Reconstitution of membranes simulating glycosignaling domain and their susceptibility to lyso-GM3

GM3 ganglioside at the surface of mouse melanoma B16 cells is clustered and organized with signal transducer molecules c-Src, Rho A, and focal adhesion kinase (FAR) to form a membrane unit separable from caveolae, which are enriched in cholesterol and caveolin but do not contain GM3 or the above three signal transducers, The GMS-enriched membrane units are involved in GM3-dependent cell adhesion coupled with activation of c-Src, Rho A, and FAK and are termed the glycosphingolipid signaling domain or the glycosignaling domain (GSD), In order to assess the essential components that display GSD function, membranes with properties similar to those of GSD were reconstituted using GM3, sphingomyelin, and c-Src, with or without other lipid components. The reconstituted membrane thus prepared displayed GM3-dependent adhesion to plates coated with Gg3 or anti-GM3 antibody, resulting in enhanced c-Src phosphorylation (c-Src phosphorylation response). This response in reconstituted membrane depends on GM3 concentration and was not observed when GM3 was absent or replaced with other gangliosides GM1 or GD1a, or with LacCer, The GM3-dependent c-Src phosphorylation response was enhanced when cholesterol and phosphatidylcholine were added. Although GM3, sphingomyelin, and c-Src are essential for GSD function, a small quantity of cholesterol and phosphatidylcholine may act as an auxiliary factor to stabilize membrane. GSD function in terms of GM3-dependent adhesion and signaling was blocked in the presence of lyso-GM3 or its analogue but not psychosine, lactosyl-sphingosine, or lyso-phosphatidylcholine. Such susceptibility of reconstituted GSD to lyso-GM3 and other lyse compounds is the same as GSD of original B16 cells. Thus, functional organization of the reconstituted membrane closely simulates that of GSD in B16 cells, which is based on clustered GM3 organized with c-Src as the essential components.