Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain.
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.
Department of Neuroscience, University of Edinburgh, Edinburgh, UK.
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Grant
D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D000076122:Disks Large Homolog 4 Protein; D051528:Guanylate Kinases; D007163:Immunosorbent Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051379:Mice; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D016548:Prosencephalon / Q000737:chemistry*; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; C121001:NR2B NMDA receptor; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D016194:Receptors, N-Methyl-D-Aspartate; C033652:postsynaptic density proteins; D051528:Guanylate Kinases