Immunoseparation of sphingolipid-enriched membrane domains enriched in Srcfamily protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody

Rat cerebellar granule cells differentiated in culture were fed [1-H-3]sphi ngosine, allowing the metabolic radiolabelling of all cell sphingolipids an d phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched mem brane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-3H]sphingosine-fe d cells by sucrose gradient centrifugation. This fraction was enriched in t he Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-an chored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolip id-enriched membrane fraction were subjected to immunoprecipitation with an ti-GD3 ganglioside monoclonal antibody R24, under experimental conditions d esigned to preserve the integrity of the domain. The radioactive lipid comp osition of the immunoprecipitates; obtained from the cell lysate and from t he sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fr action. In fact, the immunoprecipitates contained, together with GD3 gangli oside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcho line were detected in the immunoprecipitates by qualitative TLC analysis fo llowed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were assoc iated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. Thes e data suggest that a membrane domain containing cholesterol, phosphatidylc holine, sphingolipids and proteins can be separated from the total cell mem branes by anti-GD3 antibody immunoprecipitation, and that the association o f c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is media ted by the interaction with a complex lipid environment, rather than by spe cific interactions with a single sphingolipid species.