Study of the Structure-Function of IPィイD23ィエD2 Receptor/CaィイD12+ィエD1 Release Channel and IPィイD23ィエD2/CaィイD12+ィエD1 Signaling

IPD23D2受体/CaD12+D1释放通道结构功能及IPD23D2/CaD12+D1信号传导的研究

• 批准号: 10490007
• 负责人: FURUICHI Teiichi
• 金额: $ 8.32万
• 依托单位: The Institute of Physical and Chemical Research (RIKEN) (1999)The University of Tokyo (1998)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10490007/
• 关键词: IPィイD23ィエD2 receptor; CaィイD12+ィエD1 signaling; Calmodulin; Ligand binding; Apoptosis; Single channel recording; Cytokinesis; Caspase-3; セカンドメッセンジャー; カルシウムチャネル; イノシトール1,4,5-三リン酸; IP_3誘導Ca^<2+>放出

1. We found that type 1 IPィイD23ィエD2 receptor-channel, which is abundant in mouse cerebellar microsomal membrane fractions, is composed of five folding domains unsusceptible to limited trypsin digestion. By trypsin digestion, the N-terminal IPィイD23ィエD2-binding region, middle regulatory region and C-terminal channel region were split into 2,3, and one domains, respectively. Trypsin sensitive sites are mainly localized at the alternative splicing sites and regulatory sites such as a calmodulin-binding site, and seem to be exposed to surface of its molecule as a relatively relaxed structure.2. These five domains generated by trypsin digestion assembled each other as a relatively stable complex through protein-protein interaction, and retained sufficient activities for IPィイD23ィエD2 binding and IPィイD23ィエD2-induced CaィイD12+ィエD1 release. Thus, IPィイD23ィエD2 receptor on CaィイD12+ィエD1 store membranes has a relatively compact folding higher structure, coupling of IPィイD23ィエD2 ligand binding with chann … More el opening may occur through such a tight domain-domain interaction.3. Of two trypsin-resistant domains containing the IPィイD23ィエD2 binding region, the N-terminal domain had no IPィイD23ィエD2 binding activity, whereas the C-terminal domain exhibited low affinity. On the other hand, by just mixing these two domains expressed individually in E. coli cells we could reconstituted a high-affinity binding activity. These results indicate that these two individual domains can form an active IPィイD23ィエD2 ligand-binding pocked through a protein-protein interaction. We have developed a system to efficiently produce a large amount of soluble protein with high-affinity IPィイD23ィエD2 binding activity in E. coli cells.4. We analyzed cerebellar type 1 IPィイD23ィエD2 receptor-channel reconstituted into planar lipid bilayers by a single channel recording. As to a biphasic CaィイD12+ィエD1 sensitivity of cerebellar IPィイD23ィエD2 receptor that is thought to deeply be related to intracellular CaィイD12+ィエD1 dynamics, we clarified that an inhibitory phase by about 0.5 μM or higher CaィイD12+ィエD1 concentrations is dependent on CaィイD12+ィエD1-activated calmodulin. These data indicate that calmodulin plays an indispensable role in feedback regulation in intracellular CaィイD12+ィエD1 dynamics.5. We demonstrated that the local movement of IPィイD23ィエD2 receptor-sensitive intracellular CaィイD12+ィエD1 stores occur during cell division and appears to be involved in CaィイD12+ィエD1 signaling required for cytokinesis.6. We elucidated that upon induction of T-cell apoptosis the type 1 IPィイD23ィエD2 receptor but not type 2 and type 3 is proteolyzed in dependence on caspase-3, thereby resulting in inactivation of IPィイD23ィエD2 receptor-channel activity. Less

1.我们发现，1型IP β-D23 β-D2受体通道，这是丰富的小鼠小脑微粒体膜组分，是由5个折叠结构域不受限制的胰蛋白酶消化。经胰蛋白酶消化后，将N-端IP蛋白D2, 3结合区、中间调控区和C-端通道区分别拆分为2、3和1个结构域。胰蛋白酶的敏感位点主要位于选择性剪接位点和钙调素结合位点等调控位点，并以相对松弛的结构暴露于其分子表面.胰蛋白酶消化产生的这5个结构域通过蛋白质-蛋白质相互作用相互组装成相对稳定的复合物，并保留了足够的活性，可与IP受体D23结合，以及IP受体D23诱导的Ca受体D12+ Ca受体D1释放。因此，在Ca ~（2+）D_（12+）D_（1+）储存膜上的IP_（23）D_ ...更多信息 EL打开可以通过这种紧密的畴-畴相互作用发生。两个胰蛋白酶抗性结构域含有IP蛋白酶D23受体D2结合区域，N-末端结构域没有IP蛋白酶D23受体D2结合活性，而C-末端结构域表现出低亲和力。另一方面，通过将在E.大肠杆菌细胞，我们可以重建一个高亲和力的结合活性。这些结果表明，这两个单独的结构域可以通过蛋白质-蛋白质相互作用形成活性IP结合蛋白D23结合蛋白D2配体结合的凹坑。我们已经开发了一种系统，以有效地产生大量的可溶性蛋白，具有高亲和力的IP连接蛋白D23连接蛋白D2结合活性在E。大肠杆菌细胞。4.我们分析了小脑1型IP β-D23 β-D2受体通道重组成平面脂质双层的单通道记录。关于小脑IP β D23受体的双相Ca ~（2+）D12+ Ca ~（2+）D1敏感性，我们阐明了约0.5 μM或更高浓度的Ca ~（2+）D12+ Ca ~（2+）D1的抑制相依赖于Ca ~（2+）D12 + Ca ~（2+）D1激活的钙调素。这些数据表明，钙调素在细胞内Ca ~（2+）D_12 + Ca ~（2+）D_1动态的反馈调节中起着不可或缺的作用.我们证明了在细胞分裂过程中，IP β D23 β D2受体敏感的细胞内Ca β D12+ β D1储存的局部运动发生，并且似乎参与了细胞分裂所需的Ca β D12+ β D1信号传导。我们阐明了在诱导T细胞凋亡时，依赖于半胱天冬酶-3，1型IP β D23 β D2受体而不是2型和3型IP β D23 β D2受体被蛋白水解，从而导致IP β D23 β D2受体通道活性失活。少

项目成果

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Yoshikawa,F.: "Trypsinized cerebellar IP_3 receptor: structural and functional coupling of cieaved ligand binding and channel domains." J.Biol.Chem.274. 316-327 (1999)

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