The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDARdependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A-and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.development ͉ glutamate receptor ͉ lateral mobility N MDA receptors (NMDARs) are heterotetrameric cation channels composed of NR1 and NR2͞3 subunits (1). NMDARs are assembled early in the endoplasmic reticulum, and both NR1 and NR2 subunits are necessary for their association and their successful cell surface targeting (2). In addition to glutamate and glycine, NMDARs require membrane depolarization to open with high probability (3), making this receptor a pre-and postsynaptic activity coincident detector involved in the induction of Hebbian synaptic plasticity. The functional properties of NMDARs depend also on the subunit composition, and such subunit heterogeneity of synaptic NMDARs is thought to play an important role during synaptic development, maturation, and plasticity processes (4). During synaptic development, the subunit composition of synaptic NMDARs changes from heterodimers containing predominantly NR2B subunits at early stages to heterodimers containing NR1͞NR2B, NR1͞NR2A, and NR1͞NR2A͞NR2B subunits at mature stage (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). This change often is associated with the refinement of neuronal connections within cortical areas, although this model has been challenged and, thus, is likely incomplete (4). The pathways by which neurons differentially traffic NR2A-and NR2B-containing NMDARs remain, however, an open question of crucial importance to understand the shaping of synaptic maturation and plasticity.Changes in NR2 subunit composition of NMDARs within synapses can be triggered by mechanisms that include differences in insertion (15), internalization (16, 17), and͞or lateral diffusion. Interestingly, NMDARs diffuse laterally at the neuronal surface (18,19). In immature neurons, synaptic NMDARs are replaced rapidly by extrasynaptic ones through lateral diffusion (18), suggesting that surface mobility of NMDARs may be involved in shaping mature NMDAR synaptic components. In this study, we i...

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using highresolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A-or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions. lateral diffusion | glutamate receptor | trafficking | biomimetic multivalent ligand | development T he identification of the cellular mechanisms involved in the regulation of glutamate receptor trafficking is crucial to our understanding of synaptic maturation and plasticity. One common paradigm of these processes is the activation of the calciumpermeable postsynaptic NMDA receptors (NMDARs). In the neocortex, the most abundant types of NMDARs are composed of NR1 subunits associated with NR2A (enriched in synapses) and/or NR2B subunits (1). Rapid changes in the synaptic 2A/2B NMDAR ratio have been reported during connection refinements and synaptic plasticity (2), and several key molecular interactions have been shown to control the trafficking of intracellular and membrane NMDARs (3-6).The intracellular proteins that interact with the C terminus of the subunits, through direct binding or modification of the phosphorylation state, are likely candidates for regulating the synaptic retention of NMDARs. Indeed, intracellular domains of NR2 subunits provide a binding motif for proteins of the postsynaptic density such as PSD-95 and SAP102 (7-10). The binding of the NR2B subunit C terminus to PDZ domain-containing scaffold proteins regulates, in part, the synaptic retention of this receptor (8,9,(11)(12)(13)(14). For the 2A-NMDARs, which make up the majority of synaptic NMDARs, the role of such interactions in synaptic retention remains controversial. Indeed, long-term expression of NR2A subunits with a truncated or mutated C terminus does not affect synaptic NMDAR currents in cerebellar or hippocampal neurons (9, 15), whereas deletion of the NR2A subunit C terminus sequence significantly reduces NMDAR synaptic signaling (11,14,16,17). Currently, there is no simple explanation for this discrepancy, and the use of long-term expression of exogenous NR subunits and lack of good pharmacological tools to discriminate betwee...

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co‐immunoprecipitated with assembled NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1‐1a, NR1‐2a, NR1‐4bc‐Myc, or NR2 subunit transfections revealed that co‐association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1‐2ac‐Myc‐trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N‐terminal domains. Anti‐APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti‐NR1 or anti‐NR2A antibodies co‐immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co‐expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.

N-Methyl-D-aspartate (NMDA) receptors are a subclass of the excitatory, ionotropic L-glutamate neurotransmitter receptors. They are important for normal brain function being both primary candidates for the molecular basis of learning and memory and in the establishment of synaptic connections during the development of the central nervous system. NMDA receptors are also implicated in neurological and psychiatric disorders. Their dysfunction which is primarily due to either hypo- or hyper-activity is pivotal to these pathological conditions. There is thus a fine balance between NMDA receptor-mediated mechanisms in normal brain and those in diseased states where receptor homeostasis is perturbed. Receptor activity is due in part to the number of surface expressed receptors. Understanding the assembly and trafficking of this complex, heteromeric, neurotransmitter receptor family may therefore, be pivotal to understanding diseases in which their altered activity is evident. This article will review the current understanding of the mechanisms of NMDA receptor assembly, how this assembly is regulated and how assembled receptors are trafficked to their appropriate sites in post-synaptic membranes where they are integral components of a macromolecular signalling complex.