Spatial and functional heterogeneity of sphingolipid-rich membrane domains

Authors:
Kiyokawa E, Baba T, Otsuka N, Makino A, Ohno S and Kobayashi T
In:
Source: J Biol Chem
Publication Date: (2005)
Issue: 280(25): 24072-24084
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Cells used in publication:
Jurkat
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Abstract
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 (TCR) activation and cross-linking of GM1, cross-linking of sphingomyelin induces 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 TCR or GM1. Our study therefore elucidates the spatial and functional heterogeneity of lipid rafts