SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN-domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN-domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN-domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1/RIAM/talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

Mutations in SHANK3, coding for a large scaffold protein of excitatory synapses in the CNS, are associated with neurodevelopmental disorders including autism spectrum disorders and intellectual disability (ID). Several cases have been identified in which the mutation leads to truncation of the protein, eliminating C-terminal sequences required for post-synaptic targeting of the protein. We identify here a patient with a truncating mutation in SHANK3, affected by severe global developmental delay and intellectual disability. By analyzing the subcellular distribution of this truncated form of Shank3, we identified a nuclear localization signal (NLS) in the N-terminal part of the protein which is responsible for targeting Shank3 fragments to the nucleus. To determine the relevance of Shank3 for nuclear signaling, we analyze how it affects signaling by β-catenin, a component of the Wnt pathway. We show that full length as well as truncated variants of Shank3 interact with β-catenin via the PDZ domain of Shank3, and the armadillo repeats of β-catenin. As a result of this interaction, truncated forms of Shank3 and β-catenin strictly co-localize in small intra-nuclear bodies both in 293T cells and in rat hippocampal neurons. On a functional level, the sequestration of both proteins in these nuclear bodies is associated with a strongly repressed transcriptional activation by β-catenin owing to interaction with the truncated Shank3 fragment found in patients. Our data suggest that truncating mutations in SHANK3 may not only lead to a reduction in Shank3 protein available at postsynaptic sites but also negatively affect the Wnt signaling pathway.