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NMDA receptors comprise a family of ionotropic glutamate receptors (1) with properties that indicate they have a central role in synaptic plasticity and memory formation (2). These receptors are formed by assembly of the principal subunit NR1 (3) with different modulatory NR2 subunits (NR2A-D) (4), A conspicuous structural feature of the NR2 subunits is their extended, intracellular COOH-terminal sequence distal to the last transmembrane region (5), which may anchor the receptors or assemble a signal-transducing complex for the voltage-dependent Ca sup 2+ entry through the glutamate-activated ion channel (6). Thus, we set out to identify intracellular proteins that bind to the NR2 subunits at synapses.
We used the two-hybrid system (7) to identify such cellular targets for the COOH-terminal domain of NR2 subunits. A bait consisting of the yeast GAL4 DNA binding domain (amino acids 1 to 147) fused to the entire COOH-terminal domain (627 amino acids) of NR2A (8) was expressed from a shuttle vector introduced into yeast strain YPB2 (9). This strain was transformed with a rat brain complementary DNA (cDNA) expression library (10) constructed in plasmid pGAD (9) to produce proteins tagged an the NH sub 2 -terminus with the GAL4 activation domain. Library clones activating the expression of both the selection marker HIS3 and the LacZ reporter gene were sequenced. Our screen identified a plasmid (pGAD-PSD) with nearly the entire coding region (10) for the prominent postsynaptic density protein PSD-95 (11, 12).
To determine whether other NR2 subunits also interact with PSD-95, we constructed baits consisting of COOH-terminal NR2 sequences tagged with the GAL4 DNA binding domain. These were tested for reporter gene activation after cotransfection of yeast with pGAD-PSD. We found that COOH-terminal sequences of NR2B (Fig. 1A) and NR2D (8) also interacted with PSD-95. (Figure 1A omitted) Dissection of the COOH-terminal NR2B sequences (Fig. 1A) revealed that activation of reporter genes depended on the presence of the seven amino acids at the COOH-terminus. Indeed, a bait constructed from synthetic DNA encoding only the COOH-terminal seven residues (8) showed activity, identifying these residues as those that mediate the interaction of NR2B with PSD-95. These residues are conserved (Fig. 1A) in the otherwise divergent cytoplasmic tails of the NR2 subunits (4). A similar sequence (PSVSTVV) (13) occurs at the COOH-termini of...