| Research Abstract |
Previous studies have shown that neurotrophins such as nerve growth factor (NGF) and their cognate receptors play important roles in the maintainance of neurons in the central and peripheral nervous system. The detailed molecular mechanism of the regulation of neurotrophin receptor, however, remained to be elucidated. Our previous studies have shown that Trk, a high affinity functional receptor for NGF, is regulated positively by endogenous acid glycosphingolipids, ganglioside GM1, by its direct binding to the Trk receptor. In this project, we tried to understand the molecular mechanism for the positve regulation of Trk by GM1 and found the facts listed below ; In the first, rat pheochromocytoma cell line PC12 cells were pre-incubated with various concentrations of D-PDMP, an inhibitor for glucosylceramide synthase activity, for one week in order to deplete cellualr gangliosides including GM1. Then, these cells were stimulated with NGF.To our surprise, these cells failed to respond to NGF in terms of morphological differentiation and the autophosphorylation of the Trk protein. These results strongly indicated that endogenous gangliosides, especially GM1, are indespensable for the normal function of Trk, which further verifys our previous results. In the next, we transfected human trk cDNA into PC12 cells and got a stable transfectant overexpressing Trk protein. Trk-immunoprecipitates from these transfectants were subjected to in-situ V8 proteinase mapping. The results strongly suggested that GM binding sites in the Trk protein reside at the juxtamembrane regions. Confocal laser microscopic examinations and sucrose density ultra-centrifugation examination suggested that Trk is present in the glycolipid-enriched microdomains of the plasma membranes in association wIth GM1.
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