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Biochemical and molecular mechanism for the activation of neurotrophin receptors by Ganglioside GM1

神经节苷脂GM1激活神经营养素受体的生化和分子机制

基本信息

批准号：
09670648
负责人：
MUTOH Tatsuro
金额：
$ 1.98万
依托单位：
Fukui Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670648/
关键词：
Trk GM1 signal transduction tyrosine kinase D-PDMP GEM O-PDMP signal transduction NGF GM1 PDMP

项目摘要

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.

以往的研究表明，神经营养因子如神经生长因子（NGF）及其同源受体在中枢和外周神经系统神经元的发育中起重要作用。然而，神经营养因子受体调控的详细分子机制仍有待阐明。我们以前的研究表明，Trk，一个高亲和力的功能性受体的神经生长因子，正调控的内源性酸性鞘糖脂，神经节苷脂GM 1，通过其直接结合的Trk受体。本研究试图了解GM 1对Trk正调控的分子机制，发现以下事实：首先，大鼠嗜铬细胞瘤细胞系PC 12细胞与不同浓度的D-Paddy（一种葡萄糖神经酰胺合酶活性抑制剂）预孵育一周，以消耗包括GM 1在内的细胞神经节苷脂。然后，这些细胞与神经生长因子刺激，令人惊讶的是，这些细胞没有响应神经生长因子的形态分化和磷酸化的Trk蛋白。这些结果有力地表明，内源性神经节苷脂，特别是GM 1，是不可缺少的Trk的正常功能，这进一步验证了我们以前的结果。然后将人trk cDNA转染PC 12细胞，获得了稳定表达Trk蛋白的转染细胞。对来自这些转染子的Trk-免疫沉淀物进行原位V8蛋白酶作图。结果表明，GM的Trk蛋白的结合位点位于质膜区域。共聚焦激光显微镜检查和蔗糖密度超离心检查表明，Trk是存在于富含糖脂的微区的质膜与GM 1协会。