Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells

Masashi Nishio, Satoshi Fukumoto, Keiko Furukawa, Akiko Ichimura, Hiroshi Miyazaki, Susumu Kusunoki, Takeshi Urano, Koichi Furukawa

Research output: Contribution to journalArticle

100 Citations (Scopus)

Abstract

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 125I-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 125I-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. p75NTR 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.

Original languageEnglish
Pages (from-to)33368-33378
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number32
DOIs
Publication statusPublished - Aug 6 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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