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Regulation of NMDA Receptors by an Associated Phosphatase-Kinase Signaling Complex

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Science  02 Jul 1999:
Vol. 285, Issue 5424, pp. 93-96
DOI: 10.1126/science.285.5424.93

Abstract

Regulation of N-methyl-d-aspartate (NMDA) receptor activity by kinases and phosphatases contributes to the modulation of synaptic transmission. Targeting of these enzymes near the substrate is proposed to enhance phosphorylation-dependent modulation. Yotiao, an NMDA receptor–associated protein, bound the type I protein phosphatase (PP1) and the adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) holoenzyme. Anchored PP1 was active, limiting channel activity, whereas PKA activation overcame constitutive PP1 activity and conferred rapid enhancement of NMDA receptor currents. Hence, yotiao is a scaffold protein that physically attaches PP1 and PKA to NMDA receptors to regulate channel activity.

The molecular organization of the postsynaptic density (PSD) is thought to be essential for the fidelity and precision of synaptic signaling events. Clustering and immobilization of neurotransmitter receptors and ion channels is maintained by an intricate system of protein-protein interactions (1). For example, NMDA receptors are clustered and coupled to the cytoskeleton through association with PDZ domain–containing proteins, α-actinin, and neurofilaments (2). Many signaling pathways converge on the NMDA receptor (3), allowing the regulation of channel activity in response to the generation of second messengers such as Ca2+ and cAMP (4, 5). PKA and PP1 activities modulate NMDA receptor function and appear to act in opposition to each other (5, 6). Individual targeting or anchoring proteins such as AKAP79 and spinophillin localize the kinase and phosphatase at the PSD (7, 8).

A two-hybrid screen for proteins that bind the NMDA receptor subunit isoform NR1A identified a protein called yotiao that interacts with the COOH-terminal C1 exon cassette of the ion channel (9). We isolated cDNAs encoding fragments of yotiao by an interaction cloning strategy to identify A-kinase anchoring proteins (AKAPs) (10) and confirmed that the protein bound NR1A (11). Expression of full-length yotiao fused to green fluorescent protein (GFP) in HEK 293 cells (12) resulted in detection of a ∼210-kD protein that bound the type II regulatory subunit of PKA (RII), as assessed by overlay assay (Fig. 1A). Immunoprecipitations with antiserum to yotiao from brain extracts also isolated an RII binding protein and were enriched by a factor of 10.5 ± 2 (n = 3) for PKA catalytic subunit activity (Fig. 1, B and C). We mapped the RII binding site to a region between residues 1229 and 1480 by screening a family of recombinant yotiao fragments (Fig. 1D) in the overlay assay (Fig. 1, E and F). Residues 1440 to 1457 appear to include the principal determinants for RII interaction because a synthetic peptide encompassing this region blocked RII binding (Fig. 1F). These findings indicate that yotiao functions to anchor PKA to NMDA receptors.

Figure 1

Identification of yotiao as an AKAP. (A) Binding of full-length yotiao to RII. Lysates prepared from HEK 293 cells transfected with control vector or a yotiao-GFP fusion construct (12) were subjected to RII overlay assay as described (23). (B) Immunoprecipitation of yotiao. Preimmune or immune (αYotiao) sera were used for immunoprecipitation from rat brain extracts as described (24). Immunoprecipitated complexes were blotted and subjected to RII overlay assay (23). (C) Coprecipitation of PKA activity with yotiao immune sera. Immunoprecipitated material from rat brain extracts was incubated with cAMP (10 mM) and assayed for kinase activity (24) in the absence (control) and presence (+PKI) of 4 μM PKA inhibitor PKI (15). Values show activity relative to that associated with preimmune serum and are expressed as means ± SEM of three separate experiments. (D) Schematic representation of recombinant yotiao fragments (25). Abbreviations for amino acid residues: A, Ala; E, Glu; F, Phe; I, Ile; K, Lys; L, Leu; M, Met; Q, Gln; S, Ser; and V, Val. (E andF) Mapping of the RII binding domain on yotiao. Fragments of yotiao were blotted and assayed for RII binding by overlay assay in the absence (E) and presence (F) of a yotiao peptide encompassing residues 1440 to 1457 (10 μM). Equal amounts of protein were present in each lane, as determined by staining of protein on the membrane. Relative molecular mass standards (in kilodaltons) are at the left in (A), (B), and (E).

Because PP1 activity participates in the regulation of NMDA receptors (6), we conducted experiments to address whether the phosphatase also associated with yotiao. Immunoprecipitation of yotiao from brain extracts resulted in the copurification of PP1 (Fig. 2A). In overlay assays, PP1 bound to recombinant fragments (Fig. 2B) expressed in Escherichia coli (Fig. 2C) encompassing residues 1171 to 1229 of yotiao (Fig. 2, C and D) and the PP1 targeting inhibitor peptide Gm (13) blocked PP1 binding to yotiao (Fig. 2E). A hallmark of some PPI targeting subunits is the presence of a Lys-Val-X-Phe (KVXF) motif that binds to an allosteric site on the catalytic subunit of the phosphatase (13). Although yotiao contains this motif, it is not essential for interaction between yotiao and PP1. Binding of yotiao had no effect on PP1 activity toward an NR1A receptor fragment, and it enhanced activity by a factor of 1.9 ± 0.2 (n = 6) toward phosphorylasea with a median effective concentration (EC50) of 52 nM (Fig. 2F). These results show that yotiao is not an inhibitor of PP1 activity, hence yotiao may target active PP1 to the NMDA receptor.

Figure 2

PP1 targeting by yotiao. (A) Coimmunoprecipitation of PP1 with yotiao. Immunoprecipitations from rat brain extracts were done as described (24) with affinity-purified antibodies to yotiao or control immunoglobulin G (IgG). Precipitates were blotted and probed with antibodies to PP1, glutamic acid decarboxylase (GAD), or yotiao (26). (B) Schematic representation of recombinant yotiao fragments (25). (C to E) Direct interaction of PP1 with yotiao in overlay assays. Bacterial extracts expressing recombinant fragments of yotiao were separated by SDS-PAGE, transferred to a membrane, stained with Coomassie blue (C), and subjected to PP1 overlays (26) in the absence (D) and presence (E) of 10 μM Gm peptide (13). Relative molecular mass standards (in kilodaltons) are at the left. (F) Yotiao does not inhibit PP1 catalytic activity. Phosphatase activity was assayed (27) using phosphorylase a as substrate in the presence of increasing concentrations of recombinant yotiao fragment (residues 808 to 1385). Phosphatase activity was also assayed using PKA-phosphorylated GST-NR1A fusion protein (NR1A; solid bars) in the absence (control) or presence (yotiao) of 500 nM recombinant yotiao fragment (residues 808 to 1385) (27). The number of times each experiment was performed is indicated.

Currents through NMDA receptors are enhanced after activation of PKA (5) or inhibition of PP1 (6). We made whole-cell recordings of transfected HEK 293 cells (14) that expressed NMDA receptors alone or NMDA receptors and yotiao. Application of the cell-permeant cAMP analog 8-(4-chlorophenylthio)-cAMP (8-CPT cAMP) enhanced NMDA currents to a greater extent in cells expressing yotiao (54.9 ± 11.0%; n = 10; P < 0.01) than in control cells (16.1 ± 4.6%; n = 8) (Fig. 3, A and B). The effect of cAMP was inhibited by introduction of the PKA inhibitor peptide PKI 5-24 (15) or the RII anchoring inhibitor peptide Ht31 (16) through the patch pipette. These results indicate that an anchored pool of PKA was required for augmentation of the current (Fig. 3B).

Figure 3

Facilitation by yotiao of cAMP-dependent modulation of recombinant NMDA receptor currents. (A) Whole-cell recordings from HEK 293 cells expressing heteromeric NMDA receptors consisting of NR1A and NR2A (14). Currents were evoked every 30 s by 500-ms applications of glutamate (1 mM) in the presence of glycine (100 μM). Examples of the time course for modulation of recombinant NMDA receptor currents by the cell-permeant cAMP analog 8-CPT cAMP are shown for a cell in which yotiao was expressed (solid circles) and a control cell (open squares). Currents were normalized to the peak of the first sweep recorded from each cell. Representative traces are included from a yotiao-expressing cell (1) before, (2) during, and (3) after 8-CPT cAMP treatment. (B) Bar graph of the percent increases in peak whole-cell current from control cells, yotiao-expressing cells, yotiao-expressing cells in the presence of PKI peptide (10 μM), and yotiao-expressing cells in the presence of Ht31 peptide (10 μM) upon application of 8-CPT cAMP. Significant differences (*P < 0.01 compared to control; **P < 0.01 compared to yotiao) and the number of times each experiment was performed are indicated. (C) Bar graph of the percent increases in peak whole-cell current in response to application of 8-bromo-cAMP (8-Br cAMP; 100 μM). Solid bar, cells expressing NR1A; open bars, cells expressing NR1C. Expression of yotiao is indicated. Significant differences (*P < 0.01 compared to NR1A) and the number of times each experiment was performed are indicated.

Yotiao did not facilitate the cAMP-dependent modulation of NMDA receptors containing the NR1C subunit, which lacks the C1 exon (Fig. 3C). This suggests that yotiao selectively regulates NMDA receptors containing the C1 exon.

To test for a role of yotiao-dependent anchoring of PP1 in the modulation of NMDA receptor activity, we dialyzed Gm peptide into cells through the patch pipette. A significantly greater time-dependent increase in NMDA receptor currents (41.1 ± 10.2%;n = 7; P < 0.01) was observed in yotiao-expressing cells relative to control cells (9.0 ± 3.6%;n = 7). The effect of the inhibitor plateaued within 5 to 10 min of establishing the whole-cell configuration, whereas no effect was observed with NMDA receptors containing NR1C (Fig. 4). Application of the phosphatase inhibitor okadaic acid at 1 μM (but not 10 nM) produced an effect similar to that of Gm peptide on NR1A in yotiao-expressing cells (28.5 ± 3.6%; n = 4; P < 0.05) relative to control cells (2.3 ± 6.9%; n = 4) (Fig. 4B). These results indicate that tonic PP1 activity associated with yotiao may negatively regulate NMDA receptors. The extent of the increase in current conducted by the NMDA receptor was related to the initial extent of current desensitization (17). This relation was observed whether we activated PKA or displaced PP1. Thus, yotiao-mediated localization of PKA and PP1, the balance of enzyme activities, and the initial state of the channel all appear to contribute to the modulation of current flow through the NMDA receptor.

Figure 4

Influence of yotiao on regulation of NMDA receptor activity by PP1 activity. (A) Time course of normalized peak NMDA receptor currents from a control and yotiao-expressing cell during whole-cell dialysis of Gm peptide (10 μM). Traces shown below correspond to the first and last sweep from the indicated cells after establishment of the whole-cell configuration. (B) Bar graph summarizing percent increase of peak whole-cell current for cells expressing NR1A (solid bars) and cells expressing NR1C (open bars). Expression of yotiao and the use of Gm or okadaic acid (OA, 1 μM) are indicated. Significant differences (**P < 0.01, *P < 0.05) and the number of times each experiment was performed are indicated.

Under resting conditions, targeting of a constitutively active phosphatase may favor dephosphorylation of the channel or a closely associated protein. However, when intracellular concentrations of cAMP are increased, PKA may be released from anchored sites, thus shifting the equilibrium in favor of phosphorylation, which in turn results in enhancement of current flow through the NMDA receptor.

Subcellular targeting of phosphatases and kinases is achieved through various mechanisms. Sometimes both enzymes interact with each other (18), but complex formation more often requires an intermediary protein (19). Scaffold proteins such as sterile 5, Pbs-2, and Jip-1 immobilize successive members in a kinase cascade such that signals can be efficiently transduced from one kinase to the next (20). In contrast, multivalent scaffold proteins such as AKAP79, PTG, and InaD coordinate the location of several signaling enzymes and thus integrate diverse signals at a specific intracellular site (7, 21). Yotiao facilitates the dynamic regulation of an individual phosphoprotein by assembling a signaling complex that contains a kinase and phosphatase with opposing activities and is attached to the substrate. Interestingly, the gene encoding yotiao is alternatively spliced to yield a family of proteins, including the recently identified AKAP350. This splice variant of yotiao also contains the interaction domains for PP1, NR1A, and PKA and therefore may also mediate the assembly of a macromolecular complex involved in regulating NMDA receptor function. Because a number of ion channels appear to be modulated by closely associated kinases and phosphatases (22), other structural elements similar to yotiao may exist.

  • * These authors contributed equally to this article.

  • To whom correspondence should be addressed. E-mail: scott{at}ohsu.edu

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