Analysis of signal transduction mechanism by GPI-anchored neuronal cell adhesion molecule TAG-1

我们已经证明，抗神经节苷脂GD 3抗体共免疫沉淀GPI锚定的神经元细胞粘附分子TAG-1、src家族激酶林恩、Csk（C末端src激酶）结合蛋白Cbp和三聚体G蛋白Goα。此外，我们已经证明，抗GD 3抗体治疗导致激活的林恩，这是目前在培养的大鼠小脑颗粒神经元的脂筏部分。当颗粒细胞上的TAG-1与抗TAG-1抗体交联时，也观察到林恩的活化。由于GD 3已经存在于大脑发育的早期阶段，我们假设通过这些相互作用的分子的信号传导受到发育调节。Western blotting分析表明，发育中小脑（出生后4 d）的总酪氨酸（Tyr）磷酸化水平高于成年小脑，且磷酸化蛋白在发育中小脑的脂筏组分中高度积累。因此，我们比较了林恩和Cbp在发育中的小脑（出生后4-7天）和在成人小脑中的磷酸化水平。活性形式的林恩和酪氨酸磷酸化形式的Cbp，这是能够结合到Csk，高度积累在脂筏组分制备从发展中的小脑相比，成人的脂筏组分。林恩和Cbp在CHO细胞中的过表达导致Cbp的磷酸化，这表明Cbp是林恩的底物。此外，TAG-1、林恩、Cbp在从发育中的小脑制备的生长锥级分中高度浓缩。这些结果表明，这些信号分子与发育中小脑生长锥上的脂筏功能相关。

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.