亚基的GABA_A受体。行为学分析表明，与野生型动物相比， ...更多信息 空气和腹腔注射地西泮诱导的突变小鼠的镇静和抗焦虑作用显著降低。这些结果表明PRIP对GABA_A受体的功能，特别是对作用于γ2亚单位的药物的反应是必需的。此外，我们已经使用PRIP^<-/->小鼠研究了PRIP在促进GABA_A受体磷酸依赖性调节中的作用，PRIP结合并灭活PP 1。在野生型动物中，cAMP依赖性蛋白激酶对含有β3亚基的GABA_A受体的强烈磷酸化和功能调节是明显的，这在PRIP^<-/->小鼠中减弱。与对照相比，PRIP^<-/->小鼠表现出增强的PPl活性。此外，PRIP能够直接与GABA_A受体p亚基相互作用，而且这些蛋白被发现是PP 1的底物。最后，磷酸化的PRIP对苏氨酸94促进PP 1/PRIP复合物的解离，提供了一个本地的机制激活PP 1。总之，这些结果表明PRIP在控制GABAA受体活性中的重要作用，通过调节亚基磷酸化，从而调节这些受体介导的神经元抑制的功效。少

项目成果

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Kanematsu, T. et al.: "Role of the PLC-related, catalytically inactive protein p130 in GABA_A receptor function"The EMBO Journal. 21. 1004-1011 (2002)

Kanematsu, T. 等人：“PLC 相关催化失活蛋白 p130 在 GABA_A 受体功能中的作用”EMBO 杂志。

Uji, A.et al.: "Molecules interacting with PRIP-2, a novel Ins(1,4,5)P_3 binding protein type 2"Life Sciences. 72. 443-453 (2002)

Uji, A. 等人：“与 PRIP-2 相互作用的分子，一种新型 Ins(1,4,5)P_3 结合蛋白 2 型”生命科学。