title: Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B.
abstract: The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.
authors.affiliation: Department of Pediatrics, Medical University of Vienna , Währinger Gürtel 18; 1090 Vienna, Austria.
authors.forename: Maryam
authors.initials: M
authors.lastname: Ghafari
authors.forename: Harald
authors.initials: H
authors.lastname: Höger
authors.forename: Soheil
authors.initials: S
authors.lastname: Keihan Falsafi
authors.forename: Nina
authors.initials: N
authors.lastname: Russo-Schlaff
authors.forename: Arnold
authors.initials: A
authors.lastname: Pollak
authors.forename: Gert
authors.initials: G
authors.lastname: Lubec
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D011494:Protein Kinases; D016194:Receptors, N-Methyl-D-Aspartate; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor