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.

先前的研究表明，神经营养因子如神经生长因子（NGF）及其同源受体在中枢和周围神经系统神经元的维持中发挥着重要作用。然而，神经营养素受体调节的详细分子机制仍有待阐明。我们之前的研究表明，Trk 是 NGF 的高亲和力功能性受体，通过与 Trk 受体直接结合，受到内源性酸性鞘糖脂、神经节苷脂 GM1 的正向调节。在本项目中，我们试图了解GM1正向调节Trk的分子机制，并发现以下事实：首先，将大鼠嗜铬细胞瘤细胞系 PC12 细胞与不同浓度的 D-PDMP（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 GM1 在内的细胞神经节苷脂。然后，用NGF刺激这些细胞。令我们惊讶的是，这些细胞在形态分化和Trk蛋白自身磷酸化方面未能对NGF做出反应。这些结果强烈表明内源性神经节苷脂，尤其是GM1，对于Trk的正常功能是不可或缺的，这进一步验证了我们之前的结果。接下来，我们将人trk cDNA转染到PC12细胞中，得到了过表达Trk蛋白的稳定转染子。对这些转染子的 Trk 免疫沉淀物进行原位 V8 蛋白酶图谱分析。结果强烈表明 Trk 蛋白中的 GM 结合位点位于近膜区域。共聚焦激光显微镜检查和蔗糖密度超速离心检查表明，Trk 存在于与 GM1 相关的质膜富含糖脂的微区中。