||The postsynaptic density (PSD) is a protein-rich thickening in the postsynaptic membrane and contains mainly scaffold, cytoskeletal and signaling proteins that structurally and functionally interact with glutamate receptors and other postsynaptic membrane proteins. The molecular mechanisms regulating the assembly of PSD proteins and their association with synapses are still largely unknown. I investigated the molecular mechanisms of an important scaffold protein, Shank1, in synapse targeting and assembly by examining its interactions with the neuronal proteins GKAP (guanylate kinase-associated protein) and PSD-95. The data demonstrate that the SH3 domain of Shank1 specifically binds to the Shank1 ankyrin repeat domain, thus allowing Shank1 to multimerise via a novel form of intermolecular interactions. This form of multimerisation regulates Shank1 accumulation and stability in synapses as well as in hippocampal neuronal culture. Surprisingly, in both COS cells and hippocampal neurons, GKAP (which binds to the Shank PDZ domain) forms insoluble aggregates with Shank that co-localise with Hsp70 and neurofilaments, two markers of the aggresomes. However, both Shank1 and GKAP are organized in clusters in neurons and COS cells when they are overexpressed and can associate with PSD-95. Therefore, our data suggest that the formation of the PSD-95/GKAP/Shank complex is required for Shank1 targeting to synapses.