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.
Highlights
NMDA receptors are organized via scaffold proteins into macromolecular signaling complexes at synapses
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 postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) scaffold proteins
Refinement of the Second post-synaptic density (PSD)-95-binding Site within the NMDA Receptor NR2A C-terminal Domain—We reported previously that the second non-ESDV PSD-95-binding site within the NMDA receptor NR2A C-terminal domain mapped to NR2A(1382–1420) (Fig. 1A)
Summary
NMDA receptors are organized via scaffold proteins into macromolecular signaling complexes at synapses. 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 the function of NMDA receptor subtypes at synapses. They are found predominantly at extra-synaptic sites but can be found within synapses due in part to their enhanced lateral mobility compared with NR2A receptors [3] This specialized distribution of the different NMDA receptor subclasses is thought to be mediated via their association with the post-synaptic density (PSD)-95 membrane-associated guanylate kinase (MAGUK) family of scaffold proteins (reviewed in Ref. 4). We demonstrate definitively that the interactions of NMDA receptors with these second PSD-95 binding domains are direct
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