Intracellular signal transduction mechanims of neuronal survival and death

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Our previous studies have shown that gangliosideGM1 (GM1), a major constiuent of membrane raft, regulates high affinity nerve growth factor receptor, Trk, which is an essential factor for neuronal differentiation and survival. We constructed mutant human trk cDNA which is lacking possible N-glycosylation site and transfected into PCnnr cells which lacks Trk expression. We have been able to get the stable transfectant which overexpress mutant trk cDNA.This mutant clone did not show any response to NGF in terms of tyrosine autophosphorylation response, neurite outgrowth, and the prevention of serum-deprivation-induced apoptosis. These mutant Trk protein is not located in the lipids raft but rather distributed in higher density fractions of sucrose density gradient ultracentrifugation. To validate the above data, we also examined the effect of cholesterol kilating agents such as β-methyl cyclodextrin and filipin on NGF-induced Trk autophosphorylation response, because these agents has been reported to disrupt raft structure. We did not observe any reponses to NGF in cells pretreated with these agents. These data strongly suggest that GM1 and membrane raft structure are important factors for the normal function of the Trk receptor protein.