Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ.
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.
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA.
Rodney P
RP
Guttmann
Set
S
Sokol
Dana L
DL
Baker
Kelly L
KL
Simpkins
Yina
Y
Dong
David R
DR
Lynch
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D016291:Dizocilpine Maleate / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D018691:Excitatory Amino Acid Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; D016291:Dizocilpine Maleate; C033668:calpastatin; D010447:Peptide Hydrolases; D002154:Calpain; D002118:Calcium