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  • Research Article
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In vitro selection of peptides acting at a new site of NMDA glutamate receptors

Nature Biotechnology volume 14, pages 986–991 (1996)Cite this article

Abstract

Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands. For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel. Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease. Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway. There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself. To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries. The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain. We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC. Using biochemical binding analysis and patch clamp electro-physiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.

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References

  1. Choi, D.W. 1990. Possible mechanisms limiting N-methyl-D-aspartate receptor overactivation and the therapeutic efficacy of N-methyl-D-aspartate antagonists. Stroke 21: III20–22.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Hollmann, M. and Heinemann, S. 1994. Cloned glutamate receptors. Annu. Rev. Neurosci. 17: 31–108.

    Article  CAS  PubMed  Google Scholar 

  4. Monaghan, D.T., Bridges, R.J., and Cotman, C.W. 1989. The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Annu. Rev. Pharmacol. Toxicol. 29: 365–402.

    Article  CAS  PubMed  Google Scholar 

  5. Nakanishi, S. 1992. Molecular diversity of glutamate receptors and implications for brain function. Science 258: 597–603.

    Article  CAS  PubMed  Google Scholar 

  6. Moriyoshi, K., et al. 1991. Molecular cloning and characterization of the rat NMDA receptor. Nature 354: 31–37.

    Article  CAS  PubMed  Google Scholar 

  7. Johnson, J.W., and Ascher, P. 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325: 529–531.

    Article  CAS  PubMed  Google Scholar 

  8. Mayer, M.L., Westbrook, G.L., and Guthrie, P.B. 1984. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309: 261–263.

    Article  CAS  PubMed  Google Scholar 

  9. McGurk, J.F., Bennett, M.V., and Zukin, R.S. 1990. Polyamines potentiate responses of N-methyl-D-aspartate receptors expressed in xenopus oocytes. Proc. Natl. Acad. Sci. USA 87: 9971–9974.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A., and Prochiantz, A. 1984. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462–465.

    Article  CAS  PubMed  Google Scholar 

  11. Peters, S., Koh, J., and Choi, D.W. 1987. Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons. Science 236: 589–593.

    Article  CAS  PubMed  Google Scholar 

  12. Westbrook, G.L. and Mayer, M.L. 1987. Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neuronsNature 328: 640–643.

  13. Wong, E.H., et al. 1986. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc. Natl. Acad. Sci. USA 83: 7104–7108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Smith, G.P. 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317.

    Article  CAS  PubMed  Google Scholar 

  15. Scott, U.K. and Smith, G.P. 1990. Searching for peptide ligands with an epitope library. Science 249: 386–390.

    Article  CAS  PubMed  Google Scholar 

  16. Devlin, J.J., Panganiban, L.C., and Devlin, P.E. 1990. Random peptide libraries: a source of specific protein binding molecules. Science 249: 404–406.

    Article  CAS  PubMed  Google Scholar 

  17. Cwirla, S.E., Peters, E.A., Barrett, R.W., and Dower, W.J. 1990. Peptides on phage: a vast library of peptides for identifying ligands. Proc. Natl. Acad. Sci. USA 87: 6378–6382.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Whitehorn, E.A., Tate, E., Yanofsky, S.D., Kochersperger, L., Davis, A., Mortensen, R.B., et al. 1995. A generic method for expression and use of “tagged” soluble versions of cell surface receptors. Bio/Technology 13: 1215–1219.

    CAS  Google Scholar 

  19. Chazot, R.L., Cik, M., and Stephenson, F.A. 1992. Immunological detection of the NMDAR1 glutamate receptor subunit expressed in embryonic kidney 293 cells and in rat brain. J. Neurochem. 59: 1176–1178.

    Article  CAS  PubMed  Google Scholar 

  20. Barrett, R.W., et al. 1992. Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage. Anal. Biochem. 204: 357–364.

    Article  CAS  PubMed  Google Scholar 

  21. Hollmann, M., O'Shea-Greenfield, A., Rogers, S.W., and Heinemann, S. 1989. Cloning by functional expression of a member of the glutamate receptor family. Nature 342: 643–648.

    Article  CAS  PubMed  Google Scholar 

  22. Hollmann, M., et al. 1993. Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron 10: 943–954.

    Article  CAS  PubMed  Google Scholar 

  23. Haack, J.A., et al. 1990. Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-D-aspartate antagonist activity. J. Biol. Chem. 265: 6025–6029.

    CAS  PubMed  Google Scholar 

  24. Huettner, J.E. and Bean, B.P. 1988. Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels. Proc. Natl. Acad. Sci. USA 85: 1307–1311.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mori, H., Masaki, H., Yamakura, T., and Mishina, M. 1992. Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature 358: 673–675.

    Article  CAS  PubMed  Google Scholar 

  26. Monyer, H., et al. 1992. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256: 1217–1221.

    Article  CAS  PubMed  Google Scholar 

  27. Tingley, W.G., Roche, K.W., Thompson, A.K., and Huganir, R.L. 1993. Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain. Nature 364: 70–73.

    Article  CAS  PubMed  Google Scholar 

  28. Kutsuwada, T., et al. 1992. Molecular diversity of the NMDA receptor channel. Nature 358: 36–41.

    Article  CAS  PubMed  Google Scholar 

  29. Lucas, D.R. and Newhouse, J. 1957. The toxic effect of sodium L-glutamate on the inner layers of retina. Arch. Ophthalmol. 58: 193–204.

    Article  CAS  Google Scholar 

  30. Choi, D.W. and Rothman, S.M. 1990. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13: 171–182.

    Article  CAS  PubMed  Google Scholar 

  31. Miller, R.J., Murphy, S.N., and Glaum, S.R. 1989. Neuronal Ca2+ channels and their regulation by excitatory amino acids. Ann. NY Acad. Sci. 568: 149–158.

    Article  CAS  PubMed  Google Scholar 

  32. Young, A.B., Penney, J.B., Dauth, G.W., Bromberg, M.B., and Gilman, S. 1983. Glutamate or aspartate as a possible neuretransmitter of cerebral corticofugal fibers in the monkey. Neurology 33: 1513–1516.

    Article  CAS  PubMed  Google Scholar 

  33. Rothstein, J.D., et al. 1991. Excitatory amino acids in amyotrophic lateral sclerosis: an update. Ann. Neurol. 30: 224–225.

    Article  CAS  PubMed  Google Scholar 

  34. Rothstein, J.D., Martin, L.J., and Kuncl, R.W. 1992. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N. Engl. J. Med. 326: 1464–1468.

    Article  CAS  PubMed  Google Scholar 

  35. Rothstein, J.D., Jin, L., Dykes-Hoberg, M., and Kuncl, R.W. 1993. Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. Proc. Natl. Acad. Sci. USA 90: 6591–6595.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rothstein, J.D., et al. 1994. Localization of neuronal and glial glutamate transporters. Neuron 13: 713–725.

    Article  CAS  PubMed  Google Scholar 

  37. Storm-Mathisen, J. and Ottersen, O.P. 1987. Tracing of neurons with glutamate or gamma-aminobutyrate as putative transmitters. Biochem. Soc. Trans. 15: 210–213.

    Article  CAS  PubMed  Google Scholar 

  38. Storm-Mathisen, J. and Ottersen, O.P. 1990. Immunocytochemistry of glutamate at the synaptic level. J. Histochem. Cytochem. 38: 1733–1743.

    Article  CAS  PubMed  Google Scholar 

  39. O'Brien, R.J. and Fischbach, G.D. 1986. Modulation of embryonic chick motoneuron glutamate sensitivity by intemeurons and agonists. J. Neurosci. 6: 3290–3296.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tsai, G.C., et al. 1991. Reductions in acidic amino acids and N-acetylaspartylglu-tamate in amyotrophic lateral sclerosis CNS. Brain Res. 556: 151–156.

    Article  CAS  PubMed  Google Scholar 

  41. Shaw, P.J. 1994. Excitotoxicity and motor neurone disease: a review of the evidence. J. Neurol. Sci. 124: 6–13.

    Article  PubMed  Google Scholar 

  42. Shaw, P.J. 1994. Excitotoxicity and motor neurone disease: a review of the evidence. J. Neurol. Sci. 124: 6–13.

    Article  PubMed  Google Scholar 

  43. Lipton, S.A. and Stamler, J.S. 1994. Actions of redox-related congeners of nitric oxide at the NMDA receptor. Neuropharmacology 33: 1229–12233.

    Article  CAS  PubMed  Google Scholar 

  44. Li, M., Jan, Y.N., and Jan, L.Y. 1992. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. Science 257: 1225–1230.

    Article  CAS  PubMed  Google Scholar 

  45. Guzman, L.M., Belin, D., Carson, M.J., and Beckwith, J. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177: 4121–4130.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author notes

  1. Min Li: min_li@qmail.bs.jhu.edu.

Authors and Affiliations

  1. Departmentof Physiology and Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD, 21205, USA

    Min Li, Weifeng Yu & Chien-Huan Chen

  2. Department of Molecular Biology, Aflymax Research Institute, 4001 Miranda Ave., Polo Alto, CA, 94304, USA

    Steve Cwirla, Erik Whitehorn, Emily Tate, Ron Raab, Meire Bremer & Bill Dower

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Li, M., Yu, W., Chen, CH. et al. In vitro selection of peptides acting at a new site of NMDA glutamate receptors. Nat Biotechnol 14, 986–991 (1996). https://doi.org/10.1038/nbt0896-986

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