SAITO Yumiko The Tokyo Metropolitan Institute of Medical Sciencen, Department of Molecular Bi, 遺伝情報研究部門, 研究員 (00215568)
MARUYAMA Kei National Institute for Physiological Science, Laboratory for Neurochemistry, Ass, 生理学研・神経化学, 助教授 (30211577)
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¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1997 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
We have identified a novel neuron-specific molecule, N23K and its splicing form, N27K by using subtraction and plus/minus method with mouse NS2OY neuroblastoma cell line. Their expression were dramatically but transiently increased during neurite outgrowth when the cells were treated with dbcAMP.Noteworthy, in vivo mouse brain, the localization of these molecules was dynamically changed during cortical development. Surprisingly, by chance, it was found that N23K/N27K are precursor molecules of a novel opioid, orphanin FQ, which was independently found by other two groups at the same time. Thus, N23K/N27K may have bifunctional role both in developmental stage arid in adult. In order to examine the direct role of N23K/N27K in neuronal development, N23K-, N27K-, and N23K + N27K- stably overexpressing NS2OY cells were established and analyzed their morphology systematically. As expected, these all three transfectants initiated neuronal outgrowth without dbcAMP treatment. Orphanin FQ is known to bind to G-protein coupled receptor LC-7. Since LC-7 is coupled to Gi-protein, intracellular cyclic AMP level is efficiently inhibited by this peptide. In wild NS2OY cells, orphanin FQ and other two peptides produced from the precursor had no influence for morphological change, amounts of cyclic AMP, and a calcium influx. Further, no difference in cyclic AMP content and expression level of LC-7 were detected between mock-transfected NS2OY cells and all N23K/N27K transfectants. Analysis of truncated mutation in relation to neurite outgrowth revealed that the C-terminus in N23K/N27K protein is crucial for translation efficiency. These results suggest that N23K/N27K protein itself or unknown processing product including the C-terminal portion plays a direct role for neurite outgrowth, but not for orphanin FQ-LC7 coupling pathway.