Protein kinase CK2 in postsynaptic densities: phosphorylation of PSD-95/SAP90 and NMDA receptor regulation

Protein kinase CK2 (CK2) is highly expressed in rat forebrain where its function is not well understood. Subcellular distribution studies showed that the catalytic subunit of CK2 (CK2alpha) was enriched in postsynaptic densities (PSDs) by 68%. We studied the putative role of CK2 activity on N-methyl-D-aspartate receptor (NMDAR) function using isolated, patch-clamped PSDs in the presence of 2 mM extracellular Mg(2+). The usual activation by phosphorylation of the NMDARs in the presence of ATP was inhibited by the selective CK2 inhibitor 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB). This inhibition was voltage-dependent, i.e., 100% at positive membrane potentials, while at negative potentials, inhibition was incomplete. Endogenous CK2 substrates were characterized by their ability to use GTP as a phosphoryl donor and susceptibility to inhibition by DRB. Immunoprecipitation assays and 2D gels indicated that PSD-95/SAP90, the NMDAR scaffolding protein, was a CK2 substrate, while the NR2A/B and NR1 NMDAR subunits were not. These results suggest that postsynaptic NMDAR regulation by CK2 is mediated by indirect mechanisms possibly involving PSD-95/SAP90.     

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in XENOPUS: oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2（HDAC2）在成年C57BL/6小鼠海马内的分布及其与突触后致密区（PSD）蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸（NMDA）受体亚单位1（NR1）、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating

Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs.     

PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high-affinity, voltage-independent Zn(2+) inhibition. However, it has been reported that Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition. The post-synaptic density protein PSD-95 interacts with the C-terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD-95 eliminates the Src-induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn(2+). Our results reveal that the absence of Src-induced potentiation of PSD-95-coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn(2+). These results indicate that PSD-95 functionally modulates NR1/NR2A channels and explain why Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition.     

Spatiotemporal Expression of PSD-95 and nNOS After Rat Sciatic Nerve Injury

Neuronal nitric oxide synthase (nNOS) has been implicated to influence peripheral nerve lesion and regeneration. Post-synaptic density-95 (PSD-95) is one of nNOS-anchoring proteins and plays an important role in specifying the sites of reaction of NO in nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of PSD-95 and nNOS. At gene levels, PSD-95 mRNA diminished shortly after crush, and significantly elevated from 2 days to 2 weeks, whereas nNOS decreased progressively post-operation, reached the valley at 1 day, and markedly up-regulated from 1 to 2 weeks after SNC. The expression of both molecules returned to the control level at 4 weeks post-injury. At protein levels, PSD-95 and nNOS underwent the similar changes as their gene expression except for a time lag during up-regulating. At their peak expression, PSD-95 co-labeled with nNOS in Schwann cells (SCs) of sciatic nerve within 0.5 mm from the lesion site, but had few colocalization in axons. In addition, the interaction between PSD-95 and nNOS enhanced significantly at 2 weeks after SNC. These results suggest a correlation of PSD-95 up-regulation with nNOS in reactive SCs of crushed sciatic nerve, which may lead to understanding the function of PSD-95 during peripheral nerve regeneration. Shangfeng Gao and Min Fei contributed equally to this work.     

Coexpression of postsynaptic density-95 protein with NMDA receptors results in enhanced receptor expression together with a decreased sensitivity to L-glutamate

Coexpression in human embryonic kidney (HEK) 293 cells of the postsynaptic density-95 protein (PSD-95) with NMDA receptor NR2A or NR2B single subunits or NR1-1a/NR2A and NR1-1a/NR2B subunit combinations induced an approximately threefold increase in NR2A and NR2B subunit expression. Deletion of the NR2 C-terminal ESDV motifs resulted in the loss of this increase following coexpression of NR1-1a/NR2A(Trunc) and NR1-1a/NR2B(Trunc) with PSD-95. Characterisation of the radioligand binding properties of [(3)H]MK-801 to NR1-1a/NR2A receptors with or without PSD-95 showed that PSD-95 induced a threefold increase in B:(max) values and an apparent approximately fivefold decrease in affinity in the presence of 10 microM: L-glutamate. In the presence of 1 mM: L-glutamate, the K:(i) for MK-801 binding to NR1-1a/NR2A with PSD-95 was not significantly different from that for NR1-1a/NR2A without PSD-95. The EC(50) value for the enhancement of [(3)H]MK-801 binding by L-glutamate to NR1-1a/NR2A was 1.8 +/- 0.4 (n = 4) and 8.9 (mean of n = 2) microM: in the absence and presence of PSD-95, respectively. Thus, coexpression of PSD-95 with NR1-1a/NR2A results in a decreased sensitivity to L-glutamate and an enhanced expression of NR2A and NR2B subunits. Deletion studies show that this effect is mediated via interaction of the C-terminal ESDV motif of the NR2 subunit with PSD-95.     

Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95

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.     

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro . The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.     

Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain

Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85-90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways.     

Frontal cortical afferents facilitate striatal nitric oxide transmission in vivo via a NMDA receptor and neuronal NOS-dependent mechanism

Striatal nitric oxide (NO) signaling plays a critical role in modulating neural processing and motor behavior. Nitrergic interneurons receive synaptic inputs from corticostriatal neurons and are activated via ionotropic glutamate receptor stimulation. However, the afferent regulation of NO signaling is poorly characterized. The role of frontal cortical afferents in regulating NO transmission was assessed in anesthetized rats using amperometric microsensor measurements of NO efflux and local field potential recordings. Low frequency (3 Hz) electrical stimulation of the ipsilateral cortex did not consistently evoke detectable changes in striatal NO efflux. In contrast, train stimulation (30 Hz) of frontal cortical afferents facilitated NO efflux in a stimulus intensity-dependent manner. Nitric oxide efflux evoked by train stimulation was transient, reproducible over time, and attenuated by systemic administration of either the NMDA receptor antagonist MK-801 or the neuronal NO synthase inhibitors 7-nitroindazole and NG-propyl-L-arginine. The interaction between NO efflux evoked via train stimulation and local striatal neuron activity was assessed using dual microsensor and local field potential recordings carried out concurrently in the contralateral and ipsilateral striatum, respectively. Systemic administration of the non-specific NO synthase inhibitor methylene blue attenuated both evoked NO efflux and the peak oscillation frequency (within the delta band) of local field potentials recorded immediately after train stimulation. Taken together, these observations indicate that feed-forward activation of neuronal NO signaling by phasic activation of frontal cortical afferents facilitates the synchronization of glutamate driven oscillations in striatal neurons. Thus, NO signaling may act to amplify coherent corticostriatal transmission and synchronize striatal output.     

NMR evaluation of interactions between substituted-indole and PDZ1 domain of PSD-95

We synthesized small organic molecules designed as PDZ ligands. These indole-based compounds were evaluated for their interaction with the PDZ1 domain of the post-synaptic density 95 (PSD-95) protein. Three molecules were found to interact with the targeted PDZ protein by NMR. One of them showed chemical shift perturbations closely related to the natural ligands.     

Nω-硝基-L-精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N-甲基-D-天冬氨酸受体表达上调

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase,NOS）抑制剂——N^ω-硝基-L-精氨酸甲酯（L-NAME）对吗啡镇痛耐受形成的影响,并通过观察脊髓和中脑神经元型NOS（nNOS）和N-甲基-D-天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague-Dawley大鼠平均分为6组（每组6只）：1组为对照组,皮下注射生理盐水1ml;2、3、4、5和6组为处理组,分别皮下注射L-NAME10mg／kg、L-NAME20mg／kg、吗啡10mg／kg、L-NAME10mg／kg＋吗啡10mg／kg、L-NAME20mg／kg＋吗啡10mg／kg,每天2次。在注射前测量大鼠的热甩尾潜伏期（tail-flick latency,TFL）基础值,随后每天第一次给药50min后测量其TFL。第8天最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑,采用RT-PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示,2组大鼠第1天至第7天的TFL与基础值相比无显著差异;3组第7天时的TFL仍显著高于基础值;4组的TFL在第1天时最高,第2至第6天期间逐渐降低,第6天时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势,虽然第7天时较第1天有所降低,但是仍然显著高于基础值;6组的TFL变化趋势与5组相同。PT—PCR分析结果显示,与1组相比,3组脊髓和中脑的nNOS mRNA表达显著降低,但NR1A mRNA和NR2A mRNA表达无显著改变;4组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著高于1组。与4组相比,6组的nNOS mRNA、NR1A mRNA和NR2A mRNA表达均显著降低。结果提示,吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加,联合应用L—NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调,延缓吗啡镇痛耐受的形成。本研究结果提示,脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。     

PSD-95与脑缺血的关系及其调控机制研究

PSD-95（突触后密度蛋白-95）在突触后密度区含量丰富,具有复杂的结构域,与膜受体、离子通道、细胞粘附因子和信号分子等相互作用聚集成大分子复合物,在突触的可塑性、学习记忆、大脑的病理生理紊乱等起重要作用。PSD-95与脑缺血神经元损伤和凋亡的分子机制有密切联系。脑缺血再灌注后PSD-95在缺血侧皮层的变化表现为PSD．95阳性细胞数的减少和细胞形态的受损改变。抑制NMDA受体活性的治疗策略包括破坏受体本身、钙离子通道阻滞剂、破坏PSD-95／NMDAR相互作用、破坏PSD-95／nNOS相互作用、nNOS抑制剂药物干预。已有研究发现在大鼠大脑中动脉栓塞模型中抑制PSD-95复合体之间的相互作用可以改善脑缺血。实验性的PSD-95抑制剂减少了短时间和长时间局部脑缺血大鼠的梗死面积、并恢复相应的运动功能治疗脑缺血。本文重点研究PSD-95与脑缺血的关系及其调控机制。     

PSD-95 与脑缺血的关系及其调控机制研究

PSD-95(突触后密度蛋白-95)在突触后密度区含量丰富,具有复杂的结构域,与膜受体、离子通道、细胞粘附因子和信号分子 等相互作用聚集成大分子复合物,在突触的可塑性、学习记忆、大脑的病理生理紊乱等起重要作用。PSD-95 与脑缺血神经元损伤 和凋亡的分子机制有密切联系。脑缺血再灌注后PSD-95 在缺血侧皮层的变化表现为PSD-95 阳性细胞数的减少和细胞形态的受 损改变。抑制NMDA 受体活性的治疗策略包括破坏受体本身、钙离子通道阻滞剂、破坏PSD-95/NMDAR 相互作用、破坏 PSD-95/nNOS相互作用、nNOS抑制剂药物干预。已有研究发现在大鼠大脑中动脉栓塞模型中抑制PSD-95 复合体之间的相互作 用可以改善脑缺血。实验性的PSD-95 抑制剂减少了短时间和长时间局部脑缺血大鼠的梗死面积、并恢复相应的运动功能治疗脑 缺血。本文重点研究PSD-95 与脑缺血的关系及其调控机制。     

Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.     

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.     

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCγ and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.     

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95

Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.