Synaptic MAGUK multimer formation is mediated by PDZ domains and promoted by ligand binding

Chem Biol. 2013 Aug 22;20(8):1044-54. doi: 10.1016/j.chembiol.2013.06.016.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Flow Cytometry / methods
  • Fluorescence
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / metabolism
  • Guanylate Kinases / chemistry
  • Guanylate Kinases / metabolism*
  • Ligands
  • Microscopy, Confocal / methods
  • Models, Molecular
  • PDZ Domains*
  • Protein Binding
  • Protein Multimerization
  • Synapses / metabolism*

Substances

  • Fluorescent Dyes
  • Ligands
  • Guanylate Kinases