Proteolysis of theN-Methyl-d-Aspartate Receptor by Calpain in Situ
| Autor: | David A. Lynch, Dana L. Baker, Rodney P. Guttmann, Kelly L. Simpkins, Set Sokol, Yina Dong |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
DNA
Complementary Cell Survival Proteolysis Blotting Western Excitotoxicity Cysteine Proteinase Inhibitors Biology Transfection medicine.disease_cause Receptors N-Methyl-D-Aspartate Calcium in biology Cell Line medicine Humans Receptor Calpastatin Pharmacology medicine.diagnostic_test Calpain Calcium-Binding Proteins Glutamate receptor Molecular biology biology.protein Molecular Medicine NMDA receptor Calcium Dizocilpine Maleate Excitatory Amino Acid Antagonists Peptide Hydrolases |
| Zdroj: | Journal of Pharmacology and Experimental Therapeutics. 302:1023-1030 |
| ISSN: | 1521-0103 0022-3565 |
| DOI: | 10.1124/jpet.102.036962 |
| Popis: | 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 (125)I-MK-801 ((125)I-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. |
| Databáze: | OpenAIRE |
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