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.

NMDA receptors are a subclass of excitatory, ionotropic glutamate receptors. They are composed of obligatory NR1 subunits co-assembled with NR2 subunits of which there are four types yielding four major NMDA receptor subclasses NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D (reviewed in e.g. Cull-Candy et al. 2001). NMDA receptors are clustered at synapses via their interaction with the scaffolding protein, post-synaptic density (PSD)-95 (Kornau et al. 1995). The association between NMDA receptors and PSD-95 is mediated via the motif, ES(D/E)V that is common to all NR2 subunit C-termini. A PSD-95 binding motif, threonine serine valine valine, is also present in the C2' exon of NR1-3a,b and NR1-4a,b splice variants. Co-transfection of PSD-95 with NMDA NR1/NR2A or NR1/NR2B NMDA receptor clones has been shown to enhance in an ES(D/E)Vdependent manner, the expression of NR2A and NR2B subunits resulting in an increased cell surface expression of assembled NR1/NR2A and NR1/NR2B subtypes (Rutter and Stephenson 2000;Rutter et al. 2002;Lin et al. 2004). Received August 8, 2007; accepted September 18, 2007. Address correspondence and reprint requests to F. Anne Stephenson, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK. E-mail: anne.stephenson@pharmacy.ac.uk 1 The present address of Michalis Papadakis is the Acute Stroke Programme, Nuffield Department of Clinical Medicine, Level 7, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.Abbreviations used: ESDV, glutamate, serine, aspartate, valine; MAGUK, membrane associated guanylate kinase; PBS, phosphate buffered saline; PDZ, post-synaptic density protein (PSD95), Drosophila disc large tumour suppressor (DlgA), and zo-1 protein; PSD, postsynaptic density; SAP, synapse associated protein. AbstractNMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C a...

N-Methyl-D-aspartate receptors are a subclass of ligandgated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an ϳ3 ؋ 10 4 decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors. N-Methyl-D-aspartate (NMDA)2 receptors are a subclass of the family of excitatory, ionotropic L-glutamate neurotransmitter receptors. They are important due to the pivotal role they play in synaptic transmission, synaptic plasticity, and synaptogenesis during the development of the central nervous system. They are activated by the binding of the co-agonists, L-glutamate and glycine, and the alleviation of voltage-dependent blockade by magnesium ions. Receptor activation results within milliseconds in the opening of an integral ion channel that has a high permeability for calcium ions. The dysfunction of NMDA receptors is implicated in a broad spectrum of neurodegenerative and psychiatric disorders. This is primarily due to the permeability properties of the NMDA receptor channel; overactivation results in inappropriate increases in intracellular calcium ion concentration and excitotoxic neuronal cell death (reviewed in Ref. 1).NMDA receptors are a family of ligand-gated, heteromeric, integral membrane proteins. There are seven NMDA receptor genes encoding the NR1, NR2A-NR2D, and NR3A-NR3B subunits. Functional NMDA receptors are formed from the coassembly of the obligatory NR1 subunit with NR2 and/or NR3 subunits. The quaternary ...

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Neuropilin tolloid-like 1 (Neto1), is a CUB domain-containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/ GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1HA was shown to co-immu- Neuropilin tolloid-like 1 (Neto1) and Neuropilin tolloid-like 2 (Neto2) are complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane proteins that were recently identified as novel components of glutamatergic neurotransmitter receptor complexes. Neto1 and Neto2 have both been described as auxiliary subunits of kainate receptors since they were shown to alter the trafficking, channel kinetics and pharmacology of this glutamate receptor subtype in a subunitdependent manner ( Abbreviations used: AMPA, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid; APP, amyloid precursor protein; CUB, complement C1r/C1s, Uegf, Bmp1; ELISA, enzyme linked immunoadsorbent assay; GluN1, GluN2A, GluN2B, NMDA receptor NR1 subunit, NR2A subunit, NR2B subunit; Neto1, neuropilin tolloid-like 1; NMDA, N-methyl-D-aspartate; PSD-95, post-synaptic density 95; SAP102, synapse associated protein 102. 554