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

我们发现抗神经节苷脂GD3抗体免疫共沉淀了GPI锚定的神经细胞黏附分子Tag-1、src家族蛋白Lyn、C端src蛋白结合蛋白CBP和三聚体G蛋白Goα。此外，我们还证明了抗GD3抗体处理导致了存在于培养的大鼠小脑颗粒神经元脂支组分中的Lyn的激活。当颗粒细胞上的TAG-1与抗TAG-1抗体交联时，也观察到LYN的激活。由于GD3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发出的信号受到发育调节。Western blotting分析显示，发育中的小脑(出生后4d)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的小脑脂筏组分中高度积聚。因此，我们比较了发育中的小脑(出生后4-7天)和成年小脑中Lyn和CBP的磷酸化水平。与成年小脑脂筏组分相比，发育小脑制备的脂筏组分中活性形式的Lyn和结合CSK的酪氨酸磷酸化形式的CBP具有较高的积累量。在CHO细胞中过表达Lyn和CBP导致CBP的磷酸化，这表明CBP是Lyn的底物。此外，TAG-1、LYN、CBP在发育中的小脑的生长锥部分中高度集中。这些结果表明，这些信号分子在功能上与发育中的小脑生长锥体上的脂筏有关。

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.