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

基本信息

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

项目摘要

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IPI D23E D2受体通道（在小鼠小脑微粒体膜分数中都很丰富）由五个折叠域组成，无法易于胰蛋白酶消化。通过胰蛋白酶消化，N末端IPI D23E D2结合区域，中间调节区域和C末端通道区分别分为2,3和一个域。胰蛋白酶敏感的位点主要定位于替代剪接位点和调节位点，例如钙调蛋白结合位点，并且似乎将其作为相对放松的结构暴露于其分子的表面。2。这五个由胰蛋白酶消化产生的五个结构域通过蛋白质 - 蛋白质相互作用互相组装，作为相对稳定的复合物，并保留了IPIY D23E D2结合和IPIY D23E D2 D2诱导的CAIY D12+IE D1 D1释放的足够活性。这就是CAII D12+II D1上的IPIY D23E D2受体具有相对紧凑的折叠结构，IPII D2配体与CHANN的结合偶联……可能通过这种紧密的结构域相互作用而发生更多的EL开放。3。在包含IPII D23I D2结合区域的两个耐胰蛋白酶耐药结构域中，N末端结构域没有IPI D23I D2结合活性，而C末端结构域暴露了低亲和力。另一方面，仅将这两个域在大肠杆菌细胞中单独表示，我们可以重构高亲和力的结合活性。这些结果表明，这两个域可以通过蛋白质 - 蛋白质相互作用形成活跃的IPIY D23E D2配体结合。我们已经开发了一个系统，可以有效地生产大量的固体蛋白，并在大肠杆菌细胞中具有高亲和力的IPIY D23E D2结合活性。4。我们通过单个通道记录分析了小脑1型IPI D23E D2受体渠道，将其重构为平面脂质双层。 As to a biphasic Cai D12+E D1 sensitivity of cerebellar IPi D23E D2 receptor that is thought to deeply be related to intracellular Cai D12+E D1 dynamics, we clared that an inhibitory phase by about 0.5 μM or higher Cai D12+E D1 concentrations is dependent on Cai D12+E D1-activated calmodulin.这些数据表明，钙调蛋白在细胞内CAIY D12+IE D1动力学中的反馈调节中起着必不可少的作用。5。我们证明了IPIY D23 D2受体敏感的细胞内CAIY D12+IE D1商店的局部运动发生在细胞分裂过程中，并且似乎参与了细胞因子所需的CAIY D12+IE D1信号传导。6。我们阐明了诱导T细胞凋亡后，1型IPIY D23 D2受体，但2型和3型和3型的蛋白质有利为依赖caspase-3，从而导致IPI D23E D23E D2受体通道活性灭活。较少的