Synaptic MAGUK Multimer Formation Is Mediated by PDZ Domains and Promoted by Ligand Binding

To examine the scaffolding properties of PSD-95, we have taken advantage of established ligand/PDZ domain interactions and developed a cell-based assay for investigating protein complex formation.. This assay enables quantitative analysis of PDZ domain-mediated protein clustering using bimolecular fluorescence complementation (BiFC). Two nonfluorescent halves of EYFP were fused to C-terminal PDZ ligand sequences to generate probes that sense for PDZ domain binding grooves of adjacent (interacting) molecules. When these probes are brought into proximity by the PDZ domains of a multiprotein scaffold, a functional fluorescent EYFP molecule can be detected. We have used this system to examine the properties of selected PSD-95 variants and thereby delineated regions of importance for PSD-95 complex formation. Further analysis led to the finding that PSD-95 multimerization is PDZ domain-mediated and promoted by ligand binding.