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.

Proteomic Analysis of Postsynaptic Protein Complexes Underlying Neuronal Plasticity

“…Interestingly, this type of approach uncovered multiple novel phosphorylation sites on GluN2A and GluN2B. 55 Moreover, coupling proteomics approaches to biochemical preparations that isolate different subcellular fractions can allow for tracking of specific phosphorylation sites in different parts of the neuron. This type of approach can also be used to determine how different pathological or developmental conditions impact phosphorylation at multiple sites at one time.…”

Roles of subunit phosphorylation in regulating glutamate receptor function

“…Multiple tyrosine sites within the GluN2A CT (842, 1292, 1325, and 1387) and GluN2B CT (1252, 1336, and 1472) are phosphorylated by Src and/or Fyn NRTKs (Nakazawa et al, 2001; Vissel et al, 2001; Yang and Leonard, 2001; Taniguchi et al, 2009). Other phosphorylation sites may exist according to a recent study, although their functional relevance has not been determined (Ghafari et al, 2012). …”

Reduced cortical neurotransmitter receptor complex levels in fetal Down syndrome brain

“…Total membrane fractions (Ghafari et al 2012b) were obtained from cortical tissue; suspended in lysis buffer containing 1 % Triton X100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0), 10 mM NaF, 10 mM Na 3 VO4 (Sigma-Aldrich)and a protease inhibitor cocktail (Roche, Mannheim, Germany); and placed on a rotation shaker for 1 h at 4 °C. Total membrane fractions (Ghafari et al 2012b) were obtained from cortical tissue; suspended in lysis buffer containing 1 % Triton X100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0), 10 mM NaF, 10 mM Na 3 VO4 (Sigma-Aldrich)and a protease inhibitor cocktail (Roche, Mannheim, Germany); and placed on a rotation shaker for 1 h at 4 °C.…”

“…The volume was reduced to approximately 20 µL, to which 20 µL of HPLCgrade water (Sigma, Germany) was added (Ghafari et al 2012b). The gel pieces were first washed with 50 mM ammonium bicarbonate and then washed twice with washing buffer (50 % 100 mM ammonium bicarbonate/50 % acetonitrile) for 30 min each (with vortexing).…”