Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells
Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells
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DOI：
10.1074/jbc.m403816200
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发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Furukawa, K
中科院分区：
文献类型：
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作者：
Nishio, M;Fukumoto, S;Furukawa, K
神经节苷脂GM 1被认为具有神经营养因子样活性。为了分析内源性产生的GM 1的作用，用GM 1/GD 1b/GA 1合酶基因转染大鼠嗜铬细胞瘤细胞系PC 12，显示GM 1的表达水平增加。令我们惊讶的是，GM 1（+）-转染细胞（GM 1（+）细胞）在神经生长因子（NGF）刺激后没有显示神经突形成。在GM 1（+）细胞中，NGF处理后几乎未检测到TrkA的自磷酸化和ERK 1/2的激活。I-125-NGF与PC 12细胞的结合在GM 1（+）细胞和对照之间几乎相等。然而，在双（磺基琥珀酰亚胺基）辛二酸酯3的交联分析和I-125-NGF结合试验中，GM 1（+）细胞中NGF处理后TrkA二聚体的形成明显受到抑制。细胞裂解液的蔗糖密度梯度分离显示，在GM 1（+）细胞中，主要位于脂筏部分的TrkA移动到非筏部分。p75（NTR）和Ras在GM 1（+）细胞中也从筏转移到非筏部分，而flotillin和GM 1持续存在于脂筏中。TrkA激酶活性差异调节时，GM 1加入体外激酶测定系统，表明抑制/增强作用的GM 1对神经生长因子信号的基础上的浓度。光漂白后荧光恢复的测量显示，GM 1（+）细胞的膜流动性降低。这些结果表明，过表达的GM 1通过调节脂筏的性质以及NGF受体和相关信号分子的细胞内定位来抑制NGF/ TrkA介导的分化信号。
Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/ GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1(+)-transfectant cells (GM1(+) cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1(+) cells. Binding of I-125-NGF to PC12 cells was almost equivalent between GM1(+) cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1(+) cells in both cross-linking analysis with Bis(sulfosuccinimidyl) suberate 3 and I-125-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1(+) cells. p75(NTR) and Ras also moved from the raft to non-raft fraction in GM1(+) cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1(+) cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/ TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.