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| Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain |
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| Author(s): Husi H, Grant SGN |
| Source: JOURNAL OF NEUROCHEMISTRY Volume: 77 Issue: 1 Pages: 281-291 Published: APR 2001 |
| Times Cited: 47 References: 33 |
| 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 e[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. |
| Document Type: Article |
| Language: English |
| Reprint Address: Grant, SGN (reprint author), Univ Edinburgh, Dept Neurosci, Edinburgh, Midlothian Scotland |
Addresses:
1. Univ Edinburgh, Dept Neurosci, Edinburgh, Midlothian Scotland |
| Publisher: BLACKWELL SCIENCE LTD, P O BOX 88, OSNEY MEAD, OXFORD OX2 0NE, OXON, ENGLAND |
| Subject Category: Biochemistry & Molecular Biology; Neurosciences |
| IDS Number: 417KY |
| ISSN: 0022-3042 |
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