NMDA receptors are a family of glutamate-gated ion channels that regulate various CNS functions such as synaptic plasticity and learning. However hypo- or hyper-activation of NMDA receptors is critically involved in many neurological and psychiatric conditions such as pain, stroke, epilepsy, neurodegeneration, schizophrenia, and depression. Thus, it is important to identify mechanisms (such as by targeted ubiquitination) that regulate the levels of individual subtypes of NMDA receptors. In this study, we used a series of tagged, carboxy terminal constructs of GluN2D to identify associating proteins from rat brain. Of seven different GluN2D C-terminal fragments used as bait, only the construct containing amino acids 983-1097 associated with an E3 ubiquitin ligase, Nedd4. A direct interaction between GluN2D and Nedd4 was confirmed both in vivo and in vitro. This association is mediated by an interaction between GluN2D's C-terminal PPXY motif and the 2nd and 3rd WW domains of Nedd4. Of the four GluN2 subunits, Nedd4 directly interacted with GluN2D and also weakly with GluN2A. Nedd4 coexpression with GluN2D enhances GluN2D ubiquitination and reduces GluN1/GluN2D NMDA receptor responses. These results identify Nedd4 as a novel binding partner for GluN2D and suggest a mechanism for the regulation of NMDA receptors that contain GluN2D subunits through ubiquitination-dependent downregulation. This article is part of the Special Issue entitled 'Glutamate Receptor-Dependent Synaptic Plasticity'.

N-methyl-d-aspartate receptors (NMDARs) have multiple prominent roles in CNS function but their excessive or insufficient activity contributes to neuropathological/psychiatric disorders. Consequently, a variety of positive and negative allosteric modulators (PAMs and NAMs, respectively) have recently been developed. Although these modulators bind to extracellular domains, in the present report we find that the NMDAR's intracellular C-terminal domains (CTDs) significantly influence PAM/NAM activity. GluN2 CTD deletion robustly affected NAM and PAM activity with both enhancing and inhibiting effects that were compound-specific and NMDAR subunit-specific. In three cases, individual PAMs became NAMs at specific GluN2-truncated receptors. In contrast to GluN2, GluN1 CTD removal only reduced PAM activity of UBP684 and CIQ, and did not affect NAM activity. Consistent with these findings, agents altering phosphorylation state or intracellular calcium levels displayed receptor-specific and compound-specific effects on PAM activity. It is possible that the GluN2's M4 domain transmits intracellular modulatory signals from the CTD to the M1/M4 channel gating machinery and that this site is a point of convergence in the direct or indirect actions of several PAMs/NAMs thus rendering them sensitive to CTD status. Thus, allosteric modulators are likely to have a marked and varied sensitivity to post-translational modifications, protein-protein associations, and intracellular ions. The interaction between PAM activity and NMDAR CTDs appears reciprocal. GluN1 CTD-deletion eliminated UBP684, but not pregnenolone sulfate (PS), PAM activity. And, in the absence of agonists, UBP684, but not PS, was able to promote movement of fluorescently-tagged GluN1-CTDs. Thus, it may be possible to pharmacologically target NMDAR metabotropic activity in the absence of channel activation.