Postsynaptic Shank proteins as effectors of Ras family G-proteins

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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.