Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Abstract

NR2C-containing N-methyl-D-aspartate NMDA2 receptors are glutamate-gated ion channels that are critical for synapse formation, synaptic plasticity, and learning and memory (1, 2). They are members of the larger family of ionotropic glutamate receptors that also include AMPA and kainate receptors. Endogenous NMDA receptors are heteromeric channels containing two NR1 subunits combined with additional NR2 (NR2A-D) and NR3 (NR3A-B) subunits (2, 3). In contrast to the widespread distribution for NR1, NR2 subunits display unique spatiotemporal expression patterns throughout the central nervous system (4) and confer distinct pharmacological and functional properties on NMDA receptors (2). NMDA receptors in the cerebellum are composed of NR1 and NR2A-C subunits; the NR2C subunit is notably enriched in cerebellar granule cells and is up-regulated developmentally such that in the adult, NR1, NR2A, and NR2C are the predominant NMDA receptor subunits assembling to form functional receptors (2, 5). Moreover, NR2C-containing receptors have unique properties of low conductance and reduced sensitivity to Mg 2ϩ compared with NR2A-or NR2B-containing receptors (2,4,6). Despite this, the specific regulation of NR2C-containing receptors has not been studied extensively.Phosphorylation is an important mechanism regulating glutamate receptors and synaptic plasticity and occurs primarily in the cytosolic COOH-terminal domain of the receptor subunits. Phosphorylation of AMPA receptors by PKA and CaMKII directly affects channel function (7-9), receptor trafficking to synapses (10), and is critical for the expression of long term potentiation and long term depression (11, 12). Phosphorylation is an important regulator of NMDA receptor function and trafficking (13-15); however, the direct phosphorylation of NR2C has not been reported. Interestingly, NR2C contains a prototypic PKA phosphorylation site in the distal COOH-terminal region that is not conserved in NR2A and NR2B, suggesting that phosphorylation at this location might play a specific role in regulating NR2C-containing NMDA receptor complexes.In this study, we have investigated the regulation of NR2C by phosphorylation. We demonstrate that Ser 1244 is phosphorylated by both PKA and PKC in vitro and also on native NR2C in neurons. This serine is just upstream of the PDZ binding domain, which is conserved in NR2 subunits and is critical for NMDA receptor binding to PSD-95. We demonstrate that NR2C interacts directly with PSD-95; however, we find no effect of Ser 1244 phosphorylation on PSD-95 binding to NR2C. Unlike the PKA regulation of AMPA receptors (10), we find no effect of Ser 1244 phosphorylation on surface expression of NR1/NR2C NMDA receptors. However, we find that this site alters NMDA receptor channel function, because a phosphomimetic mutation (S1244E) accelerates channel kinetics. Thus, we find that this phosphorylation site in the distal COOH terminus regulates NR2C channel function.

Reciprocal signalling between NR2 subunits of the NMDA receptor and neuregulin1 and their role in schizophrenia

“…including PSD-95, synapse associated protein (SAP)102, PSD-93 and SAP97, with PSD-95 being the most studied (Kornau et al, 1995, Muller et al, 1996, Niethammer et al, 1996, Bassand et al, 1999, Sans et al, 2000, Chen et al, 2006, Sato et al, 2008, Cousins et al, 2009. These proteins form the DLG (disks large homolog) subfamily of the membrane-associated guanylate kinases (MAGUKs), which have three PDZ domains, a src homology 3 (SH3) domain and a guanylate kinase domain (GUK) (Montgomery et al, 2004).…”

Mitotic Inhibition of GRASP65 Organelle Tethering Involves Polo-like Kinase 1 (PLK1) Phosphorylation Proximate to an Internal PDZ Ligand

“…In other words, adjoining residues including Ser 189 may adopt a conformation in the folded PDZ2 domain that exposes the IGYGYL sequence, and phosphorylation of Ser 189 may induce a conformational change in the ligand that blocks its access to the binding groove. This mode of regulation has been suggested for the NMDA receptor subunit NR2C, which is phosphorylated on a serine adjacent to its PDZ ligand sequence that binds PSD-95 (45). A final possibility is that Ser 189 phosphorylation alters orientation of GRASP65 on the membrane.…”

Subunit-specific Regulation of N-Methyl-d-aspartate (NMDA) Receptor Trafficking by SAP102 Protein Splice Variants

“…Cells were transiently transfected with plasmids using the calcium phosphate method as described previously (18). Cells were harvested 24 h after transfection for Western blot analysis.…”

“…Yeast Two-hybrid Assay-The yeast two-hybrid assay was performed as described previously (18). Briefly, pBHA and pGAD10/pGADT7 constructs were co-transformed into the L40 yeast strain.…”