Postsynaptic Shank proteins as effectors of Ras family G-proteins

突触后柄蛋白作为 Ras 家族 G 蛋白的效应子

• 批准号: 407143299
• 负责人: Professor Dr. Hans-Jürgen Kreienkamp
• 金额: --
• 依托单位: Institut für Humangenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/407143299?language=en
• 关键词: Postsynaptic; Shank; proteins; effectors; Ras

Mutations in SHANK3 are associated with autism spectrum disorders in humans. However, the pathological relevance of many missense mutations found in patients is unclear, as none of them has been correlated with a molecular defect of the postsynaptic Shank3 protein. Using biochemical approaches, expression in neurons and X-ray structural analysis, we have identified an N-terminal domain of Shank3 which on one side exerts intramolecular, regulatory control over the ankyrin repeat region (Ank), and on the other hand binds with high affinity to active (GTP-bound) G-proteins of the Ras family (H-, K-, N-, R-Ras, Rap1, Rap2). Functionally this leads e.g. to inhibition of integrin activation by Rap1. Our observation that binding to Ras and Rap is destroyed by patient derived mutations (R12C, L68P) shows for the first time a clear molecular defect for SHANK3 missense mutations. As Ras and Rap variants affect formation, homeostasis and plasticity of synapses, we will investigate here to what extent Shank3 mediates these effects of small G-proteins at synapses. For this purpose, we will study how Ras family members affect structure and conformation, as well as the biochemical interactions of the Shank3 protein. In cultured neurons we will analyze how active G-proteins influence the localization of Shank3 in dendritic spines and so-called nano-clusters. Furthermore we want to clarify how active Ras or Rap affect the role of Shank3 in formation and plasticity of the postsynaptic density, dendritic spines and synapses. Patient derived mutations will provide excellent negative controls; furthermore we will be able to validate our results in a mouse line carrying the L68P mutation in the Shank3 gene.

Shank3中的突变与人类的自闭症谱系障碍有关。但是，在患者中发现的许多错义突变的病理相关性尚不清楚，因为它们都与突触后尚克3蛋白的分子缺陷没有相关。使用生化方法，在神经元和X射线结构分析中的表达，我们确定了Shank3的N末端结构域，该结构域在一侧施加了分子内的调节性控制对Ankyrin重复区域（ANK），另一方面则与高高的结合在高度上结合与RAS家族的活性（GTP结合）G蛋白的亲和力（H-，K-，N-，RAS，RAP1，RAP2）。从功能上讲，这引起了例如用Rap1抑制整联蛋白的激活。我们的观察结果是，与患者衍生突变（R12C，L68P）破坏了与RAS和RAP的结合，这首先显示出Shank3错义突变的明显分子缺陷。随着RAS和RAP变体影响形成，稳态和突触的可塑性，我们将在这里调查Shank3在突触下小G蛋白的这些影响的程度。为此，我们将研究RAS家族成员如何影响结构和构象，以及Shank3蛋白的生化相互作用。在培养的神经元中，我们将分析活性G蛋白如何影响Shank3在树突状刺和所谓的纳米簇中的定位。此外，我们要阐明活性Ras或RAP如何影响Shank3在突触后密度，树突状刺和突触的形成和可塑性中的作用。患者衍生的突变将提供出色的阴性对照；此外，我们将能够在携带shank3基因的L68P突变的小鼠系中验证我们的结果。