NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1

Abstract

Phosphorylation regulates the function of ligand-gated ion channels such as the N-methyl d-aspartate (NMDA) receptor. Here we report a mechanism for modulation of the phosphorylation state and function of the NMDA receptor via an inhibitory scaffolding protein, RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. Peptides that disrupt the interactions between RACK1, NR2B, and Fyn induce phosphorylation and potentiate NMDA receptor-mediated currents. Therefore, RACK1 is a regulator of NMDA receptor function and may play a role in synaptic plasticity, addiction, learning, and memory.

Figure 1

RACK1 binds Fyn kinase. (a) Immunoprecipitation was performed with monoclonal IgM anti-RACK1 antibodies (lane 1) or with mouse IgM antibodies (lane 3). Membranes were probed with anti-RACK1 (Lower) or anti-Fyn antibodies (Upper). The presence of Fyn and RACK1 in NG108–15 homogenate was verified by Western blot analysis (50 μg, lane 2) (n = 3). (b) Increasing concentrations of RACK1 or MBP were blotted onto nitrocellulose membrane by using a slot-blot apparatus and overlaid with Fyn (75 units, 0.32 pmol/min/unit). Binding was detected with anti-Fyn antibodies (n = 3). Histogram (Right) shows densitometric analysis of binding. (c) Immunoprecipitations were carried out by using anti-RACK1 antibodies (lane 1). Membranes were probed with the antibodies indicated. The presence of the proteins in hippocampal homogenate was verified by Western blot analysis (50 μg, lane 2) (n = 3).

Figure 2

ctNR2B shares sequences of homology with Fyn and interacts with RACK1. (a) Sequences of homology between NR2B and Fyn were identified with blast search (National Center for Biotechnology Information) and confirmed by the alignment of both sequences (NR2B: rat, accession no. Q00960; Fyn: rat, accession no. NP_036887) with macvector Ver. 6.5.3 (Oxford). Lower-case letters represent amino acids that are different (nonidentical and nonhomologous). (b) 0.5 μg of MBP-ctNR2B (a.a. 1086–1482), MBP-ctNR1, MBP-ctNR2BΔN (a.a. 1171–1482), and MBP were blotted onto a nitrocellulose membrane by using a slot-blot apparatus and overlaid with RACK1 (500 ng). RACK1 binding was detected with anti-RACK1 antibodies. Histogram (Bottom) shows densitometric analysis of binding normalized to binding of RACK1 to ctNR2B ± SD (n = 3).

Figure 3

RACK1 associates with ctNR2B and Fyn. (a) [³⁵S]methionine-labeled proteins were generated in rabbit reticulocyte lysates programmed with the appropriate cDNA [RACK1 (a.a. 1–317), Fyn (a.a. 1–537), ctNR2B (a.a. 839–1482)]. Interaction of the proteins was determined by coimmunoprecipitation from the lysates with anti-RACK1 (lane 2), anti-Fyn (lane 3), and anti-NR2B (lane 4) antibodies. An aliquot of the triple translation reaction is shown in lane 1. Control immunoprecipitations are shown in lanes 5 and 6 (n = 6). (b) [³⁵S]methionine-labeled proteins were generated as in a. Interactions were determined by coimmunoprecipitation from the lysates with anti-NR2B (lane 1) and anti-Fyn (lane 2) antibodies. An aliquot of the double translation reaction is shown in lane 3 (n = 2). (c) [³⁵S]methionine-labeled Fyn and RACK1 were generated as in a. Empty vector control (lanes 1 and 3) or unlabeled ctNR2B (a.a. 839-1482; lanes 2 and 4) were added to the reaction mixture. Complexes were immunoprecipitated with anti-RACK1 antibodies (lanes 3 and 4) (n = 2). (d) Immunoprecipitations were carried out by using mc anti-NR2B (lane 1), anti-RACK1 (lane 2), and anti-GluR2 (lane 3). Controls included monoclonal anti-NR2B antibodies alone (0.25 μg, lane 5) as well as immunoprecipitations using mouse IgG (lane 4) and mouse IgM antibodies (data not shown). Membranes were probed with the antibodies shown that included polyclonal anti-NR2B antibodies. The presence of RACK1, Fyn, NR2B, and GluR2 in hippocampal homogenate was verified by Western blot analysis (lane 6) (n = 6).

Figure 4

Identification of the binding sites of RACK1, Fyn, and NR2B. (a) Five hundred nanograms of MBP-ctNR2BΔN (a.a. 1172–1482) and MBP-ctNR2BΔC (a.a. 839–1170) were blotted onto nitrocellulose membrane by using a slot-blot apparatus and overlaid with 500 ng of RACK1. Binding was detected with anti-RACK1 antibodies. Histogram (Right) shows densitometric analysis of binding (n = 3). (b) RACK1 (500 ng) was blotted onto nitrocellulose membrane and incubated with MBP-ctNR2B (a.a. 1086–1482; 500 ng) or Fyn (75 units, 0.32 pmol/min/unit) in the absence (c) or presence of peptides F1, N1, R14, and R7 (100 μM). Membranes were probed with anti-NR2B or anti-Fyn antibodies. Binding is presented as mean percent of control ± SD of three experiments. Results compared with R7 are statistically significant (P < 0.01). (c) MBP-ctNR2B (a.a. 1086–1482; 500 ng) was blotted as described in a and overlaid with RACK1 (500 ng) and increasing concentrations of peptides R14 (0–250 μM) and N2 (0–500 μM). Binding was detected with anti-RACK1 antibodies and presented as mean percent of control ± SD of three experiments. (d) R14, R7, F1, scrambled F1, N1, and N2 (10 μl of 10 mM) or vehicle (10 μl of 20% DMSO) were blotted onto nitrocellulose membrane. The membranes were incubated with RACK1 (3 μg, 1), MBP-ctNR2B (a.a. 1086–1482; 3 μg, 2), and Fyn (25 units, 0.32 pmol/min/unit, 3). The membranes were probed with anti-RACK1 (1), anti-NR2B (2), and anti-Fyn antibodies (3) (n = 3, except for N2 peptide, n = 2). (e) Yeast coexpressing RACK1ΔC (1–60) and ctNR2B (a.a. 839–1482) or RACK1ΔC (a.a. 1–60), and Fyn gave a positive result compared with yeast containing RACK1ΔC (1–60) and pGAD alone (n = 3).

Figure 5

RACK1 inhibits the phosphorylation of ctNR2B by Fyn, and peptides derived from RACK1, Fyn, or ctNR2B restore phosphorylation. (a) MBP-ctNR2B (a.a. 1086–1482; 750 ng) was incubated in the absence (lane 1) or the presence (lanes 2–6) of increasing concentrations of RACK1 or 1 μM MBP (lane 8) and 15 units of Fyn kinase (lanes 1–6 and 8). The presence and concentrations of MBP-ctNR2B (a.a. 1086–1482), Fyn, and RACK1 were verified by Western blot analysis (data not shown) (n = 5). (b) Kinase assays were performed in the presence of RACK1 (500 ng) and in the presence or absence of R7, N1 (100 μM), and N2 (500 μM). Data presented as mean percent of control ± SD (n = 3). Results compared with N1 or N2 are statistically significant (P < 0.01).

Figure 6

RACK1 inhibits NMDA receptor-mediated EPSCs, whereas peptides enhance NMDA receptor-mediated currents. (a) I_t/I₁ [the average size of the EPSCs for each minute (I_t) divided by the average size of the EPSCs of the first minute (I₁)] was plotted for recordings made by using standard whole-cell voltage–clamp configuration from CA1 pyramidal neurons. EPSCs were measured in the absence (Δ; n = 5) or presence of recombinant Tat-RACK1 (●; 15 μg/ml, n = 4) or RACK1 fragment that does not contain the putative Fyn/NR2B-binding site Tat-RACK1ΔN (⧫; 15 μg/ml, n = 4) in the intracellular solution. The bar histogram on the right is the mean ratio ± SD of amplitude of the current recorded at 15 min after the start of the recording (I₁₅) divided by that of the initial current (I₁). Top Right shows representative current tracers taken at the time indicated using one neuron perfused with Tat-RACK1. (b) EPSCs were recorded in the absence (Δ; n = 5), or presence of the Fyn-derived peptide, F1 (■; 500 μM; n = 5), the scrambled F1 peptide (500 μM; n = 5, shown only in the bar histogram) and the RACK1-derived peptides, R14 (⧫; 500 μM; n = 5) and R7 (●; 500 μM; n = 5). The bar histogram on the right is the mean ratio ± SD of I₁₀/I₁. Top Right shows representative current tracers taken at the time indicated using one neuron perfused with F1 and another perfused with R14. (c) EPSCs were recorded in the absence (Δ, n = 5) or presence (⧫, n = 5) of RACK1-derived peptide R14 (100 μM), and in the presence of R14 (100 μM) and PP2 (50 nM) (●, n = 5) or in presence of only PP2 (□, n = 5) (50 nM) in the bath solution. The bar histogram on the right is the mean ratio ± SD of I₁₅/I₁. Top Right shows representative current tracers taken at the time indicated using one neuron perfused with R14 and another perfused with R14 and PP2. (d) Before the recordings, PP2 (50 nM) was bath applied for 20 min. EPSCs were recorded in the presence of Tat-RACK1 (●; 15 μg/ml, n = 5) or RACK1-derived peptide R14 (500 μM; Δ; n = 4). (e) AMPA receptor-mediated EPSCs were measured in the absence (○; n = 4), or presence of recombinant Tat-RACK1 (Δ; 15 μg/ml, n = 4), or RACK1-derived peptide, R14 (■; 500 μM; n = 5) in the intracellular solution.