Involvement of gangliosides in glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid rafts.
The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.
Kasahara
K
K
Department of Biochemical Cell Research, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. kasahara@rinshoken.or.jp
Watanabe
K
K
Takeuchi
K
K
Kaneko
H
H
Oohira
A
A
Yamamoto
T
T
Sanai
Y
Y
D000818:Animals; D001483:Base Sequence; D015816:Cell Adhesion Molecules, Neuronal; D002478:Cells, Cultured; D058969:Contactin 2; D017931:DNA Primers; D005732:Gangliosides / Q000502:physiology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D015398:Signal Transduction
D015816:Cell Adhesion Molecules, Neuronal; C548676:Cntn2 protein, rat; D058969:Contactin 2; D017931:DNA Primers; D005732:Gangliosides; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; C026226:ganglioside, GD3