Affinity purification of PSD-95-containing ... - Wiley Online Library

Journal of Neurochemistry, 2003, 87, 1255–1261

doi:10.1046/j.1471-4159.2003.02091.x

Affinity purification of PSD-95-containing postsynaptic complexes Lucia Vinade,* Michael Chang,* Michelle L. Schlief,* Jennifer D. Petersen,* Thomas S. Reese,* Jung-Hwa Tao-Cheng and Ayse Dosemecià *Laboratory of Neurobiology and Electron Microscopy Facility, National Institute of Neurological Disorders and Stroke/National Institutes of Health, Bethesda, Maryland, USA àMarine Biological Laboratory, Program in Molecular Physiology, Woods Hole, Massachusetts, USA

Abstract 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 a-amino3-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-95containing scaffold through interaction of GluR1 with SAP-97. Keywords: affinity purification, anchoring proteins, glutamate receptors, postsynaptic density, PSD-95. J. Neurochem. (2003) 87, 1255–1261.

The postsynaptic density (PSD) is a protein complex that appears as an electron-dense structure underneath the postsynaptic membrane at the synaptic contact zone. The PSD is involved in anchoring and organizing key elements of the postsynaptic response, such as channels, neurotransmitter receptors and transduction molecules. Thus, knowledge of the composition of the PSD and how it changes with activity is crucial for an understanding of synaptic function and plasticity. Preparations enriched in PSDs are obtained based on the insolubility of this structure in relatively mild detergents. A classical preparation (Cohen et al. 1977) involves treatment of synaptosomal membrane fractions with Triton X-100 and further fractionation by density gradient centrifugation. PSDs isolated by this method retain their original morphology as observed by electron microscopy (EM). This Triton-derived fraction has been widely used for the identification of

components of the PSD (Walsh and Kuruc 1992; Walikonis et al. 2000). A major concern in the use of PSD preparations for the elucidation of PSD composition is the presence of contamReceived May 20, 2003; revised manuscript received August 20, 2003; accepted August 21, 2003. Address correspondence and reprint requests to Dr Lucia Vinade, Laboratory of Neurobiology/National Institute of Neurological Disorders and Stroke/National Institutes of Health, 9000 Rockville Pike, Building 36/2A21, Bethesda, MD-20892, USA. E-mail: [email protected] Abbreviations used: AMPA, a-amino-3-hydroxy-5-methylisoxazole4-propionate; CaMKII, calcium/calmodulin-dependent protein kinase II; EM, electron microscopy; GFAP, glial fibrillary acidic protein; GluR, glutamate receptor; GRIP, glutamate receptor interacting protein; MAGUK, membrane-associated guanylate kinase; NLS, N-lauryl-sarcosinate; NF, neurofilament; PSD, postsynaptic density; SAP, synapse associated protein; SDS, sodium dodecyl sulfate; SynGAP, synapticRDS-GTpase activating protein.

2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 1255–1261

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inants. The consistent detection of glial fibrillary acidic protein (GFAP) in Triton X-100-derived PSD fractions (Walsh and Kuruc 1992; Walikonis et al. 2000) is an indicator of the problem. Indeed, all membrane-free, detergent-insoluble protein complexes have densities similar to that of PSDs and therefore co-fractionate during separation protocols based on centrifugation. One striking example of such contaminants is calcium/calmodulin-dependent protein kinase II (CaMKII) clusters (Dosemeci et al. 2000). CaMKII is recognized as a component of the PSD, and its enrichment in PSD fractions compared with parent fractions is generally regarded as an indication of PSD enrichment. However, our recent immunoEM studies revealed that a major portion of the CaMKII present in the Triton-derived PSD fraction is actually contributed by contaminating CaMKII clusters of non-synaptic origin (Dosemeci et al. 2000). An alternative method for the isolation of PSDs involves treatment of the Triton-derived PSD fraction with the stronger detergent N-lauryl-sarcosinate (NLS) (Cho et al. 1992). This approach is proposed to remove several peripherally attached proteins, contaminants as well as certain genuine PSD components, leaving a core PSD complex (Kennedy 1993). On the other hand, our recent EM studies have demonstrated that contaminating CaMKII clusters are insoluble in NLS and therefore become particularly enriched in the NLS-derived PSD fraction (Dosemeci et al. 2000). In the present study, we developed an affinity-based strategy to purify further the Triton-derived PSD fraction. This protocol involves use of magnetic beads coated with an antibody against PSD-95, a protein belonging to the membrane-associated guanylate kinase (MAGUK) family and considered to be a marker for PSDs. Affinity-based purification is a suitable approach because contaminating material with similar densities cannot be eliminated by density gradient centrifugation. Furthermore, antibodycoated beads are collected with a magnet rather than by centrifugation to avoid non-specific co-sedimentation of particulate contaminants. PSD-95 is a suitable target for affinity purification of PSDs because it is present in the majority of PSDs at asymmetric synapses of cortical neurons (Valtschanoff et al. 1999; Aoki et al. 2001) and any PSD-95 not located in synapses (Aoki et al. 2001) is essentially eliminated in the parent PSD fraction prepared from synaptosomes. The presynaptic pool of PSD-95 has been reported to be either non-existent (Hunt et al. 1996) or relatively small (Aoki et al. 2001). It should be noted that the affinity purification strategy selects for complexes containing PSD-95. Any other protein complexes present in the postsynaptic thickening will not be co-purified unless they are physically attached to the PSD-95 scaffold. Thus, analysis of the affinity-purified preparation should clarify whether certain postsynaptic proteins that do not bind directly to PSD-95 are part of the main PSD complex. The present study focuses on a-amino-3-hydroxy-

5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors. These receptors are observed at the postsynaptic thickening by immunoEM (reviewed in Nusser 2000) and are present in Triton-derived PSD fractions. On the other hand, AMPA receptors do not interact directly with PSD-95 and therefore might be present as physically separate complexes. We analyzed the affinity-purified PSD fraction for its content of AMPA receptors and AMPA receptor-binding proteins to clarify whether the AMPA receptors are anchored to the PSD-95-containing scaffold.

Materials and methods Materials Dynabeads coated with secondary antibody (M-450) were purchased from Dynal (Oslo, Norway). Antibodies against PSD-95 (MA1-046) and SAP-97 (PA1-741) were from ABR (Golden, CO, USA), anti-a-CaMKII (6G9 2) and anti-spectrin (240/235E) were from Chemicon International, Inc. (Temecula, CA, USA), antiGRIP-1, anti-GluR1 and anti-GluR2/3 were from Upstate (Lake Placid, NY, USA), anti-GFAP was from Dako A/S (Copenhagen, Denmark) and Sigma (St Louis, MO, USA), anti-synapsin Ia/b and anti-synGAP were from Santa Cruz Biotech., Inc. (Santa Cruz, CA, USA), and anti-neurofilament (NF-L) was from Sigma. Preparation of Triton X-100-derived PSD fraction The PSD fraction was prepared by the method of Cohen et al. (1977) and Carlin et al. (1980), with modifications as described in Dosemeci et al. (2000), using frozen brains from adult Sprague– Dawley rats (collected and frozen within 2 min of death by PelFreeze Biologicals, Rogers, AR, USA). Briefly, a synaptosomal fraction was obtained and treated with 0.5% Triton X-100 to release PSDs. Detergent-insoluble pellets were collected and further fractionated by sucrose density centrifugation. The material from the 1.5/2.1-M sucrose interface was treated with 0.5% Triton X-100/ 75 mM KCl and collected on a 2.1-M sucrose cushion. Protein concentration was estimated by the method of Peterson (1977). Affinity purification of the PSD fraction During all the incubation and washing steps described below magnetic beads were continuously mixed with a rotary mixer and the beads were collected by placing the tubes in magnetic racks. Magnetic beads with covalently attached anti-mouse secondary antibody (Dynabeads M-450 goat anti-mouse IgG) were washed twice for 5 min in solution A (2% bovine serum albumin, 0.01% Tween-20 in Tris-buffered saline, pH 7.4) and then incubated with PSD-95 antibody (2 · 107 beads per 4 lL antibody) in solution A for 1 h at room temperature (25–30C). The supernatant was discarded and the beads were washed in solution A (twice for 20 min) to remove unbound antibody. Before incubation with magnetic beads, the parent Triton X-100derived PSD fraction (20 lg protein per mL in solution A) was briefly sonicated using a probe sonicator [twice for 45 s, at the lowest power setting (< 5 W)] with the tube held in ice. This step was necessary for proper resuspension of the particulate material. Resuspended PSD fraction (20 lg protein) was incubated with


Affinity-purified postsynaptic densities

PSD-95 antibody-coated beads (2 · 107 beads) in a final volume of 1 mL for 2 h at 4C. The beads were then collected and washed twice for 5 min in 1 mL solution A, three times for 10 min and three times for 20 min in 1 mL 0.01% Tween-20/Tris-buffered saline. In a few experiments all six washes with Tween were for 20 min. The washed beads with attached PSDs were treated with either sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, 5% b-mercaptoethanol, 125 mM Tris-HCl, pH 6.8] for electrophoresis or with 4% glutaraldehyde for EM. Electrophoresis and immunoblotting Proteins were separated by SDS-polyacrylamide gel electrophoresis using 7.5% mini-gels. Protein staining was either with Coomassie Brilliant Blue R-250 or with Daiichi Silver Stain II kit (Owl Separation Systems, Inc., Portsmouth, NH, USA). For immunoblotting, proteins were transferred to nitrocellulose using a Trans-Blot system (Bio-Rad, Hercules, CA, USA). Membranes were processed by blocking with 2% bovine serum albumin for 2 h and then incubating with for 1 h with primary antibody (1 : 5000 antiPSD-95; 1 : 5000 anti-SAP-97; 1 : 100 anti-CaMKII; 1 : 1000 anti-GRIP1; 1 : 1000 anti-GluR1; 1 : 1000 anti-GluR2/3; 1 : 500 anti-GFAP from Dako or 1 : 100 from Sigma; 1 : 100 anti-synapsin I; 1 : 100 anti-synGAP; 1 : 2000 anti-NF-L). After washing, the membranes were incubated with alkaline phosphatase-conjugated secondary antibody (1 : 5000 anti-mouse or 1 : 10 000 anti-rabbit) for 1 h, washed and treated with alkaline phosphatase substrate. Membranes were then dried and scanned. Analysis of the intensity of the bands and measurement of the total area of the peaks obtained were performed using the public domain NIH Image program (developed at the US National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/).

profiles or others. After decoding, ‘other’ counts were eliminated for PSD-95-coated beads, but not for control beads in order not to undercount the control. EM immunocytochemistry and rotary-shadowed replicas PSDs were immobilized on 5-mm2 glass coverslips by incubating each coverslip in a 15-lL droplet of PSD fraction (1 mg protein per mL). Following immobilization, samples were immunogold labeled for PSD-95 (1 : 100 dilution; secondary antibody 1 : 100 anti-mouse conjugated to 10 nm gold; Ted Pella, Redding, CA, USA) and for spectrin (1 : 10 dilution; secondary antibody 1 : 100 anti-rabbit conjugated to 20 nm gold). Next, immunogold-labeled samples were mounted on a freezing stage and preserved by rapid freezing on a Life Cell Slam Freezing machine (CF100, The Woodlands, TX, USA) and processed as described in Dosemeci et al. (2000).

Results

Analysis of the Triton X-100-derived PSD fraction PSD fractions were prepared by conventional techniques, which included treatment of synaptosomes with 0.5% Triton X-100 to release PSDs and further purification by sucrose density gradient centrifugation and a second extraction with 0.5% Triton X-100/75 mM KCl. Thin-section electron micrographs of this fraction show that it contains many structures with the characteristic elongated shape of PSDs in (a)

Thin-section EM of Triton X-100-derived PSD fraction The PSD fraction was centrifuged 250 000 g for 1 h and the pellet was fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for at least 30 min at room temperature, washed and treated with 1% osmium tetroxide for 1 h, en bloc mordanted with 0.25% uranyl acetate in acetate buffer for 30 min to overnight, washed and dehydrated through a graded series of ethanol and propylene oxide, and embedded in epoxy resin. Thin sections were cut and counterstained with uranyl acetate and lead citrate. Pre-embedding immunogold labeling of the fraction was as described in Tanner et al. (1996). Briefly, the pellet was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at room temperature for 45 min, washed, and further cut into several pieces and labeled with antibodies against PSD-95 and CaMKII. Controls included omitting the primary antibody. Thin-section EM of affinity-purified PSD fraction Magnetic beads with attached material were fixed with 4% glutaraldehyde as above in a microfuge tube. At the end of fixation, the supernatant was removed and samples were mixed and embedded with 10% low melting point agarose. The agaroseembedded pellets were cut into 1-mm cubes, then processed for conventional thin-section EM as above. Random grid openings were scored for the number of attached PSDs per bead. Grids from control and PSD-95-coated beads were counted blind. All attached materials were included in the counts and categorized into either PSD-like

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(c)

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Fig. 1 Electron microscopic and biochemical characterization of Triton X-100-derived PSD fraction prepared by conventional methods. (a) Thin-section electron micrograph of pelleted fraction shows numerous elongated structures recognizable as PSDs (large arrows) as well as round structures that were previously characterized as CaMKII clusters (small arrows). (b) Coomassie blue-stained gel showing the electrophoretic protein staining pattern of the PSD fraction. The positions of molecular weight markers (in KDa) are indicated on the left. (c, d) Enlarged electron micrographs of the fraction immunogold-labeled for PSD-95 (c) and CaMKII (d). Small arrow in (d) indicates a CaMKII cluster. Silver-enhanced gold label appears as black grains.



Fig. 2 Identification of contaminating elements in the Triton X-100derived PSD fraction in rotary-shadowed replicas. The PSD fraction was adhered to glass and labeled for PSD-95 (10 nm gold, yellow pseudocolor) and for the spectrin isoform 240/235E (20 nm gold, red

pseudocolor). PSDs show a fairly uniform labeling for PSD-95. Particulate contaminants that do not label for PSD-95 include filament networks labeled for spectrin, neurofilaments and CaMKII clusters.

cross-section (Fig. 1a, large arrows). By pre-embedding immunoEM, these elongated structures are shown to label for PSD-95 (Fig. 1c) further confirming their identity as PSDs. ImmunoEM also reveals labeling of the same structures for CaMKII (Fig. 1d). Even though the PSD is the major constituent of this preparation, other contaminants are evident. CaMKII clusters, 100-nm spherical structures of non-synaptic origin (Dosemeci et al. 2000), are indicated by small arrows in Figs 1(a) and (d). The electrophoretic protein profile of the PSD fraction (Fig. 1b) is similar to that reported by other groups (Walikonis et al. 2000) with a major 50-kDa band corresponding to a-CaMKII. Other contaminating structures in the Triton X-100derived PSD fraction were identified by EM via immunogold labeling and rotary shadowing. The electron micrograph in Fig. 2 illustrates the different structures identified by this approach. These include PSDs labeled with an antibody against PSD-95 CaMKII clusters (Dosemeci et al. 2000), neurofilaments, and filament networks labeled with an antibody against spectrin.

GFAP relative to PSD-95 have been reduced significantly by affinity purification (Fig. 3b). For a quantitative assessment of the immunoblots, densitometric scans were obtained and the percentage co-purification of selected proteins with PSD-95 was evaluated (Table 1). Exclusive co-purification is assumed when the ratio of the peak area for the selected protein to the peak area for PSD-95 remains the same after affinity purification (100% co-purification). The percentage co-purification ranged from 6 to 19% (six experiments) for GFAP, indicating significant removal of the contaminant (Table 1). The depletion of other proteins thought to be contaminants of the PSD fraction was also tested by comparison of immunoblots from the parent and the affinity-purified fractions. Synapsin I, a presynaptic protein (De Camilli et al. 1983) that is commonly detected in Triton-derived PSD fractions, (Walikonis et al. 2000) and NF-L, a component of neurofilaments shown in the present study to contaminate the Triton-derived PSD fraction (Fig. 2), appear to be virtually eliminated upon affinity purification (Fig. 3b). In addition, the marked decrease in the relative levels of CaMKII (Fig. 3b) suggests that contaminating CaMKII clusters have also been removed. The variability of the values obtained for the percentage co-purification of CaMKII (13–48%) in four experiments is attributed to differences in the relative levels of PSD-associated versus cluster-associated CaMKII in the different parent Triton-derived PSD preparations used. Furthermore, steps during affinity purification, dilution and sonication of the Triton-derived PSD fraction cause some dissociation of CaMKII (data not shown), and therefore may contribute to the decrease in CaMKII levels in affinity-purified samples. SynGAP, a protein described to bind to PSD-95 directly (Kim et al. 1998), co-purifies more substantially with PSD95, although the co-purification is still not exclusive

Characterization of the affinity-purified PSD fraction Identification of numerous contaminants in the Triton X-100-derived preparation prompted us to develop an affinity-based technique for further purification of PSDs (Fig. 3). As described in detail above, the technique is based on selective binding of PSDs to magnetic beads coated with an antibody against PSD-95 and separation from the rest of the suspended particulate material by collection of the beads on a magnet. One criterion to assess the success of the purification protocol was to track the depletion of the non-neuronal protein GFAP. Comparison of the immunoblots from the parent and affinity-purified fractions shows that levels of



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Fig. 3 Affinity purification of Triton X-100-derived PSD fraction using magnetic beads coated with PSD-95 antibody. Fractions were mixed with antibody-coated magnetic beads. The beads were collected on a magnetic rack and the suspension containing unattached material was discarded. The beads were then washed several times to remove nonspecifically attached material before biochemical and electron microscopic analysis. Parallel controls were carried out using beads without the PSD-95 antibody coating. The upper panels show silver-stained gels (a) and immunoblots (b) corresponding to material bound to magnetic beads with (affin. purif.) or without (control) the PSD-95 antibody coating (yields from 20 lg parent fraction protein). Note that relative sensitivities of different proteins to Coomassie blue and to silver stain are different so that the protein profiles of the Triton-derived PSD fraction in Figs 1(b) and 3(a) appear different. The two faint bands that appear on the control lane in (a) correspond to the secondary antibody and bovine serum albumin. These bands are more prominent than other bands in Coomassie-stained gels (not shown). The righthand lanes correspond to the parent Triton X-100-derived PSD fraction (5 lg protein). The lower panels show thin-section electron micrographs (c and d) of PSDs attached to magnetic beads.

(Fig. 3b). Lack of complete co-purification suggests the existence of detergent-insoluble complexes containing synGAP but not PSD-95. This conclusion is further supported by the recent finding of a novel isoform of synGAP that does not interact with PSD-95 (Li et al. 2001). Consistent with the biochemical analysis, thin sections of affinity-purified materials typically show individual PSDs attached to the PSD-95 antibody-coated magnetic beads (Figs 3c and d). Quantitative analyses were carried out on three sets of control and PSD-95 antibody-coated beads. Every thin-sectioned bead in randomly chosen grid openings was evaluated for attached materials. Of the control beads, 6% (seven of 114) showed attached material, whereas 79% (63 of 80) of the PSD-95 antibody-coated beads showed at

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Table 1 Percentage co-purification of various proteins following affinity purification Protein

Percentage co-purification with PSD-95

GFAP a-CaMKII GluR1 GluR2/3 SAP-97 GRIP

12 ± 2 (6) 28 ± 8 (4) 32 ± 5 (5) 42 ± 6 (4) 29 ± 8 (5) 4 ± 0.2 (6)

Values are mean ± SEM (n). Immunoblots were scanned and areas of densitometric peaks corresponding to the indicated proteins and PSD-95 were determined (denoted as ‘area X’ and ‘area PSD-95’, respectively). Percentage copurification was then calculated using the following formula: [(area X/area PSD-95) parent fraction‚(area X/area PSD-95) affinity-purified fraction]·100 Co-purification is 100% (exclusive co-purification) when the ratio of the peak areas remains the same after affinity purification.

least one PSD-like profile attached (17 beads with no PSD attached; 29 beads with one PSD attached; 19 beads with two PSDs attached; 10 beads with three PSDs attached; five beads with four or more PSDs attached). As only a thin cross-section of each bead is sampled, the total number of PSDs attached to beads must actually be higher. Altogether, these results indicate specific attachment of PSDs to the antibody-coated beads. AMPA receptors and anchoring proteins in the affinity-purified PSD fraction AMPA receptors are observed at the postsynaptic thickening by immunoEM (reviewed in Nusser 2000) and are present in Triton-derived PSD fractions. However, it is still possible that these receptors exist as detergent-insoluble complexes that are physically separate from the PSD-95 scaffold, because they do not bind to PSD-95 directly. Analysis of the affinity-purified fraction should enable us to determine whether AMPA receptors are physically associated with the PSD-95-based complex. Immunoblots with an antibody against the AMPA receptor subunits GluR1 and GluR2/3 indicate that these proteins are present in the affinity-purified fraction (Fig. 4). However,

Fig. 4 Glutamate receptors and anchoring proteins in the affinitypurified fraction. Immunoblots corresponding to the affinity-purified (affin. purif.) fraction (yield from 20 lg parent fraction protein) and to the parent Triton X-100-derived PSD fraction (5 lg protein) are shown.



these receptors did not show exclusive co-purification with PSD-95 (32 and 42% respectively; Table 1). These results suggest the existence of a pool of AMPA receptor complexes that are physically separate from the PSD-95-containing scaffold. The affinity-purified fraction was also immunopositive for SAP-97 (Fig. 4; 29% copurification), a GluR1-binding protein (Leonard et al. 1998). Although SAP-97 and PSD-95 belong to the same MAGUK family, the immunopositive labeling with the SAP-97 antibody cannot be due to cross-reactivity with PSD-95 because bands corresponding to PSD-95 and SAP-97 are separated under the electrophoretic conditions used. In contrast, GRIP1, an anchoring protein for GluR2/3 (Dong et al. 1997) does not appear to co-purify with PSD-95 (Fig. 4); its values for percentage co-purification (4%) are even lower than those for GFAP (12%). Discussion

Biochemical analysis of isolated PSDs and immunoEM of intact tissue are complementary approaches for the identification of PSD components. ImmunoEM has the advantage of yielding information on the in situ structure, but the detection of minor components, especially those that are not exclusively located in the PSD, may be problematic owing to low signal-to-noise ratios. Biochemical analysis on the other hand can detect proteins even when present in trace amounts and ultimately allows quantification. However, because PSD fractions contain elements that do not originate from PSDs, the approach can yield false positives. Indeed, analysis of the conventional Triton-derived PSD fraction by immunolabeling and rotary shadowing revealed numerous contaminants, mostly of cytoskeletal origin. In the present study we took advantage of the consistent and selective presence of PSD-95 in PSDs to separate them from contaminating materials. An affinity-based purification protocol was devised using magnetic beads coated with a PSD-95 antibody. Because many proteins detected in the parent Triton-derived PSD fraction, such as spectrin and CaMKII, can be found in PSDs as well as in other cellular compartments, the success of the purification protocol was assessed by monitoring the elimination of the glial protein GFAP. The results show a significant reduction in this glial contaminant upon affinity purification (Fig. 3b, Table 1). In addition, two neuronal proteins, synapsin I and NF-L, thought to be contaminants in the Triton-derived PSD fraction, are virtually eliminated (Fig. 3b). Relative levels of another protein, CaMKII, considered to be a major element of the PSD, are also significantly reduced in the affinity-purified samples (Fig. 3, Table 1). This is mostly attributed to the elimination of contaminating CaMKII clusters from the parent PSD fraction. Electron microscopic analysis of the structures adhering to beads reveals only

structures resembling PSDs. Thus, biochemical and electron microscopic analysis of the affinity-purified PSD fraction indicates effective removal of contaminating materials. Although the affinity purification strategy results in a significant reduction in contaminants, it should be noted that the protocol, which involves dilution, sonication and extensive washing with salt solutions, might also result in the removal of certain loosely attached PSD components. Thus the purified fraction is likely to represent the PSD-95-based scaffold along with covalently/strongly attached components. Because the affinity-purified PSDs very much resemble PSDs observed in situ (Fig. 3), we conclude that many of the major original scaffolding components have been retained during fractionation. The affinity purification strategy adopted in this study selects for PSD-95-containing PSD complexes but any other postsynaptic complexes that are not physically attached to the main scaffold are eliminated. This allowed us to test whether AMPA receptors and anchoring proteins are associated with the PSD-95-containing main scaffold. Immunoblotting with specific antibodies (Fig. 4) indicated that AMPA receptors (GluR1 and GluR2/3) are associated with the PSD-95-containing scaffold. Although AMPA receptors do not bind directly to PSD-95, the present demonstration of their association with the PSD-95 scaffold is in agreement with the growing body of evidence on the role of PSD-95 in the localization of AMPA receptors. Indeed, changes in the expression/localization of PSD-95 appear to modulate synaptic localization of AMPA receptors (El-Husseini et al. 2000, 2002). Detection of the AMPA receptor (GluR1)-binding protein SAP-97 in the affinity-purified fraction indicates its association with the PSD-95-containing main complex. The finding that only a subpopulation of SAP-97 co-purifies with PSD-95 is in agreement with the results of Sans et al. (2001) who used our affinity purification method to isolate PSDs with attached membranes from a synaptic membrane fraction. However, although a smaller proportion of SAP-97 than GluR1 fractionates with PSD-95 when PSDs with attached membranes are isolated (Sans et al. 2001), the relative ratios are similar when membrane-free PSDs are isolated (Table 1). Thus, contrary to the conclusion of Sans et al. (2001), the results from the present study suggest that the subpopulation of GluR1 associated with the PSD-95 scaffold is anchored through SAP-97. However, it should be noted that SAP-97 may not be the only protein that anchors AMPA receptors to the PSD-95 scaffold. Another candidate is Stargazin, a protein that binds to both PSD-95 and GluR1 (Schnell et al. 2002). The fact that AMPA receptors did not co-purify exclusively with PSD-95 suggests that a subpopulation of detergent-insoluble AMPA receptors is present as complexes independent of PSD-95. Furthermore, GRIP, an AMPA receptor (GluR2/3)-binding protein (Dong et al. 1997; Osten



et al. 2000), is virtually absent in our affinity-purified fraction. These observations are in agreement with the findings of Srivastava et al. (1998) that GRIP and the related protein AMPA receptor binding protein (ABP) (GRIP2) do not bind PSD-95. Thus, the present findings support the conclusions of Srivastava et al. (1998) that GRIP–AMPA receptor complexes are physically separate from the PSD-95 scaffold. Acknowledgements We thank Virginia Tanner Crocker and Rita Azzam for technical assistance with EM. This work was supported by the National Institutes of Health Intramural Program and National Science Foundation Grant 9817317 to A.D.

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