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Research ArticleNEUROPHARMACOLOGY

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

Rodney P. Guttmann, Set Sokol, Dana L. Baker, Kelly L. Simpkins, Yina Dong and David R. Lynch
Journal of Pharmacology and Experimental Therapeutics September 2002, 302 (3) 1023-1030; DOI: https://doi.org/10.1124/jpet.102.036962
Rodney P. Guttmann
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Set Sokol
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Dana L. Baker
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Kelly L. Simpkins
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Yina Dong
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David R. Lynch
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Abstract

N-Methyl-d-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of 125I-MK-801 (125I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.

Footnotes

  • This work was supported by Grants DA07130, NS01789, NS39126, MH14654, and NS1084 from the National Institutes of Health; a Beeson Fellowship from the American Federation for Aging Research; and Grant 9920365U from the American Heart Association (R.P.G.). Support for DNA sequencing was provided through the Molecular Genetics Core of the Mental Retardation Research Center Grant HD26979.

  • DOI: 10.1124/jpet.102.036962

  • Abbreviations:
    NMDA
    N-methyl-d-aspartate
    NR1
    NMDA receptor subunit 1
    HEK
    human embryonic kidney
    MK-801
    dizocilpine
    DTT
    dithiothreitol
    HBSS
    HEPES-buffered saline solution
    GFP
    green fluorescence protein
    PAGE
    polyacrylamide gel electrophoresis
    Suc-LLVY-AMC
    succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosyl-aminomethyl coumarin
    AMPA
    α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
    • Received April 4, 2002.
    • Accepted May 3, 2002.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 302 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 302, Issue 3
1 Sep 2002
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Research ArticleNEUROPHARMACOLOGY

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

Rodney P. Guttmann, Set Sokol, Dana L. Baker, Kelly L. Simpkins, Yina Dong and David R. Lynch
Journal of Pharmacology and Experimental Therapeutics September 1, 2002, 302 (3) 1023-1030; DOI: https://doi.org/10.1124/jpet.102.036962

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Research ArticleNEUROPHARMACOLOGY

Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ

Rodney P. Guttmann, Set Sokol, Dana L. Baker, Kelly L. Simpkins, Yina Dong and David R. Lynch
Journal of Pharmacology and Experimental Therapeutics September 1, 2002, 302 (3) 1023-1030; DOI: https://doi.org/10.1124/jpet.102.036962
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