The goal of this proposal is to understand the structural basis of signaling complex assembly and organization at the synapse. At excitatory synapses, glutamate receptors and their downstream effectors are clustered at a complex cellular structure known as the postsynaptic density (PSD). This assembly is thought to allow rapid and efficient neurotransmission. A central organizer of the PSD is the PSD-95 family of membrane-associated guanylate kinase (MAGUK) proteins. The scaffolding function of PSD-95 and other MAGUK proteins is consistent with their modular structure: they comprise multiple protein-protein interaction domains including one or three PDZ domains, an SH3 domain and a region homologous to yeast guanylate kinase (GK). These domains participate in an array of interactions required for organization of the PSD. In addition, homologous MAGUK proteins play a similar role in organizing other cell-cell signaling junctions. Our long-term goal is to understand the assembly of the PSD at atomic resolution. Our strategy is to first focus on PSD-95, and to dissect the structural basis of its individual interactions. In earlier work, we characterized interactions of PDZ domains. Here we address functional interactions of the SH3 and GK domains. Genetic studies indicate that the SH3 and GK domains of MAGUKs are essential for proper cellular signaling. However, the molecular roles of these domains have remained elusive. Recent studies have revealed that the SH3 and GK domains form a strong intramolecular interaction, and that mutations that disrupt this interaction lead to dysregulation. Given that SH3 and SH2 modules in the Src family of kinases participate in regulatory intramolecular interactions, the intramolecular SH3/GK interaction in PSD-95 may play a similar role. We now propose a combined structural and functional analysis of this SH3/GK interaction. This work will clarify the important but poorly understood role of these domains in cell differentiation and synaptic organization. Our specific aims will be to determine the crystal structure of the SH3-GK complex and to analyze the mechanism and role of this interact/on in complex assembly both in vitro and in vivo.