Postsynaptic Shank proteins as effectors of Ras family G-proteins

SHANK 3的突变与人类自闭症谱系障碍有关。然而，在患者中发现的许多错义突变的病理相关性尚不清楚，因为它们中没有一个与突触后Shank 3蛋白的分子缺陷相关。使用生物化学方法，在神经元中的表达和X射线结构分析，我们已经确定了一个N-末端结构域的Shank 3的，一方面施加分子内，调节控制的锚蛋白重复区（Ank），另一方面结合高亲和力的活性（GTP结合）的G-蛋白的Ras家族（H-，K-，N-，R-Ras，Rap 1，Rap 2）。在功能上，这导致例如Rap 1对整联蛋白活化的抑制。我们观察到与Ras和Rap的结合被患者来源的突变（R12 C，L 68 P）破坏，这首次显示了SHANK 3错义突变的明确分子缺陷。由于Ras和Rap变体影响突触的形成、稳态和可塑性，我们将在这里研究Shank 3在多大程度上介导小G蛋白在突触中的这些作用。为此，我们将研究Ras家族成员如何影响结构和构象，以及Shank 3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Shank 3在树突棘和所谓的纳米簇中的定位。此外，我们希望阐明活性Ras或Rap如何影响Shank 3在突触后密度、树突棘和突触的形成和可塑性中的作用。患者来源的突变将提供极好的阴性对照;此外，我们将能够在Shank 3基因中携带L 68 P突变的小鼠系中验证我们的结果。

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.