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 adhesio n kinase (FAR) to form a membrane unit separable from caveolae, which are e nriched in cholesterol and caveolin but do not contain GM3 or the above thr ee signal transducers, The GMS-enriched membrane units are involved in GM3- dependent cell adhesion coupled with activation of c-Src, Rho A, and FAK an d are termed the "glycosphingolipid signaling domain" or the "glycosignalin g domain" (GSD), In order to assess the essential components that display G SD function, membranes with properties similar to those of GSD were reconst ituted using GM3, sphingomyelin, and c-Src, with or without other lipid com ponents. The reconstituted membrane thus prepared displayed GM3-dependent a dhesion to plates coated with Gg3 or anti-GM3 antibody, resulting in enhanc ed c-Src phosphorylation (c-Src phosphorylation response). This response in reconstituted membrane depends on GM3 concentration and was not observed w hen 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, sphingomyeli n, and c-Src are essential for GSD function, a small quantity of cholestero l and phosphatidylcholine may act as an auxiliary factor to stabilize membr ane. GSD function in terms of GM3-dependent adhesion and signaling was bloc ked in the presence of lyso-GM3 or its analogue but not psychosine, lactosy l-sphingosine, or lyso-phosphatidylcholine. Such susceptibility of reconsti tuted GSD to lyso-GM3 and other lyse compounds is the same as GSD of origin al B16 cells. Thus, functional organization of the reconstituted membrane c losely simulates that of GSD in B16 cells, which is based on clustered GM3 organized with c-Src as the essential components.