title: Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.
abstract: N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.
authors.affiliation: University College London School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Cousins
authors.forename: F Anne
authors.initials: FA
authors.lastname: Stephenson
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003902:Detergents / Q000494:pharmacology; D000076122:Disks Large Homolog 4 Protein; D005982:Glutathione Transferase / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000502:physiology*
chemical_list: D003902:Detergents; D000076122:Disks Large Homolog 4 Protein; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C501449:Dlg4 protein, rat; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor