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Free keywords:
Amino Acid Sequence
Animals
COS Cells
Cercopithecus aethiops
Flow Cytometry/methods
Fluorescence
Fluorescent Dyes/analysis/metabolism
Guanylate Kinase/chemistry/*metabolism
Ligands
Microscopy, Confocal/methods
Models, Molecular
*PDZ Domains
Protein Binding
Protein Multimerization
Synapses/*metabolism
Abstract:
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.
