Identification and Verification of Novel Rodent Postsynaptic Density Proteins*S

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.

[1]  R. Tsien,et al.  Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons , 1988, Neuron.

[2]  S. J. Smith,et al.  The post-synaptic density: putative involvement in synapse stabilization via cadherins and covalent modification by ubiquitination. , 1995, Biochemical Society transactions.

[3]  Taka-Aki Sato,et al.  Identification of mRNA/Protein (mRNP) Complexes Containing Purα, mStaufen, Fragile X Protein, and Myosin Va and their Association with Rough Endoplasmic Reticulum Equipped with a Kinesin Motor* , 2002, The Journal of Biological Chemistry.

[4]  Rosemary L Martin,et al.  Cytohesins and centaurins control subcellular trafficking of macromolecular signaling complexes: regulation by phosphoinositides and ADP-ribosylation factors. , 2002, Biological research.

[5]  J. Yates,et al.  The application of mass spectrometry to membrane proteomics , 2003, Nature Biotechnology.

[6]  V. Ferrans,et al.  ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Da-Zhi Wang,et al.  Activation of Cardiac Gene Expression by Myocardin, a Transcriptional Cofactor for Serum Response Factor , 2001, Cell.

[8]  M. Constantine-Paton,et al.  NMDA receptor-mediated control of protein synthesis at developing synapses , 2000, Nature Neuroscience.

[9]  V. Piëch,et al.  Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons , 2001, Nature Neuroscience.

[10]  J. Yates,et al.  DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. , 2002, Journal of proteome research.

[11]  M. Mayford,et al.  Cellular and molecular mechanisms of memory: the LTP connection. , 1999, Current opinion in genetics & development.

[12]  M. Rogawski,et al.  Kainate receptor-mediated heterosynaptic facilitation in the amygdala , 2001, Nature Neuroscience.

[13]  P Siekevitz,et al.  Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities , 1980, The Journal of cell biology.

[14]  P. Osten,et al.  Mutagenesis Reveals a Role for ABP/GRIP Binding to GluR2 in Synaptic Surface Accumulation of the AMPA Receptor , 2000, Neuron.

[15]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[16]  A. Nairn Faculty Opinions recommendation of Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. , 2003 .

[17]  A. Reynolds,et al.  ARVCF localizes to the nucleus and adherens junction and is mutually exclusive with p120(ctn) in E-cadherin complexes. , 2000, Journal of cell science.

[18]  T. Boeckers,et al.  The postsynaptic density , 2006, Cell and Tissue Research.

[19]  H. Kasai,et al.  Structure–stability–function relationships of dendritic spines , 2003, Trends in Neurosciences.

[20]  T. Yamauchi Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density. , 2002, Mass spectrometry reviews.

[21]  J. McCabe,et al.  Cullin-5 is ubiquitous in the rat brain , 2003, Neuroscience Letters.

[22]  E. Schuman,et al.  Ubiquitin-Mediated Proteasome Activity Is Required for Agonist-Induced Endocytosis of GluRs , 2003, Current Biology.

[23]  E. Ziff Enlightening the Postsynaptic Density , 1997, Neuron.

[24]  E. Kandel,et al.  An Unbiased cDNA Library Prepared from Isolated Aplysia Sensory Neuron Processes Is Enriched for Cytoskeletal and Translational mRNAs , 2003, The Journal of Neuroscience.

[25]  G. Courtois,et al.  The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination , 2003, Nature.

[26]  R. Treisman,et al.  Actin Dynamics Control SRF Activity by Regulation of Its Coactivator MAL , 2003, Cell.

[27]  Karim Nader,et al.  Memory consolidation of Pavlovian fear conditioning: a cellular and molecular perspective , 2001, Trends in Neurosciences.

[28]  M. Taoka,et al.  Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry. , 2002, Biochemical and biophysical research communications.

[29]  M. Kennedy,et al.  Signal-processing machines at the postsynaptic density. , 2000, Science.

[30]  M. J. Walsh,et al.  The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing , 1992, Journal of neurochemistry.

[31]  G. Crabtree,et al.  NFAT Signaling Choreographing the Social Lives of Cells , 2002, Cell.

[32]  M. Mayford,et al.  Disruption of Dendritic Translation of CaMKIIα Impairs Stabilization of Synaptic Plasticity and Memory Consolidation , 2002, Neuron.

[33]  R. Beavis,et al.  A method for assessing the statistical significance of mass spectrometry-based protein identifications using general scoring schemes. , 2003, Analytical chemistry.

[34]  C. Winters,et al.  Glutamate-induced transient modification of the postsynaptic density , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P Siekevitz,et al.  The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure , 1977, The Journal of cell biology.

[36]  C. Cotman,et al.  PROTEINS OF THE POSTSYNAPTIC DENSITY , 1974, The Journal of cell biology.

[37]  M. Czech Dynamics of phosphoinositides in membrane retrieval and insertion. , 2003, Annual review of physiology.

[38]  M. Molloy,et al.  Membrane proteins and proteomics: Un amour impossible? , 2000, Electrophoresis.

[39]  M. Ehlers Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system , 2003, Nature Neuroscience.

[40]  Eckart D Gundelfinger,et al.  Proteomics Analysis of Rat Brain Postsynaptic Density , 2004, Journal of Biological Chemistry.

[41]  R. Aebersold,et al.  Mass spectrometry-based proteomics , 2003, Nature.

[42]  Trent P Munro,et al.  Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein* , 2002, The Journal of Biological Chemistry.

[43]  S. Grant,et al.  Proteomic analysis of NMDA receptor–adhesion protein signaling complexes , 2000, Nature Neuroscience.

[44]  N. Nomura,et al.  Construction and characterization of human brain cDNA libraries suitable for analysis of cDNA clones encoding relatively large proteins. , 1997, DNA research : an international journal for rapid publication of reports on genes and genomes.

[45]  Albert B. Reynolds,et al.  A core function for p120-catenin in cadherin turnover , 2003, The Journal of cell biology.

[46]  M. Kennedy,et al.  Identification of Proteins in the Postsynaptic Density Fraction by Mass Spectrometry , 2000, The Journal of Neuroscience.

[47]  F. van Roy,et al.  Nuclear localization of the p120(ctn) Armadillo-like catenin is counteracted by a nuclear export signal and by E-cadherin expression. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Yoshiya Oda,et al.  Identification of activity‐regulated proteins in the postsynaptic density fraction , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[49]  H. Ploegh,et al.  Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. , 2002, Chemistry & biology.

[50]  Xunsheng Chen,et al.  The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia. , 2000, Journal of cell science.

[51]  J. Donaldson Multiple Roles for Arf6: Sorting, Structuring, and Signaling at the Plasma Membrane* , 2003, Journal of Biological Chemistry.

[52]  Takashi Yamauchi,et al.  Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography‐tandem mass spectrometry , 2003, Journal of neurochemistry.

[53]  D. N. Perkins,et al.  Probability‐based protein identification by searching sequence databases using mass spectrometry data , 1999, Electrophoresis.

[54]  E. Kandel,et al.  Cognitive Neuroscience and the Study of Memory , 1998, Neuron.

[55]  E. Kandel,et al.  Toward a molecular definition of long-term memory storage. , 1996, Proceedings of the National Academy of Sciences of the United States of America.