Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells.
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.
Nishio
Masashi
M
Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan.
Fukumoto
Satoshi
S
Furukawa
Keiko
K
Ichimura
Akiko
A
Miyazaki
Hiroshi
H
Kusunoki
Susumu
S
Urano
Takeshi
T
Furukawa
Koichi
K
D000818:Animals; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005677:G(M1) Ganglioside / Q000235:genetics / Q000502:physiology*; D005700:Galactosyltransferases / Q000235:genetics; D051147:Ganglioside Galactosyltransferase; D005732:Gangliosides / Q000235:genetics; D015870:Gene Expression; D006028:Glycosphingolipids / Q000235:genetics; D006801:Humans; D008560:Membrane Fluidity / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D020800:Receptor, Nerve Growth Factor / Q000032:analysis* / Q000502:physiology; D020917:Receptor, trkA / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
C011258:G(A1) ganglioside; D005732:Gangliosides; D006028:Glycosphingolipids; D020800:Receptor, Nerve Growth Factor; C033851:ganglioside, GD1b; D005677:G(M1) Ganglioside; D020932:Nerve Growth Factor; D005700:Galactosyltransferases; D051147:Ganglioside Galactosyltransferase; D020917:Receptor, trkA; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases