GM1 promotes TrkA‐mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA.

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA–GM1 oligosaccharide complex at the cell surface. To study the TrkA–GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross‐linked TrkA–GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA–GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent‐resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by 'flopping' down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1. The plasma‐membrane GM1‐TrkA interaction is an absolute milestone but the precise molecular mechanism has not been precisely defined. By the use of photoactivable GM1, we demonstrated that only GM1 having the photoactivable group at the external galactose unit crosslinks TrkA. We propose that GM1‐TrkA interaction occurs thanks to the TrkA extracellular portion, present in the fluid plasma‐membrane microenvironment, that lying on the cell surface, reaches the GM1 oligosaccharide moiety, whose ceramide is inserted within the rigid plasma‐membrane microdomain. [ABSTRACT FROM AUTHOR]