SUMMARY Postsynaptic densities (PSDs) are membrane semienclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/ PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.

Shank and SAPAP (synapse-associated protein 90/postsynaptic density-95-associated protein) are two highly abundant scaffold proteins that directly interact with each other to regulate excitatory synapse development and plasticity. Mutations of SAPAP, but not other reported Shank PDZ domain binders, share a significant overlap on behavioral abnormalities with the mutations of Shank both in patients and in animal models. The molecular mechanism governing the exquisite specificity of the Shank/SAPAP interaction is not clear, however. Here we report that a sequence preceding the canonical PDZ domain of Shank, together with the elongated PDZ BC loop, form another binding site for a sequence upstream of the SAPAP PDZ-binding motif, leading to a several hundred-fold increase in the affinity of the Shank/SAPAP interaction. We provide evidence that the specific interaction afforded by this newly identified site is required for Shank synaptic targeting and the Shank-induced synaptic activity increase. Our study provides a molecular explanation of how Shank and SAPAP dosage changes due to their gene copy number variations can contribute to different psychiatric disorders.Shank | SAPAP | PDZ | synapse | specific interaction P ostsynaptic density (PSD) at excitatory synapses refers to disk-shaped, densely packed mega-protein assemblies located beneath postsynaptic membranes (1-5). A set of highly abundant scaffold proteins, including DLGs (disc large proteins including PSD-95, PSD-93, and SAP102), SAPAP (synapse-associated protein 90/postsynaptic density-95-associated protein; also known as GKAP or DLGAP), and Shank, are known to be critical for the formation, stability, and neuronal activity-dependent dynamic regulations of . In addition to orchestrating PSD formation, these scaffold proteins also serve to control the trafficking and clustering of receptors on the plasma membranes and to interface with actin cytoskeletons at synapses (15,18,19). Mutations of DLGs, SAPAP, and Shank have been found to cause or associate with various psychiatric disorders, including autism spectrum disorder (ASD), depression, and schizophrenia (20-28), further supporting the importance of these proteins in normal brain development and functions.Electron and superresolution light microscopy imaging studies (4,5,18,(29)(30)(31)(32) have revealed that proteins in PSDs form distinct layers along the axodendritic axis of synapses with a sequential order of membrane-spanning glutamate receptors and cell adhesion molecules, DLGs, the SAPAP and Shank scaffolds, and actin cytoskeletons. Such a distinct layered structure in PSDs depends critically on the specific interactions between the scaffold proteins. For example, the phosphorylation-dependent interaction between DLG GK domain and the N-terminal repeating sequences of SAPAP positions SAPAP beneath DLGs (33), and the interaction between the SAPAP PDZ-binding motif (PBM) and Shank PDZ domain functions to place Shank at a deeper layer in PSD (7).The critical roles of the PSD scaffold proteins in norma...

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