Endoplasmic reticulum‐associated degradation of the NR1 but not the NR2 subunits of the N‐methyl‐D‐aspartate receptor induced by inhibition of the N‐glycosylation in cortical neurons

Abstract

The N-methyl-D-aspartate receptor (NMDAR) is fundamental to normal and pathological functioning of neurons. The receptor subunits are N-glycosylated proteins synthesized in the endoplasmic reticulum (ER) that fold, mature, and oligomerize as they transit through the secretory pathway. Although the early processes of biogenesis are fundamental to NMDAR expression and function, our knowledge of them is nevertheless limited. Additionally, the investigation of NMDAR synthesis is highly relevant, in that ER dysfunction, frequently associated with acute and degenerative brain diseases, might alter this process. We characterize here the effect of ER stress produced by inhibition of N-glycosylation on NMDAR synthesis and function. We use first heterologous systems of NMDAR expression in which NR1 and NR2A subunits are synthesized in nonneuronal cells. The function of these NMDARs as Ca2+ channels is repressed by tunicamycin, because of the inhibition of NR1, but no NR2A, synthesis. The regulation of NR1 is relevant to the central nervous system, in that a dramatic decrease in synthesis of this subunit and assembly of NMDARs is observed in cortical neurons treated with tunicamycin. The inhibition of NR1 synthesis is not due to changes in levels of mRNA but associated with the earliest stages in NMDAR biogenesis. The inhibition of N-glycosylation activates ER-specific stress responses in neurons, which include the ER-associated degradation (ERAD) mechanism responsible for differential and extremely efficient degradation of nonglycosylated NR1 by the proteasome after ubiquitination. Because this is an obligatory NMDAR component, the significant sensitivity of NR1 to ER stress will have important consequences on receptor function.

NMDARs Mediate the Role of Monoamine Oxidase A in Pathological Aggression

“…The observed alterations may result from functional impairments of this enzymatic machinery; accordingly, previous studies showed that, in cortical neurons, the inhibition of the first enzymatic step of N -glycosylation enhances the degradation of nonglycosylated NR1, but not NR2A subunits (Gascón et al, 2007). The extensive N -glycosylation of NR1 (based on the attachment of high-mannose oligosaccharide side-chains onto 12 Asparagine sites) is essential for its oligomerization with NR2 subunits (Chazot et al, 1995; Standley and Baudry, 2000); thus, the increased expression in NR2A and NR2B may be paralleled by a reduced number of functional NR1 subunits.…”

Molecular Determinants Controlling NMDA Receptor Synaptic Incorporation

“…Some of the differences in amino acid sequence in the loops occur on or around predicted N-glycosylation sites, a mechanism used by chaperone proteins in the Golgi to identify and move proteins to distinct trafficking vesicles (Martinez-Maza et al, 2001; Nathanson, 2008). GluN2 subunits are glycosylated proteins (Clark et al, 1998) and glycosylation can affect the NMDA receptor’s assembly and function (Chazot et al, 1995; Everts et al, 1997; Standley and Baudry, 2000; Gascon et al, 2007). In polarized cells, a single glycosylation site can target proteins to the apical or basolateral membrane.…”

Ubiquitination in Postsynaptic Function and Plasticity

“…During assembly of heteromeric NMDA receptors, GluN1 subunits containing high-mannose glycans are ubiquitinated and degraded through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway during changes in synaptic activity (Gascon et al 2007, Kato et al 2005). This process is regulated by a dendritic spine–localized F-box ubiquitin E3 subunit, SCF Fbx2 , that binds specifically to high-mannose glycans (Yoshida et al 2002).…”