Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Affinity purification of PSD-95-containing postsynaptic complexes

A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

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.     

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.     

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.     

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.     

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.     

Phosphorylation state of postsynaptic density proteins

The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.     

Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.     

Preparation of postsynaptic density fraction from hippocampal slices and proteomic analysis

Hippocampal slices offer an excellent experimental system for the study of activity-induced changes in the postsynaptic density (PSD). While studies have documented electrophysiological and structural changes at synapses in response to precise manipulations of hippocampal slices, parallel biochemical and proteomic analyses were hampered by the lack of subcellular fractionation techniques applicable to starting tissue about three orders of magnitude smaller than that used in conventional protocols. Here, we describe a simple and convenient method for the preparation of PSD fractions from hippocampal slices and the identification of its components by proteomic techniques. The "micro PSD fraction" obtained following two consecutive extractions of a synaptosomal fraction with Triton X-100 shows a significant enrichment in the marker protein PSD-95. Thin section electron microscopy shows PSDs similar to those observed in situ. However, other particulate material, especially myelin, and membrane vesicles are also present. The composition of the PSD fraction from hippocampal slices was analyzed by 2D LC/MS/MS. The proteomic approach which utilizes as little as 10microg total protein allowed the identification of >100 proteins. Many of the proteins detected in the fraction are the same as those identified in conventional PSD preparations including specialized PSD-scaffolding proteins, signaling molecules, cytoskeletal elements as well as certain contaminants. The results show the feasibility of the preparation of a PSD fraction from hippocampal slices of reasonable purity and of sufficient yield for proteomic analyses. In addition, we show that further purification of PSDs is possible using magnetic beads coated with a PSD-95 antibody.     

Isolation and major components of core structure of postsynaptic density

The core structure of postsynaptic density (PSD-core) was prepared from rat cerebral synaptosomes by application of the isolation procedure of synaptic junctions (SJ) after trypsinization, which dissociated pre- and post-synaptic structures. The PSD-core was considered to consist mainly of cytoplasmic part of postsynaptic structure, and lack the proteins localized on the external surface of the synaptic plasma membrane, such as receptors for neurotransmitters, Con A-binding proteins and connecting molecule(s) between pre- and post-synaptic structures. The PSD-core proteins which increased greatly in their contents compared with those of SJ prepared from synaptosomes (Syn-SJ) were 120 k Mr Con A-binding protein (Con A-BP) and 30 k Mr protein. Electron microscopic histochemistry suggested that 120 k Con A-BP localized widely in the main structure of the PSD-core. Protein of 30 k Mr was not extracted from PSD-core with 6 M urea, whereas actin, major PSD protein, and tubulin were easily extractable. The 30 k Mr protein was the most resistant one to trypsinization in the SJ fraction. The results suggest that the 30 k Mr protein plays an important role in stabilization and integrity of the postsynaptic density.     

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.     

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.     

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.     

Interaction of brain somatostatin receptors with the PDZ domains of PSD-95

Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95kDa (PSD-95) and PSD-93. The existence of the SSTR4/PSD-95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD-95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD-95, the association may serve to localize the receptor to postsynaptic sites.     

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.