title: Phosphorylation and assembly of glutamate receptors after brain ischemia.
abstract: OBJECTIVE
  Overassembly of synaptic glutamate receptors leads to excitotoxicity. The goal of this study is to investigate phosphorylation and assembly of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors after brain ischemia with reperfusion (I/R).
  
  
  METHODS
  Rats were subjected to 15 minutes of global ischemia followed by 0.5, 4, and 24 hours of reperfusion. Phosphotyrosine peptides of glutamate receptors in synaptosomal fraction after I/R were identified and quantified by state-of-the-art immuno-affinity purification of phosphotyrosine peptides followed by liquid chromatography/mass spectrometry/mass spectrometry analysis (immunoaffinity purification-coupled liquid chromatography/mass spectrometry/mass spectrometry). Glutamate receptor phosphorylation and synaptic assembly after I/R were studied by biochemical methods.
  
  
  RESULTS
  Numerous phosphotyrosine-sites of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate were upregulated by approximately 2- to 37-fold after I/R. A core glutamate receptor kinase, Src kinase, was significantly activated. GluR2/3 and NR2A/B were rapidly clustered from extrasynaptic to synaptic membrane fractions after I/R. GluR2/3 was then translocated into the intracellular pool, whereas NR2A/B remained in the synaptic fraction for as long as 24 hours. Consistently, trafficking-related phosphorylation of GluR2/3-S880 was significantly but transiently upregulated, whereas NR2A/B-Y1246 and NR2A/B-Y1472 were significantly and persistently upregulated after I/R.
  
  
  CONCLUSIONS
  Phosphorylation of glutamate receptors at synapses may lead to overassembly of glutamate receptors, probably via activation of Src family kinases, after I/R. This study provides global proteomic information about glutamate receptor tyrosine phosphorylation after brain ischemia.
authors.affiliation: University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Ailan
authors.initials: A
authors.lastname: Guo
authors.forename: Chunli
authors.initials: C
authors.lastname: Liu
authors.forename: Micheal
authors.initials: M
authors.lastname: Comb
authors.forename: Bingren
authors.initials: B
authors.lastname: Hu
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000737:chemistry / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*; D015424:Reperfusion / Q000379:methods; D013569:Synapses / Q000378:metabolism / Q000473:pathology
chemical_list: D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; C464437:NR3A NMDA receptor; C104723:glutamate receptor ionotropic, AMPA 3; C104722:glutamate receptor ionotropic, AMPA 2