Mass Spectrometrical Identification of Hippocampal NMDA Receptor

Article pubs.acs.org/jpr

Mass Spectrometrical Identification of Hippocampal NMDA Receptor Subunits NR1, NR2A−D and Five Novel Phosphorylation Sites on NR2A and NR2B Maryam Ghafari,† Harald Höger,‡ Soheil Keihan Falsafi,† Nina Russo-Schlaff,† Arnold Pollak,† and Gert Lubec*,† †

Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18; 1090 Vienna, Austria Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Brauhausgasse 34, A-2325 Himberg, Austria

‡

S Supporting Information *

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. KEYWORDS: NMDA receptor, NR1, NR2A, NR2B, NR2C, NR2D, immunoprecipitation, phosphorylation site, ion trap

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receptors.10,12 NMDA receptor subunits contain a long extracellular N-terminal domain, three true transmembrane segments, a re-entrant pore loop, and an intracellular Cterminal domain of variable length. The C-terminal domain is the most divergent region of the protein when comparing NMDA receptor subunits, consistent with playing a critical role in the diversity conferred on NMDA receptors by different subunit compositions. Whereas the N-terminal domain and extracellular loop form the ligand-binding pocket,13 the C-terminal tail regulates receptor interactions with a variety of cytosolic proteins. These protein−protein interactions dictate the precise intracellular trafficking and localization of NMDA receptors. In addition, different NMDA receptor subunits can couple receptors to distinct intracellular signaling complexes. For example, NR2B specifically interacts with the protein SynGAP, which is a Ras GTPase-activating protein demonstrated to selectively inhibit NMDA-stimulated ERK signaling.14 Also, NR2A and NR2B bind to active calcium/calmodulin-dependent protein kinase II (CaMKII) with different affinities,15 which

INTRODUCTION N-Methyl-D-aspartate (NMDA) receptors are a calcium permeant, depolarization-dependent subtype of ionotropic glutamate receptors that mediate a wide variety of physiological and pathological processes in the central nervous system, including development of proper neuronal circuits,1 certain learning tasks,2,3 forms of synaptic modification4−6 and excitotoxicity.7,8 Three families of genes (NR1, NR2 and NR3) have been identified that encode the NMDA receptor subunits.9 Functional NMDA receptors are tetramers composed of two essential NR1 subunits assembling with two NR2 subunits or, in some cases, an NR2 and an NR3 subunit.10 A unique feature of NMDA receptors is that receptor activation requires the binding of the coagonist glycine in addition to glutamate.11 Therefore, functional NMDA receptors require both an NR1 subunit, which contains the glycine binding site, and an NR2 subunit, which binds to glutamate. In addition to the formation of diheteromeric receptors (e.g., NR1/NR2B), there is compelling evidence for the existence of triheteromeric NMDA receptors (e.g., NR1/NR2A/NR2B). Many studies have demonstrated that NR2 and NR3 subunits confer distinct electrophysiological properties to the NMDA © 2012 American Chemical Society

Received: November 4, 2011 Published: February 15, 2012 1891

dx.doi.org/10.1021/pr201099u | J. Proteome Res. 2012, 11, 1891−1896

Journal of Proteome Research

Article

results in different forms of synaptic plasticity.16 Finally, the Ctermini of NMDA receptor subunits are substrates for posttranslational modifications such as phosphorylation. Phosphorylation regulates many cellular processes including protein activity, localization and mobility. In addition, phosphorylation is an important regulator of many protein−protein interactions. Direct phosphorylation of ionotropic glutamate receptors is a key mechanism regulating channel function and receptor localization at synapses. Many serine/threonine phosphorylation sites have been identified in NMDA receptor subunits, which are substrates for cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), protein kinase B (PKB), CaMKII, cyclin-dependent kinase-5 (Cdk5), and casein kinase II (CKII). These kinases can regulate intracellular trafficking or channel properties of NMDA receptors, resulting in changes in synaptic strength underlying many forms of synaptic plasticity.17 Given the importance of NMDA-R phosphorylations in the rat hippocampus for receptor function, it was the aim of the study to find additional phosphorylation sites using a gel-based proteomic approach.

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pieces were washed with 50 mM ammonium bicarbonate and then two times with washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) for 30 min each with vortexing. An aliquot of 100 μL of 100% acetonitrile was added to the tube to cover the gel pieces completely and the mixture was incubated for 10 min. Gel pieces were dried completely using a SpeedVac concentrator. Reduction of cysteine residues was carried out with a 10 mM dithiothreitol (DTT) solution in 100 mM ammonium bicarbonate pH 8.6 for 60 min at 56 °C. After discarding the DTT solution, the same volume of a 55 mM iodoacetamide (IAA) solution in 100 mM ammonium bicarbonate buffer pH 8.6 was added and incubated in darkness for 45 min at 25 °C to achieve alkylation of cysteine residues. The IAA solution was replaced by washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) and washed twice for 15 min each with vortexing. Gel pieces were washed and dried in 100% acetonitrile followed by dryness in SpeedVac. Dried gel pieces were reswollen with 12.5 ng/μL trypsin (Promega, Germany) solution reconstituted with 25 mM ammonium bicarbonate or 12.5 ng/μL chymotrypsin (Roche Diagnostics, Mannheim, Germany) solution buffered in 25 mM ammonium bicarbonate. Gel pieces were incubated for 16 h (overnight) at 37 (trypsin) or 25 °C (chymotrypsin). The supernatant was transferred to new 0.5 mL tubes, and peptides were extracted with 50 μL of 0.5% formic acid/20% acetonitrile for 20 min in a sonication bath. This step was repeated two times. Samples in extraction buffer were pooled in 0.5 mL tubes and evaporated in a SpeedVac concentrator. The volume was reduced to approximately 20 μL and then 20 μL HPLC grade water (Sigma, St. Louis, MO) was added for nano-LC−ESI− (CID/ETD)-MS/MS analysis via high capacity ion trap (HCT; Bruker, Bremen, Germany). The extracted peptides were subsequently analyzed by nano-LC−ESI−(CID/ETD)-MS/ MS.20

EXPERIMENTAL SECTION

Immunoprecipitation of NR1 from Rat Hippocampi

Total hippocampal membrane fractions,18,19 from hippocampi of Sprague−Dawley rats (male, 12 weeks of age, Division of Laboratory Animal Science and Genetics, Medical University of Vienna) were 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 Na3VO4 and protease inhibitor cocktail (Roche, Mannheim, Germany) on a rotation shaker for 1 h at 4 °C. After centrifugation at 15300× g, at 4 °C for 10 min, the supernatant was incubated with affinity purified goat antibody against NR1 (glutamate (NMDA) receptor zeta 1; Santa Cruz, Santa Cruz, CA) and subsequently incubated with protein G agarose beads (GE Healthcare, Uppsala, Sweden) for 4 h at 4 °C with gentle rotation. After five times of washing with the same lysis buffer, proteins bound were denatured with sample buffer containing 125 mM Tris (pH 6.8), 4% SDS, 20% Glycerol, 10% Beta-mercaptoethanol, 0.02% Bromophenol blue at 95 °C for 3 min.

Phophatase Treatment

NR2A and NR2B spots were cut, destained and dried as shown above. The dried spots were incubated in a solution of 0.5 μL of calf intestine alkaline phosphatase (New England Biolabs, Ipswich, MA) in the presence of 100 mM ammonium bicarbonate for 1 h at 37 °C. The spots were then washed using 100 mM ammonium bicarbonate, shrunk in ACN and dried in a SpeedVac.21 Samples were then processed as given above for mass spectrometical analysis.

Western Blots

Samples were loaded onto 10% SDS-polyacrylamide gels, electrophoresed, and subsequently transferred to PVDF membranes (Pall, Ann Harbor, MI). After blocking of membranes for 1 h with 5% nonfat dry milk in 0.1% TBST (100 mM Tris-HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), membranes were incubated with diluted goat primary antibodies against NR1 (glutamate receptor zeta 1 antibody; 1:5000, Santa Cruz, Santa Cruz, CA), NR2A (rabbit-NMDAR2A antibody; 1:10 000, Abcam, Cambridge, U.K.), NR2B (rabbitNMDAR2B antibody, 1:500, Abcam, Cambridge, U.K.) and detected with horseradish peroxidase-conjugated antirabbit IgG (1:10000, Abcam, Cambridge, U.K.) or antigoat IgG (1:5,000, Abcam, Cambridge, U.K.). Membranes were developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Uppsala, Sweden).

Nano-LC−ESI−CID/ETD-MS/MS

The HPLC used was an Ultimate 3000 system (Dionex, Sunnyvale, CA) equipped with a PepMap100 C-18 trap column (300 μm × 5 mm) and PepMap100 C-18 analytic column (75 μm × 150 mm). The gradient was (A = 0.1% FA in water, B = 0.08% FA in ACN) 4−30% B from 0 to 105 min, 80% B from 105 to 110 min, 4% B from 110 to 125 min. An HCT ultra ETD II (Bruker Daltonics, Bremen, Germany) was used to record peptide spectra over the mass range of m/z 350−1500, and MS/MS spectra in information-dependent data acquisition over the mass range of m/z 100−2800. Repeatedly, MS spectra were recorded followed by four data-dependent CID MS/MS spectra and four ETD MS/MS spectra generated from four highest intensity precursor ions. An active exclusion of 0.4 min after two spectra was used to detect low abundant peptides. The voltage between ion spray tip and spray shield was set to 1500 V. Drying nitrogen gas was heated to 150 °C and the flow rate was 10 L/min. The collision energy was set automatically according to the mass and charge state of the peptides chosen

In-gel Digestion of Proteins and Peptides

Spots picked from the SDS gel (bands 1 and 2) that were recognized by the corresponding antibodies against the NR1, NR2A and NR2B subunits were put into a 1.5 mL tube. Gel 1892



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Figure 1. (a) Pattern of the immunoprecipitation (IP) with Coomassie-staining on an SDS-PAGE. Bands corresponding to NR1, NR2A and NR2B, PKC gamma and calcium/calmodulin-dependent protein kinase 2 beta as shown by mass spectrometry. (b) Immunoblotting results on the IP SDSPAGE: Lane 1 of NR1 immunoreactivity shows the presence of the light and heavy chain of the immunoglobulin and the eluted NR1 subunit between 100 and 150 kDa. Lane 2 represents the unbound supernatant of the IP and lane 3 represents the starting solution used for the IP. Clear enrichment of NR1 is shown in lane 1. Analogy results from IP for (c) NR2A and (d) NR2B.

Table 1. Receptor Accession Numbers, Names, Mascot Protein Score and Sequence Coverages by Trypsin and Chymotrypsin, Expected Molecular Weight, Number of Total Amino Acids and Total Sequence Coverages accession number P35439 NR1 NMDZ1 Q00959 NR2A NMDE1 Q00960 NR2B NMDE2 Q00961 NR2C NMDE3 Q62645 NR2D NMDE4

enzyme

Mascot protein sore

sequence coverage, %

molecular weight (expected), kDa

total amino acids

total sequence coverage, %

Trypsin Chymotrypsin Trypsin Chymotrypsin Trypsin Chmotrypsin Chymotrypsin

957 845 1438 509 1461 927 51

51.59 54.26 41.59 43.54 47.36 45.54 24.89

105

938

78.14

165

1464

69.36

166

1482

71.05

135

1237

24.89

Chymotrypsin

62

22.37

143

1323

22.37

0.2 Da for peptide tolerance, 0.2 Da for fragment mass tolerance, modification 1 of carbamidomethyl(C) and modification 2 of methionine oxidation. Searches for unknown mass shifts, for amino acid substitution and calculation of significance were selected on advanced PTM-explorer search strategies. A list of 172 common modifications including phosphorylation and hydroxylation was selected and added to virtually cleaved and fragmented peptides searched against experimentally obtained MS/MS spectra. Positive protein identification was first of all listed by spectra view and subsequently each identified peptide was considered significant based on the ioncharge status of peptide, b- and y-ion fragmentation quality, ion score (>200) and significance scores (>80). Protein identification and modification information returned were manually inspected and filtered to obtain confirmed protein identification and modification lists.21

for fragmentation. Multiple charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted and peak lists were generated by DataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany). MASCOT searches were done by using the MASCOT 2.2.06 (Matrix Science, London, U.K.) against latest UniProtKB database for protein identification. Searching parameters were set as follows: enzyme selected as trypsin or chymotrypsin with three maximum missing cleavage sites, species taxonomy: limited to Rat, a mass tolerance of 0.2 Da for peptide tolerance, 0.2 Da for MS/MS tolerance, ions score cutoff lower than 15, fixed modification of carbamidomethyl (C) and variable modification of oxidation (M), deamidation (N, Q), and phosphorylation (S, T, Y). Positive protein identifications were based on a significant MOWSE score. After protein identification, an error-tolerant search was done to detect unspecific cleavage and unassigned modifications. Protein identification and modification information returned were manually inspected and filtered to obtain confirmed protein identification and modification lists The values lower than 0.1 (or higher than score 27) were selected for high significance.19,21 PTM searches were done using Modiro software with following parameters: enzyme selected as used with three maximum missing cleavage sites, a peptide mass tolerance of

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RESULTS AND DISCUSSION NR1 and NR2A-D subunits were observed on immunoprecipitation (Figure 1) using an antibody against NR1 in a complex consisting of at least NR1, NR2A, NR2B. Western blotting using the antibodies against NR1, NR2A and NR2B showed the presence of the three subunits in the IP. NR1 and NR2A-D were identified by mass spectrometry and this finding significantly extends protein sequence information in the rat 1893



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available by mass spectrometrical techniques so far.22 Moreover, two protein kinases, PKC gamma and Calcium/ calmodulin-dependent protein kinase type 2 subunit beta were coprecipitating and identified by mass spectrometry. Table 1 shows protein names and accession numbers from UniProtKB, the enzymes used, sequence coverages, expected molecular weight, number of amino acids and the total sequence coverage from combining trypsin and chymotrypsin data. Data on peptide identification, scores, mass errors (delta), sequence conflicts and the list of peptides used for identification are given in the supplemental Table 1 (a-z1). The major finding of the current study is represented by the detection of five novel phosphorylation sites on serines S511 from NR2A and S886, S917, S1303 and S1323 from NR2B. Representative spectra are shown in Supplemental Figure 1, Supporting Information, the list of serine phosphorylations that were validated by phosphatase treatment, published in refs 8 and 23−25 or predicted is given in Table 2. The probable

While no mass spectrometrical data exist on these five phosphorylation sites, four of them have been predicted by analogy (http://www.phosphosite.org/). As shown in Table 3, we observed a series of phosphorylation sites, but only five could be verified by phosphatase treatment and are herein reported as confirmed results. There is, however, experimental evidence for S1303 phosphorylation on synthetic NMDA-R peptides in the rat as shown by Liao et al. and Omkumar et al. have shown by mass spectrometry that a recombinant C-terminal peptide from NR2B containing S1303 was phosphorylated by CaMKII; subsequently, they compared this peptide with NR2B on HPLC.8,23 It is well-documented that NMDA receptor subunits can be regulated by phosphorylation on serine residues. PKA and PKC can directly phosphorylate serine residues on NR1, NR2A and NR2B subunits.26,27 NR2A and NR2B-containing receptors are regulated by PKC: Sequence alignment of NR2A and NR2B shows that S1303 and S1323 of NR2B are analogous to the PKC substrates, S1291 and S1312 on NR2A. And indeed, PKC gamma and Calcium/calmodulin-dependent protein kinase type 2 subunit beta were coprecipitating using an antibody against NR1, that also coprecipitated the other subunits (Figure 1). Identification data for the protein kinases are shown in Supplemental Table 2 (Supporting Information). Studies in oocytes show that phosphorylation of S1303 and S1323 is required for PKC potentiation of NR1/NR2B receptor currents.8 Calcium-influx-dependent phosphorylation of recombinant rat NR2B on S1303 by CaMKII inhibits receptor-kinase interactions and promotes slow dissociation of preformed CaMKII-NR2B complexes.24 Studies in neostriatum showed that enhanced dopamine D2 receptor-NR2B interaction disrupts the association of Ca2+/calmodulin-dependent protein kinase II with NR2B during cocaine treatment, reduces S1303 phosphorylation thus mediating NMDA receptormediated currents in striatal neurons.25 It appears that CaMKII phosphorylation of S1303 regulates NMDA receptors in a different way from PKC phosphorylation of the same site. Although there is no doubt that phosphorylation of NR2B at

Table 2. Kinases Corresponding to the Phosphorylation Sites in NR2A and NR2B subunit

phosphorylation site

NR2A

S511

NR2B NR2B NR2B NR2B

S886 S917 S1303 S1323

corresponding kinasesa AKT1, ATM, PKG, RSK, CDK, STK4, CHK1, PDK, DNAPK Cdk5 P38MAPK, Cdk5 CaMKII, PKC, RSK, PKA PKC

a

ATM, Ataxia telangiectasia mutated; CaMKII, Ca2+/calmodulin dependent kinases; CDK, Cyclin-dependent kinase; Cdk5, Cyclindependent kinase 5; CHK1, Serine/Threonine-protein kinase CHK1; DNA-PK, DNA-dependent protein kinase; p38 MAPK, p38mitogenaktivierte Proteinkinasen; PDK, phosphoinositide-dependent kinase; PKA, cAMP-dependent protein kinase; PKC, Protein Kinase C; PKG, cGMP-dependent protein kinase; RSK, Ribosomal S6 kinase; STK4, Serine/Threonine kinase 4.

corresponding protein kinases for these phosphorylation sites are suggested from two databases (http://www.cbs.dtu.dk/ services/NetPhosK and http://kinasephos2.mbc.nctu.edu.tw).

Table 3. Characterization of Phosphopeptides as Handled by Mascot or Modiro Softwares Verified Means Abolishment of the Shift Following Phosphatase Treatment subunit

enzyme

position

observed

Mr (expt)

NR2A NR2A NR2A NR2A NR2B NR2B NR2B NR2B

Trypsin Trypsin Trypsin Trypsin Trypsin Trypsin Trypsin Chymotrypsin

S511 S882 S1291 S1459 S886 S917 S1477 S886

785.4200 664.3415 774.9300 543.3300 776.7300 1086.6064 929.4000 844.0300

1568.8254 1990.0027 1547.6770 1084.6454 2327.1682 2171.1982 928.3927 2529.0682

NR2B NR2B NR2B NR2A NR2A NR2A NR2A

Trypsin Trypsin Chymotrypsin Trypsin Trypsin Trypsin Trypsin

S886 S1303 S1323 S511 S882 S1291 S1459

776.73 639.32 636.08 785.48 664.34 522.64 543.33

776.6751 639.3076 635.9942 785.3708 664.315 522.5751 543.2329

Z

sequence

Mascot 2 R.AVMAVGSLTINEER.S 3 K.KKSPDFNLTGSQSNMLK.L 2 R.QHSYDNILDKPR.E 2 K.KMPSIESDV 3 R.QSVMNSPTATMNNTHSNILR.L 2 K.NMANLSGVNGSPQSALDFIR.R 1 K.LSSIESDV. 3 Y.SCIHGVAIEERQSVMNSPTATM.N Modiro 3 R.QSVMOxNSHPO3PTATMOxNNTHSNILR.L 4 R.RQHSHPO3YDTFVDLQKEEAALAPR.S 3 F.VDLQKEEAALAPRSVSHPO3L.K 2 R.AVMAVGS HPO3LTINEER.S 3 K.KKSHPO3PDFNLTGSQSNMOxLK.L 3 R.QHSHPO3YDNILDKPR.E 2 K.KMPSHPO3IESDV.− 1894

ion score/sign

verified

48 37 54 34 43 67 27 46

+ − − − + + − −

349/99 386/83.2 231/99 309/100 278/99.3 341/80.3 237/99.9

+ + + + − − −



Article

Notes

S1303 can take place, the physiologically relevant kinase could be PKC or CaMKII or both. NR2B is also phosphorylated by casein kinase II on S1480 within the PDZ domain binding site at the extreme C-terminus, and phosphorylation of S1480 disrupts the interaction between NR2B and PSD-95.28 PKC can potentiate NR2A-containing receptor currents via phosphorylation of NR2A on S1291 and S1312. 29,30 S1416, another PKC target in NR2A, is phosphorylated by PKC in vitro.31 Phosphorylation of S1416 decreases the binding affinity of CaMKII for NR2A, providing a molecular mechanism for a direct cross talk between CaMKII and PKC signaling pathways. In addition to PKC, Cyclindependent kinase 5 also phosphorylates NR2A,32 which enhances NMDA receptor activity, and inhibition of this phosphorylation protects CA1 pyramidal neurons from ischemic insults.33 By measuring NMDA receptor currents from NR1/NR2A expressed in HEK-293 cells, S900 and S929 were identified as putative phosphorylation sites based on alanine scanning mutagenesis analysis, but the relevant kinase(s) remains to be identified. Using NetPhosK PKC and cdc2 could be predicted as protein kinases phosphorylating S900 and PKC, PKA and cdc2 could be predicted to phosphorylate S929. Dephosphorylation of S900 and S929 by protein phosphatase IIb (calcineurin) modulates desensitization of NR1/NR2A-containing NMDA receptors.34 In addition, other PTMs were proposed as shown in the Supplemental Table 1 that were not verified by other methods (Supporting Information). Amino Acid substitutions/sequence conflicts were observed as shown in Supplemental Table 3 (Supporting Information). Several sequence conflicts have been reported so far (http:// www.uniprot.org), and we are adding a series of new amino acid substitutions. One of them, a sequence conflict 1430VT to SA of NR2B was reported before.35

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the contribution by the Verein zur Durchführung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin “Unser Kind”.

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CONCLUSION Taken together, we detected five novel serine phosphorylation sites in rat hippocampus that may be involved in a series of NMDA functions in analogy to those of other serine phosphorylations reported so far. These sites were predicted but not experimentally shown to occur in rat hippocampus and the corresponding protein kinases that could have been phosphorylating these sites were already reported partially forming the basis for the predictions of the phosphosites determined. Herein, the protein chemical basis for functional studies on the biological relevance of serine phosphorylations is provided.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Tables 1−3 and Supplementary Figure 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

(1) Constantine-Paton, M.; Cline, H. T.; Debski, E. Patterned activity, synaptic convergence, and the NMDA receptor in developing visual pathways. Annu. Rev. Neurosci. 1990, 13, 129−154. (2) Morris, R. G.; Anderson, E.; Lynch, G. S.; Baudry, M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 1986, 319, 774−776. (3) Sakimura, K.; Kutsuwada, T.; Ito, I.; Manabe, T.; Takayama, C.; Kushiya, E.; Yagi, T.; Aizawa, S.; Inoue, Y.; Sugiyama, H.; et al. Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 1995, 373, 151−155. (4) Collingridge, G. L.; Bliss, T. V. Memories of NMDA receptors and LTP. Trends Neurosci. 1995, 18, 54−56. (5) Tsien, J. Z.; Huerta, P. T.; Tonegawa, S. The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell 1996, 87, 1327−1338. (6) Ito, I.; Futai, K.; Katagiri, H.; Watanabe, M.; Sakimura, K.; Mishina, M.; Sugiyama, H. Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit. J. Physiol. 1997, 500 (Pt 2), 401−408. (7) Choi, D. W. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1988, 1, 623−634. (8) Liao, G. Y.; Wagner, D. A.; Hsu, M. H.; Leonard, J. P. Evidence for direct protein kinase-C mediated modulation of N-methyl-Daspartate receptor current. Mol. Pharmacol. 2001, 59, 960−964. (9) Cull-Candy, S.; Brickley, S.; Farrant, M. NMDA receptor subunits: diversity, development and disease. Curr. Opin. Neurobiol. 2001, 11, 327−335. (10) Cull-Candy, S. G.; Leszkiewicz, D. N. Role of distinct NMDA receptor subtypes at central synapses. Sci. STKE 2004, 2004, re16. (11) Erreger, K.; Chen, P. E.; Wyllie, D. J.; Traynelis, S. F. Glutamate receptor gating. Crit. Rev. Neurobiol. 2004, 16, 187−224. (12) Chen, B. S.; Roche, K. W. Regulation of NMDA receptors by phosphorylation. Neuropharmacology 2007, 53, 362−368. (13) Furukawa, H.; Gouaux, E. Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligandbinding core. Embo J. 2003, 22, 2873−2885. (14) Kim, M. J.; Dunah, A. W.; Wang, Y. T.; Sheng, M. Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking. Neuron 2005, 46, 745−760. (15) Strack, S.; Colbran, R. J. Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. J. Biol. Chem. 1998, 273, 20689−20692. (16) Barria, A.; Malinow, R. NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron 2005, 48, 289−301. (17) Lee, H. K. Synaptic plasticity and phosphorylation. Pharmacol. Ther. 2006, 112, 810−832. (18) Ghafari, M.; Falsafi, S. K.; Hoeger, H.; Lubec, G. Hippocampal levels of GluR1 and GluR2 complexes are modulated by training in the multiple T-Maze in C57BL/6J mice. Brain Struct. Funct. 2011, DOI: 10.1007/s00429-00011-00335-00428. (19) Kang, S. U.; Heo, S.; Lubec, G. Mass spectrometric analysis of GABAA receptor subtypes and phosphorylations from mouse hippocampus. Proteomics 2011, 11, 2171−2181. (20) Kang, S. U.; Fuchs, K.; Sieghart, W.; Lubec, G. Gel-based mass spectrometric analysis of recombinant GABA(A) receptor subunits

AUTHOR INFORMATION

Corresponding Author

*Univ. Prof. Dr. Gert Lubec, Dept. of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria. Tel: +43-1-40400-3215. Fax: +43-1-40400-6065. Email: [email protected]. 1895



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representing strongly hydrophobic transmembrane proteins. J. Proteome Res. 2008, 7, 3498−3506. (21) Chen, W. Q.; Priewalder, H.; John, J. P.; Lubec, G. Silk cocoon of Bombyx mori: proteins and posttranslational modifications--heavy phosphorylation and evidence for lysine-mediated cross links. Proteomics 2010, 10, 369−379. (22) Reyes-Montano, E. A.; Lareo, L. R.; Chow, D. C.; Perez-Gomez, G. Immunolocalization and biochemical characterization of N-methylD-aspartate receptor subunit NR1 from rat brain. Protein J. 2006, 25, 95−108. (23) Omkumar, R. V.; Kiely, M. J.; Rosenstein, A. J.; Min, K. T.; Kennedy, M. B. Identification of a phosphorylation site for calcium/ calmodulindependent protein kinase II in the NR2B subunit of the Nmethyl-D-aspartate receptor. J. Biol. Chem. 1996, 271, 31670−31678. (24) Strack, S.; McNeill, R. B.; Colbran, R. J. Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. J. Biol. Chem. 2000, 275, 23798−23806. (25) Liu, X. Y.; Chu, X. P.; Mao, L. M.; Wang, M.; Lan, H. X.; Li, M. H.; Zhang, G. C.; Parelkar, N. K.; Fibuch, E. E.; Haines, M.; Neve, K. A.; Liu, F.; Xiong, Z. G.; Wang, J. Q. Modulation of D2R-NR2B interactions in response to cocaine. Neuron 2006, 52, 897−909. (26) Leonard, A. S.; Hell, J. W. Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites. J. Biol. Chem. 1997, 272, 12107−12115. (27) Tingley, W. G.; Ehlers, M. D.; Kameyama, K.; Doherty, C.; Ptak, J. B.; Riley, C. T.; Huganir, R. L. Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J. Biol. Chem. 1997, 272, 5157−5166. (28) Chung, H. J.; Huang, Y. H.; Lau, L. F.; Huganir, R. L. Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J. Neurosci. 2004, 24, 10248−10259. (29) Grant, E. R.; Guttmann, R. P.; Seifert, K. M.; Lynch, D. R. A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters. Neurosci. Lett. 2001, 310, 9−12. (30) Jones, M. L.; Leonard, J. P. PKC site mutations reveal differential modulation by insulin of NMDA receptors containing NR2A or NR2B subunits. J. Neurochem. 2005, 92, 1431−1438. (31) Gardoni, F.; Bellone, C.; Cattabeni, F.; Di Luca, M. Protein kinase C activation modulates alpha-calmodulin kinase II binding to NR2A subunit of N-methyl-D-aspartate receptor complex. J. Biol. Chem. 2001, 276, 7609−7613. (32) Li, B. S.; Sun, M. K.; Zhang, L.; Takahashi, S.; Ma, W.; Vinade, L.; Kulkarni, A. B.; Brady, R. O.; Pant, H. C. Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12742−12747. (33) Wang, J.; Liu, S.; Fu, Y.; Wang, J. H.; Lu, Y. Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors. Nat. Neurosci. 2003, 6, 1039−1047. (34) Krupp, J. J.; Vissel, B.; Thomas, C. G.; Heinemann, S. F.; Westbrook, G. L. Calcineurin acts via the C-terminus of NR2A to modulate desensitization of NMDA receptors. Neuropharmacology 2002, 42, 593−602. (35) Sullivan, J. M.; Traynelis, S. F.; Chen, H. S.; Escobar, W.; Heinemann, S. F.; Lipton, S. A. Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron 1994, 13, 929−936.

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Mass Spectrometrical Identification of Hippocampal NMDA Receptor

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