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.

FIGURE 1.

The second non-ESDV, PSD-95α-binding site within the NMDA receptor C-terminal tail of NR2A subunits maps to amino acids 1382–1389 and is direct, demonstration by yeast two-hybrid interaction and in vitro GST pulldown assays. A, schematic diagram showing the NR2A C-terminal region containing the ESDV PDZ binding domain and the amino acid sequence of the second PSD-95 binding domain with the putative SH3 binding domain highlighted and the arrow denoting the position of the new truncation. B, HEK 293 cells were co-transfected in parallel with pCISNR1-1a, pCISNR1-1a/pCISNR2A, pCISNR1-1a/pCISNR2A¹⁴⁶⁰, pCISNR1-1a/pCISNR2A^EADV, pCISNR1-1a/pCISNR2A¹⁴²⁰, pCISNR1-1a/pCISNR2A¹³⁸⁹, and pCISNR1-1a/pCISNR2A¹³⁸² + pGW1PSD-95α^c-Myc clones. Transfected cells were harvested 24 h post-transfection; cell homogenates were prepared and detergent-solubilized; immunoprecipitations were carried out using anti-NR1 C2 or anti-nonimmune IgG antibodies, and immune pellets were analyzed by immunoblotting using anti-NR1 C2, anti-NR2A(44–58), or anti-c-Myc antibodies to detect NR1, NR2A, and PSD-95 as shown. The format of the immunoblots is identical for all NR1/NR2A/PSD-95α^c-Myc combinations where lane 1 is the detergent-solubilized sample; lane 2 is the anti-nonimmune IgG control pellet, and lane 3 is the anti-NR1 C2 immune pellet. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections. C, Y189 S. cerevisiae yeast cells transformed with pGBKT7 NR1-1a or pGBKT7 NR2A C-terminal bait constructs and AH109 S. cerevisiae cells transformed with pGADT7-PSD-95α^c-Myc were mated and grown on −Leu/−Trp/−His/−Ade agar plates, and the number of diploid colonies were counted after 7 days of incubation at 30 °C, all as described under “Experimental Procedures.” The figure depicts the bait and fish constructs and the resulting diploid colonies. The numbers of diploid colonies were semi-quantified as a percentage of the number of colonies obtained for the NR2A full-length C-terminal domain, thus ++++ = 100%; +++ = 80–90%; ++ = 40–79%; + = <10%, and − = 0 colonies. The results are the means of at least n = 3 independent mating assays for all combinations shown. D, histogram showing the percentages of diploid activity (means ± S.E.) where the full-length NR2A C-terminal peptide is 100% (912 ± 68 colonies, n = 21); ***, p < 0.01; ****, p < 0.005; *****, p < 0.001. E, immunoblots resulting from GST in vitro pulldown assays. The NR2A C-terminal constructs were immobilized on glutathione-agarose, incubated with purified His-PSD-95α^c-Myc and washed, and bound proteins were eluted and analyzed by immunoblotting, all as described under “Experimental Procedures.” The antibodies used for the immunoblotting were anti-c-Myc to detect PSD-95α^c-Myc; anti-NR2A(1381–1394) to detect NR2A, and anti-NR2A(1252–1391) antibodies because NR2A¹³⁸² will not be detected by anti-NR2A(1381–1394) antibodies. The results shown are representative of n = 5 independent assays.

FIGURE 2.

Identification of the SH3 binding domain motif, PSDPYK, as the second PSD-95α-binding site within the NR2A C-terminal domain, demonstration by co-immunoprecipitation and yeast two-hybrid interaction assays. A, HEK 293 cells were co-transfected in parallel with pCISNR1-1a/pCISNR2A, pCISNR1-1a/pCISNR2A^ASDA, pCISNR1-1a/pCISNR2A¹⁴⁶⁰, pCISNR1-1a/pCISNR2A^1460-ASDA, pCISNR1-1a/pCISNR2A¹⁴²⁰, pCISNR1-1a/pCISNR2A^1420-ASDA, pCISNR1-1a/pCISNR2A¹³⁸² + pGW1PSD-95α^c-Myc clones. Transfected cells were harvested 24 h post-transfection and cell homogenates were prepared and detergent-solubilized; immunoprecipitations were carried out using anti-NR1 C2 or anti-nonimmune IgG antibodies; immune pellets were analyzed by immunoblotting using anti-NR1 C2, anti-NR2A(44–58), or anti-c-Myc antibodies to detect NR1, NR2A, and PSD-95α^c-Myc as shown. The format of the immunoblots is identical for all NR1/NR2A/PSD-95α^c-Myc combinations where lane 1 is detergent-solubilized sample; lane 2 is anti-nonimmune IgG control pellet, and lane 3 is anti-NR1 C2 immune pellet. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections. B, Y189 S. cerevisiae yeast cells transformed with pGBKT7 NR2A C-terminal bait constructs as shown and AH109 S. cerevisiae cells transformed with pGADT7-PSD-95α^c-Myc were mated and grown on −Leu/−Trp/−His/−Ade agar plates, and the number of diploid colonies counted after 7 days of incubation at 30 °C are all as described under “Experimental Procedures.” The figure depicts the bait and fish constructs and the resulting diploid colonies. The number of diploid colonies was semi-quantified as a percentage of the number of colonies obtained for the NR2A full-length C-terminal domain and thus ++++ = 100%; +++ = 80–90%; ++ = 40–79%; + = <10%, and − = 0 colonies. C, histogram showing the percentages of diploid activity where the full-length NR2A C-terminal peptide is 100%, and as in Fig. 1, 912 ± 68 colonies (n = 21). The values are the means ± S.E. for n = 6; ****, p < 0.005; *****, p < 0.001.

FIGURE 3.

Peptide inhibition of the co-immunoprecipitation of NR2A and PSD-95 from detergent extracts of adult rat brain. Detergent extracts of adult rat brain were prepared, and triplicate samples were co-incubated with anti-NR2A(44–58 Cys) or nonimmune Ig (duplicates) and peptides encompassing the NR2A SH3 binding domain (SH3), the ESDV site (ESDV), or the respective scrambled (scr) control peptides, SH3scr and ESDVscr overnight at 4 °C. Protein A-Sepharose was added for precipitation of antigen·antibody complexes, and immune pellets were analyzed by immunoblotting all as described under “Experimental Procedures.” A is immunoblotting with anti-NR2A(44–58 Cys) and anti-PSD-95 antibodies; B is a histogram summary of the NR2A immunoblots; C is a histogram summary of the PSD-95 immunoblots. The immunoblots shown are representative of n = 6 immunoprecipitations from n = 6 independent detergent-solubilized preparations. The values are the means ± S.E.; *, p < 0.05; **, p < 0.025; ***, p < 0.01; *****, p < 0.001.

FIGURE 4.

Mapping the NMDA receptor NR2A binding domains within PSD-95α by yeast two-hybrid interaction assays. A, schematic diagram showing the PSD-95 constructs highlighting the protein domains of PSD-95. B–E, Y189 S. cerevisiae yeast cells were transformed with pGBKT7 NR2A C-terminal bait constructs, and AH109 S. cerevisiae cells were transformed with pGADT7-PSD-95α^c-Myc constructs as shown. Cells were mated and grown on −Leu/−Trp/−His/−Ade agar plates, and the number of diploid colonies counted after 7 days incubation at 30 °C all as described under “Experimental Procedures.” The number of diploid colonies were semi-quantified as a percentage of the number of colonies obtained for the NR2A full-length C-terminal domain, and thus ++++ = 100%; +++ = 80–90%; ++ = 40–79%; + = <10%, and − = 0 colonies. C and E are histogram summaries, where the values shown are the means ± S.E. for n = 3 (C) and n = 4 (E).

FIGURE 5.

Refinement of a novel binding domain that contributes to the association of the NR2B C-terminal tail with PSD-95α, demonstration by co-immunoprecipitation and yeast two-hybrid interaction assays. A, schematic diagram showing the NR2B C-terminal region highlighting the ESDV PDZ binding domain, the amino acid sequence of the second PSD-95 binding domain, and the arrow denoting the position of the new truncation. Also highlighted are the potential protein binding domain sites that are mutated, i.e. PRSP → ARSA and TDI → ADA. B and C, HEK 293 cells were co-transfected in parallel with pCISNR1-1a/pCISNR2B^FLAG, pCISNR1-1a/pCISNR2B^FLAG/1478, pCISNR1-1a/pCISNR2B^FLAG/EADV, pCISNR1-1a/pCISNR2B^FLAG/1149, pCISNR1-1a/pCISNR2B^FLAG/1120, pCISNR1-1a/pCISNR2B^FLAG/1086, pCISNR1-1a/pCISNR2B^{FLAG/1157-ARSA}, and pCISNR1-1a/pCISNR2B^{FLAG/1157-ADA} + PSD-95α^c-Myc clones. Transfected cells were harvested 24 h post-transfection; cell homogenates were prepared and detergent-solubilized; immunoprecipitations carried out using anti-NR1 C2 or anti-nonimmune IgG antibodies, and immune pellets were analyzed by immunoblotting using anti-NR1 C2, anti-FLAG, or anti-c-Myc antibodies to detect NR1, NR2B, and PSD-95 as shown. The format of the immunoblots is identical for all NR1/NR2B/PSD-95α^c-Myc combinations where lane 1 is detergent-solubilized sample; lane 2 is anti-nonimmune IgG control pellet, and lane 3 is anti-NR1 C2 immune pellet. The immunoblots are representative of at least n = 5 immunoprecipitations from n = 5 independent transfections. D and E, Y189 S. cerevisiae yeast cells transformed with pGBKT7 NR2B C-terminal bait constructs and AH109 S. cerevisiae cells transformed with pGADT7-PSD-95α^c-Myc were mated and grown on −Leu/−Trp/−His/−Ade agar plates, and the number of diploid colonies counted after 7 days of incubation at 30 °C are all as described under “Experimental Procedures.” D depicts the bait and fish constructs and the resulting diploid colonies. The number of diploid colonies were semi-quantified as a percentage of the number of colonies obtained for the NR2B full-length C-terminal domain, thus ++++ = 100%; +++ = 80–90%; ++ = 40–79%; + = <10%, and − = 0 colonies. E, a histogram showing the percentages of diploid activity (means ± S.E., n = 6) where the full-length NR2B C-terminal peptide is 100% (422 ± 54 colonies, n = 18); *, p < 0.05; **, p < 0.025; *****, p < 0.001.

FIGURE 6.

Proposed mechanisms for the interaction between NMDA receptor NR2A C-terminal tails and PSD-95. The figure shows possible models for the association between PSD-95 and NR2A subunits within an assembled tetrameric NMDA receptor. A, PSD-95 binds to one NR2A subunit via both the ESDV and non-ESDV site leading to a ratio of NR2A/PSD-95, 1:1. B, PSD-95 binds to one NR2A via the ESDV site and to the second NR2A via the non-ESDV site leading to a ratio of NR2A/PSD-95, 2:1. C, NR2A/PSD-95 ratio is 1:2 because two molecules of PSD-95 bind to one NR2A, one via the ESDV site and the other via the non-ESDV site.