|Budget Amount *help
¥13,000,000 (Direct Cost : ¥13,000,000)
Fiscal Year 1999 : ¥5,600,000 (Direct Cost : ¥5,600,000)
Fiscal Year 1998 : ¥7,400,000 (Direct Cost : ¥7,400,000)
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
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