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

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 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.

Polyglutamine-Modulated Striatal Calpain Activity in YAC Transgenic Huntington Disease Mouse Model: Impact on NMDA Receptor Function and Toxicity

“…Calpain activation results in cleavage of the C-terminal tail (around amino acid 1030) of the NR2B subunit of NMDARs (Bi et al, 1998a;Guttmann et al, 2001Guttmann et al, , 2002Simpkins et al, 2003). We hypothesized that the elevated calpain activity levels we found in YAC128 striatal neurons would be associated with an increased proportion of calpain-cleaved NR2B.…”

N-Methyl-d-aspartate Receptor- and Calpain-mediated Proteolytic Cleavage of K+-Cl− Cotransporter-2 Impairs Spinal Chloride Homeostasis in Neuropathic Pain

“…It seems that protein phosphatase 1-mediated KCC2 dephosphorylation cannot fully explain the profound reduction in the KCC2 protein level in the spinal cord by nerve injury. It has been shown that NR2 subunits are substrates of calpain and that activation of NMDARs can activate calpain, which in turn down-regulates the NMDAR function (57,58). However, PSD-95 clustering and direct association of NR2 and PSD-95 may prevent calpain cleavage of NMDARs (59).…”

Mechanistic Role of Calpains in Postischemic Neurodegeneration

“…Classic NMDA-type glutamate receptors are heteromeric proteins composed of two NR1 subunits and two NR2 subunits, selected from subtypes NR2A, NR2B, NR2C, and NR2D. Of these, NR2A, -2B, and -2C are known to undergo Cterminal proteolysis by calpain in vitro (Guttmann et al, 2002), a truncation that does not appear to prevent any aspects of receptor function. However, only the NR2B subunit has been (Xu et al, 2007) shown to be cleaved by calpain in mature neurons (Dong et al, 2006).…”