Proteomic Analysis of an Interactome for Long-Form AMPA Receptor

Feb 4, 2010 - Identification of GABAC Receptor Protein Homeostasis Network Components from Three Tandem Mass Spectrometry Proteomics Approaches. Ya-Ju...
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Proteomic Analysis of an Interactome for Long-Form AMPA Receptor Subunits Sandra D. Santos, Bruno Manadas, Carlos B. Duarte, and Ana Luı´sa Carvalho* Center for Neuroscience and Cell Biology and Department of Life Sciences, University of Coimbra, 3004-517 Coimbra, Portugal Received August 28, 2009

Glutamate receptors of the AMPA-type mediate fast excitatory synaptic transmission in the central nervous system and play key roles in synaptic plasticity. The binding of these receptors to a variety of proteins is known to regulate their targeting to the synapse and consequently to modulate synaptic strength, as well as to modify receptor characteristics. In this study, a proteomic screening was conducted in order to identify new binding partners for GluR4 AMPA receptor subunit. Immunoprecipitation of GluR4 and associated proteins was performed using rat cerebellum lysates and an heterologous systems overexpressing GluR4 AMPA receptor subunit. Isolated immuno-complexes were resolved by 1-D SDS-PAGE, and analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS). This approach led to the identification of several interactors, most of which are novel AMPA receptor partners, namely, cytoskeleton proteins, motor proteins, RNA processing proteins which are part of neuronal RNA granules, and kinases, among others. This study unravels new constituents of the macromolecular complex of long-form calcium-permeable AMPA receptors. Keywords: AMPA receptors • GluR4 • Cerebellum • Interacting proteins • Proteomics

Introduction The majority of excitatory transmission in the central nervous system is mediated by R-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. They coassemble as heterotetrameric structures formed by combinations of four glutamate receptor (GluR) subunits, termed GluR1 through GluR41,2 or GluA1-A4, according to the last recommendation of the nomenclature committee of the International Union of Pharmacology.3 The regulation of AMPA receptor (AMPAR) function is critical for the long-lasting changes in synaptic strength that are considered to underlie higher brain functions such as learning and memory.4,5 Protein-protein interactions, together with post-translational modification of the receptors, regulate AMPAR function by either modulating the trafficking and localization of the receptors at the synapses, or modifying the channel properties of the receptors.6 AMPAR subunits bind to a variety of proteins through their C-terminal intracellular domain,6-8 which influences their targeting to the postsynaptic membrane of excitatory synapses. Moreover, transmembrane proteins, such as transmembrane AMPAR regulatory proteins (TARPS)9 and cornichons,10 are important determinants of AMPAR behavior. Subunit-specific receptor trafficking is studied intensively because the number and subunit composition of AMPARs regulate synaptic strength.5,11,12 Synaptic activity is required for synaptic insertion of the long-tailed GluR1-containing * Corresponding author: Ana Luı´sa Carvalho, Center for Neuroscience and Cell Biology & Department of Life Sciences, University of Coimbra. 3004517 Coimbra, Portugal. Tel: +351 239 104397. Fax: +351 239 822776. E-mail: [email protected].

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AMPARs in thalamic, hippocampal and cortical neurons,13-17 whereas spontanenous synaptic activity drives GluR2L or GluR4-containing receptor to synapses.15,18,19 In contrast, AMPARs composed of short-tailed subunits (GluR2, GluR3 and GluR4c) cycle between synaptic and extrasynaptic sites in a manner independent of activity.14,20,21 In the mature hippocampus, receptors composed of GluR1 + GluR2 and GluR2 + GluR3 predominate,22,23 whereas GluR4 is expressed and functional at synapses in the immature hippocampus,19 and in other mature brain regions, such as the cerebellum,24 the thalamus15 and the cortex.17 GluR4 mediates transmission with fast kinetics,25 increasing action potential timing precision.17 So far, little is known about how spontaneous synaptic activity triggers GluR4 synaptic delivery and few GluR4-interacting proteins have been identified. Furthermore, its C-terminal domain lacks the ES(V/I)KI consensus sequence present in the other AMPAR subunits, through which they interact with PDZ domain-containing proteins such as GRIP/ ABP, PICK1 and SAP97.26 The mechanisms that regulate synaptic targeting of GluR4 are unknown, and probably depend on its interaction partners. Therefore, we used a proteomic approach that combined coimmunoprecipitation experiments, 1-D electrophoresis and identification of proteins by mass spectrometry, in order to identify new interactors for AMPARs, specifically for the GluR4 subunit.

Experimental Procedures Reagents. Protein A Sepharose CL-4B, the alkaline phosphatase-conjugated anti-rabbit and anti-mouse secondary antibodies, and the ECF immunodetection substrate, were 10.1021/pr900766r

 2010 American Chemical Society

Proteomics of AMPA Receptor Complexes obtained from GE Healthcare (Carnaxide, Portugal). The BCA assay kit was purchased from Pierce, as part of Thermo Fisher Scientific (Rockford, IL). Normal rabbit and mouse IgGs, and the anti-hnRNP K antibody, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-myc antibody was acquired from Cell Signaling (Danvers), the anti-Na+/K+ATPase R1 antibody was obtained from Millipore (Madrid, Spain), and the anti-PSD 95 antibody was from Affinity BioReagents (Golden, CO). The anti-GluR4 antibody27 was a gift from Dr. Richard Huganir (Johns Hopkins University School of Medicine, Baltimore, MD), the sheep anti-GluR1 N-terminal antibody28 was a gift from Dr. Andrew Irving (University of Dundee, Dundee, Scotland), and the GluR1 contruct was a gift from Dr. Juan Lerma (Instituto Neurociencias Alicante, Alicante, Spain); the anti-Dead Box 3 antibody29 was a gift from Dr. Wayne Sossin (McGill University, Montreal, Canada) and the anti-Syncrip antibody and construct30 were given to us by Dr. Kerstin Duning (University Hospital Mu ¨ nster, Mu ¨ nster, Germany). The secondary conjugated antibodies (anti-rabbit-Alexa 488, anti-sheep-Alexa 568 and anti-mouse-Alexa 647) were purchased from Molecular Probes (Leiden, The Netherlands). All other reagents were from Sigma (Sintra, Portugal) or from Merck (Darmstadt, Germany). Rat Cerebellum Total Extracts. Rat cerebellum was homogeneized in a Potter-Elvehjem homogeneizer with 10 vol (10× weight) of ice-cold 10 mM Tris-HCl buffer, pH 7.4, containing 320 mM sucrose. The homogenate was centrifuged at 700g, for 10 min, at 4 °C. The pellet was rehomogenized in the same buffer and centrifuged at 700g, for 10 min, at 4 °C. Both supernatants were pooled, supplemented with protease inhibitors (0.2 mM PMSF, 100 mM DTT, 1 µg/mL each of chymostatin, pepstatin, antipain, and leupeptin) and subjected to protein quantification by the BCA method. The protein was aliquoted and frozen at -20 °C, until needed. HEK 293 Cell Cultures: Maintenance and Transfection. Human embryonic kidney (HEK) 293FT cells or HEK 293 cells constitutively expressing GluR4 (HEK-GluR4,31 a kind gift of Dr. EL Barsoumian, Nippon Boehringer Ingelheim Co, Kawanishi, Japan) were grown in D-MEM, supplemented with 10% (v/v) FBS, 0,1 mM NEAA, 6 mM L-glutamine, 1 mM sodium pyruvate (in MEM) and 500 µg/mL Geneticin. Cells were maintained at 37 °C, in a humidified atmosphere containing 5% CO2. Transient transfection of HEK 293FT cells was carried out using Lipofectamine reagent (Invitrogen, Barcelona, Spain) with a modified version of the manufacturer’s protocol, as follows: the culture medium was replaced for OptiMEM (Invitrogen, Barcelona, Spain) supplemented with 10% FBS, and lipid-DNA complexes were added to each cell plate; after overnight incubation, transfection medium was replaced with normal HEK 293FT cell medium and expression was left to occur for 24-48 h of cell growth. Hippocampal Cultures. Hippocampal sandwich cultures with glial feeders were used. Briefly, hippocampi were dissected from E18 rat embryos and dissociated using trypsin (0.25%) and trituration. Neurons were plated at a final density of 1-5 × 104 cells/dish on poly-D-lysine-coated coverslips in 60 mm culture dishes in neuronal platting medium (MEM supplemented with 10% horse serum, 0.6% glucose and 1 mM pyruvic acid). After 2-4 h, coverslips were flipped over an astroglial feeder layer in Neurobasal medium supplemented with B27 supplement (1:50 dilution), 25 µM glutamate, 0.5 mM glutamine and 0.12 mg/mL gentamycin. The neurons grew face down over

research articles the feeder layer but were kept separate from the glia by wax dots on the neuronal side of the coverslips. To prevent the overgrowth of the glia, neuron cultures were treated with 5 µM cytosine arabinoside after 3 days in vitro (DIV). Cultures were maintained in Neurobasal medium for 2 weeks, feeding the cells once per week by replacing one-third of the media per dish. Immunoprecipitation Assays. Proteomic screenings were performed using receptor complexes immunoprecipitated from rat whole cerebellum lysates and from lysates of HEK 293 cells constitutively expressing GluR4 [HEK-GluR4].31 Four milligrams of protein from rat cerebellum lysates or 6 mg from HEK-GluR4 cell lysates was solubilized in Immunoprecipitation Buffer [IPB: 10 mM Tris (pH 7.0), 50 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100 and protease inhibitors (0.2 mM PMSF, 100 mM DTT, 1 µg/mL each of chymostatin, pepstatin, antipain, and leupeptin or Complete mini protease inhibitor mixture, Roche Diagnostics)]. The samples were sonicated with a probe sonicator, on ice, for 30 s. The insoluble material was removed by centrifuging the sample at 13 000g during 10 min, at 4 °C. The supernatant was transferred to a tube containing 100 µL of 50% slurry of protein A sepharose beads suspended in IPB. The tube was rotated at 4 °C, for 1 h; this step preabsorbs any protein that may stick nonspecifically to the protein A sepharose beads. After a 5 min centrifugation step, the sedimented sepharose beads were discarded. The supernatant was split into two tubes, one that was incubated with 4 µg of anti-GluR4 antibody and another that was incubated with the same amount of nonimmune rabbit IgGs. This incubation step was performed at 4 °C, for 3 h. The samples were then incubated with 100 µL of 50% slurry of protein A sepharose beads and rotated for 2 h at 4 °C. Several washing steps were performed in order to minimize nonspecific binding: 2× IPB + 1% Triton, 3× IPB + 1% Triton + 500 mM NaCl and 2× IPB. The proteins were eluted by boiling the beads in 50 µL of SDS sample buffer (125 mM Tris, pH 6.8, 100 mM glycine, 4% SDS, 200 mM DTT, 40% glycerol, 3 mM sodium orthovanadate, and 0.01% bromophenol blue) for 5 min, whenever 1-D gel was the next step, or the beads were frozen at -80 °C if liquid digestion of samples was performed, before LC-MS/MS. Proteins were separated by SDS-PAGE in 7.5% or 12% polyacrylamide gels. After staining the gel with Coomassie blue, the bands of interest were excised and proteins were analyzed by mass spectrometry, as described below. To probe for specific interactions, we used 2 mg of protein from rat cerebellum lysates, or 1 mg of protein from HEK-GluR4 or transfected HEK 293FT cell extracts and performed the immunoprecipitation assays as indicated above, using 3 µg of antibody against the protein of interest or nonimmune mouse IgGs. Proteins were separated by SDS-PAGE in 7.5% polyacrylamide gels, followed by Western-blot analysis. Protein Identification by Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS). Liquid digestion (applied to protein complexes immobilized on beads): 1 µL of 110 mM DTT was added to 30 µL of sample, followed by a sonication step for 10 min, at 37 °C. Next, 1 µL of 600 mM iodoacetamide was added and samples were again sonicated for 10 min, at room temperature. NH4HCO3 was then added to a final concentration of 10 mM, and the samples were trypsin digested overnight (0.5 µg Trypsin/10 µg protein), at 37 °C, with swirling at 400 rpm. Peptides were dried by rotary evaporation under vacuum, and resuspended in 2% acetonitrile, 0.1% formic acid. Journal of Proteome Research • Vol. 9, No. 4, 2010 1671

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Table 1. Proteins Coimmunoprecipitated with GluR4 in Rat Cerebellum Lysates (Figure ) or in HEK-GluR4 Cells (Figure 2), Identified by Mass Spectrometry

In-gel digestion: protein in-gel digestion was performed as previously described, with slight modifications.32 Briefly, gel bands were sliced in small pieces and destained with 50 mM ammonium bicarbonate and 30% acetonitrile. In-gel digestion was performed overnight at room temperature with 30 1672

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µL of trypsin (10 ng/µL) in 10 mM ammonium bicarbonate. Peptides were extracted with 30%, 50%, and 98% acetonitrile in 1% formic acid, pooled, dried by rotary evaporation under vacuum, and resuspended in 2% acetonitrile and 0.1% formic acid.

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Table 2. Proteins Coimmunoprecipitated with GluR4 in Rat Cerebellum Lysates (Figure ) or in HEK-GluR4 Cells (Figure 2), Identified by Mass Spectrometry, And Also Detected in the Control Conditionsa

a The specificity of the co-purification with GluR4 was evaluated using the relative peptide query (rPQ) score,10 as well as a label-free quantification method91 (see Supplementary Materials and Methods and Supplementary Figure 1 for details) to determine the ratio of peak area for each peptide detected in the immunoprecipitation condition and in the control condition (IPR4/Control). Proteins with rPQ g 2 and/or mean area ratio g 2 were considered as specifically co-purifying with GluR4. For each protein, average ratio ( SEM of all matching ions is presented.

Protein identification experiments were carried out on a hybrid quadrupole/linear ion-trap mass spectrometer (4000 QTrap; Applied Biosystems/MDS Sciex) using electrospray source and a dual gradient pump (Ultimate 3000; Dionex). The mass spectrometer was programmed for information dependent acquisition33 scanning full spectra, followed by an enhanced resolution scan to determine the ion charge states, and set the appropriate collision energy for fragmentation. The IDA cycle was programmed to perform 8 MS/MS on multiple charged ions (+1 to +4) and performed two repeats before adding ions to the exclusion list for 60 s (mass spectrometer operated by Analyst 1.4.1). Peptides were eluted into the mass spectrometer with a binary gradient (300 nL/min 2% acetonitrile, 0.1% formic acid to 98% acetonitrile, 0.1% formic acid in a multiple step gradient for 50 min) (Ultimate 3000, Dionex), using a nanoelectrospray source. Peptide identification was performed with Protein Pilot software (v2.0.1, Applied Biosystems/MDS Sciex)34,35 against the Swiss-Prot database. Positive identifications were considered when protein score was above 1.3 (95%).36 Protein identification based on single peptide hit had a minimum individual score of 95% and a minimum sequence tag of 3 amino acids (4 consecutive peaks in the MS/ MS spectrum). Immunocytochemistry. Surface GluR1-containing receptors were labeled by incubating the neurons for 10 min at room temperature with the GluR1 N-terminal antibody diluted in culture medium, after which the cells were fixed in 4% sucrose/ paraformaldehyde and permeabilized with PBS + 0.25% Triton X-100. The neurons were then incubated in 10% BSA for 30 min at 37 °C to block nonspecific staining and incubated with an anti-sheep secondary antibody (45 min, 37 °C). After washing 6 times in PBS, cells were incubated with anti-Dead box 3 or anti-Syncrip antibodies, together with the anti-PSD

95 antibody (2 h, 37 °C). After washing 6 times in PBS, cells were incubated with the appropriate secondary antibodies (45 min, 37 °C). The coverslips were mounted in a fluorescent mounting medium from DAKO (Glostrup, Denmark). Imaging was performed on a Zeiss Axiovert 200 M microscope, using a 63× 1.4 NA oil objective.

Results and Discussion Several proteins have been reported to regulate receptor trafficking and synaptic plasticity through direct or indirect interactions with AMPARs.6 Most of the known AMPAR binding partners were identified in yeast two-hybrid screenings, or by coimmunoprecipitation studies for candidate proteins. However, combining affinity isolation of native protein complexes and mass spectrometry is powerful for revealing interacting proteins which directly or indirectly associate with a protein of interest. Recently, proteomic analysis showed that AMPARs are coassembled with a new family of proteins, the cornichons, which affect receptor surface expression and channel gating.10 A previous study using similar methodology identified five new AMPAR interactors.37 In the present work, immunoprecipitation assays, performed in a large scale, allowed us to identify by mass spectrometry, with high confidence, 17 AMPAR subunit interactors, most of which are novel interactors (Tables 1 and 2). In this study, we used two different systems to purify GluR4 in complex with interacting proteins, rat cerebellum lysates and HEK 293 cells stably expressing GluR4. The adult rat cerebellum is enriched in GluR4,24 and therefore, we could isolate large amounts of GluR4 and associated proteins from this preparation; on the other hand, HEK 293 cells stably expressing GluR4 (HEK-GluR4) express GluR4 at high levels,31 and are a useful Journal of Proteome Research • Vol. 9, No. 4, 2010 1673

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Figure 1. Immunoprecipitation of GluR4 using rat cerebellum extracts. Two milligrams of rat cerebellum lysates was incubated with 4 µg of anti-GluR4 antibody coupled to Protein A Sepharose (a, IPR4) or 4 µg of rabbit IgGs coupled with Protein A Sepharose (b, negative control). Purified proteins were resolved by SDS-PAGE and stained with Coomassie Blue. The numbered arrows indicate the bands that were excised, digested and analyzed by liquid chromatography coupled with tandem mass spectrometry. The list of proteins on the right refers to proteins that were identified in the respective band specifically in the IPR4 (lane a).

system for identifying GluR4 interactors that are not neuronspecific. GluR4 was immunoprecipitated from rat cerebellum lysates, and immuno-complexes immobilized in Protein A beads were either removed using Laemmli solution or directly subjected to tryptic digestion followed by LC-MS/MS. In parallel, as a negative control, the same procedure was undertaken but using rabbit IgG-coupled protein A-Sepharose. As shown in Figure 1, several proteins specifically coimmunoprecipitated with GluR4 from the rat cerebellum lysate. The indicated protein bands were excised from the gel in both the immunoprecipitation lane (Figure 1, lane a) as well as in the negative control (Figure 1, lane b). The tryptic peptides obtained from each gel slice were analyzed by mass spectrometry. Proteins identified in the GluR4 immunoprecipitation lane (Figure 1, lane a) with two or more peptides and with a confidence level g95%, but absent in the negative control (Figure 1, lane b), can be considered specific interactors for GluR4, and are listed in Table 1. In the duplicate of this experiment, isolated immuno-complexes were not eluted from protein A-Sepharose, and were instead digested with trypsin directly from the beads (liquid digestion). Tryptic peptides were analyzed by LC-MS/MS, both for the GluR4 immunoprecipitation sample and for the negative control, and proteins identified specifically in the GluR4 immunoprecipitation, with two or more peptides and a confidence level g95%, and absent in the control sample, are listed in Table 1. Several proteins that were identified by peptides from the in-gel digested proteins (Figure 1) were not detected using the tryptic peptides obtained from the immobilized protein (e.g., CaMKII γ chain and GluR1). Conversely, some proteins identified using the tryptic peptides obtained after digesting the immobilized proteins (liquid digestion) could not be identified in the gel bands (e.g., spectrin). The combination of the two approaches proves useful in revealing novel interactors, as well as in excluding false positive interactors. GluR4 and associated proteins purified by immunoprecipitation from HEK-GluR4 cell extracts, similarly to the proteins purified from rat cerebellum lysates, were separated by SDSPAGE (Figure 2), followed by in-gel digestion. In this case, the 1674

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Figure 2. Immunoprecipitation of GluR4 using lysates from HEK 293 cells constitutively expressing GluR4 (HEK-GluR4). Three milligrams of protein lysate was incubated with 4 µg of antiGluR4 antibody coupled with Protein A Sepharose (a) or with uncopled Protein A Sepharose beads (b, negative control). Purified proteins were resolved by SDS-PAGE and stained with Coomassie Blue. Numbered arrows indicate the bands that were excised and analyzed by LC-MS/MS. The list of proteins on the right refers to proteins that were identified in the respective band in the IPR4 (lane a).

protein bands specifically detected in the GluR4 immunoprecipitation lane as well as a band from the negative control (uncopled beads) were excised from the gel and the gel slabs were subjected to tryptic digestion. Peptide sequence information obtained in the mass spectrometry analysis was used to search protein databases, and the proteins identified by two or more peptides, with a confidence level g95%, are listed in Table 1. The identified proteins were categorized according to their cellular functions (Figure 3) as “RNA processing proteins”

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Figure 3. Schematic representation of AMPA Receptor interactors in the postsynaptic density (PSD), in dendritic shafts and glia cells.

(heterogeneous nuclear ribonuclear proteins, hnRNPs, and RNA helicases), “Motor proteins” (myosin Va), “Metabolic proteins” (the CAD multifunctional protein of the pyrimidine pathway), “Kinases” (CaMKII γ chain), “Axonal growth and guidance proteins” (Dihydropyrimidinase-related protein 2) and “Cytoskeleton proteins” (tubulin-R and -β, actin, vimentin). Other proteins of these protein families were concomitantly detected in the eluates of the GluR4 antibody and control IgG pools. The specificity of copurification with GluR4 was judged from quantitative comparison between the amounts of each protein in the immunoprecipitation sample and in the control sample, using two different approaches (Table 2). These analyses resulted in the identification of several additional motor proteins (myosin-10 and myosin-9), cytoskeletal proteins (spectrin R and β chains, R-internexin), and a metabolic protein (ADP/ATP translocase) as specific GluR4 interactors (Table 2, see Supplementary Figure 1 for detailed analysis of the specificity of copurification of Myosin-10 with GluR4). In addition to these proteins, which were identified with high confidence, some interesting proteins were identified with a smaller confidence level (one peptide only), namely, drebrin, hnRNP K, IP3K, NCam, Shank 2, 14.3.3ε and R-actinin4, among others, and are listed in Table 3. The proteins identified with only 1 peptide with a confidence level g95% have yet to be validated as GluR4 interactors, for example, by repeating the immunoprecipitation of GluR4, performing Western blot and

analyzing the specific coimmunoprecipitation of those proteins with GluR4. So far we have validated the interaction between hnRNP K and GluR4 by coimmunoprecipitating GluR4 together with hnRNP K from rat cerebellum lysates, as well as from HEKGluR4 cells (Figure 4). In the immunoprecipitation assays for GluR4 performed using rat cerebellum lysates, GluR1 was the only other AMPAR subunit detected. This suggests that most of GluR4 in the adult rat cerebellum is present as an heteromeric complex with GluR1, as previously found.24 Since heteromeric receptor complexes comprised of GluR1 and GluR4 were isolated, the identified binding partners may interact with both GluR1 and GluR4, or with either of these receptor subunits. GluR1 and GluR4 subunits are intensely expressed in cerebellar Bergmann glia,38-40 where they form Ca2+-permeable AMPARs with an important role in generating and maintaining the appropriate structural and functional association between the neuronal elements of glutamatergic synapses in the cerebellum and Bergmann glia.39 Bergmann glia extend processes around the synapses between parallel fiber and climbing fiber terminals and Purkinje cell dendrites in the cerebellar cortex, and glutamate released at these synapses activates AMPARs on Purkinje cells and GluR1/GluR4-containg Ca2+-permeable AMPARs on Bergmann glia cells.41 The generated Ca2+ signal is necessary to maintain the partnership between Bermann glia and the synaptic elements, and to regulate innervation of Journal of Proteome Research • Vol. 9, No. 4, 2010 1675

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Table 3. Proteins Coimmunoprecipitated with GluR4 in Rat Cerebellum Lysates (Figure ) or in HEK-GluR4 Cells (Figure 2), Identified by Mass Spectrometry with Only 1 Peptide with a Confidence Level g 95%

Purkinje cells by climbing fibers.39 A recent study shows that these Ca2+-permeable AMPARs containing long-form subunits 1676

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of AMPARs are regulated by γ5,42 an atypical transmembrane AMPAR regulatory protein (TARP), but little else is known about

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Figure 4. RNA granule proteins interact with AMPA Receptors GluR1 and GluR4 subunits. (A) GluR4 subunit coimmunoprecipitated with hnRNP K immunopurified from rat cerebellum (a) or HEK-GluR4 cell lysates (b). (B) GluR1 coimmunoprecipitated with Syncrip immunopurified from HEK 293 FT cells transfected with both GluR1 and Syncrip-myc, when a specific antibody for myc was used in the immunoprecipitation assay. (C) Syncrip labeling colocalizes with live cell labeling for surface GluR1 in cultured hippocampal neurons 15 DIV. Syncrip partially colocalizes with GluR1 and PSD95, indicating that it partially distributes to AMPAR-containing synapses (as indicated by arrows in figure), as well as colocalizes with extrasynaptic AMPARs (* in figure). (D) DEAD box 3 labeling in hippocampal neurons in culture shows a punctuate distribution, which colocalizes with synaptic cell surface GluR1 (arrows), as well as with extrasynaptic GluR1 (*). Scale bars: 10 µm; inset, 5 µm.

their regulation. Besides their role in Bergmann glia, Ca2+permeable AMPARs play important roles for instances in the hippocampus,43 where they are transiently incorporated in synapses during long-term potentiation. Moreover, they are crucial players in excitotoxicity, both in acute conditions, such as stroke, and chronic neurodegenerative conditions, such as amyotrophic lateral sclerosis.44 The interacting partners found

in the present study are potential candidates for modulating the behavior of this type of receptors. Proteins related with RNA regulation were found in the GluR4-containing immuno-complexes purified from HEKGluR4 cells (RNA helicases, DEAD box helicases 3 and 9, and members of the hnRNP family, SYNCRIP, hnRNP M and K) and also from rat cerebellum lysates (hnRNP K), suggesting a Journal of Proteome Research • Vol. 9, No. 4, 2010 1677

research articles possible involvement of AMPARs in regulating their function. The interaction of hnRNP K with GluR4 was validated using both rat cerebellum extracts and HEK-GluR4 cells lysates, since GluR4 coimmunoprecipitated with hnRNP K in both systems (Figure 4A). Synaptotagmin-binding cytoplasmic RNA interacting protein (Syncrip) and the RNA helicase DEAD box 3 are core components of large mRNA transport granules in neurons, localize to dendrites, and were proposed to play a role in the regulation of the translation of mRNA molecules associated with the transport granules.45 To test whether Syncrip interacts with GluR1 AMPAR subunit, we transfected HEK 293 FT cells with plasmids encoding GluR1 and myc-tagged Syncrip, and used the anti-myc antibody to immunoprecipitate Syncrip-myc from this system. GluR1 was found to coimmunoprecipitate with Syncrip-myc (Figure 4B), indicating that in addition to interacting with GluR4 (Table 1) Syncrip also binds to GluR1. To test whether Syncrip colocalizes with AMPARs present at the surface of neurons, we performed in vivo staining of GluR1 in rat hippocampal neurons maintained in culture for 15 days, using an antibody against the extracellular N-terminal region of GluR1. Neurons were then fixed, permeabilized, and stained with an antibody against Syncrip, as well as an antibody against the postsynaptic protein PSD95, to label glutamatergic synapses (Figure 4C). Our data indicate that Syncrip colocalizes with GluR1-containing AMPARs at the cell surface of hippocampal neurons, at both synaptic (positive for PSD95 labeling) and extrasynaptic (negative for PSD95 labeling) sites. The same strategy was used to test whether DEAD box 3 colocalizes with cell surface AMPARs (Figure 4D), and the data show extensive colocalization of DEAD box 3 punctuate staining with both cell surface GluR1 and PSD95. Our results indicate that Syncrip and DEAD box 3 can localize in proximity to synaptic sites, and that these two RNA processing proteins, present in RNA granules, cocluster with GluR1-containing AMPA receptors at the surface of hippocampal neurons. These observations lead us to speculate that the coupling of RNA granules to AMPARs may facilitate regulation of localized translation of transcripts present in RNA granules in dendrites, in response to synaptic activity. In fact, local protein synthesis in neuronal dendrites is essential for transducing neural activity into persistent changes in synaptic connectivity,46 and has been shown in response to different paradigms that change synaptic efficacy (e.g., see refs 47 and 48). Among the proteins identified in the screening we found three myosin molecules: Va, 9 and 10. Myosins are a superfamily of motor proteins that generate force along actin filaments. Some members of the myosin family have been shown to participate in AMPAR traffic. Myosin VI plays a role in AMPAR endocytosis,49 while myosin Vb is necessary for dendritic surface expression of AMPARs,50 and responds to a localized influx of Ca2+ ions through NMDA receptors in spines. Myosin Vb also associates with recycling endosomes, and triggers local exocytosis of AMPARs in spines during LTP.51 Correia and co-workers recently found that myosin Va directly interacts with AMPARs and with Rab11 and is also responsible for AMPAR trafficking during LTP.52 Accordingly, we detected myosin Va in the immunoaffinity purified GluR4 complexes (Table 1). Myosin 10 contains a FERM domain through which it binds to β-integrins, providing a motor based link between the actin cytoskeleton and integrins.53 Given the role of β-integrins in regulating AMPAR function,54,55 the interaction between myosin 10 and AMPAR subunits is of potential interest. Myosin 956 is one of two 1678

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Santos et al. myosin motors (along with myosin VI) to be minus end directed. Therefore, it would be informative to evaluate possible roles of myosin 9 in AMPAR endocytosis, and in the traffic of endosomes containing AMPARs. The dihydropyrimidinase-related protein 2 (DRP-2), another protein found to interact with GluR4 (Table 1), is involved in the formation of neuronal connections and consequently in the maintenance of neuronal communication. DRP-2 is a pathfinding and guidance protein for axonal outgrowth, which interacts with and modulates collapsin, a protein responsible for the elongation and guidance of dendrites.57 Recently, it was shown in our laboratory that the brain-derived neurotrophic factor regulates the protein levels of DRP-2 and of hnRNP K in hippocampal neurons.58 Two kinases were identified in the screening for GluR4 interactors: Calcium/calmodulin-dependent protein kinase type II γ chain (CaMKIIγ, Table 1) and Inositol-trisphosphate 3-kinase A (IP3K, Table 3). CaMKII is a multifunctional enzyme that has been implicated in a multitude of Ca2+-regulated biological processes, including synaptic transmission, gene transcription, and growth control.59,60 The γ isoform of CaMKII is ubiquitously expressed and has been described to be present at the PSD.37 This kinase can phosphorylate both AMPARs and NMDA receptors61 and it is known to accumulate in the PSD region after a local Ca2+ increase, and to bind directly to the NR2B subunit.62,63 Our results support the idea that CaMKII exerts its functions at synapses by regulating synaptic AMPARs and/or NMDARs since it appears to be part of both glutamate receptor complexes. A lipid kinase, IP3K, which phosphorylates inositol 1,4,5trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4), was identified in this screening (Table 3). Another lipid kinase, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), has been previously shown to be complexed with AMPARs at synapses.64 Activation of the AMPAR-associated PI3K is required for the increased cell surface expression of AMPARs and LTP.64 Moreover, synaptojanin, a polyphosphoinositide phosphatase, which dephosphorylates PI(4,5)P2 was found to be involved in AMPAR internalization.65 These evidence together suggest a role for phospholipids turnover in AMPAR traffic, and reinforce the idea that the association of IP3K with AMPARs may be functionally relevant. SynGAP, a Ras GTPase-activating protein found in our screening for GluR4 interactors (Table 3), is highly enriched at the postsynaptic density of excitatory synapses, and is part of the NMDA receptor multiprotein complex through interaction with PSD95.66-68 Interestingly, mutant mice for SynGAP showed an increase in the number of synaptic AMPAR clusters, suggesting that SynGAP regulates AMPAR synaptic targeting. Furthermore, SynGAP heterozygous mutant mice have a specific defect in hippocampal long-term potentiation (LTP), suggesting that regulation of synaptic Ras signaling by SynGAP is important for glutamate receptor trafficking and for the expression of LTP.69 These observations are in agreement with the role proposed for Ras in the synaptic delivery of AMPARs during LTP.15,70 The detection of SynGAP in the GluR4 immunoaffinity isolation from rat cerebellum suggests that SynGAP may associate with AMPAR complexes, and thereby regulate the traffic of AMPAR subunits through a negative effect on Ras. The Na,K-ATPase (NKA), an heterodimer composed of a catalytic R subunit (ATPase activity) and a regulatory β subunit (required for enzymatic activity), which maintains ion gradients

Proteomics of AMPA Receptor Complexes

research articles vimentin knockout animals show cerebellar defects, such as impaired motor coordination, changes in the morphology of Bergmann glia, and stunting of Purkinje cell dendrites.82 Interestingly, morphological changes in Bergmann glia and Purkinje cells were also observed when Ca2+-permeable AMPARs in Bergmann glia were converted into Ca2+-impermeable channels.39 Our observation that GluR4/GluR1-containing Ca2+permeable AMPARs in the cerebellum associate with vimentin suggests that the phenotype observed for vimentin knockout mice may be at least partially accounted for by disturbance of this interaction.

Figure 5. Na+/K+ ATPase interacts with AMPAR subunits GluR1 and GluR4 in rat cerebellum lysates. (A) GluR4 and GluR1 subunit coimmunoprecipitated with NAK from rat cerebellum lysates when a specific antibody for NAK R1 subunit was used in the immunoprecipitation assay. (B) NAK R1 subunit coimmunoprecipitated with GluR1 immunopurified from rat cerebellum.

in neurons, was identified in our study as an AMPAR interactor (Table 3). In another recent study,71 an association between GluR1 and GluR2 subunits and the R1 subunit of NKA was also described, and shown to be mediated by the C-terminal region of the GluR2 subunit. Interestingly, we found that the GluR1 and GluR4 AMPAR subunits coimmunoprecipitate with NKA (Figure 5A), and that NKA coimmunoprecipitates with GluR1 (Figure 5B) immunopurified from rat brain cerebellum. Zhang and colleagues showed that NKA inhibition induces a reduction in AMPAR cell-surface expression, as well as total protein abundance, indicating that NKA regulates AMPAR turnover, being in this way involved in regulating synaptic strength. Several proteins isolated in this study are cytoskeleton proteins, namely, actin, tubulin (R and β), vimentin and R-internexin. The proteomic study conducted by Collins and colleagues also detected R-tubulin as an AMPAR interactor.37 Notably, tubulin molecules were detected in all the immunoaffinity screenings performed in our study (Table 1). Other reports have shown that mGluR1a,72 mGluR1b73 and mGluR574 interact with β-tubulin, whereas mGluR7 was found to interact with R-tubulin.75 These observations are not surprising, given the role played by microtubules in glutamate receptor trafficking in neurons. Tubulin molecules are likely to associate with extrasynaptic AMPARs at the plasma membrane, or with intracellular pools of receptors. However, recent studies indicate that dynamic microtubules can invade dendritic spines,76,77 which are rich in actin, and which formation and morphology are regulated by actin filaments.78 Invading microtubules modulate actin dynamics within dendritic spines, directly influencing spine morphology and function.76,77 The significance of these interactions remains to be studied. Vimentin and R-internexin belong to the intermediate filament protein family, a dynamic component of the cytoskeleton characterized by rapid movement and dynamic exchange of subunits.79 Vimentin is involved in multiple cellular functions including adhesion, membrane trafficking and scaffolding of protein complexes at the cell membrane.80 In the cerebellum, Bergmann glia astrocytes continue to express vimentin together with glial fibrillary acidic protein (GFAP) in adults,81 and

Alpha-internexin is well characterized as a PSD protein, and is postulated to be essential for the early development and organization of the PSD.83 Moreover, R-internexin was described to interact with the 4.1 band protein,84 a known GluR185 and GluR486 interactor. Several members of the spectrin superfamily were identified in the immunoprecipitation assays (Tables 2 and 3): spectrin R and β chains and R-Actinin-4. Spectrins play a critical role in preserving the morphological integrity and organization of cell membranes.87 These interactions with GluR4 could support the notion that AMPARs are anchored via spectrin to the actin cytoskeleton since spectrin is an actin-binding protein known to anchor various membrane proteins to the actin microfilament network.88 The R-actinins are a family of closely related proteins that cross-link actin filaments and tether proteins to the cytoskeleton. We identified R-actinin-1 in eluates of immunoprecipitated GluR4 from rat whole brain extracts, by MALDI Peptide mass fingerprint analysis (data not shown). Alpha-actinin-1 was previously identified through an yeast-two hybrid assay as a GluR4 interactor,89 whereas R-actinin-4, which was identified in our GluR4 immunoprecipitation screenings from rat cerebellum lysates (Table 3), although at low confidence, was also found to be a mGluR537 and mGluR1b interactor.73 These proteins contain two calponin homology domains, four spectrin repeats and two EF hands. Nuriya and collaborators described that GluR4 interacts with R-Actinin-1 and IQGAP1, and colocalizes with both proteins in dendrites.89 These two interactions occur at the same region of the GluR4 C-terminus, and phosphorylation of Ser842 by PKA differentially regulates those interactions; the interaction between R-Actinin-1 and GluR4 is disrupted by Ser842 phosphorylation, whereas the interaction with IQGAP1 is preserved.89 Alpaactinin-4 forms a ternary complex with densin-180 and CaMKIIR, which are enriched at the postsynaptic density of excitatory synapses.90 In fibroblasts, this protein is involved in the recycling of internalized membrane receptors,33 and therefore, it may contribute to the regulation of AMPARs endocytic trafficking and recycling. Concluding Remarks. In this study, the purification of native GluR4-containing AMPAR complexes, combined with mass spectrometry identification of the copurified proteins, led to the identification of several novel binding partners for AMPAR subunits. For the RNA binding proteins hnRNP K, SYNCRIP and DEAD box 3, further analyses validated the interactions, and indicated that SYNCRIP and DEAD box 3 colocalize with synaptic AMPARs at the surface of cultured hippocampal neurons. For some of the identified proteins, validation of the interactions using independent methodologies is warranted, to confirm that these proteins are bona fide interactors of AMPARs in vivo. Additionally, future studies are necessary to Journal of Proteome Research • Vol. 9, No. 4, 2010 1679

research articles address the consequences of the identified interactions in the regulation of physiological and pathological functions of AMPARs.

Acknowledgment. This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT: POCI/SAU-NEU/58955/2004 and PTDC/ BIA-BCM/71789/2006). S.D.S. was a recipient of a doctoral fellowship (SFRH/BD/11826/2003), and B.M. (SFRH/BPD/ 26456/2006) is a recipient of a postdoctoral fellowship from FCT. We would like to thank Vera M. Mendes from the CNC Proteomics Unit for assistance in Mass Spectrometry. Supporting Information Available: Supplementary Materials and Methods; label free peptide quantification. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Boulter, J.; Hollmann, M.; O’Shea-Greenfield, A.; Hartley, M.; Deneris, E.; Maron, C.; Heinemann, S. Molecular cloning and functional expression of glutamate receptor subunit genes. Science 1990, 249 (4972), 1033–7. (2) Keinanen, K.; Wisden, W.; Sommer, B.; Werner, P.; Herb, A.; Verdoorn, T. A.; Sakmann, B.; Seeburg, P. H. A family of AMPAselective glutamate receptors. Science 1990, 249 (4968), 556–60. (3) Collingridge, G. L.; Olsen, R. W.; Peters, J.; Spedding, M. A nomenclature for ligand-gated ion channels. Neuropharmacology 2009, 56 (1), 2–5. (4) Malenka, R. C.; Nicoll, R. A. Long-term potentiation--a decade of progress. Science 1999, 285 (5435), 1870–4. (5) Kessels, H. W.; Malinow, R. Synaptic AMPA receptor plasticity and behavior. Neuron 2009, 61 (3), 340–50. (6) Santos, S. D.; Carvalho, A. L.; Caldeira, M. V.; Duarte, C. B. Regulation of AMPA receptors and synaptic plasticity. Neuroscience 2009. (7) Sheng, M.; Sala, C. PDZ domains and the organization of supramolecular complexes. Annu. Rev. Neurosci. 2001, 24, 1–29. (8) Shepherd, J. D.; Huganir, R. L. The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu Rev Cell Dev Biol 2007, 23, 613–43. (9) Ziff, E. B. TARPs and the AMPA receptor trafficking paradox. Neuron 2007, 53 (5), 627–33. (10) Schwenk, J.; Harmel, N.; Zolles, G.; Bildl, W.; Kulik, A.; Heimrich, B.; Chisaka, O.; Jonas, P.; Schulte, U.; Fakler, B.; Klocker, N. Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 2009, 323 (5919), 1313–9. (11) Malinow, R.; Malenka, R. C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 2002, 25, 103–26. (12) Collingridge, G. L.; Isaac, J. T.; Wang, Y. T. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 2004, 5 (12), 952–62. (13) Hayashi, Y.; Shi, S. H.; Esteban, J. A.; Piccini, A.; Poncer, J. C.; Malinow, R. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 2000, 287 (5461), 2262–7. (14) Harms, K. J.; Tovar, K. R.; Craig, A. M. Synapse-specific regulation of AMPA receptor subunit composition by activity. J. Neurosci. 2005, 25 (27), 6379–88. (15) Kielland, A.; Bochorishvili, G.; Corson, J.; Zhang, L.; Rosin, D. L.; Heggelund, P.; Zhu, J. J. Activity patterns govern synapse-specific AMPA receptor trafficking between deliverable and synaptic pools. Neuron 2009, 62 (1), 84–101. (16) Takahashi, T.; Svoboda, K.; Malinow, R. Experience strengthening transmission by driving AMPA receptors into synapses. Science 2003, 299 (5612), 1585–8. (17) Zhu, J. J. Activity level-dependent synapse-specific AMPA receptor trafficking regulates transmission kinetics. J. Neurosci. 2009, 29 (19), 6320–35. (18) Kolleker, A.; Zhu, J. J.; Schupp, B. J.; Qin, Y.; Mack, V.; Borchardt, T.; Kohr, G.; Malinow, R.; Seeburg, P. H.; Osten, P. Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors. Neuron 2003, 40 (6), 1199–212. (19) Zhu, J. J.; Esteban, J. A.; Hayashi, Y.; Malinow, R. Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity. Nat Neurosci 2000, 3 (11), 1098–106.

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