| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We have shown that ant-ganglioside GD3 antibody coimmunoprecipitated GPI-anchored neuronal cell adhesion molecule TAG-1, src-family kinase Lyn, Csk (C-terminal src kinase) binding protein Cbp, and trimeric G protein Goα. Furthermore, we have demonstrated that anti-GD3 antibody treatment resulted in activation of Lyn that was present in the lipid raft fraction of cultured rat cerebellar granule neurons. Activation of Lyn has been also observed when TAG-1 on the granule cells were cross-linked with anti-TAG-1 antibody. Since GD3 is already present in early stages of brain development, we hypothesized that signaling through these interacting molecules were subject to developmental regulation. Western blotting analysis showed that total tyrosine (Tyr) phosphorylation level was higher in the developing cerebellum (postnatal day 4) than that in the adult one, and the phosphorylated proteins were highly accumulated in the lipid raft fraction of the developing cerebellum. Therefore, we compared phosphorylation level of Lyn and Cbp in the developing cerebellum (postnatal day 4-7) and that in the adult cerebellum. Active form of Lyn and the Tyr-phosphorylated form of Cbp, that is capable of biding to Csk, were highly accumulated in the lipid raft fraction prepared from the developing cerebellum compared to the lipid raft fraction of the adult one. Overexpression of Lyn and Cbp in CHO cells resulted in phosphorylation of the Cbp, which indicates that Cbp is a substrate of Lyn. In addition, TAG-1, Lyn, Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. These results suggest that these signaling molecules functionally associate with the lipid rafts on growth cones in developing cerebella.
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