Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD‐95 and SAP97

Abstract

The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins. Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif. Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.

Intracellular protein interaction mapping with FRET hybrids

“…An identical ligand preference was identified with FRET hybrids. Four C-terminal residues of the ligand PDZ2-3 (STVV-COOH) identified in the current study with FRET hybrids are identical to those from the NR1 subunit of NMDA receptor, although Y2H assays have not identified this interaction (40). Finally, short peptides derived from the C terminus of NMDA receptor (NR2) have been shown to prevent neuronal death after stroke in a rat model (41).…”

An NMDA Receptor ER Retention Signal Regulated by Phosphorylation and Alternative Splicing

“…In the case of NMDA receptors, NR2 C-terminal domains interact with PDZ domains of the synaptic scaffolding protein postsynaptic density-95 and related family members to target and anchor NMDA receptor complexes at synapses Sheng and Lee, 2000). Unlike the PDZ-binding motifs of NR2 subunits (IESDV), the binding partners and cellular function of the consensus C2Ј PDZ-binding motif (VSTVV) of NR1 remain unknown [Bassand et al (1999), but see Kornau et al (1995)]. …”

In vivo blockade of N-methyl-d-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex

“…Among the NMDA receptor subunits, the NR1 is necessary for NMDA receptor function, but the NR2 subunits may be relatively more central in determining NMDA receptor localization (Mori et al, 1998;Steigerwald et al, 2000;Mohrmann et al, 2002) because their C-termini are longer and permit interaction with postsynaptic density (PSD) scaffolding proteins, such as PSD-95 (Niethammer et al, 1996;Bassand et al, 1999;Sheng and Pak, 2000). Among the NR2 subunits, the NR2A and NR2B subtypes are the most prevalent in the cerebral cortex (Watanabe et al, 1992), and of the two, the NR2B subunits are shown to be particularly influential in learning and memory (Tang et al, 1999;Tang and Schuman, 2002) as they prolong NMDA receptor currents, thus allowing greater Ca influx (Mori and Mishina, 1995;Dingledine et al, 1999).…”

“…In vitro and fewer in vivo preparations have been used to demonstrate that NMDA receptors, too, are removed from or inserted into synaptic membrane (Rao and Craig, 1997;Liao et al, 1999;Quinlan et al, 1999;Heynen et al, 2000;Barria and Malinow, 2002;Grosshans et al, 2002;Tovar and Westbrook, 2002). However, since nearly all of these studies have used neonatal tissues, whether or not such dynamic properties of NMDA receptor subunits persist at mature synapses, in vivo, is a topic that remains relatively unexplored.Among the NMDA receptor subunits, the NR1 is necessary for NMDA receptor function, but the NR2 subunits may be relatively more central in determining NMDA receptor localization (Mori et al, 1998;Steigerwald et al, 2000;Mohrmann et al, 2002) because their C-termini are longer and permit interaction with postsynaptic density (PSD) scaffolding proteins, such as PSD-95 (Niethammer et al, 1996;Bassand et al, 1999;Sheng and Pak, 2000). Among the NR2 subunits, the NR2A and NR2B subtypes are the most prevalent in the cerebral cortex (Watanabe et al, 1992), and of the two, the NR2B subunits are shown to be particularly influential in learning and memory (Tang et al, 1999;Tang and Schuman, 2002) as they prolong NMDA receptor currents, thus allowing greater Ca influx (Mori and Mishina, 1995;Dingledine et al, 1999).…”