Calcium/Calmodulin‐Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Gardoni, Fabrizio; Caputi, Antonio; Cimino, Mauro; Pastorino, Lucia; Cattabeni, F.; Luca, M.M.G. Di

doi:10.1046/j.1471-4159.1998.71041733.x

Cited by 167 publications

(110 citation statements)

References 37 publications

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“…These included: 1) the NMDAR subunit NR2B (24), 2) the AMPAR subunit GluR1 (25), 3) the synaptic scaffold protein PSD-95 (26,27), and 4) Stargazin, a protein that is necessary for the trafficking of AMPARs to the plasma membrane and also modifies their biophysical properties (15,28,29). We began by examining phosphorylation of the NR2B subunit of the NMDAR, a known substrate and binding partner for CaMKII (30,31). GFP-NR2B-Vim (Fig.…”

Section: Resultsmentioning

confidence: 99%

Substrate Localization Creates Specificity in Calcium/Calmodulin-dependent Protein Kinase II Signaling at Synapses

Tsui

Malenka

2006

Journal of Biological Chemistry

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Calcium/calmodulin-dependent protein kinase II (CaMKII), a major component of the postsynaptic density (PSD) of excitatory synapses, plays a key role in the regulation of synaptic function in the mammalian brain. Although many postsynaptic substrates for CaMKII have been characterized in vitro, relatively little is known about their phosphorylation in vivo. By tagging synaptic proteins with a peptide substrate specific for CaMKII and expressing them in cultured neurons, we have visualized substrate phosphorylation by CaMKII at intact synapses. All substrates tested were strongly phosphorylated by CaMKII in HEK293 cells. However, activity-dependent phosphorylation of substrates at synapses was highly selective in that the glutamate receptor subunits NR2B and GluR1 were poorly phosphorylated whereas PSD-95 and Stargazin, proteins implicated in the scaffolding and trafficking of AMPA receptors, were robustly phosphorylated. Phosphatase activity limited phosphorylation of Stargazin but not NR2B and GluR1. These results suggest that the unique molecular architecture of the PSD results in highly selective substrate discrimination by CaMKII.Calcium-calmodulin-dependent protein kinase II (CaMKII) 2 is a major effecter for calcium-dependent signaling in neurons. It has been implicated in dendritic filapodial extension (1), presynaptic plasticity (2), retrograde signaling to the presynaptic terminal (3), and most notably, the phenomenon of long-term potentiation (LTP) (4, 5), the leading model for a synaptic mechanism underlying learning and memory (6). Although several candidates have been proposed to mediate the synaptic consequences of activating CaMKII (7), it has been difficult to characterize how synaptic proteins are phosphorylated by CaMKII when incorporated into functioning synapses. This is a particularly important and challenging issue because biochemical and proteomic approaches to identifying CaMKII substrates in the postsynaptic density (PSD), the electron dense postsynaptic specialization that contains glutamate receptors and associated signaling machinery, have found at least 28 potential substrate proteins (8). Furthermore, it is unknown whether stimulus-dependent substrate specificity is an inherent property of the PSD and whether the source of the calcium that activates CaMKII and protein phosphatase activity influence synaptic substrate phosphorylation.Some recent evidence suggests that the intricate scaffolding of proteins within different levels of the PSD may influence CaMKII signal transduction. First, electron microscopy studies on isolated PSDs have revealed both a laminar and heterogeneous distribution of CaMKII on the cytosolic face, suggesting that CaMKII may be specifically positioned to exert its activity on nanodomains within this macromolecular complex (9). Second, CaMKII interactions with the NMDA receptor (NMDAR) subunit NR2B (10) and the Drosophila homolog of CASK, Cmg (11), a member of the MAGUK family of synaptic scaffolding proteins (12), have both been found to influence its state o...

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Section: Resultsmentioning

confidence: 99%

Substrate Localization Creates Specificity in Calcium/Calmodulin-dependent Protein Kinase II Signaling at Synapses

Tsui

Malenka

2006

Journal of Biological Chemistry

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“…To isolate PSDs from rat hippocampus, a modification of the method of Gardoni et al 59 was used. Animals were killed and hippocampi (from 15 animals) were rapidly dissected and pooled.…”

Section: Discussionmentioning

confidence: 99%

SNAP-25 regulates spine formation through postsynaptic binding to p140Cap

et al. 2013

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Synaptosomal-associated protein of 25 kDa (SNAP-25) is a member of the Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) protein family, required for exocytosis of synaptic vesicles and regulation of diverse ion channels. Here, we show that acute reduction of SNAP-25 expression leads to an immature phenotype of dendritic spines that are, consistently, less functional. Conversely, over-expression of SNAP-25 results in an increase in the density of mature, Postsynaptic Density protein 95 (PSD-95)-positive spines. The regulation of spine morphogenesis by SNAP-25 depends on the protein's ability to bind both the plasma membrane and the adaptor protein p140Cap, a key protein regulating actin cytoskeleton and spine formation. We propose that SNAP-25 allows the organization of the molecular apparatus needed for spine formation by recruiting and stabilizing p140Cap.

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“…PSD and Triton-insoluble Fraction Preparation-Striatal PSD were isolated according to Carlin et al (20) with minor modifications as described previously (21). Briefly, the tissue was homogenized in icecold 0.32 M sucrose containing 1 mM Hepes, 1 mM MgCl 2 , 1 mM NaHCO 3 , 0.1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Complete, Roche Diagnostic, Milano) at pH 7.4 (buffer A) and centrifuged at 1000 ϫ g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Dopamine D1 Receptor Trafficking and Desensitization by Oligomerization with Glutamate N-Methyl-D-aspartate Receptors

Fiorentini

Gardoni

Spano

et al. 2003

Journal of Biological Chemistry

205

179

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Activation of dopamine D 1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D 1 receptors are co-localized with N-methyl-D-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D 1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D 1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D 1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D 1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D 1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D 1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D 1 receptor complex was translocated to the plasma membrane. These data suggest that D 1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D 1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D 1 receptor desensitization and cellular trafficking. Dopaminergic fibers originating in the substantia nigra and cortical glutamatergic neurons extensively interact in the striatum to drive the physiological functions of this structure from motor planning to reward seeking and procedural learning (1, 2). The critical importance of dopamine in this system is such that the degeneration of nigral dopaminergic neurons leads to the motor and cognitive deficits of Parkinson's disease (3).At the cellular level, nigral and cortical fibers converge on the medium spiny projection neurons (4), where dopamine D 1 -and D 2 -like receptors are coexpressed to high degree with glutamate NMDA 1 and non-NMDA receptor channels (5-8). From a functional point of view, it is well established that dopamine modulates the firing pattern of these neurons. In particular, there is evidence that dopamine, while attenuating the responses mediated by non-NMDA receptors, potentiates those associated with activation of NMDA receptors (2). The D 1 receptor appears to be involved in these interactions. In fact, activation of D 1 receptors in medium spiny neurons enhances NMDA-induced whole cell currents (2, 9) and is a critical requirement for the formation of NMDA-mediated long-term potentiation at corticostriatal...

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Calcium/Calmodulin‐Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Cited by 167 publications

References 37 publications

Substrate Localization Creates Specificity in Calcium/Calmodulin-dependent Protein Kinase II Signaling at Synapses

Substrate Localization Creates Specificity in Calcium/Calmodulin-dependent Protein Kinase II Signaling at Synapses

SNAP-25 regulates spine formation through postsynaptic binding to p140Cap

Regulation of Dopamine D1 Receptor Trafficking and Desensitization by Oligomerization with Glutamate N-Methyl-D-aspartate Receptors

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