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J. Biol. Chem., Vol. 279, Issue 32, 33368-33378, August 6, 2004

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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, and Koichi Furukawa||

From the Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, the Department of Pediatric Dentistry, Nagasaki University School of Dentistry, Sakamoto, Nagasaki 852-8588, and the ^¶Department of Neurology, Kinki University School of Medicine, Ohno-Higashi, Sayama, Osaka 589-8511, Japan

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. 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.

Received for publication, April 6, 2004 , and in revised form, May 14, 2004.

^* This work was supported by Grants-in-aid for Scientific Research of Priority Areas 10178104, 10152223, and 10470029, and COE (Center of Excellence) Research from the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 81-52-744-2070; Fax: 81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.

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