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

以往的研究表明，神经生长因子(NGF)等神经营养因子及其同源受体在中枢和外周神经系统神经元的维持中发挥着重要作用。然而，神经营养素受体调控的详细分子机制仍有待阐明。我们以前的研究表明，Trk是一种高亲和力的NGF功能性受体，通过与Trk受体的直接结合，受到内源性酸性神经节苷脂GM1的正调控。在本项目中，我们试图了解GM1对Trk正向调控的分子机制，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预先孵育一周，以耗尽包括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蛋白的自磷酸化方面对NGF没有反应。这些结果有力地表明，内源性神经节苷脂，尤其是GM1，对于Trk的正常功能是不可忽视的，这进一步验证了我们以前的结果。接下来，我们将人Trk基因导入PC12细胞，获得了稳定高表达Trk蛋白的转染体。对这些转染体的Trk免疫沉淀物进行V8蛋白的原位定位。结果表明，Trk蛋白中的GM结合部位位于膜旁区域。激光共聚焦显微镜和蔗糖密度超速离心法检测表明，Trk与GM1结合存在于细胞膜糖脂丰富的微区中。

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.