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Keywords:

  • excitatory synapse;
  • liquid chromatography;
  • mass spectrometry;
  • postsynaptic density;
  • protein composition;
  • proteomics

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell–cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.

Abbreviations used
AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate

BIR

baculovirus inhibitor of apoptosis repeat

CaM

kinase II, Ca2+/calmodulin-dependent protein kinase II

CRMP

collapsin response mediator protein

GABA

γ-aminobutyric acid

GAP

GTPase-activating protein

GEF

GTP-GDP exchange factor

GFAP

glial fibrillary acidic protein

GPI

glycosyl phosphatidylinositol

LC

liquid chromatography

LTP

long-term potentiation

MAP

1, microtubule-associated protein 1

MAP kinase

mitogen-activated protein kinase

MS

mass spectrometry

NMDA

N-methyl-D-aspartic acid

NRC

NMDA-receptor multiprotein complexes

PAGE

polyacrylamide gel electrophoresis

PI

phosphatidyl inositol

PKA

cAMP-dependent protein kinase

PLC

phospholipase C

PP

protein phosphatase

PSD

postsynaptic density

SDS

sodium dodecyl sulphate

TGF

transforming growth factor

VDAC

voltage-dependent anion channel protein

The postsynaptic density (PSD) is a tiny, amorphous structure located beneath the postsynaptic membrane of synapses in the central nervous system (CNS) and plays an important role in synaptic plasticity (for reviews see Kennedy 1993; Kennedy 1997; Ziff 1997; Kennedy 1998; Kennedy 2000; Luscher et al. 2000; Sheng 2001; Sheng and Sala 2001; Yamauchi 2002). The most prominent PSDs are Type I PSDs associated with excitatory glutamatergic synapses. The PSD contains specific receptors for the neurotransmitter glutamate and many others, as well as numerous receptor-associated proteins and scaffold proteins that organize signal transduction pathways.

Within the PSD may lie clues to the mechanisms of the most intriguing of brain functions, including activity-dependent changes in synaptic strengthening and higher functions such as learning and memory. The total number of proteins in the PSD may be as high as several hundred, if one includes those proteins that are only weakly accumulated in, or associated with, the PSD. Many attempts have been made to identify and characterize the molecular constituents of the PSD (Kelly and Cotman 1978; Walsh and Kuruc 1992; Langnaese et al. 1996; Kennedy 1997; Ziff 1997; Kennedy 1998; Walikonis et al. 2000; Yoshimura et al. 2000). Protein sequencing and immunoblotting identified several proteins in the solubilized Triton-extracted PSD fraction. Some synaptic-signalling ‘machines’ were also identified, in particular, molecules such as glutamate receptors and associated proteins. A change of PSD components was demonstrated after cerebral ischemia (Hu et al. 1998) and kainate-induced seizures (Wyneken et al. 2001). However, the entire composition of the PSD has not fully been elucidated.

NMDA-receptor multiprotein complexes (NRC), isolated from mouse brain, have been analysed by immunoblotting and mass spectrometry (Husi et al. 2000). This study revealed that NRC comprised 77 proteins organized into receptor, adaptor, signalling, cytoskeletal, and novel types, of which 19 participate in NMDA receptor-mediated cell signalling, and several proteins encoded by an activity-dependent gene (Husi et al. 2000). Previous study also showed that one of the major PSD constituents is the α isoform of CaM kinase II (Kennedy et al. 1983; Goldenring et al. 1984; Kelly et al. 1984), which plays important roles in synaptic plasticity. CaM kinase II translocates from cytosol to the PSD by autophosphorylation, binds to the NMDR-receptor (Strack and Colbran 1998; Yoshimura et al. 1999), and phosphorylates many proteins in the PSD fraction as revealed by proteomic analysis (Yoshimura et al. 2000; Yoshimura et al. 2002).

In the present study, we performed a comprehensive analysis of the protein constituents of the PSD fraction using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale 2DLC with high-resolution hybrid MS and an automated data analysis system (Mawuenyega et al. 2003). We identified 492 proteins, including numerous known and unknown constituents of the PSD, as well as many ‘hypothetical’ proteins that had not been characterized. This study will thereby allow an overview of the protein composition of the PSD.

Purification of PSD

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

The PSD was purified from rat forebrain as described previously (Yoshimura and Yamauchi 1997). Forebrain (about 15 g) was dissected and immediately homogenized in 0.32 m sucrose. The homogenate was centrifuged to remove the nuclear fraction, and the postnuclear fraction was centrifuged to isolate the crude mitochondrial fraction. The mitochondrial fraction was then fractionated by centrifugation with a density gradient from 1.0 to 1.4 m sucrose to separate the synaptosomal fraction from the myelin and mitochondrial fractions. The synaptosomal fraction was treated with 0.5% Triton X-100, and the Triton X-100 insoluble fraction was obtained by centrifugation. Purified PSD (about 6 mg) was finally obtained by centrifugation with a density gradient from 1.5 to 2.1 m sucrose.

Two-dimensional gel electrophoresis and identification of PSD protein

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

Two-dimensional gel electrophoresis was performed as described previously (Hirabayashi 1981; Yamauchi et al. 1995). The first dimensional isoelectric focusing was performed in a 1% agarose gel (2.6 × 180 mm) with a pH 3–10 gradient at 500 V for 4 h and then at 700 V for 15 h at 4°C, and the second dimensional SDS gel electrophoresis was performed with 5–15% acrylamide gradient gels in a standard slab-gel (20 × 13 cm) at 30 mA for 3 h and then 70 mA for 2 h at room temparature. Gels were stained with Coomassie blue.

Some of the major Coomassie-stained protein spots were excised from the 2D-gel, in gel digested with trypsin and subjected to protein sequencing analysis or LC-MS/MS analysis as described previously (Yoshimura et al. 2000; Yoshimura et al. 2002).

Protein identification by automated 2DLC-tandem mass spectrometry and data analysis

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

The purified PSD (6 mg proteins) was dissolved in 1 mL of 7.0 m guanidine-HCl buffered with 500 mm Tris-HCl (pH 8.0) containing 10 mm EDTA. The preparation was reduced by the addition of 1 mm dithiothreitol (DTT) and was S-carbamoylmethylated with 10 mm iodoacetamide under a nitrogen atmosphere as described previously (Mawuenyega et al. 2003). The S-carbamoylmethylated proteins were dialysed against 10 mm Tris-HCl (pH 8.0) to remove excess reagents, and then digested overnight at 37°C with sequence grade modified trypsin (Promega, Madison, USA) at an enzyme-substrate ratio of 1 : 25 (w/w). The digests were acidified to pH 2 by addition of an aliquot of concentrated HCl and precipitates that formed were removed by centrifugation. The supernatant was adjusted to pH 8 with aqueous ammonia, and subjected immediately to analysis with an automated micro scale 2DLC-MS/MS system (Isobe et al. 2003).

The 2DLC-MS/MS analysis was performed as described (Mawuenyega et al. 2003). The peptide mixture was separated by a combination of first-dimensional anion-exchange LC on a bioassist-Q column (2 mmID × 35 mmL, 5 µm particles, TOSOH, Tokyo) and second-dimensional reversed phase LC on a Mightysil-C18 column (320 µm ID × 100 mm L, 3 µm particles, Kanto Chemicals, Tokyo), which were synchronized by a computer program. The eluted peptides were sprayed directly into a quadrupole time of flight (Q-TOF) hybrid mass spectrometer (Q-Tof-2, Micromass Ltd, Manchester, U.K). The total analysis time for a single 2DLC-MS/MS process was 16 h.

The large volume of MS/MS data was acquired with the software MassLynx (Micromass, Manchester, UK), converted to text files listing mass values of the parent ions and intensities and masses of fragment ions using ProteinLynx software (Micromass), and processed with the MASCOT algorism (Matrix Science Ltd, London, UK) to assign peptides in the NCBI non-redundant sequence database using a taxonomic restriction, ‘mammalia’. The database search was performed with parameters as described by Mawuenyega et al. (2003). The results were extracted using an in-house program ‘stem’ and imported into Microsoft Excel (Seatlle, WA, USA) for further analysis. We first screened the candidate peptides based on a probability-based Mowse score and finally selected the ‘hit’ peptides under the criteria described below in the Results section.

Identification of proteins in the PSD fraction

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

To examine the complexity of the PSD fraction, proteins of this fraction were separated by 2D-gel electrophoresis, and stained with Coomassie blue. As shown in Fig. 1, about 80 proteins were detected using 200 µg of total protein. Major proteins were identified by protein sequencing or LC-MS/MS, and indicated by arrows. This PSD preparation was then analysed using an integrated liquid chromatography (LC)-based protein identification system, which has the potential to identify ∼1000 proteins in a single analytical run (Mawuenyega et al. 2003). The analysis was repeated three times, and in each case, the MS/MS data was processed to assign candidate peptides in the NCBI genome database. We basically selected the candidate peptide, whose probability-based Mowse score (total score) exceeded a threshold that indicated significant homologuey (p < 0.05), and referred to it as a ‘hit’ according to the manufacturer's definition (Matrix Science, Ltd). Furthermore, we set stricter criteria for the protein assignment: (1) Peptide ions with a y-or-b ion series of 2 or more was considered as a ‘hit’ candidate. (2) Proteins, whose identity score exceeded 10 (p < 0.005), were referred to as ‘assigned’. (3) If the protein was assigned with a single peptide candidate having a identity score lower than 10, the original MS/MS spectrum was carefully inspected to confirm that the assignment was based on three or more y- or b-series ions. (4) For all candidates, if the individual candidate carried multiple modifications, its MS/MS spectrum was visually inspected for confirmation. In cases where all modifications were evident from the MS/MS signals, the peptides were included among the ‘hit’ peptides.

image

Figure 1. Proteins in the PSD fraction revealed by two-dimensional (2D)-gel electrophoresis and 2D-display. (a) 2D-gel electrophoresis of the PSD fraction. PSD protein (200 µg) was subjected to 2D-gel electrophoresis and stained with Coomassie blue. Some of the major Coomassie-stained protein spots were identified; 1, α and β fodrin; 2, NMDA-receptor 2B subunit; 3, densin 180; 4, SAPAP; 5, neurofilament M protein; 6, GluR1; 7, SAP−97; 8, α actinin; 9, PSD-95/SAP-90; 10, HSP70; 11, neurofilament L protein; 12, TOAD-64; 13, α internexin; 14, β CaM kinase II; 15, IRSp58; 16, IRSp53; 17 α and β tubulin; 18, GFAP; 19, α CaM kinase II; 20, homer 1b; 21, actin; 22, VDAC. (b) Theoretical 2D-gel representation of data assuming that identified peptides correspond to full-length proteins. PSD proteins with an identification number greater than 5 are shown. Their predicted molecular weight and pI values are indicated. The region in the box indicates the region measured in 2D-gel electrophoresis. Proteins are numbered the same as in (a).

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Using these criteria, we assigned ∼1800 hit peptides in each analysis, and in experiments performed in triplicate, a total of 5264 peptides including repetitive assignments of the same peptide. Removing the redundant identification of the same single protein from different portions of the protein sequence, these assigned peptides were attributed to 492 proteins. The proteins identified thus far are listed in supplementary Table 1. About 25% of PSD proteins were identified in the rat database, with remaining proteins identified by searching the more complete mouse and human databases. This approach is feasible because of the relatively high sequence conservation between rat, mouse and human protein.

Table 1.  Identification of high molecular weight proteins in the PSD fraction
ProteinaPropertyMr (kDa)Identification NoGi numberb
  1. Proteins with a molecular mass of more than 300 kDa are shown. aUnderline shows proteins previously known in the PSD fraction bh, m, and r immediately after the Gi number indicates the database of human, mouse and rat, respectively.

Dystonin β isoformhemidesmosomal protein838.4919882221m
Macrophin620 kDa actin-binding protein623.52815011904h
Dystonin α isoformhemidesmosomal protein617.5919882219m
Plectinintermediate filament-binding protein535.128213540714r
Dynein, heavy-chainminus-ended microtubule motor534.4513384736m
BRUCEBIR-ubiquitin-conjugated enzyme534.4110048468m
Piccoloaczonin, scaffold526.43715277340m
Ankyrin 3spectrin-binding protein482.31710947056h
Ankyrin 2spectrin-binding protein435.96310947052h
BassoonZn finger, CAG glutamine repeat420.41489506427r
Protocadherin 16cell adhesion molecule346.7116933557h
TrioPTPRF-interacting protein326.916005922h
Dystonin ε isoformhemidesmosomal protein303.7219882219m
MAP 1amicrotubule-associated protein300.82413591886r

Previous study indicated that the number of ‘hit’ peptides used to identify a particular protein with this LC-based protein identification technology roughly parallels the relative abundance of each protein in the sample mixture (Mawuenyega et al. 2003). About 150 proteins, with peptide hits of more than 5, are shown in Fig. 1(b) with their calculated molecular weight (Mr) and isoelectric point (pI) assuming that identified peptides correspond to full-length proteins. The most acidic protein found in this protein subset was calmodulin (pI 3.93) while the most basic was ribosome L3 protein (pI 11.39). The smallest protein was calmodulin (Mr = 16.8 kDa) and the largest was dystonin β isoform (Mr = 838.4 kDa). Proteins in Coomassie staining 2D-gel migrated to a similar molecular size to that predicted, but to a relatively acidic region as compared with their pI, except tubulin (spot No.17) (Fig. 1a). These results suggests that the LC-based protein identification technology could assign a wide variety of proteins with respect to Mr and pI and appeared to have covered almost all proteins detected on the 2D-gel.

In fact, many large proteins with a molecular weight of more than 300 kDa were identified in this study (Table 1). These included many cytoskeletal and membrane-associated proteins such as brain ankyrins, an intermediate filament-binding protein plectin, a hemidesmosomal protein dystonin, and a cross-linker of micro- and actin-filaments, macrophin. Trio is also a large protein with a potential role in the intracellular targeting of proteins, which consists of three enzyme domains, two EGF domains, a protein serine/threonine kinase domain, and a spectrin-like domain. Cadherin 16, a molecule for cell-cell adhesion in various cell types, was also identified. Bassoon (Dieck et al. 1998) and piccolo (alternatively called aczonin: Wang et al. 1999), which locates in the active zone of the presynaptic plasma membrane, were found in our PSD preparation in relatively large quantities.

When these proteins were divided into groups, it was found that 137 proteins were involved in signal transduction, 129 were structural proteins, 117 were proteins involved in cellular metabolism, 17 were proteins in the presynapse, and 72 were not characterized or hypothetical proteins, and 20 proteins were derived from cells other than neurones, including glial cells and blood (supplementary Table 1).

Proteins involved in signal transduction

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

The PSD serves as a general organizer of the postsynaptic signal transduction machinery, which links regulatory molecules to their targets, and coordinates developmental and activity-dependent changes in postsynaptic structures. Various proteins involved in signal transduction were found. Major proteins were glutamate receptors, CaM kinase II, PKC, calmodulin, protein phosphatase 1, various PDZ proteins and IRS p58/53, all found previously in the PSD. The most abundant receptors in our PSD preparation were NMDA receptors, including NR1, and NR2A, 2B, and 2C. From estimates made based on the identification number, NMDA receptor (total identification number, 133) roughly accounted for about 2.5% of total protein (identification number, 5264) found in the PSD fraction, and AMPA receptor 1, 2, 3, and 4 subunits (total identification number, 84), about 1.6%. CaM kinase II, known to be one of the most abundant proteins in the PSD, was estimated to account for 2.3% from total identification number of 121, consistent with a previous estimation (Yoshimura et al. 1997). In addition to these major proteins, about 70 proteins involved in signal transduction were found in this study (Table 2).

Table 2.  Proteins involved in signal transduction in the PSD fraction
Class ProteinaProperty Mr (kDa)bIdentification No.c Gi numberd
  • a

    Underline indicates protein previously known in the PSD fraction;

  • b

    b molecular weight of each isoform is shown in order, respectively;

  • c

    c the total identification number of isoforms is shown as (T);

  • d

    d h, m, and r immediately after the Gi number indicates the database of human, mouse and rat, respectively. Gi number of each subunit indicates the order.

(1) receptor
AMPA-R1, 2, 3, 4glutamate receptor102.3/99.3/100.9/101.684 (T)6680087m/8393475r/ 15147226r/8393478r
Eph receptor A4Ephrin receptor A4111.316679657m
G protein-coupled receptor 85super conserved receptor expressed in brain42.719507141h
GABA A- and B-receptorGABA receptor54.3/109.314 (T)6679907m/10835015h
Glutamate receptor δ subunitglutamate receptor114.1113259382r
Kainate receptor β2glutamate receptor, kinate98.356754074m
NMDA-R NR1glutamate receptor, NMDA106.0456680095m
NMDA-R NR2A, 2B, 2Dglutamate receptor, NMDA166.6/167.5/144.188 (T)6980982r/6980984r/ 6680101m
mGlu-R7glutamate receptor, metabotrophic103.714504147h
Transmembrane 7 superfamily 62.914507547h
(2) ion channel and transporter
Ca2+ channelL-type Ca2+ channel258.1213386500h
Ca2+ channelP/Q-type Ca2+ channel348.346680820m
Ca2+ channelvoltage-dependent Ca2+ channel β, γ subunit54.7/35.913 (T)6680824m/9506453m
K+channel Kir2.3inwardly-rectifying channel50.2616758740r
K+ channelCa2+ activated K+ channel133.716754436m
Na+ channelVoltage-gated Na+ channel a210.916981512r
Glutamate transporterneurone and astrocyte62.248394286r
(3) Protein kinase
BCDK-kinaseBranched chain keto acid dehydrogenase kinase 46.716753164m
CASKCa2+/CaM-dependent serine protein kinase (GK motif)105.616753272m
CaMKII α, β, γ, δCa2+/calmodulin -dep. protein kinase II55.7/61.1/59.6/54.6121 (T)7706286h/6671660m/ 19424316r/ 18158420m
Casein kinase I, IIα, IIβserine/threonine kinase 47.4/45.2/25.215 (T)7949025m/6681059m/ 7106277m
Cdc2-related kinasecyclin-dependent protein kinase-related kinase 56.614505569h
Double cortin and Ca2+/CaM kinase brain development84.779910164m
MAP kinase kinase 43.716678794m
MAP kinase kinase kinase kinase 141.226679060m
MAP/microtubule regulated kinaseMAP kinase family88.7416758824r
PKCα, β, γprotein kinase Cα, β, γ77.929 (T)6755078m/6679345m/ 6755080m
PKC-like 1protein kinase C-like 105.1 kinase, leucine zipper105.118394047r
ROCKRho-associated coiled-coil forming kinase161.616677761m
Serine/threonin kinase protein kinase81.1/116.1/146.25 (T) 11067437r/4507281h/5902140h
SPAKSte-20-related kinase, Pro,Ala-rich kinase 60.718394347m
Zipper protein kinaseMAP kinase kinase kinase 197.027106457m
(4) protein phosphatase
Calcineurin BCa2+/calmodulin-dep. protein phosphatase 19.418394036r
MAPK phosphataseDual specificity phosphatase53.2111528506m
PP 1, α, β,γprotein phosphatase 1, catalytic subunit α, β, γ38.2/37.9/37.625 (T)13994195m/4506005h/ 7305405m
PP 2Aprotein phosphatase 2A66.058394027m
Tyrosine phosphatasereceptor type215.6114506309h
(5) regulatory protein
Brain protein 44-like proteinapoptosis-regulating basic protein12.629055178m
CalmodulinCa2+-binding protein16.8246753244m
CDC10cell division cycle 1050.81912018296r
Cyclin G2cdk activator39.716680872m
IκB ɛnuculear factor kappa B-inhibitor protein 39.916679046m
NCK-associated protein 1apoptosis-related gene, human Nap1.130.2137305303h
Neurabin IIPP1 regulatory subunit, spinophilin89.6314211927h
Neuromodulingrowth-accentuating protein, CaM-binding protein23.716679935m
PKA, R subunit type IIprotein kinase A, regulatory subunit46.734506065h
PLC βphospholipase Cβ134.019845289m
14-3-3 Proteinβ, γ, ɛ, η, ζ, θphosphoprotein-binding protein∼28.324 (T)9055384m/9256646m/ 13928824r/6756037m/ 6756041m/6756039m
PKC & CK substrateneuronal protein50.816754974m
Prion proteinassociated with synaptic membrane28.1213173473m
Protein 9 Ka homologueousCa2+-binding protein11.916981326r
S-100 proteinCa2+-binding protein10.816677839m
STAT 3signal transducer and activator of transcription 87.816678153m
(6) G protein and related molecule
cAMP-dependent Rap1 GEF IIGEF family114.4106678153m
Centaurin α, γGAP homologue, IP3 binding43.7/90.03 (T)6806913h/7662484h
Collybistin Ibrain-specific GEF58.7113027402r
Cyclic nucleotide phosphodiesterase 47.456753476m
GDIGDP-dissociation inhibitor 151.118393425r
DOC2/DAB2 interactive protein GAP family107.4220070109h
Exchange factor for ARF6GEF to ARF71.1119705469r
G protein α, β, γTrimeric G protein40.6/38.1/8.118 (T)6754012m/6680045m/ 13384618m
H-ras proteinLow molecular G protein21.616680271m
K-ras 2 proteinLow molecular G protein21.6115718761h
N-ras proteinLow molecular G protein21.927242162m
PAK-interacting exchange factorGEF family70.9116758564r
Rab3 interacting protein 1regulating synaptic membrane exocytosis 173.6716716607m
Ras-GRF 2RAS-specific guanine nucleotide releasing factor 2136.917242199m
Ras-GAP binding proteinDnaJ chaperone protein53.1517865321m
Regulator of G-protein signalling 25.019910532m
Vav2 oncogeneRhoGEF101.116678555m
(7) scaffold protein
AKAPA kinase-anchoring protein87.716753024m
BEGAINPSD-95/SAP-90-binding protein67.51013162365r
Cateninα, β, δcadherin-associated protein106.3/86.0/140.026 (T)6753296m/6671684m/ 6679118m
Chapsin-110PSD-95 family95.28311560113r
CitronGABAergic and glutamatergic neurones187.53313929120r
GKAP/SAPAP interacting proteinSH3 and multiple ankyrin repeat domains135.21319743794h
GRIP 2Glutamate interacting protein 288.8119924071r
Homer 1b, 3AMPA receptor-binding protein40.3/39.823 (T)6754224m/16758964r
ProSAPProline-rich synapse-associated protein135.32019263336r
SAP-102synapse-associated protein94.0437949129m
SAP-90/PSD-95synapse-associated protein85.6179665227r
SAP-97SAP-90-related protein103.4166681191m
SAPAP 1, 2PSD-95/SAP-90/guanylate kinase-associated protein (2)111.3/110.1 8819923689r/16758774r
Shank 1, 3a, 3bproline-rich synapse-associated protein218.7/186.9/77.799(T) 13929054r/11067399r/ 10946786m
Similar to GK-associated proteinGKAP-like protein65.3413124765h
Somatostatin receptor-interacting protein 225.74011968152h
(8) adaptor protein
Arg/Abl-interacting protein 1, 2 multiple SH3 domain-containing74.8/125. 56(T)4502217h/16758606h
BAI1-associated protein2 (IRS p58/53) insulin-receptor substrate p58/5357.5669257197h/5453564h
c-Cbl-associated proteinSH3 domain protein76.566677935m
Copin 6dual C2 domain containing protein62.546677935m
Doc2Adisable gene family45.1112621100r
Growth factor receptor bound protein 10SH2 domain protein family71.316754064m
Interleukin 1 receptor accessory protein-like protein 10 80.817657232h
Pescadillo homologue 1BRCT domain-containing protein68.317657455h
Pleckstrinprotein kinase C substrate40.2111464971m
Pleckstrin and Sec7 domain proteinPleckstrin family70.714506161h
SH3 domain-binding glutamic acid-rich protein-like protein 12.3113899317h
TGF β1-induced anti-apoptotic factor-1 associated with Jak-3 N-terminal181.5117978507h
TNF receptor-associated factor 3 65.836755865m
Tripartite motif protein 9, 37RING box protein80.8/109.42 (T)18426848r/15147333h
TrioPTPRF, triple functional domain326.916005922h
WD repeat domain protein 123.7113878227m

Various protein kinases and phosphatases and their regulating proteins including double cortin and Ca2+/CaM kinase, cdc2 related kinase, MAP kinase family kinase, and neurabin were found. There were a lot of G-proteins and related proteins, including low molecular weight G proteins or small GTPases such as H-ras, N-ras, c-K-ras 2, and GTPase activating protein (GAP) and guaninenucleotide exchange factors (GEFs) such as centaurin, collybstin I, DOC2/DAB2 interacting protein, GDP-dissociation factor, cAMP Rap 1 GEFII and ras protein-specific guaninenucleotide-releasing factor. These results suggested that there were some unknown signalling cascade. It has been reported that MAP kinase signalling plays an important role in synaptic plasticity (Kornhaser and Greenberg 1997; Mazzucchelli et al. 2002) and that small GTPases participate in postsynaptic signalling and affect dendrite spine morphology presumably via the actions on the actin cytoskeleton (Pak et al. 2001).

In addition to major scaffold proteins in the PSD, known to contain PDZ including PSD-95/SAP-90, SAP-97, chapsyn-110, SAPAP, and shank, the scaffold proteins arg/abl-interacting protein, GKAP/SAPAP-interacting protein, and catenin, and somatostatin-receptor interacting protein were identified.

The finding of GABA receptors indicated that, in addition to classical PSDs from glutamatergic synapses, the PSD preparation used here contained postsynaptic specializations of inhibitory synapses. Collybistin I is a family of dbl-like GEF and may be an important determinant of inhibitory postsynaptic membrane formation and plasticity. As the protein compositions of inhibitory and excitatory postsynapses differ considerably, it has to be established at which type of synapse it occurs for any known protein identified in the study.

Structural proteins

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

Various membrane proteins, including cell adhesion proteins and cell-cell interacting proteins were found. Proteins for cell–cell interaction included the gap junctional channel protein connexin, cadherin 16, hemidesmsomal protein dystonin α, β and ε isoforms, and tight junction protein 1. Neuroligin 1, a postsynaptic cell-adhesion molecule of excitatory synapses, and neurexin, a synaptic cell surface protein, were also found. These proteins may be involved in synapse formation.

The finding of proteins involved in endocytosis, including clathrin, AP-1 and AP-2, COPII, and dynamin, suggested that the PSD fraction contained dynamic trafficking machinery for cell surface protein.

Motor proteins such as dynein, kinesin and myosin had relatively high identification numbers, suggesting that these proteins are involved in the transport of various component of the PSD fraction.

There were a variety of actin-binding proteins, including actin-binding LIM protein, afadin, actin-filament capping protein, cofilin, coronin, dorebrin, an enebled homologue, macrophin (620 kDa actin-binding protein, actin-cross linker), synaptopodin, tropomodulin, tropomyosin, and WASP family proteins. Various types of actin-related proteins were also found in this study. These proteins had relatively high identification numbers. These proteins might have a role in the morphological changes of the PSD and dendrite spine, because actin is the major cytoskeletal protein in the tip of the dendrite spine (Matus 2000),

Intermediate filament and related proteins, and microtubule and related proteins were also abundant. α and β Fodrin, known to be brain spectrins, were the most abundant protein in the PSD fraction, since their total identification number was 673.

In addition to these major proteins, about 70 structural proteins were found in this study (supplementary Table 1).

Proteins in presynaptic fraction

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

We found a number of proteins known to locate in the presynaptic fraction. Bassoon and piccolo bound to junctional proteins that span the synaptic cleft in vivo (Dieck et al. 1998; Wang et al. 1999). Proteins involved in the exocytosis of synaptic vesicles were found. These included synapsin I and II, SNAP-25, rab 3 effector, rabphilin 3 A, synaptotagmin, syntaxin, and an unc-13 mouse homologue (Munc-13). Munc-13 is a large, brain-specific protein with conserved C termini containing C1- and C2-domains, and is a peripheral membrane protein that is abundant in synaptosomes and localized to plasma membranes (Brose et al. 1995). Other grouping proteins found in this study, such as dynamin, NSF, vacuolar H+ pump ATPase subunit A, tubulin, actin, CASK, β-catenin, clathrin heavy chain, and brain spectrin α and β (fodrin) were also reported in the components of presynaptic particles (Phillips et al. 2001). These presynaptic proteins might be tightly bound to the PSD component in the active zone, and so were copurified with the PSD.

Proteins belonging to other groups

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

Some of the proteins found in this study apparently derive from subcellular fractions other than the PSD, such as the nucleus, mitochondrion, lysosome, and cytoplasm, or from fractions derived from glial cells. Several of these proteins, such as GFAP, might be associated tightly with the PSD fraction through protein components in the synaptic cleft or subcellular particles (Walikonis et al. 2000). Others arose probably due to insufficient purity of our PSD preparation. The method for the purification of PSD is expected to improve in the future.

We also found 31 unique ribosomal proteins and three elongation factors. There is a report that the protein translation machinery is localized beneath the synapses (Gardiol et al. 1999) and serves in protein synthesis important for synaptic plasticity (Steward and Schuman 2001; Jiang and Schuman 2002). However, we could not exclude the possibility of cytosolic contamination.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

The PSD is a tiny, amorphous structure located beneath the postsynaptic membrane and is visible under the electron microscope as tight complexes of postsynaptic junctional proteins. It is a disc-shaped subcellular organelle c. 50 nm thick and 100–900 nm in diameter apposed to postsynaptic membranes (Cotman et al. 1974), and plays an important role in synaptic plasticity (Kennedy 1993). Previous studies revealed a characteristic profile of the protein composition of the PSD fraction (Walikonis et al. 2000) or of the NMDA-receptor multiprotein complexes (Husi et al. 2000), which comprised specific cell surface receptors, receptor-associated proteins, scaffold proteins, etc. This study identified 492 proteins in the PSD fraction including proteins previously unknown in the fraction. Although additional experiments are required to confirm the site of cellular localization of each protein by, for instance, immunohistochemical means, the proteins identified in this study provide some insight into the protein composition of the PSD fraction. Here, the general profile of protein constituents strongly supports the notion that PSD is particularly rich in cell signalling proteins such as glutamate receptors and a variety of proteins involved in the protein phosphorylation cascade to form a functional signalling network of the cell, and in structural proteins, including membrane and cytoskeletal proteins. Structural proteins should have a role in stabilizing the PSD and in activity-dependent morphological changes to this important neural structure. Thus, this study provides a catalogue of the major protein sets associated with the PSD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References

This study was supported by a grant-in-aid for the National Project on Protein Structural and Functional Analysis to TY from the Ministry of Education, Culture, Sports, Science & Technology of Japan, by a grant to TY from the NOVARTIS Foundation (Japan) for the Promotion of Science, and also in part by a grant for the Integrated Proteomics System Project, Pioneer Research on the Genome Frontier to TI from the Ministry of Education, Culture, Sports, Science & Technology of Japan.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Purification of PSD
  5. Two-dimensional gel electrophoresis and identification of PSD protein
  6. Protein identification by automated 2DLC-tandem mass spectrometry and data analysis
  7. Results and Discussion
  8. Identification of proteins in the PSD fraction
  9. Proteins involved in signal transduction
  10. Structural proteins
  11. Proteins in presynaptic fraction
  12. Proteins belonging to other groups
  13. Conclusion
  14. Acknowledgements
  15. Supplementary material
  16. References
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