Ca2+/calmodulin-dependent protein kinase IIalpha clusters are associated with stable lipid rafts and their formation traps PSD-95

Relatively large number of post-synaptic density (PSD) proteins, including Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), have the potential to associate with lipid rafts. We in this study demonstrate that the CaMKIIα clusters induced by ionomycin in human embryonic kidney 293 cells, as well as unclustered CaMKIIα (Du F., Saitoh F., Tian Q. B., Miyazawa S., Endo S. and Suzuki T, 2006, Biochem. Biophys. Res. Commun 347, 814–820), were associated with lipid rafts. The CaMKIIα clusters associated with lipid raft fraction became resistant to treatment with methyl-β-cyclodextrin and subsequent cold Triton X-100, which suggests the stabilization of CaMKIIα cluster-associated lipid rafts. Next, we found that PSD-95, which is also a component of lipid raft fraction and does not interact directly with CaMKII, was trapped by stable CaMKIIα cluster-containing structure. Association of PSD-95 with CaMKIIα clusters was also observed in cultured neuronal cells. These results suggest the CaMKIIα clusters associated with the lipid rafts in the cytoplasmic region play a role in the assembly and stabilization of certain PSD proteins that have the potential to associate with lipid rafts.     

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.     

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.     

Tumor necrosis factor-α-induced neutral sphingomyelinase-2 modulates synaptic plasticity by controlling the membrane insertion of NMDA receptors

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase-2) is critical for tumor necrosis factor (TNF) α-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory post-synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase-2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA-evoked calcium flux and excitatory post-synaptic currents.     

Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95

The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.     

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.     

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.     

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.     

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.     

Antagonistic effects of TrkB and p75(NTR) on NMDA receptor currents in post-synaptic densities transplanted into Xenopus oocytes

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are essential regulators of synaptic function in the adult CNS. A TrkB-mediated effect at excitatory synapses is enhancement of NMDA receptor (NMDA-R)-mediated currents. Recently, opposing effects of TrkB and the pan-neurotrophin receptor p75(NTR) on long-term synaptic depression and long-term potentiation have been reported in the hippocampus. To further study the regulation of NMDA-Rs by neurotrophin receptors in their native protein environment, we micro-transplanted rat forebrain post-synaptic densities (PSDs) into Xenopus oocytes. One-minute incubations of oocytes with BDNF led to dual effects on NMDA-R currents: either TrkB-dependent potentiation or TrkB-independent inhibition were observed. Pro-nerve growth factor, a ligand for p75(NTR) but not for TrkB, produced a reversible, dose-dependent, TrkB-independent and p75(NTR)-dependent inhibition of NMDA-Rs. Fractionation experiments showed that p75(NTR) is highly enriched in the PSD protein fraction. Immunoprecipitation and pull-down experiments further revealed that p75(NTR) is a core component of the PSD, where it interacts with the PDZ3 domain of the scaffolding protein SAP90/PSD-95. Our data provide striking evidence for a rapid inhibitory effect of p75(NTR) on NMDA-R currents that antagonizes TrkB-mediated NMDA-R potentiation. These opposing mechanisms might be present in a large proportion of forebrain synapses and may contribute importantly to synaptic plasticity.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell-mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n−3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.     

Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

PUFAs have been shown to mediate immune re-sponse especially the functions of T cells[1]. Recent researches have demonstrated that PUFAs can in-crease membrane fluidity and modify the functions of membrane receptors and enzymes in T cell membra-ne[2,3]. M…     

不同训练方式建立大鼠空间记忆后海马结构NMDA受体表达的变化

短期强化训练能否建立可靠的空间长时记忆？用不同训练方式建立空间记忆后,大鼠海马结构NMDA受体的表达发生怎样的变化？目前尚未见明确报道。本研究应用Morris水迷宫方法分别采用以下模式对大鼠进行训练：空间长时记忆训练模式（LT组）、空间短时记忆训练模式（ST组）以及短期强化训练模式（SRT组）,对不同训练模式建立的空间记忆进行了比较,应用免疫荧光组织化学方法检测各组大鼠海马结构NMDA／NR1受体表达的变化。结果表明,Morris水迷宫训练过程中,LT和SRT组大鼠寻找站台的半均潜伏期和策略均无显著性差异：记忆检测发现,除LT组大鼠在站台所在象限的停留时间明显长于SRT组大鼠外,两组大鼠寻找站台的潜伏期和策略以及穿越站台的次数均无显著性差异。ST组大鼠海马结构NMDA／NR1的免疫反应强度与对照组相比,无显著差异。但是,LT和SRT组大鼠海马CA1区锥体细胞联及齿状回的颗粒细胞层NMDA／NR1免疫荧光反应都明显增强,两组之间比较无显著差异,但是两组分别与对照组和ST组相比均有显著性差异。上述结果提示,短期强化训练可建立与长期训练基本相同的空间长时记忆。大鼠海马结构CA1区和齿状回NMDA受体表达的增加,可能是空间长时记忆形成的机制之一。     

Splice variants of the glutamate transporter GLT1 form hetero-oligomers that interact with PSD-95 and NMDA receptors

The glutamate transporter GLT1 is expressed in at least two isoforms, GLT1a and GLT1b, which differ in their C termini. As GLT1 is an oligomeric protein, we have investigated whether GLT1a and GLT1b might associate as hetero-oligomers. Differential tagging (HA-GLT1a and YFP-GLT1b) revealed that these isoforms form complexes that could be immunoprecipitated when co-expressed in heterologous systems. The association of GLT1a and GLT1b was also observed in mixed primary cultures of rat brain and in the adult rat brain, where specific antibodies for GLT1a immunoprecipitated GLT1b and vice versa. Dual immunofluorescence in mixed cultures demonstrated the partial co-localization of both isoforms in neurons and in glial cells. Because GLT1b interacts with an organizer of post-synaptic densities, PSD-95, we examined the capacity of GLT1a to associate with this protein. GLT1a was immunoprecipitated from the rat brain in protein complexes that contained not only GLT1b but also PSD-95 and NMDAR. The interaction between GLT1a with PSD-95 and NMDAR was reproduced in transfected COS7 cells and it appears to be indirect as it requires the presence of GLT1b. These results indicate that the major isoform of the glutamate transporter, GLT1a, can acquire the capacity to interact with PDZ proteins through its inclusion in hetero-oligomers containing GLT1b.     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Monocyte lipid rafts contain proteins implicated in vesicular trafficking and phagosome formation

Lipid rafts are membrane microdomains of unique lipid composition that segregate proteins with poorly understood consequences for membrane organization. Identification of raft associated proteins could therefore provide novel insight into raft-dependent functions. Monocytes process antigens for presentation to T cells by ingesting pathogens into calcium-dependent plasma membrane invaginations called "phagosomes" which develop by sequential fusion with the endoplasmic reticulum, early and late endosomes. We investigated the protein composition of Triton X-100 insoluble low density membranes of the monocyte cell-line THP-1 by matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry. The ganglioside GM1 colocalized on the plasma membrane with the raft markers flotillin 1 and 2, which were enriched in low buoyant density fractions containing 52 identifiable proteins, 28 of which have not been reported in rafts, and nine of which are associated with the endoplasmic reticulum (ER). Remarkably, 27 of the 52 proteins are components of phagosomes, including the ER protein calnexin which we demonstrate is phosphorylated on serine 562, a switch controlling calcium homeostasis. The presence of the early and late endosome trafficking proteins Rab-1, and Rab-7 together with the late endosome protein LIMPII, indicate lipid rafts are present throughout endosome maturation. Identification of vacuolar ATP synthase, and synaptosomal-associated protein-23, proteins implicated in membrane fusion, together with the cytoskeletal proteins actin, alpha-actinin, and vimentin, and Rac 1, 2, and 3, regulators of cytoskeletal assembly, indicate monocyte lipid rafts contain the machinery to direct vesicular fusion and actin based vesicular migration throughout phagosome development.     

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.     

Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory

The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca²⁺-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation.