Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A-or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2B or NR2A, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day 7 (P7) hippocampus and P42 and 6-month-old CA1/CA2. Quantitative Western blot analysis revealed that 60 -70% of NR2A and 70 -85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A-or NR2B-specific interactions with synapseassociated proteins. Our results indicate that NR2A-or NR2B-containing NMDARs associate similarly with postsynaptic density-95 (PSD-95), synapse-associated protein 102, and PSD-93 at P42. However, NR2A-containing receptors coimmunoprecipitated a greater proportion of the synaptic proteins neuronal nitric oxide synthase, Homer, and ␤-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, collapsin response mediator protein 2, which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist primarily in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes.

The NR3A subunit of the N-methyl-D-aspartate receptor has been shown to form glutamatergic receptor complexes with NR1 and NR2 subunits and excitatory glycinergic receptor complexes with NR1 alone. We developed an antibody to NR3A and, using quantitative immunoblotting techniques, determined the degree of association between the NR3A subunit and the NR1 and NR2 subunits as well as changes in these associations during development. NR3A expression peaks between postnatal days 7 and 10 in the cortex, midbrain, and hippocampus and reaches higher maximal expression levels in these areas than in the olfactory bulb and cerebellum. Immunoprecipitation experiments with an anti-NR1 antibody demonstrated that the majority of NR3A is associated with NR1 in postnatal day 10 rat cortex (80 Ϯ 8%), decreasing by half (38 Ϯ 4%) in the adult rat cortex. Using the anti-NR3A antibody in immunoprecipitation studies, we find that 9.7 Ϯ 0.8% of NR1, 8.7 Ϯ 1.8% of NR2A, and 5.0 Ϯ 0.6% of NR2B are associated with NR3A at postnatal day 10. These values decrease by about half in adult rat cortex. The results of this study demonstrate that NR3A is expressed, distributed, and associated with other subunits in a manner that supports its role in synaptic transmission throughout the rat brain, perhaps playing different roles during development.The glutamate receptor family mediates the majority of fast excitatory synaptic transmission in the mammalian central nervous system. The ionotropic glutamate receptors are divided pharmacologically into three major groups: the Nmethyl-D-aspartate (NMDA) receptor (NMDAR), the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, and the kainate receptor. The NMDAR has received much attention because of its involvement in neuronal development, a variety of neurodegenerative diseases, and certain types of excitotoxicity (Dingledine et al., 1999;Cull-Candy et al., 2001). A voltage-dependent magnesium block and relatively high calcium permeability have implicated the NMDAR in a mechanism thought to be critical for certain types of learning and memory, namely long-term potentiation (McBain and Mayer, 1994).The NMDAR complex is a tetrameric or pentameric structure composed of at least two NR1 subunits and two or three subunits from the NR2 family (NR2A-D) (for review, see McBain and Mayer, 1994;Dingledine et al., 1999). The NR1 subunit is expressed throughout the central nervous system and is required for the formation of functional receptors.

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Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment pro®les of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII a subunit. However, synaptophysin, SAP-102, and the GABA A receptor b subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited signi®cantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of speci®c NR1 splice variants or NR2 subunits into this structure. The postsynaptic density (PSD), originally identi®ed in electron micrographs of the CNS, is the thickened, electrondense structure that lies directly apposed to the active zone at Type I, or asymmetric, synapses (Gray 1959). Recent identi®cation of a number of its component proteins has revealed various possible functions for this synaptic organelle' (for reviews see Ziff 1997; Walikonis et al. 2000). One such function is anchoring and organizing the necessary receptor and signaling proteins at the active zone in preparation for a fast response to presynaptic stimulation. Signaling proteins such as the a and b subunits of calcium/calmodulin dependent kinase II (CaMKII) are highly abundant in the PSD (Kennedy et al. 1983), as are a family of PDZ domain-containing proteins termed membrane-associated guanylate kinases (MAGUKs) (Dimitratos et al. 1999;Fanning and Anderson 1999;Garner et al. 2000). Members of the MAGUK family, including PSD-95 (Cho et al. 1992;Kistner et al. 1993), chapsyn-110/PSD-93 (Brenman et al. 1996Kim et al. 1996;Brenman et al. 1998), and SAP-102 (Lau et al. 1996;Muller et al. 1996), are highly enriched in the PSD and may serve as scaffolding proteins linking various signaling and receptor proteins and anchoring them to the cytoskeleton. Also highly enriched in the PSDs are receptor proteins such as the N-methyl-d-aspartate receptor (NMDAR) (Moon et al. 1994;Suen et al. 1998), a member of the ionotropic family of glutamate receptors that mediate the majority of excitatory transmission in the CNS. Abbreviations used: AMPA, alpha...