title: Identification of new phosphorylation sites of AMPA receptors in the rat hippocampus--A resource for neuroscience research.
abstract: OBJECTIVE
  AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmissions in the mammalian brain. A series of phosphorylation sites have been predicted or identified and knowledge on phosphorylations is mandatory for understanding receptor biology and functions.
  
  
  METHODS
  Immunoprecipitation from extracted hippocampal rat proteins was carried out using an antibody against the AMPAR GluA1 subunit, followed by identification of GluA1 and binding partners by MS. Bands from SDS-PAGE were picked, peptides were generated by trypsin and chymotrypsin digestion and identified by MS/MS (LTQ Orbitrap Velos).
  
  
  RESULTS
  Using Mascot as a search engine, phosphorylation sites S506, S645, S720, S849, S863, S895, T858, Y228, Y419, and T734 were found on GluA1; S357, S513, S656, S727, T243, T420, T741, Y 143, Y301,Y426 on GluA2; S301, S516, S657, S732, T222, and T746 were observed on GluA3; and S514, S653 was phosphorylated on GluA4.
  
  
  CONCLUSIONS
  A series of additional protein modifications were observed and in particular, tyrosine and tryptophan nitrations on GluA1 were detected that may raise questions on additional regulation mechanisms for AMPARs in addition to phosphorylations. The findings are relevant for interpretation of previous work and design of future studies using AMPAR serving as a resource for neuroscience research and indeed, phosphorylations and PTMs per se would have to be respected when neuropathological and neurological disorders are being studied.
authors: Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
authors: Maryam
authors: M
authors: Ghafari
authors: Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
authors: Soheil
authors: S
authors: Keihan Falsafi
authors: Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Himberg, Austria.
authors: Harald
authors: H
authors: Höger
authors: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors: Keiryn L
authors: KL
authors: Bennett
authors: Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
authors: Gert
authors: G
authors: Lubec
mesh_terms: D000818:Animals; D006624:Hippocampus / Q000378:metabolism*; D047468:Immunoprecipitation; D008297:Male; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000378:metabolism*
chemical_list: D018091:Receptors, AMPA
keywords: AMPA receptor; Hippocampus; LTQ-orbitrap; Phosphorylation; Rat