Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.
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.
Mesh Terms:
Animals, Binding Sites, Detergents, Disks Large Homolog 4 Protein, Glutathione Transferase, HEK293 Cells, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Protein Interaction Domains and Motifs, Protein Structure, Tertiary, Rats, Receptors, N-Methyl-D-Aspartate, Saccharomyces cerevisiae, Two-Hybrid System Techniques, src Homology Domains
Animals, Binding Sites, Detergents, Disks Large Homolog 4 Protein, Glutathione Transferase, HEK293 Cells, Humans, Immunoprecipitation, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Protein Interaction Domains and Motifs, Protein Structure, Tertiary, Rats, Receptors, N-Methyl-D-Aspartate, Saccharomyces cerevisiae, Two-Hybrid System Techniques, src Homology Domains
J. Biol. Chem.
Date: Apr. 13, 2012
PubMed ID: 22375001
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