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

The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are en coded 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 generate d by alternative splicing of the C1 and C2 exon cassettes, while four diffe rent genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors res ult from the heteromultimeric assembly of NR1 variants with distinct NR2 su bunits. The NR2B subunit interacts with post-synaptic density protein 95 (P SD-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-termin al 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. Usin g 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 d espite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the N R2A 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 th e PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interacti ons of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemi stry, we also compared the distribution of the PSD-95, NR2A and SAP97 prote ins in adult rat brain, and we show that in the cortex, hippocampus and cer ebellum, there is evidence for colocalization of these proteins.