Spatial and Functional Heterogeneity of Sphingolipid-rich Membrane Domains

• Published in: The Journal of biological chemistry Vol. 280; no. 25; pp. 24072 - 24084
• Main Authors: Kiyokawa, Etsuko, Baba, Takeshi, Otsuka, Naomi, Makino, Asami, Ohno, Shinichi, Kobayashi, Toshihide
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.06.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Cell Line; Flow Cytometry; Fluorescent Antibody Technique; G(M1) Ganglioside - metabolism; Humans; Mutagenesis; Proteins - genetics; Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Deletion; Sphingomyelin Phosphodiesterase - genetics; Sphingomyelin Phosphodiesterase - metabolism; Toxins, Biological
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M502244200

Little is known about the organization of lipids in biomembranes. Lipid rafts are defined as sphingolipid- and cholesterol-rich clusters in the membrane. Details of the lipid distribution of lipid rafts are not well characterized mainly because of a lack of appropriate probes. Ganglioside GM1-specific protein, cholera toxin, has long been the only lipid probe of lipid rafts. Recently it was shown that earthworm toxin, lysenin, specifically recognizes sphingomyelin-rich membrane domains. Binding of lysenin to sphingomyelin is accompanied by the oligomerization of the toxin that leads to pore formation in the target membrane. In this study, we generated a truncated lysenin mutant that does not oligomerize and thus is non-toxic. Using this mutant lysenin, we showed that plasma membrane sphingomyelin-rich domains are spatially distinct from ganglioside GM1-rich membrane domains in Jurkat T cells. Like T cell receptor activation and cross-linking of GM1, cross-linking of sphingomyelin induced calcium influx and ERK phosphorylation in the cell. However, unlike CD3 or GM1, cross-linking of sphingomyelin did not induce significant protein tyrosine phosphorylation. Combination of lysenin and sphingomyelinase treatment suggested the involvement of G-protein-coupled receptor in sphingomyelin-mediated signal transduction. These results thus suggest that the sphingomyelin-rich domain provides a functional signal cascade platform that is distinct from those provided by T cell receptor or GM1. Our study therefore elucidates the spatial and functional heterogeneity of lipid rafts.