| Co-Investigator(Kenkyū-buntansha) |
MATILDA Kata 英国癌研究所, チェスター ビーティーラボラトリー, 主任研究員
MATSUDA Miho Kyushu University, Faculty of Dentistry, Assistant Professor, 歯学部, 助手
YAGISAWA Hitoshi Himeji Inst.Tech, Faculty of Science, Associate Professor, 理学部, 助教授 (40192380)
KATAN Matilda Chester Beaty Laboratory, Chief Scientist
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| Research Abstract |
We isolated a new inositol 1,4,5-trisphosphate [Ins (1,4,5) P_3] binding protein with a molecular mass of 130-kDa (p130). Molecular cloning studies revealed that the protein is similar to phospholipase C-delta_1 (PLC-delta_1) but has no catalytic activity to phosphoinositides, and Thus the biological function is not yet known. To gain the insight for the function, the following studies were carried out in the present study.
1. There are several single amino acid replacements in the most likely catalytic domains, named X-and Y-domains in p130, when they compared to those of many types of PLC including PLC-delta_1. Mutant plasmids for expressing p130, in which the X-and/or Y-domains were replaced with those of PLC-delta_1, were constructed. Transfection of E.coli and subsequent assay for PLC activity will be done soon.
2. A plasmid was constructed, in which the genes encoding p130 was incorporated together with the gene for affording neomycin-resistancy. COS-1 cells were transfected with the plasmid and further cultivation in the presence of the antibiotics produced the cells which stably express p130. Modification of the cell function in response to the stimulation with bradykinin or growth factor will be examined soon, comparing non-transfected cells.
3. Plasmids containing p130 gene, but lacking various region of p130 were constructed and each plasmid was transfected into COS-1 cells in order to localize the region responsible for binding Ins (1,4,5) P_3. Cell extract lacking the pleckstrin homology domain (PH domain) of p130 failed in the binding, indicating that the PH domain is involved in the binding. The PH domain of p130 bound both Ins (1,4,5) P_3 and Ins (1,4,5,6) P_4, while that of PLC-delta_1 bound Ins (1,4,5) P_3 alone. Based on these results, we have proposed that the physiologically relevant ligands for PH domains would be inositol phosphates.
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