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Proteomic analysis of NMDA receptor–adhesion protein signaling complexes

Abstract

N-methyl-D-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter–adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

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Figure 1: Basic composition of NRC isolated from mouse brain.
Figure 2: Mass spectrometry analysis of the NRC.

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References

  1. Bliss, T. V. & Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361 , 31–39 (1993).

    CAS  PubMed  Google Scholar 

  2. Bear, M. F. & Malenka, R. C. Synaptic plasticity: LTP and LTD. Curr. Opin. Neurobiol. 4, 389– 399 (1994).

    CAS  PubMed  Google Scholar 

  3. Heresco-Levy, U. & Javitt, D. C. The role of N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission in the pathophysiology and therapeutics of psychiatric syndromes. Eur. Neuropsychopharmacol. 8, 141–152 ( 1998).

    CAS  PubMed  Google Scholar 

  4. Hollmann, M. & Heinemann, S. Cloned glutamate receptors. Annu. Rev. Neurosci. 17, 31–108 (1994).

    CAS  PubMed  Google Scholar 

  5. Sanes, J. R. & Lichtman, J. W. Can molecules explain long-term potentiation? Nat. Neurosci. 2, 597– 604 (1999).

    CAS  PubMed  Google Scholar 

  6. Pawson, T. & Scott, J. D. Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075– 2080 (1997).

    CAS  PubMed  Google Scholar 

  7. Kim, J. H. & Huganir, R. L. Organization and regulation of proteins at synapses. Curr. Opin. Cell Biol. 11, 248–254 (1999).

    CAS  PubMed  Google Scholar 

  8. Hsueh, Y. P. & Sheng, M. Anchoring of glutamate receptors at the synapse. Prog. Brain Res. 116, 123– 131 (1998).

    CAS  PubMed  Google Scholar 

  9. Kornau, H. C., Seeburg, P. H. & Kennedy, M. B. Interaction of ion channels and receptors with PDZ domain proteins. Curr. Opin. Neurobiol. 3, 368–373 (1997).

    Google Scholar 

  10. Migaud, M. et al. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439 (1998).

    CAS  PubMed  Google Scholar 

  11. Mendelsohn, A. R. & Brent, R. Protein interaction methods—toward an endgame. Science 284, 1948–1950 (1999).

    CAS  PubMed  Google Scholar 

  12. Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20, 46–50 (1998).

    CAS  PubMed  Google Scholar 

  13. Hisatsune, C., Umemori, H., Mishina, M. & Yamamoto, T. Phosphorylation-dependent interaction of the N-methyl-D-aspartate receptor epsilon 2 subunit with phosphatidylinositol 3-kinase. Genes Cells 4, 657– 666 (1999).

    CAS  PubMed  Google Scholar 

  14. Craven, S. E., El-Husseini, A. E. & Bredt, D. S. Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22, 497–509 (1999).

    CAS  PubMed  Google Scholar 

  15. Blackstock, W. P. & Weir, M. P. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol. 17, 121–127 (1999).

    CAS  PubMed  Google Scholar 

  16. O'Brien, R. J., Lau, L. F. & Huganir, R. L. Molecular mechanisms of glutamate receptor clustering at excitatory synapses. Curr. Opin. Neurobiol. 8, 364–369 (1998).

    CAS  PubMed  Google Scholar 

  17. Garcia, E. P. et al. SAP90 binds and clusters kainate receptors causing incomplete desensitization. Neuron 21, 727– 739 (1998).

    CAS  PubMed  Google Scholar 

  18. Naisbitt, S. et al. Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23, 569–582 ( 1999).

    CAS  PubMed  Google Scholar 

  19. Tu, J. C. et al. Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23, 583–592 (1999).

    CAS  PubMed  Google Scholar 

  20. Leonard, A. S., Lim, I. A., Hemsworth, D. E., Horne, M. C. & Hell, J. W. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. USA 96, 3239– 3244 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Westphal, R. S. et al. Regulation of NMDA receptors by an associated phosphatase-kinase signaling complex. Science 285, 93– 96 (1999).

    CAS  PubMed  Google Scholar 

  22. Wang, Y. T. & Salter, M. W. Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369, 233–235 (1994).

    CAS  PubMed  Google Scholar 

  23. O'Dell, T. J., Kandel, E. R. & Grant, S. G. Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nature 353, 558–560 (1991).

    CAS  PubMed  Google Scholar 

  24. Lin, S. Y. et al. Brain-derived neurotrophic factor enhances association of protein tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in the cortical postsynaptic density. Mol. Brain Res. 70, 18–25 (1999).

    CAS  PubMed  Google Scholar 

  25. Brambilla, R. et al. A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature 390, 281– 286 (1997).

    CAS  PubMed  Google Scholar 

  26. Silva, A. J. et al. A mouse model for the learning and memory deficits associated with neurofibromatosis type I. Nat. Genet. 15, 281–284 (1997).

    CAS  PubMed  Google Scholar 

  27. Gille, H. & Downward, J. Multiple ras effector pathways contribute to G(1) cell cycle progression. J. Biol. Chem. 274, 22033–22040 (1999).

    CAS  PubMed  Google Scholar 

  28. Schaeffer, H. J. & Weber, M. J. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol. Cell. Biol. 19, 2435–2444 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Mitsuuchi, Y. et al. Identification of a chromosome 3p14.3-21.1 gene, APPL, encoding an adaptor molecule that interacts with the oncoprotein-serine/threonine kinase AKT2. Oncogene 18, 4891– 4898 (1999).

    CAS  PubMed  Google Scholar 

  30. Impey, S., Obrietan, K. & Storm, D. R. Making new connections: role of ERK/MAP kinase signaling in neuronal plasticity. Neuron 23, 11– 14 (1999).

    CAS  PubMed  Google Scholar 

  31. Link, W. et al. Somatodendritic expression of an immediate early gene is regulated by synaptic activity. Proc. Natl. Acad. Sci. USA 92 , 5734–5738 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Lyford, G. L. et al. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites . Neuron 14, 433–445 (1995).

    CAS  PubMed  Google Scholar 

  33. Lin, L. L. et al. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72, 269–278 ( 1993).

    CAS  PubMed  Google Scholar 

  34. Bonventre, J. V. et al. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature 390, 622–625 (1997).

    CAS  PubMed  Google Scholar 

  35. Murase, S. & Schuman, E. M. The role of cell adhesion molecules in synaptic plasticity and memory. Curr. Opin. Cell Biol. 11, 549–553 (1999).

    CAS  PubMed  Google Scholar 

  36. Rosenmund, C. & Westbrook, G. L. Calcium-induced actin depolymerization reduces NMDA channel activity. Neuron 10, 805–814 (1993).

    CAS  PubMed  Google Scholar 

  37. Kim, C. H. & Lisman, J. E. A role of actin filament in synaptic transmission and long-term potentiation. J. Neurosci. 19, 4314–4324 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Allison, D. W., Gelfand, V. I., Spector, I. & Craig, A. M. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. J. Neurosci. 18, 2423–2436 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Fischer, M., Kaech, S., Knutti, D. & Matus, A. Rapid actin-based plasticity in dendritic spines. Neuron 20, 847–854 (1998).

    CAS  PubMed  Google Scholar 

  40. Wechsler, A. & Teichberg, V. I. Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin. EMBO J. 17, 3931–3939 ( 1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Luthl, A., Laurent, J. P., Figurov, A., Muller, D. & Schachner, M. Hippocampal long-term potentiation and neural cell adhesion molecules L1 and NCAM. Nature 372, 777–779 (1994).

    Google Scholar 

  42. Tang, L., Hung, C. P. & Schuman, E. M. A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation. Neuron 20, 1165–1175 (1998).

    CAS  PubMed  Google Scholar 

  43. Sans, N., Petralia, R. S., Wang, Y. X., Blahos, J., Hell, J. W. & Wenthold, R. J. A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J. Neurosci. 20, 1260–1271 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Grant, S. G. N. in Handbook of Molecular-Genetic Techniques for Brain and Behavior Research (eds. Crusio, W. E. & Gerlai, R. T.) 315– 328 (Elsevier, Amsterdam, 1999).

    Google Scholar 

  45. Ozonoff, S. Cognitive impairment in neurofibromatosis type 1. Am. J. Med. Genet. 89, 45–52 ( 1999).

    CAS  PubMed  Google Scholar 

  46. Fransen, E., Van Camp, G., D'Hooge, R., Vits, L. & Willems, P. J. Genotype-phenotype correlation in L1 associated diseases. J. Med. Genet. 35, 399–404 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Merienne, K. et al. A missense mutation in RPS6KA3 (RSK2) responsible for non-specific mental retardation. Nat. Genet. 22, 13– 14 (1999).

    CAS  PubMed  Google Scholar 

  48. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 ( 1996).

    CAS  PubMed  Google Scholar 

  49. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996).

    CAS  PubMed  Google Scholar 

  50. Link, A. J. et al. Direct analysis of protein complexes using mass spectrometry . Nat. Biotechnol. 17, 676– 682 (1999).

    CAS  PubMed  Google Scholar 

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Acknowledgements

Antibodies were provided by those listed in Methods. We thank T.J. O'Dell and P. Brophy for comments. H.H. and S.G. were supported by the Wellcome Trust.

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Correspondence to Seth G. N. Grant.

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Husi, H., Ward, M., Choudhary, J. et al. Proteomic analysis of NMDA receptor–adhesion protein signaling complexes. Nat Neurosci 3, 661–669 (2000). https://doi.org/10.1038/76615

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