Kainate Receptor Post-translational Modifications Differentially Regulate Association with 4.1N to Control Activity-dependent Receptor Endocytosis

Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity.     

Phosphorylation of AMPA receptors

The ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.     

Regulation of AMPA Receptor Trafficking by O-Glycosylation

The present study investigated the role of O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) in AMPA receptor trafficking. Alloxan, an inhibitor of O-GlcNAc transferase, potentiated responses of AMPA receptors composed of the GluR1 subunit expressed in Xenopus oocytes. No potentiating effect of alloxan was obtained with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. Alloxan facilitated basal synaptic transmission to approximately 120% of basal levels and enhanced Schaffer collateral-CA1 long-term potentiation (LTP) in rat hippocampal slices, especially in the late phase of the LTP. Alloxan stimulated translocation of the GluR1 and GluR2 subunit from the cytosol towards the plasma membrane in rat hippocampal slices with the LTP, although it had no effect on subcellular distribution of the NR1 subunit. Taken together, the results of the present study show that alloxan regulates AMPA receptor trafficking by inhibiting O-GlcNAcylation, to modulate hippocampal synaptic transmission and synaptic plasticity.     

A Serum Factor Potentiates ACh and AMPA Receptor Currents via Differential Signal Transduction Pathways

A serum factor is recognized to interact with a protein kinase C (PKC) pathway. Indeed, treatment with fetal bovine serum enhanced ACh-evoked currents by PKC activation in the neuronal nicotinic ACh receptors (α7) andTorpedoACh receptors expressed inXenopusoocytes. In addition, potentiation of ACh-evoked currents induced by fetal bovine serum was observed also in the mutantTorpedoACh receptors lacking potent PKC phosphorylation sites at Ser³³³on the α subunit and Ser³⁷⁷on the δ subunit; the potentiation was inhibited by the PKC inhibitor, PKC inhibitor peptide (PKCI), indicating that ACh receptor currents were enhanced by PKC activation but not by PKC phosphorylation of the receptors. On the other hand, fetal bovine serum enhanced kainate-evoked currents in oocytes expressing the α-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR1,3. The enhancement was not affected by the PKC inhibitors, PKCI or GF109203X, and instead, was inhibited by the Ca²⁺/calmodulin-dependent kinase II (CaMKII) inhibitor, KN-62. These results suggest that serum is not only involved in PKC activation but in CaMKII activation, and that thereby ACh receptor currents and AMPA receptor currents are each potentiated.     

Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.     

Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser⁹ phosphorylation). GSK3β has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a “moderate” amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32–33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3β Ser^21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.     

Regulation and Identification of Na,K-ATPase α1 Subunit Phosphorylation in Rat Parotid Acinar Cells

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser²²², Ser⁴⁰⁷), three sites (Ser²¹⁷, Tyr²⁶⁰, Ser⁴⁷) previously found from large scale proteomic screens, and two sites (Ser²³, Ser¹⁶) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser²³ and Ser¹⁶ and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca²⁺ ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser²³ α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser²³ α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser²³ α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser²³ and Ser¹⁶, respectively, the latter because ouabain itself increased Ser¹⁶ phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser¹⁶ α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.     

Phosphorylation-dependent interactions of alpha-Actinin-1/IQGAP1 with the AMPA receptor subunit GluR4

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors play key roles in excitatory synaptic transmission and synaptic plasticity in the CNS. Although a variety of proteins has been characterized to interact with AMPA receptors and regulate their function, little is known about the regulation of the AMPA receptor subunit GluR4. To understand the molecular mechanisms of GluR4 functional regulation, the yeast two-hybrid system was used to identify GluR4-interacting molecules. alpha-Actinin-1 and IQGAP1 were identified to be GluR4-specific binding partners. Both proteins interact specifically with GluR4 and co-cluster with GluR4 individually in neurons. Mapping experiments revealed that alpha-Actinin-1 and IQGAP1 bind to the same region within the C-terminus of GluR4 that contains a previously identified PKA phosphorylation site, Ser842, phosphorylation of which is regulated by synaptic activity. Interestingly, the phosphorylation of Ser842 differentially regulates interactions of GluR4 with alpha-Actinin-1 and IQGAP1; phosphorylation strongly inhibits interaction of GluR4 with alpha-Actinin-1 but has little effect on its interaction with IQGAP1. These results suggest that alpha-Actinin-1 and IQGAP1 regulate GluR4 functions via their specific associations with GluR4. In addition, our data indicate that activity-dependent phosphorylation of GluR4 may regulate its synaptic targeting through phosphorylation-dependent interactions with alpha-Actinin-1 and IQGAP1.     

Roles of Fragile X Mental Retardation Protein in Dopaminergic Stimulation-induced Synapse-associated Protein Synthesis and Subsequent α-Amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) Receptor Internalization

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.     

Distinct Subunit-specific α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking Mechanisms in Cultured Cortical and Hippocampal Neurons in Response to Oxygen and Glucose Deprivation

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca²⁺-impermeable receptors to GluA2-lacking Ca²⁺-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.     

Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus

Kainate receptors alter the excitability of mossy fiber axons and have been reported to play a role in the induction of long-term potentiation (LTP) at mossy fiber synapses in the hippocampus. These previous studies have relied primarily on the use of compounds whose selectivity is unclear. In this report, we investigate short- and long-term facilitation of mossy fiber synaptic transmission in kainate receptor knockout mice. We find that LTP is reduced in mice lacking the GluR6, but not the GluR5, kainate receptor subunit. Additionally, short-term synaptic facilitation is impaired in GluR6 knockout mice, suggesting that kainate receptors act as presynaptic autoreceptors on mossy fiber terminals to facilitate synaptic transmission. These data demonstrate that kainate receptors containing the GluR6 subunit are important modulators of mossy fiber synaptic strength.     

Regulation of AMPA receptor trafficking by N-cadherin

Dendritic spines are dynamically regulated, both morphologically and functionally, by neuronal activity. Morphological changes are mediated by a variety of synaptic proteins, whereas functional changes can be dramatically modulated by the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor trafficking. Although these two forms of plasticity appear to be highly coordinated, the connections between them are not fully understood. In this study the synaptic cell adhesion molecule N-cadherin was found to associate with AMPA receptors and regulate AMPA receptor trafficking in neurons. N-cadherin and beta-catenin formed a protein complex with AMPA receptors in vivo, and this association was regulated by extracellular Ca2+. In addition, these proteins co-clustered at synapses in cultured neurons. In heterologous cells and in cultured neurons, overexpression of wild-type N-cadherin specifically increased the surface expression level of the AMPA receptor subunit glutamate receptor 1 (GluR1) and this effect was reversed by a dominant-negative form of N-cadherin. Finally, GluR1 increased the surface expression of N-cadherin in heterologous cells. Importantly, recent studies suggest that N-cadherin and beta-catenin play key roles in structural plasticity in neurons. Therefore, our data suggest that the association of N-cadherin with AMPA receptors may serve as a biochemical link between structural and functional plasticity of synapses.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

CaMKII‐dependent phosphorylation of GluK5 mediates plasticity of kainate receptors

Calmodulin‐dependent kinase II (CaMKII) is key for long‐term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity‐dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre‐ and postsynaptic stimulation at hippocampal mossy fibre synapses induces long‐term depression of kainate receptor (KAR)‐mediated responses, which depends on Ca²⁺ influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C‐terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD‐95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR‐LTD. We propose that CaMKII‐dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.     

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

PKC SUMOylation inhibits the binding of 14–3–3τ to GluK2

Phosphorylation and SUMOylation of the kainate receptor (KAR) subunit GluK2 have been shown to regulate KAR surface expression, trafficking and synaptic plasticity. In addition, our previous study has shown that a phosphorylation-dependent interaction of 14–3–3τ and GluK2a-containing receptors contributes to the slow decay kinetics of native KAR-EPSCs. However, it is unknown whether SUMOylation participates in the regulation of the interaction between 14–3–3τ and GluK2a-containing receptors. Here we report that SUMOylation of PKC, but not GluK2, represses the binding of 14–3–3τ to GluK2a via decreasing the phosphorylation level of GluK2a. These results suggest that PKC SUMOylation is an important regulator of the 14–3–3 and GluK2a protein complex and may contribute to regulate the decay kinetics of KAR-EPSCs.     

Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression

Glutamate receptor phosphorylation has been implicated in several forms of modulation of synaptic transmission. It has been reported that protein kinase A (PKA) can phosphorylate the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR4 on Ser842, both in vitro and in vivo. Here, we studied the regulation of GluR4 phosphorylation and intracellular trafficking by PKA and by metabotropic receptors coupled to adenylyl cyclase (AC), in cultured chick retinal amacrine-like neurones, which are enriched in GluR4. The regulation of AMPA receptor activity by PKA and by metabotropic AC-coupled receptors was also investigated by measuring the [Ca2+]i response to kainate in Na(+)-free medium. Stimulation of AC with forskolin (FSK), or using the selective agonist of dopamine D1 receptors (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF38393), increased the [Ca2+]i response to kainate, GluR4 phosphorylation at Ser842 and GluR4 surface expression. Pre-incubation of the cells with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), an agonist of group II metabotropic glutamate receptors (mGluR), which are coupled to inhibition of AC, inhibited the effect of FSK and of SKF38393 on AMPA receptor activity, GluR4 phosphorylation and expression at the plasma membrane. These results indicate that there is a functional cross-talk between dopamine D1 receptors and group II mGluR in the regulation of GluR4 phosphorylation and AMPA receptor activity. Our data show that GluR4 phosphorylation at Ser842 by PKA, and its recruitment to the plasma membrane upon phosphorylation, is regulated by metabotropic receptors.     

Inhibition of Calcineurin-mediated Endocytosis and ��-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Prevents Amyloid �� Oligomer-induced Synaptic Disruption

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid β (Aβ) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Aβ oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.