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 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.
Bassand
P
P
Université René Descartes (Paris V), INSERM U-29, Paris, France.
Bernard
A
A
Rafiki
A
A
Gayet
D
D
Khrestchatisky
M
M
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001921:Brain / Q000378:metabolism; D002678:Chimera / Q000235:genetics; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D007536:Isomerism; D008297:Male; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D014018:Tissue Distribution / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000076122:Disks Large Homolog 4 Protein; C501119:Dlg1 protein, rat; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C501451:Mpp2 protein, rat; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; C033652:postsynaptic density proteins