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ィイc15ィエD2 receptor-channel,丰富在小鼠小脑微粒体膜分数,由五个折叠有限域犹豫胰蛋白酶消化。通过胰蛋白酶的酶切，将n端IP γ γ γ - 2结合区、中间调控区和c端通道区分别拆分为2个、3个和1个结构域。胰蛋白酶敏感位点主要定位于选择性剪接位点和钙调素结合位点等调控位点，并以相对宽松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-蛋白相互作用相互组装成一个相对稳定的复合体，并保留了足够的活性，以结合IP γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ。因此,IPィイc15ィエD2受体在CaィイD12 +ィエD1商店膜结构相对紧凑的折叠高,耦合的IPィイc15ィエD2配体与chann绑定 ... 更多的el开放可能发生在这样一个紧张domain-domain interaction.3。在含有IP γ γ γ - D23 γ - D2结合区的两个胰蛋白酶抗性结构域中，n端结构域不具有IP γ - D23 γ - D2结合活性，而c端结构域则表现出较低的亲和力。另一方面，通过在大肠杆菌细胞中单独表达这两个结构域，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-蛋白相互作用形成一个活性的IP γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ。我们已经开发了一种系统，可以在大肠杆菌细胞中高效地生产大量具有高亲和力的IP γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ的可溶性蛋白。我们通过单通道记录分析了由平面脂质双分子层重构的小脑1型IP γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ γ受体通道。对于被认为与细胞内ca2 + D1动态密切相关的双相ca2 + D1敏感性，我们明确了约0.5 μM或更高ca2 + D1浓度的抑制期依赖于ca2 + D1激活的钙调蛋白。这些数据表明，钙调素在细胞内钙D12+钙D1动态的反馈调节中起着不可或缺的作用。我们证明，在细胞分裂过程中，局部运动的ip_d23_d_d2受体敏感的细胞内Ca _ D12+ _ d_1储存发生，并且似乎参与细胞动力学所需的Ca _ D12+ _ d_1信号传导。我们发现，在诱导t细胞凋亡时，1型ipu - D23 - γ - D2受体而不是2型和3型依赖于caspase-3发生蛋白水解，从而导致ipu - D23 - γ - D2受体通道活性失活。少

项目成果

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