Intracellular signal transduction mechanims of neuronal survival and death

我们前期的研究表明，神经节苷GM1 （gangliosidgm1）是膜筏的主要成分，可调控高亲和神经生长因子受体Trk，而Trk是神经元分化和存活的重要因子。我们构建了缺乏可能n -糖基化位点的人trk cDNA突变体，并将其转染到缺乏trk表达的PCnnr细胞中。我们已经获得了过表达trk基因突变体的稳定的转染物。在酪氨酸自磷酸化反应、神经突生长和预防血清剥夺诱导的细胞凋亡方面，该突变克隆对NGF没有任何反应。这些突变Trk蛋白不位于脂筏中，而是分布在蔗糖密度梯度超离心的高密度部分。为了验证上述数据，我们还研究了胆固醇杀伤剂（如β-甲基环糊精和filipin）对ngf诱导的Trk自磷酸化反应的影响，因为已有报道称这些药物会破坏筏结构。我们未观察到用这些药物预处理的细胞对NGF有任何反应。这些数据强烈提示GM1和膜筏结构是Trk受体蛋白正常功能的重要因素。

Our previous studies have shown that gangliosideGM1 (GM1), a major constiuent of membrane raft, regulates high affinity nerve growth factor receptor, Trk, which is an essential factor for neuronal differentiation and survival. We constructed mutant human trk cDNA which is lacking possible N-glycosylation site and transfected into PCnnr cells which lacks Trk expression. We have been able to get the stable transfectant which overexpress mutant trk cDNA.This mutant clone did not show any response to NGF in terms of tyrosine autophosphorylation response, neurite outgrowth, and the prevention of serum-deprivation-induced apoptosis. These mutant Trk protein is not located in the lipids raft but rather distributed in higher density fractions of sucrose density gradient ultracentrifugation. To validate the above data, we also examined the effect of cholesterol kilating agents such as β-methyl cyclodextrin and filipin on NGF-induced Trk autophosphorylation response, because these agents has been reported to disrupt raft structure. We did not observe any reponses to NGF in cells pretreated with these agents. These data strongly suggest that GM1 and membrane raft structure are important factors for the normal function of the Trk receptor protein.