Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain

Holger Husi, Seth G.N. Grant

Research output: Contribution to journalArticle

92 Citations (Scopus)

Abstract

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.
Original languageEnglish
Pages (from-to)281-291
Number of pages11
JournalJournal of Neurochemistry
Volume77
Issue number1
DOIs
Publication statusPublished - 1 Apr 2001

    Fingerprint

Cite this