Morphine Administration Alters the Profile of Hippocampal Postsynaptic Density-associated Proteins

Morphine Administration Alters the Profile ofHippocampal Postsynaptic Density-associatedProteinsA PROTEOMICS STUDY FOCUSING ON ENDOCYTIC PROTEINS*

Jose A. Moron‡, Noura S. Abul-Husn‡, Raphael Rozenfeld‡, Georgia Dolios§,Rong Wang§, and Lakshmi A. Devi‡¶

Numerous studies have shown that drugs of abuse inducechanges in protein expression in the brain that arethought to play a role in synaptic plasticity. Drug-inducedplasticity can be mediated by changes at the synapse andmore specifically at the postsynaptic density (PSD), whichreceives and transduces synaptic information. To date,the majority of studies examining synaptic protein profileshave focused on identifying the synaptic proteome. Only ahandful of studies have examined the changes in synapticprofile by drug administration. We applied a quantitativeproteomics analysis technique with the cleavable ICATreagent to quantitate relative changes in protein levels ofthe hippocampal PSD in response to morphine adminis-tration. We identified a total of 102 proteins in the mousehippocampal PSD. The majority of these were signaling,trafficking, and cytoskeletal proteins involved in synapticplasticity, learning, and memory. Among the proteinswhose levels were found to be altered by morphine ad-ministration, clathrin levels were increased to the largestextent. Immunoblotting and electron microscopy studiesshowed that this increase was localized to the PSD. Mor-phine treatment was also found to lead to a local increasein two other components of the endocytic machinery,dynamin and AP-2, suggesting a critical involvement ofthe endocytic machinery in the modulatory effects of mor-phine. Because �-amino-3-hydroxy-5-methyl-4-isox-azolepropionic acid (AMPA) receptors are thought to un-dergo clathrin-mediated endocytosis, we examined theeffect of morphine administration on the association ofthe AMPA receptor subunit, GluR1, with clathrin. Wefound a substantial decrease in the levels of GluR1 asso-ciated with clathrin. Taken together, these results suggestthat, by causing a redistribution of endocytic proteins atthe synapse, morphine modulates synaptic plasticity athippocampal glutamatergic synapses. Molecular & Cel-lular Proteomics 6:29–42, 2007.

Opiates are choice analgesics in the treatment of chronicpain. However, repeated opiate administration can lead to thedevelopment of tolerance, physical dependence, and addic-tion. Opiate addiction is thought to involve the brain rewardcircuit as well as brain regions involved in learning and mem-ory, such as the hippocampus (1, 2). There is accumulatingevidence that opiates modulate synaptic transmission andplasticity in the hippocampus. For example, opiates havebeen shown to significantly alter glutamatergic transmission(3), neurogenesis (4), dendritic stability (5), and long termpotentiation (6–8). To date, however, the protein substratesinvolved in opiate-induced synaptic plasticity in the hip-pocampus are not well explored.

Recent studies have applied genomics and proteomics ap-proaches to identify changes in gene and protein expressionin the brain that may be involved in opiate addiction (9–12). Anumber of reports have shown that repeated morphine ad-ministration alters the expression of proteins involved in re-ceptor endocytosis, neurotransmission, energy metabolism,and protein degradation (11, 13–15). These effects could con-tribute to the morphine-induced neuronal changes that persistfor a long time following the cessation of drug exposure (16).However, molecular and cellular mechanisms underlyingthese long lasting changes are still not fully understood.

Because of the complexity of the central nervous system,fractionation techniques that allow the enrichment of lowerabundance proteins are usually necessary to achieve a bettercharacterization of the cellular proteome. Within the synapse,the postsynaptic site contains a high concentration of pro-teins, including receptors and their intracellular signaling com-ponents that receive and transduce synaptic information. Thispostsynaptic electron-dense structure, named the postsyn-aptic density (PSD),1 plays an important role in synaptic reg-ulation and plasticity. Although considerable efforts havebeen put toward identifying the protein components of thisfraction (17–20), none have focused on examining changes in

From the Departments of ‡Pharmacology and Biological Chemistryand §Human Genetics, Mount Sinai School of Medicine,New York, New York 10029

Received, May 18, 2006, and in revised form, October 4, 2006Published, MCP Papers in Press, October 6, 2006, DOI 10.1074/

mcp.M600184-MCP200

1 The abbreviations used are: PSD, postsynaptic density; AP-2,adaptor protein-2 �1; CaMKII, calcium/calmodulin-dependent proteinkinase II; PSD-95, postsynaptic density protein of 95 kDa; 2-DE,two-dimensional gel electrophoresis; AMPA, �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; TX-100, Triton X-100.

Research

© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 6.1 29This paper is available on line at http://www.mcponline.org

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PSD proteins in the hippocampus following drugadministration.

In this study, we examined the changes in expression levelsof PSD-associated proteins in the mouse hippocampus uponrepeated administration of morphine using quantitative pro-teomics. To measure the relative changes in the levels ofproteins, we used ICAT followed by tandem mass spectro-metric (LC-MS/MS) analysis. We found that the levels of 10proteins were altered in both forward and reverse labelingexperiments. Next we focused on clathrin heavy chain be-cause it showed the largest increase. Interestingly this in-crease was found to be local at the PSD, and this was con-firmed by Western blotting and electron microscopy. We alsofound that levels of adaptor protein-2 �1 (AP-2) and dynaminwere increased locally at the PSD after morphine treatment.Because the clathrin�dynamin�AP-2 complex modulates thelevel of cell surface receptors including �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) at glutamatergicsynapses (21), we investigated the effect of morphine on theassociation of the AMPA receptor subunit, GluR1, with clath-rin. Morphine treatment led to a decrease in the levels ofclathrin-associated GluR1, suggesting that this treatment mayalter clathrin-mediated AMPA receptor trafficking. Altogetherthese studies propose a role for morphine in regulating endo-cytic proteins at the hippocampal PSD, which in turn wouldmodulate synaptic transmission and plasticity at hippocampalsynapses.

EXPERIMENTAL PROCEDURES

Animals and Morphine Treatment—Experimental protocols involv-ing animals were approved by the Institutional Animal Care and UseCommittee at Mount Sinai School of Medicine. Four-month-old malewild-type C57BL/6JBom mice (20–25 g) were maintained on a 12-hlight/dark cycle and were allowed to acclimatize to their environmentfor 1 week prior to drug administration. Morphine sulfate (Sigma) wasprepared in 0.9% sterile isotonic saline. For each experiment, mice(n � 5 per group) were injected intraperitoneally with saline or mor-phine; in the latter case four escalating doses of morphine (5, 8, 10,and 15 mg/kg) every 12 h for 48 h as described previously (22) wereused. Animals were sacrificed 12 h following the last drug injection.The hippocampi from treated or control mice were dissected andstored at �80 °C until use.

Subcellular Fractionation and PSD Isolation—The PSD fraction wasisolated essentially as described previously (20). For each fraction-ation, hippocampi from five saline-treated or five morphine-treatedmice were combined and homogenized in 3 ml of 0.32 M sucrose, 0.1mM CaCl2 containing protease (0.1 mM PMSF, 1 �g/ml aprotinin, 1�g/ml leupeptin, 1 mM benzamidine, and 0.1 mM pepstatin) andphosphatase inhibitors (1 mM NaF and 1 mM �-glycerophosphate)from Sigma. The homogenate was brought to a final concentration of1.25 M sucrose by adding 2 M sucrose and 0.1 mM CaCl2. Thehomogenate was then placed in an ultracentrifuge tube, overlaid with1 M sucrose, and subjected to centrifugation at 100,000 � g for 3 h at4 °C. The synaptosomal fraction was collected at the 1.25 M/1 M

sucrose interface. To obtain the synaptic junctions, the synaptosomalfraction was diluted with 20 mM Tris-Cl, pH 6, 0.1 mM CaCl2 contain-ing 1% Triton X-100 (TX-100), mixed for 20 min at 4 °C, and centri-fuged at 40,000 � g for 20 min at 4 °C. The pellet containing the

isolated synaptic junctions was collected. To separate presynapticproteins from the PSD, the pellet was resuspended in 20 mM Tris-Cl,pH 8, 1% TX-100, 0.1 mM CaCl2. The mixture was again mixed for 20min at 4 °C and centrifuged at 40,000 � g for 20 min at 4 °C. Theinsoluble pellet containing the PSD fraction was collected and storedat �80 °C until use.

ICAT Labeling and Peptide Separation—ICAT labeling was per-formed according to the manufacturer’s guidelines (Applied Biosys-tems, Foster City, CA). In brief, PSD fractions were solubilized in 0.1%SDS, 200 mM Tris-Cl, pH 8.3. To reduce disulfide bonds, 5 mM

tris(2-carboxyethyl)phosphine was added, and samples were boiledfor 10 min. After cooling the samples to room temperature, samplesfrom saline-treated animals were labeled with the light (12C) ICATreagent and from morphine-treated animals were labeled with theheavy (13C) reagent for 2 h at 37 °C (forward labeling). To avoidvariability during the labeling procedure, reverse labeling was alsoperformed with a separate set of PSD fractions obtained from anindependent experiment. In this case, saline samples were labeledwith the heavy (13C) reagent, and morphine samples were labeled withthe light (12C) reagent. Following incubation with the ICAT reagent,saline and morphine samples were combined and digested overnightwith trypsin at 37 °C. Resulting peptides were separated by cation-exchange chromatography using a POROS 50 HS (4.0 � 15-mm)cation-exchange column. Peptides were eluted with 5 mM KH2PO4,pH 3, 25% acetonitrile (SCX A buffer) using a step gradient (from 40to 600 mM KCl in SCX A buffer) to collect six fractions. The labeledpeptides from each fraction were isolated using an avidin column(4.0 � 15 mm) according to the manufacturer’s instructions. Afterevaporation of the samples, the biotin tag was removed from theICAT-labeled peptides by incubation with a cleaving reagent (pro-vided by the manufacturer) for 2 h at 37 °C, and resulting peptideswere dried and stored at �80 °C until use.

Liquid Chromatography and Mass Spectrometry—For peptideidentification by MS/MS, the peptides were dissolved in 30–50 �l ofHPLC sample solvents containing water:methanol:acetic acid:triflu-oroacetic acid (70:30:0.5:0.01, v/v/v/v). Micro-HPLC-MS/MS analysiswas conducted on a QSTAR XL mass spectrometer (Applied Biosys-tems) coupled with an on-line MicroPro-HPLC system (Eldex Labo-ratories, Napa, CA). 30–50 �l of tryptic peptide solution were injectedinto a Magic C18 column (0.2 � 150 mm, 5 �m, 200 Å, MichromBioResources, Auburn, CA) that had previously been equilibrated with70% solvent A (0.5% acetic acid and 0.01% trifluoroacetic acid inwater:methanol (95:5, v/v) and 30% solvent B (0.5% acetic acid and0.01% trifluoroacetic acid in methanol:water (95:5, v/v)). Peptideswere separated and eluted from the HPLC column with a lineargradient of 30–70% solvent B in 100 min at a flow rate of 2.0 �l/min.The eluted peptides were sprayed directly into the QSTAR XL massspectrometer (2.9 kV). The QSTAR XL mass spectrometer was oper-ated in a positive ion mode. The information-dependent acquisitionmethod had a duty cycle of 9 s, selecting the three most intense ionsbetween 300 and 2000 m/z with charge states of 2–5 and thatexceeded five counts from the 1-s TOF-MS scan. Rolling collisionenergies were used to facilitate CID. Any ion within 100 ppm of aformer target ion was excluded for 60 s to eliminate repeated frag-mentation of the same peptide species.

ICAT Data Processing—Data acquired from the ICAT informationdependent acquisition experiments were analyzed by Pro ICAT soft-ware version 1.0 Service Packs 2 and 3 (Applied Biosystems). Masstolerances of 0.15 and 0.1 Da for parent ion spectra and fragment ionspectra, respectively, were used. The measured molecular masses ofparent peptides and their MS/MS data were used to search theNational Center for Biotechnology Information (NCBI) nonredundantDNA/protein sequence database (nr), which was specifically format-ted for use with Pro ICAT, with a maximum of one missed cleavage

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specified. The identification algorithms of Pro ICAT software followroutines typical to those generally used for peptide fingerprinting (23)where the patterns of sequence ions derived from MS/MS peptidefragmentation are correlated with a sequence database in accord-ance with the mass and specified mass tolerance of the precursor ion(24). The quality of data interpretation is described by the proteinconfidence, which measures how distant the identifications are fromthe next likely peptide and protein candidate. Protein confidence isexpressed in a unit called ProtScore, which is a transformation ofpercent confidence. To convert percent confidence to ProtScore, thefollowing formula is used: ProtScore � �log(1 � (percentconfidence)/100).

This formula also determines the contribution made by a singlepeptide identification to the ProtScore. The maximal contribution ofany peptide is limited. Peptides are considered to have no higherconfidence than 99.00%. This means that each peptide can contrib-ute no more than 2.0 to the ProtScore. This limitation is necessary dueto the limit in accuracy in reporting peptide confidence. In this studyprotein identifications were made based on ProtScore values �2,representing protein confidence greater than 99%.

During data processing, Pro ICAT identifies the ICAT pairs anddetermines the form of the label. Analysis of the isotope clusters fromthe light and heavy forms of the peptides only considers those peakswith corresponding peaks in the other cluster during the ICAT ratiocalculation. The ICAT ratios are calculated from the relative areas ofthe extracted precursor ion spectra. The protein ICAT ratios arecalculated from the combination of the constituent peptides whereeach charge state is treated separately.

Immunoblotting—For immunoblotting, equal amounts of total pro-tein from fractions obtained from morphine- or saline-treated animalswere resolved by 7.5% SDS-PAGE and transferred to nitrocellulosemembranes (Scheicher & Schuell) by electroblotting. Membraneswere incubated with selective antibodies: clathrin heavy chain (1:3,000, BD Biosciences), Na�/K�-ATPase �3 (1:2,000, Affinity Re-agents, Golden, CO), 14-3-3 � (1:10,000, Santa Cruz Biotechnology,Santa Cruz, CA), dynamin I (1:10,000, BD Biosciences), and the �

subunit of AP-2 (1:2,000, BD Biosciences). After incubating withsecondary antibody (1:2,000 to 1:100,000 anti-mouse, Vector Labo-ratories, Burlingame, CA, 1:1,000 to 1:2,000 anti-rabbit, AmershamBiosciences, Buckinghamshire, UK) membranes were incubated withECL detection reagents (Pierce, Rockford, IL) and exposed to ECLHyperfilm (Amersham Biosciences, Buckinghamshire, UK). Blotswere reprobed with tubulin antibody (1:50,000, Sigma, St. Louis, MO)to ensure equal loading and transfer. Band densities were determinedusing NIH ImageJ Software. Quantification was performed by meas-uring the intensity of the band with protein specific antibodies andcomparing it to that of tubulin.

Immunocytochemistry for Electron Microscopy—Animals weresacrificed 12 h following the last drug injection. Deeply anesthetizedmice were perfused with saline followed with cold fixative, containing4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphatebuffer (PB, pH 7.4). Brains were removed and postfixed in 4%paraformaldehyde for 2 h. 250 �m thick coronal Vibratome slices ofhippocampal formation were treated successively with 0.5% osmiumtetroxide, 1% uranyl acetate, dehydrated, and embedded in epoxyresin. The embedded slices were trimmed to the CA1 region ofhippocampal stratum radiatum. Thin sections were mounted ontonickel grids, etched and immunostained with an anti-clathrin antibody(1:10, BD Biosciences, San Jose, CA) visualized with 10 nm gold-labeled anti-mouse secondary (EMS, Fort Washington, PA). Sectionswere examined with a Hitachi 7000 electron microscope. For quan-titation, 25–28 fields from each animal were selected so as to containwell oriented synaptic clefts and digital images were recorded at

80,000 to 100,000 magnification. Enumeration of grains over eachpostsynaptic profile was performed on all images by an observerblinded to specimen identity, by counting particles at the postsynap-tic density (1) as well as over the remainder of each post-synapticprofile (2). The ratio of (1) to the total (1 � 2) was computed for eachprofile. Presynaptic labeling was ignored in this analysis.

Two-dimensional Gel Electrophoresis—For two-dimensional gelelectrophoresis (2-DE) total homogenate and PSD hippocampal frac-tions from saline- and morphine-treated animals were diluted in re-hydration solution, containing 7 M urea, 2 M thiourea, 4% CHAPS(3-[3-cholamidopropyl) dimethylammonio]-1-propanesulfate), and1% DTT (1,4-dithioerythritol). IPG buffer (pH 3–10, Amersham Bio-sciences, Buckinghamshire, UK) and DeStreak reagent (AmershamBiosciences, Buckinghamshire, UK) was added to the mixture, andthat was applied onto 7 cm broad IPG strips (pH 3–10). Followingrehydration for 12 h, isoelectrofocusing (IEF) was carried out on anEttan IPGPhor II (Amersham Biosciences, Buckinghamshire, UK) at20 °C according to the following voltage-time program: 500 V for30min, 1000 V for 30 min, and 5000V for 1 h. IPG strips were thenequilibrated in 1% DTT, following by 4% iodoacetamide. The seconddimension was carried out in 10% SDS-PAGE gels. The gels werethen transferred to nitrocellulose membrane by electroblotting. Im-munoblotting was performed using selective antibodies to clathrinheavy chain (1:3,000, BD Biosciences, San Jose, CA), dynamin I(1:10,000, BD Biosciences, San Jose, CA), the � subunit of AP-2(1:2,000, BD Biosciences, San Jose, CA), and tubulin (1:2,000, Sigma,St. Louis, MO). After incubating with secondary antibody (1:4,000 to1:20,000 anti-mouse, Vector Laboratories, Burlingame, CA) mem-branes were incubated with ECL detection reagents (Pierce, Rock-ford, IL) and exposed to ECL Hyperfilm (Amersham Biosciences,Buckinghamshire, UK).

Immunoprecipitation from Hippocampal Synaptic Fractions—Forimmunoprecipitation studies synaptosomal fractions from morphineor saline treated animals were resuspended in IP buffer (100 mM NaCl,5 mM EDTA, 10 mM NaHPO4, pH 7.2, and 1% TX-100) and incubatedon ice for 30 min. An anti-clathrin heavy chain N-terminal antibody(BD Biosciences, San Jose, CA) was used to immunoprecipitateclathrin. An anti-GluR1 C-terminal antibody (Chemicon, Temecula,CA) was used to immunoprecipitate GluR1. After overnight incubationat 4 °C with 2 �g (anti-clathrin) or 4 �g (anti-GluR1) of the antibodyand prewashed protein A agarose beads, the immunoprecipitateswere washed 3 times with IP buffer. Bound proteins were eluted withLaemmli loading buffer containing 2-mercaptoethanol for 30 min at60 °C, resolved in 7.5% SDS-PAGE gels and immunoblotted withantibodies to clathrin heavy chain, AP-2 (1:1,000 to 1:2,000, BDBiosciences, San Jose, CA), PSD-95 (1:10,000, Chemicon, Temecula,CA), Homer (1:500, Chemicon, Temecula, CA) and GluR1 (1:1,000,Chemicon, Temecula, CA) followed by secondary antibodies(1:20,000 to 1:2,000 anti-mouse, Vector Laboratories, Burlingame,CA; 1:2,000 anti-rabbit, Amersham Biosciences; 1:1,000 anti-rat, Am-ersham Biosciences). Immunoreactive bands were visualized withECL reagents (Pierce) on ECL Hyperfilm (Amersham Biosciences).

RESULTS

To study the effect of morphine administration on the ex-pression of PSD-associated proteins, mice were injected withescalating doses of morphine, and a fraction enriched inhippocampal PSD proteins was isolated by subcellular frac-tionation as described previously (20). This procedure allowsfor the isolation of synaptic junctions and separation of pre-synaptic proteins from the PSD, thus allowing enrichment ofPSD proteins. We confirmed this by Western blotting with


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TABLE IList of mouse hippocampal PSD proteins identified by ICAT and LC-MS/MS

Forward and reverse labeling assays were performed with PSD fractions obtained from two different and independent experiments. Dataobtained from two LC-MS/MS analyses were averaged. Data were analyzed by Pro ICAT software version 1.0 SP2 for protein identification andquantification Protein identifications were made based on ProtScore. ProtScore values �2 represent protein identification confidence greaterthan 99%. NMDA, N-methyl-D-aspartate; MAPK, mitogen-activated protein kinase; RIM, Rab3 interacting molecule; LGI, leucine gliomainactivated.

Accession no. Protein name Gene name Notea

Proteinabundance ratio(morphine/saline)

Forward Reverse

Forward Reverse ProtScore No.Pepb ProtScore No.

Pepb

Trafficking (15)56205530 Clathrin, heavy polypeptide Cltc f, r 2.24 2.77 2.15 2 14.09 1041281852 Amphiphysin Amph r 1.40 2.00 26671561 Adaptor protein complex

AP-2, �1Ap2a1 1.32 4.81 3

21594764 Syntaxin-binding protein 1 Stxbp1 f, r 1.32 1.42 2.00 3 6.00 431543349 N-Ethylmaleimide-sensitive

fusion proteinNsf 1.22 0.90 6.01 5 4.01 3

8394392 Synaptotagmin 5 Syt5 1.09 2.00 228386121 Adaptor protein complex

AP-2, �1Ap2b1 1.13 0.98 4.81 5 2.21 3

55391497 ATPase, H�-transporting,V1 subunit B

Atp6v1b 1.04 2.13 3

56205559 Myosin heavy chain 10,non-muscle

Myb 1.02 2.97 2

31560731 ATPase, H�-transporting,V1 subunit A

Atp6v1a 1.01 2.01 2

33859538 Dynamin Dnm1 0.98 4.8 322779912 Dynamin 1-like Dnm11 0.89 2.01 240254646 Adaptor protein complex

AP-2, �2Ap2a2 0.85 4.76 4

13384736 Dynein, cytoplasmic,heavy chain 1

Dynchc1 0.82 3.71 3

6754784 Myosin Va Myo5a r 0.66 6.72 6Signaling (14)

7435027 14-3-3 � Ywhaz f, r 1.54 1.61 3.50 2 4.00 36755080 Protein kinase C, � Prkcg f 1.49 4.29 36680047 G �-2 protein Gnb2 1.22 0.95 2.00 2 2.00 241393528 RIM-binding protein 2 Rimbp2 1.05 2.00 221312314 Dual specificity phosphatase 3 Dusp3 1.04 2.00 26754632 MAPK 1 Mapk1 0.99 4.74 36679345 Protein kinase C, � Prkcb 0.95 2.00 230354348 Go �-2 protein Gnao2 0.92 4.00 328916677 CaMKII � Camk2a r 0.72 7.13 41280027 Protein phosphatase 1c � Ppp1cc � r 0.71 7.95 551770143 Synaptic Ras-GTPase-

activating proteinSyngap1 r 0.70 4.02 3

29747738 Copine VI Cpne6 r 0.69 7.87 62160434 Cyclic nucleotide phosphodiesterase 1 Cnp1 f, r 0.63 0.69 2.62 3 5.16 46680045 G �-1 protein Gnb1 r 0.42 2.00 2

Receptors/channels/carriers (10)21450321 Na�/K�-ATPase �3 subunit Atp1a3 f, r 1.91 1.96 6.23 5 7.03 521311845 Mitochondrial glutamate

carrierSlc25a22 1.22 3.41 2

33859680 Solute carrier family 12 Slc12a5 1.21 0.96 2.51 3 3.45 36680095 Glutamate receptor, NMDA �1 Grin1 1.16 2.04 227369581 Solute carrier Aralar Slc25a12 1.07 1.11 1.53 1 2.01 232452028 Na�/K�-ATPase �2 subunit Atp1a2 0.98 0.96 4.11 2 2.00 220149764 Stabilin-2 Stab2 0.98 2.11 27305115 Glutamate receptor, AMPA �2 Gria2 0.90 2.04 282209601 Solute carrier family 25 Slc25a3 r 0.49 4.15 356207177 Voltage-dependent anion channel 1 Vdac1 f, r 0.47 0.61 2.19 2 2.00 2

Scaffolding/clustering (3)23468370 Homer 2 Homer2 0.99 2.00 26681195 Postsynaptic protein 95 Dlg4 0.99 0.88 3.41 2 3.23 318702315 Chapsyn-110 Dlg2 0.93 2.00 2




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TABLE I—continued

Accession no. Protein name Gene name Notea


Forward Reverse


Pepb

Cell adhesion (3)46250295 Catenin � Ctnna1 0.92 2.00 26680337 Telencephalin Icam5 0.89 0.99 3.11 2 2.00 26671684 Catenin � Ctnnb1 r 0.69 3.23 3

Cytoskeletal (19)7106421 Spectrin �2 Spnb2 1.34 2.03 37305031 Erythrocyte protein band 4.1 Epb4.113 1.21 0.90 2.10 3 2.24 36678467 Tubulin, �4 Tuba2 1.21 0.99 2.06 3 9.23 1038541878 Coronin 2b Coro2b 1.15 0.93 3.04 2 2.11 318542419 Wave1/Scar Wave1 1.12 2.71 356972196 Tubulin, �3 Tubb3 1.11 0.98 2.11 2 2.00 321307732 Actinin �2 Actn2 1.01 2.01 27106339 Tubulin, �5 Tubb5 1.09 0.96 5.22 4 8.00 631982779 Microtubule-associated protein tau Mapt 1.03 0.80 2.00 2 2.00 219909851 Debrin A Drba 1.02 3.40 26678944 Microtubule-associated protein 2 Mtap2 1.02 1.05 2.01 3 4.25 39790221 Acitn-related protein 2/3 complex Arpe1a 1.01 3.10 222122615 ARP1 actin-related protein 1 homolog

BActr1b 0.99 2.00 2

418683 Neurofilament-L Nefl 0.99 0.89 2.01 3 2.00 249019113 Actin-binding LIM protein Ablim2 0.95 2.53 328175828 Similar to ankyrin 2 neuronal Ank2 0.90 2.32 351706071 Spectrin �2 Spna2 0.88 6.51 57949100 Contactin-associated protein Cntnap1 0.85 2.01 437604188 Septin 5 Sept5 f 0.79 2.01 2

Regulatory (10)23956286 Neuritin 1 Nrn1 1.17 2.74 357242925 Heat shock protein 1, � Hspcb 1.11 1.12 2.52 2 2.01 274008507 Similar to heat shock cognate 71

proteinLOC610241 1.10 1.13 4.00 2 2.10 2

51457 Heat shock-like protein Hsp86 0.99 1.13 2.10 2 2.21 39938002 Leucine-rich repeat LGI family Lgi1 0.99 0.82 2.23 3 2.49 251705246 Similar to 60 S ribosomal protein L4 Rpl4a 0.99 2.00 238173913 Tu translation elongation factor Tufm 0.98 2.27 26678631 Ring finger protein 103 Rnf103 0.93 2.70 326024336 Ribosomal protein S27 Rps27 0.92 2.00 250403588 60 S ribosomal protein L18a Rpl18a 0.90 2.00 2

Metabolic (16)21450321 M2 type pyruvate kinase Pkm2 f, r 1.71 2.30 3.71 4 2.24 219353360 Cytochrome c oxidase, subunit V1b Cox6b1 1.15 1.16 2.03 2 2.70 319527334 NADH dehydrogenase Fe-S protein 5 Ndufs5 1.03 2.00 218079339 Aconitase 2 Aco2 1.02 1.01 2.00 2 2.00 22078522 Dihydrolipoamide dehydrogenase Dld 1.02 0.98 2.70 3 2.00 231560653 Phosphofructokinase Pfk1 0.99 2.70 319526878 Pyrroline-5-carboxylate reductase Pycr2 1.01 2.00 215214580 NADH dehydrogenase 1 � Ndufa8 0.94 1.14 2.16 2 2.87 352350626 Cytochrome c1 Cyc1 0.90 2.00 256206902 NADH dehydrogenase Fe-S protein 1 Ndufs1 0.84 2.14 36680027 Glutamate dehydrogenase 1 Glud1 f, r 0.81 0.65 2.35 4 4.00 330316354 Hexokinase I Hk1 f 0.78 2 251711114 Glyceraldehyde-3-phosphate

dehydrogenaseGapdh 0.82 2.01 2

29747888 Aldolase A Aldoa r 0.76 6.27 719683937 Creatine kinase mitochondrial Ckmt1 f, r 0.76 0.64 3.16 5 7.19 6483918 Glutamate-ammonia ligase Glu1 f, r 0.73 0.69 6.01 6 5.51 5

Other (12)26080422 Glial fibrillary acidic protein Gfap 1.21 0.97 2.14 2 2.00 251761782 Tripartite motif protein � LOC434218 1.11 0.89 3.10 3 2.15 2543294 Oligodendrocyte-myelin glycoprotein Omgp 1.05 0.82 2.00 2 3.16 33980175 Thy 1.2 antigen Thy1 1.02 2.00 2625314 Golli-myelin basic protein Mbp 1.01 2.41 330425330 Limbic system-associated membrane

proteinLsamp 0.99 2.00 2




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markers of PSD, such as PSD-95 and CaMKII. Next we useddifferential isotopic labeling with the ICAT reagent to quanti-tate differences in protein levels by mass spectrometry. ThePSD proteins from saline- and morphine-treated animals werelabeled using ICAT reagents and subjected to LC-MS/MSanalysis. The acquired MS/MS spectra were analyzed by da-tabase searching and stringent data filtering (see “Experimen-tal Procedures”). Protein identifications were made based onProtScore values equal to or higher than 2.0 (representingprotein confidence higher than 99%). Overall we identified102 proteins, the majority representing signaling, trafficking,and cytoskeletal proteins (Table I). Furthermore these repre-sented postsynaptic proteins involved in synaptic signaling(such as GluR1, Homer, PSD-95, CaMKII, and protein kinaseC �) indicating that this fractionation protocol enriches pro-teins in the PSD.

Quantitative analysis showed that of the 23 proteins thatwere altered by morphine treatment 10 proteins showed con-sistent changes in levels (in both forward and reverse labelingexperiments). Upon averaging forward and reverse protein

abundance ratios, we obtained a value of 1.10 with a standarddeviation of 0.29. Therefore, changes were considered to besubstantial when exhibiting a ratio of either �1.39 or �0.81(see Table II). A typical spectrum is shown in Fig. 1; a robustincrease in Na�/K�-ATPase �3 levels was observed when thestudies were carried out by forward labeling (Fig. 1A, leftpanel), and this ratio was reversed when the labeling wasreversed (Fig. 1A, right panel). Fig. 1B shows the MS/MSanalysis that identified the 591.85 m/z peak as a heavy ICAT-labeled tryptic peptide CHATILLQGK (where H representsheavy) of Na�/K�-ATPase �3. As a control, we found that thepeaks corresponding to tubulin �5 were not changed in eitherforward (morphine/saline) or reverse (saline/morphine) label-ing (Fig. 1C, left and right panels, respectively).

Among the proteins that showed consistent and substantialchanges, clathrin heavy chain levels were the most dramati-cally increased (more than 2-fold) upon morphine treatment(Table II). Two other proteins that showed a substantial in-crease were Na�/K�-ATPase �3 and 14-3-3 � (Table II). ICATanalysis also revealed decreases in several proteins, including

TABLE IIQuantitation of changes in mouse hippocampal PSD protein levels upon morphine treatment by ICAT and LC-MS/MS

Forward and reverse labeling was performed with PSD fractions obtained from two different and independent experiments. Data obtainedfrom two LC-MS/MS analyses were averaged. Data were analyzed by Pro ICAT software version 1.0 SP2 (Applied Biosystems) for proteinidentification and quantification. Protein identifications were made based on the ProtScore. ProtScore values �2 represent protein identifi-cation confidence greater than 99%.

Protein NCBI accessionno. Gene name


ProtScore

Forward Reverse Forward Reverse

Clathrin, heavy polypeptide 56205530 Cltc 2.24 2.77 2.15 14.09Na�/K�-ATPase � 3 subunit 21450321 Atp1a3 1.91 1.96 6.23 7.03M2-type pyruvate kinase 1405933 Pkm2 1.71 2.30 3.71 2.2414-3-3 � 7435027 Ywhaz 1.54 1.61 3.50 4.00Syntaxin-binding protein 1 21594764 Stxbp1 1.32 1.42 2.00 6.00Glutamate dehydrogenase 1 6680027 Glud1 0.81 0.65 2.35 4.00Glutamate-ammonia ligase 483918 Glu1 0.73 0.69 6.01 5.51Creatine kinase, mitochondrial 1 19683937 Ckmt1 0.76 0.64 3.16 7.192�,3�-Cyclic-nucleotide 3�-phosphodiesterase 1 2160434 Cnp1 0.63 0.69 2.62 5.16Voltage-dependent anion channel 1 56207177 Vdac1 0.47 0.61 2.19 2.00

TABLE I—continued

Accessionno. Protein name Gene name Notea


Forward Reverse


Pepb

6677905 Golgi apparatus protein 1 Glg1 0.97 1.12 2.10 2 2.40 251829863 SET binding factor 1 Sbf1 0.97 2.53 256800539 Cytoplasmatic FMR1-

interacting proteinCyfip2 0.95 2.59 3

1915913 Ulip2 protein Ulip2 0.87 0.91 2.11 2 2.00 24218976 Link protein Link 0.83 2.17 2758630 Neurocan Ncan 1.01 8.59 7a Proteins that showed substantial changes (with a ratio of either �1.39 or �0.81) in both forward and reverse (f, r) labeling experiments upon

morphine administration are shown in bold in the first two columns of the table. Proteins that showed substantial changes in either forward (f)or reverse (r) labeling are shown in italics in the first two columns of the table.

b No. Pep, number of nonredundant matching peptides found per protein, which were considered in the calculation of the ProtScore.




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FIG. 1. Representative spectra of Na�/K�-ATPase �3 (A and B) and tubulin (C) following ICAT-LC-MS/MS. A, PSD proteins isolatedfrom the hippocampi of mice treated with saline and morphine were labeled with “light” (L) and “heavy” (H) ICAT reagents, respectively (forwardlabeling, left panel). A pair of peaks corresponding to triply charged ions with heavy/light ratio greater than 1 (heavy/light � 1) was detected.A reversed heavy/light ratio (heavy/light � 1) of this pair of peaks was detected in reverse labeling (right panel) where PSD proteins from saline-and morphine-treated mice were labeled with heavy and light ICAT reagents, respectively. B, MS/MS analysis of 591.85 ion and databasesearch revealed a heavy ICAT-labeled tryptic peptide corresponding to the amino acid sequence of CHATILLQGK of Na�/K�-ATPase �3.Peptide fragment ions corresponding to peptide fragment of “y” ions were labeled as yn in the figure. C, the peak ratio of heavy/light-labeledpeaks corresponding to tubulin �5 tryptic peptide was not changed in both forward (left panel) and reverse (right panel) labeling.




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voltage-dependent anion channel 1, a protein enriched in thePSD that has been shown to be involved in learning andsynaptic plasticity in the hippocampus (25). Morphine treat-ment also decreased the levels of glutamate-ammonia ligaseand glutamate dehydrogenase 1 (Table II), enzymes that reg-ulate glutamate levels.

Using Western blotting, we verified that the changes in theirrelative abundance upon morphine treatment were consistentwith those observed by ICAT analysis. We found significantincreases in levels of clathrin heavy chain, Na�/K�-ATPase�3, and 14-3-3 � in the PSD fraction of morphine- as com-pared with saline-treated animals (Fig. 2). In contrast, tubulinlevels showed no changes, consistent with the ICAT analysisdata (see Table I and Fig. 1C). Interestingly the levels of theseproteins in the total hippocampal homogenate were not al-tered by morphine. Thus, it appears that there is a localincrease of these proteins at the PSD upon morphinetreatment.

We further examined the effects of morphine on the localincrease of clathrin using pre-embedding immunogold elec-tron microscopic immunocytochemistry. For this, we focusedon stratum radiatum of CA1 hippocampus and examined theultrastructural localization of clathrin at dendritic synapses. Todetermine whether there were changes in the levels of clathrinat the PSD in response to morphine, the number of goldparticles associated with the PSD versus the total number ofparticles in 25–28 postsynaptic profiles was compared insections from saline- and morphine-treated mice (Fig. 3A). Wefound a significant increase in the proportion of PSD-associ-ated clathrin immunogold labeling in morphine-treated mice(Fig. 3B), supporting our data from ICAT and Western blotting

analysis and indicating a local increase of clathrin at the PSD.We next analyzed the effect of morphine on the levels of

two other key endocytic molecules: AP-2 and dynamin. West-ern blot analysis indicated that morphine treatment signifi-cantly increased the levels of dynamin and AP-2 in the PSDfraction (Fig. 4, A and B, respectively). Although these pro-teins were identified in the ICAT analysis, the changes seenupon morphine administration were found to be not statisti-cally significant. However, Western blotting analysis, a moresensitive technique for detecting protein levels, revealed thattheir levels were significantly increased specifically at the PSDbut not in the total hippocampal homogenate. This is consist-ent with the notion that morphine treatment leads to a recruit-ment of dynamin and AP-2, along with clathrin, to the PSD.These data indicate that morphine administration causes aredistribution of proteins involved in endocytosis at thepostsynaptic membrane to the PSD.

To study whether morphine treatment also affects the post-translational modification states (i.e. extent of phosphoryla-tion) of these endocytic proteins, we subjected samples ofhippocampal homogenate and PSD fractions from saline- ormorphine-treated animals to 2-DE and performed Westernblot analysis. We found that morphine treatment led to asubstantial change in the post-translation modification stateof dynamin (Fig. 4C, top panel); this change was observableonly at the PSD fraction (and not in the total homogenate).Indeed morphine treatment caused a shift in the pI of dynaminisoforms to a lower pI. This is consistent with an increase inthe extent of phosphorylation of dynamin. Phosphorylation ofdynamin is thought to down-regulate the formation of clathrin-coated pits, leading to a decrease in the extent of endocytosis

FIG. 2. Western blot analysis to validate results from ICAT-MS/MS. PSD-associated and total homogenate proteins from saline- (SAL)and morphine-treated (MOR) animals were analyzed by Western blotting using antibodies against clathrin heavy chain (HC) (A), Na�/K�-ATPase �3 (B), 14-3-3 � (C), and tubulin (to ensure equal protein loading and transfer). The levels of clathrin heavy chain, Na�/K�-ATPase �3,and 14-3-3 � levels were normalized relative to tubulin levels. Data represent mean values � S.E. (**, p � 0.01 relative to saline-treated animals;t test; n � 3). Levels of clathrin heavy chain, Na�/K�-ATPase, and 14-3-3 � were significantly increased in the PSD fraction of morphine- versussaline-treated animals. Significant changes were not observed in the total homogenate.




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(26). To confirm the effects of morphine on the phosphoryla-tion state of dynamin, membranes were stripped and blottedwith an anti-tubulin antibody. The isoelectric point of tubulinwas not altered by morphine treatment in either total homo-genate or the PSD (Fig. 4C, bottom panel). We also found thatmorphine treatment did not alter the post-translational statesof clathrin or AP-2 (data not shown). Thus it appears thatmorphine administration leads to increased phosphorylationof some but not all endocytic proteins.

We next examined the extent of association of clathrinwith PSD-95 and Homer, two components of the PSD. Thelevel of clathrin associated with PSD-95 and Homer wasdrastically increased upon morphine treatment (Fig. 5A,bottom panels). In contrast, the level of clathrin associatedwith AP-2 remained unaltered (Fig. 5A, middle panel). Theseresults suggest that besides redistributing endocytic pro-teins to the PSD morphine treatment leads to the associa-tion of clathrin with components of the PSD; this could alterthe extent of endocytosis of synaptic proteins includingreceptors. Because AMPA receptors are thought to undergoclathrin-dependent endocytosis (21), we next investigated

the effect of morphine on the association of the GluR1AMPA receptor subunit with clathrin. The level of clathrinassociated with GluR1 was significantly decreased uponmorphine treatment (Fig. 5B), although the total level ofGluR1 was increased by this treatment (not shown). Takentogether, these results show that the selective associationof clathrin with endogenous GluR1 is significantly de-creased upon morphine treatment.

DISCUSSION

In this study, we analyzed proteins enriched in the PSDfraction of mouse hippocampus. A number of studies havepreviously analyzed proteins in the PSD fraction obtainedfrom whole brain (19, 27–29), forebrain (18, 30, 31), or cere-bellum (30) using different proteomics techniques such asone-dimensional gel electrophoresis (19, 27), 2-DE (18), mul-tidimensional protein identification technology (MudPIT) (28,31), ICAT (18, 30) or IMAC technology (29). When exploringthe PSD, the majority of studies to date have used fraction-ation protocols that yield PSD fractions containing presynap-tic proteins (32–34). To minimize presynaptic protein contam-

FIG. 3. Electron micrographic analy-sis to validate results from MS/MS. A,representative electron micrographs ofclathrin heavy chain in the hippocampalCA1 region of saline- and morphine-treated animals. Filled arrowheads pointto immunogold particles for clathrinheavy chain, and open arrows point tothe PSD in saline-treated and morphine-treated animals. Scale bar, 100 nm. B,quantitative analysis of clathrin immuno-labeling from synapses representing aratio of clathrin particles associated withPSD to those in the postsynaptic profileshows a significant increase in mor-phine-treated sections. Data representmean values � S.E. (**, p � 0.01 relativeto saline-treated animals; t test; n �25–28 synapses per treatment).




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ination in our samples, we used a recently developedsubcellular fractionation protocol that allows the separation ofsynaptosomal preparations into two distinct fractions of pro-teins, a soluble fraction containing presynaptic particles andan insoluble fraction that corresponds to the PSD (20). Al-though this method allows for the selective enrichment ofpostsynaptic density proteins, the requirement of consider-able amounts of starting material for the isolation of PSD

makes this method less suitable for studies in smaller brainregions or specific brain nuclei.

We identified components of the mouse hippocampal PSDfraction using a proteomics approach that combines ICATmethodology with LC-MS/MS. The list of proteins generatedin this study shows considerable overlap with other previouslyreported datasets of PSD proteins, indicating that our datasetconsists of representative PSD proteins (18, 19, 31). However,

FIG. 4. Western blot analysis of hippocampal homogenate and PSD from saline- (SAL) or morphine-treated (MOR) animals.Quantification of dynamin (A) and AP-2 (B) was performed relative to tubulin levels. Data represent mean values � S.E. (*, p � 0.05; **, p �0.01 relative to saline-treated animals; t test; n � 3). C, PSD fractions and total homogenate from mice treated with saline or morphine wereseparated by high resolution 2-DE and probed with antibodies to dynamin or tubulin. Morphine treatment induced an increase in the intensityof the spots at lower pI and a decrease at higher pI (indicated by arrowheads) in the PSD fraction (and not in total homogenate).




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the total number of proteins identified in our study is 5–10-fold lower than that identified in larger scale, non-brain region-specific studies of the PSD (27, 29). For example, using IMACtechnology, Trinidad et al. (29) recently identified 1,264 uniqueproteins in the PSD fraction obtained from whole mouse brain,and Collins et al. (27) identified 620 PSD-associated proteinsby performing a MS-based analysis of membrane-associatedguanylate kinase signaling complexes from whole mousebrain.

The variability between different PSD datasets is likely dueto differences in species/strains, brain regions, sample prep-aration, and method of analysis. For example, in a recentstudy Cheng et al. (30) compared the PSD protein profilesfrom rat forebrain and cerebellum using ICAT technologycoupled with LC-MS/MS and found a number of proteins toexhibit statistically significant differences between these twobrain regions, suggesting a marked heterogeneity of PSDprofiles between brain regions. With the aim to further explorethe PSD subproteome from specific brain regions, a recentreport studied the PSD protein profile from rat hippocampalslices using two-dimensional LC-MS/MS (35). The list of pro-teins identified in the latter study overlaps significantly (morethan 70 proteins) with our list of mouse hippocampal PSDproteins. When compared with another dataset of proteinsidentified by ICAT technology in the synaptosomal fraction(11), our dataset contains a higher proportion of proteinsinvolved in signaling and scaffolding as well as receptors andchannels. Together the datasets from these studies provide adatabase of hippocampal PSD proteins that will be useful forfunctional studies related to synaptic plasticity, learning, andmemory.

Our dataset includes proteins that have been reported to behighly abundant in the PSD (such as CaMKII�, PSD-95, andsynaptic Ras) as determined using the absolute quantification(AQUA) strategy (28). Our dataset also includes proteins thatare of moderate abundance (such as chapsyn-110, septin, andthe GluR1 subunit of AMPA receptors) and proteins of lowabundance in this fraction (such as Homer 2 and protein kinaseC �), suggesting that this dataset covers a range of proteinsregardless of their abundance in the PSD. The presence ofthese proteins, which are known to modulate synaptic signaling,at the PSD is in agreement with a critical role for the PSD as ascaffolding and signaling component of the synapse.

To avoid variability during the labeling procedure, we car-ried out independent forward and reverse labeling of PSDfractions and combined the results from these two distinctsets of studies. Of the total of 102 proteins, only 38 wereidentified in both forward and reverse experiments. This islikely due to the fact that current LC-MS/MS technology canonly achieve 30% reproducibility when samples with mod-erate complexity are repeatedly analyzed by this technique(36–39). This is largely due to the fact that the MS/MS spec-trometer fragments and consequently identifies only a smallfraction of the peptides captured at the first stage of any givenMS scan. In addition, there are some issues that can affectprotein coverage when using ICAT technology. These includethe absence of cysteine residues in some proteins, the lowabundance of cysteine-labeled peptides, the low ionizationefficiency during MS/MS, or the fact that only a subset of ionscan be selected for MS/MS when many peptides are co-eluted (30). We minimized these limitations by combiningresults from different experiments.

FIG. 5. Morphine induces the association of clathrin with components of the PSD, decreasing the levels of clathrin-associated GluR1.A, analysis of the association between clathrin and components of the PSD, PSD-95 and Homer. Hippocampal synaptosomal fractions frommorphine- or saline-treated animals were immunoprecipitated with clathrin antibody. Clathrin (upper), AP-2 (middle), PSD-95 (middle lower),and Homer (lower) were detected in the immunoprecipitate by Western blotting. B, analysis of the association between clathrin and GluR1.Hippocampal synaptosomal fractions from morphine- or saline-treated animals were immunoprecipitated with GluR1 antibody. Clathrin heavychain (upper) or GluR1 (middle) were detected in the immunoprecipitate by Western blotting. As a control, Western blotting was carried out onthe synaptosomal fraction using antibody to clathrin (lower). IB, immunoblot; IP, immunoprecipitate; HC, heavy chain; SAL, saline; MOR,morphine.




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We also report here the quantitative comparison of differ-entially expressed PSD proteins from mouse hippocampusupon morphine administration using ICAT technology. Wefound that several proteins were regulated in the morphine-treated animals; these represent trafficking proteins, signalingproteins, carriers/channels, and proteins involved in energymetabolism. Morphine-induced changes in these proteinshave also been observed in other studies (11, 13). In one ofthese studies, Prokai et al. (11) examined protein expressionin synaptic membranes obtained from cortex using ICATmethodology. They found that chronic morphine exposureinduced a decrease in the levels of the integral membraneproteins Na�/K�-ATPase �3 subunit and clathrin. We foundthat morphine administration induced an increase in the levelsof Na�/K�-ATPase �3 subunit and clathrin at the PSD. Thiscould be due to differences in the extent of fractionation; weused purified PSDs, whereas Prokai et al. (11) used intactsynaptic membranes. Thus, the changes observed in the lat-ter case would represent a result of morphine effects on boththe presynaptic as well as PSD fraction. By separating pre-synaptic proteins from the postsynaptic components, wewere able to specifically focus on changes in levels of theseproteins at a distinct subcellular location (i.e. PSD).

In the present study, we showed that morphine treatmentinduced a dramatic increase in the levels of clathrin in thehippocampal PSD fraction and increased the association ofclathrin with PSD-95 and Homer. This is, to our knowledge,the first report demonstrating that morphine treatment leadsto increases in the levels of clathrin and its interactions withcomponents of the PSD; previous studies reported clathrin tobe localized in endocytic zones that are in close proximity butdistinct from the PSD in this brain region (40, 41). We alsofound that other endocytic molecules such as dynamin andAP-2 were increased at the PSD by morphine treatment.Although these proteins were identified by ICAT technology,they did not show statistically significant increases. This islikely due to the presence of more than one isoform of thesame protein (caused by post-translational modification or thepresence of different subunits), which cannot be distinguishedat the peptide level when analyzed by mass spectrometry.The fact that we found that morphine treatment induced therecruitment of dynamin and AP-2 to the PSD suggests apossible role for the translocation of these proteins in thestructure and maintenance of the PSD. These proteins areusually expressed in endocytic zones that are independentfrom the PSD (41). Recent studies have suggested that dy-namin may localize at the PSD in association with compo-nents of the PSD, such as Homer, the mGluR5 receptorsubunit, and shank (42), where it could participate in thedevelopment and modulation of synaptic plasticity.

In addition to increasing their levels at the PSD, morphinealso altered the post-translational states of some endocyticproteins. It has been suggested that phosphorylation/dephos-phorylation of endocytic proteins regulates their interactions,

resulting in the regulation of clathrin-mediated endocytosis(43). This is supported by the findings that these proteinsundergo dephosphorylation during the maturation of clathrin-mediated endocytosis. Indeed the switch from the phospho-rylated state of the endocytic proteins to the dephosphoryla-ted state after nerve terminal depolarization is thought totrigger clathrin-mediated endocytosis (44). We found thatmorphine treatment led to an increase in the phosphorylationstate of dynamin. This would inhibit its association with part-ner proteins and thus down-regulate clathrin-mediated endo-cytosis of synaptic vesicles (45).

In neuronal dendrites and spines, clathrin cycles betweenpools in the plasma membrane and cytosol. The plasmamembrane pools constitute active endocytic zones that lieadjacent to but spatially segregated from the PSD (40). For areceptor or other membrane protein to be internalized, it mustfirst be translocated to specialized endocytic areas. At thesesites clathrin-dependent endocytosis occurs via the assemblyof a coated pit. After the binding of coated pit components tothe membrane, the coated pits invaginate and are pinched offfrom the membrane in a dynamin-dependent manner to formclathrin-coated vesicles (46). By modulating the levels andphosphorylation states of these endocytic proteins, morphinetreatment could modulate the extent of clathrin-mediated en-docytosis, and this could have an impact on the levels ofreceptors at the synapse.

Previous reports have shown that AMPA receptor endocyto-sis is a clathrin- and dynamin-dependent process that involvesdirect binding of the clathrin�dynamin�AP-2 complex to the re-ceptors themselves (21, 47, 48). In this work, we showed thatmorphine treatment significantly decreased the specific associ-ation of clathrin with the AMPA receptor subunit, GluR1. There-fore, one could hypothesize that morphine treatment decreasesthe rate and extent of receptor endocytosis at the synapse byrecruiting clathrin and other key endocytic proteins away fromthe endocytic zone to the PSD, thus disabling the associationbetween clathrin and GluR1. Such a notion is in agreement withthe findings from a recent study that found an increase in theproportion of immunogold particles for GluR1 on the synapticplasma membrane in the amygdala of rats self-administeringmorphine (49). Taken together, these results support the ideathat plasticity at excitatory synapses contributes to the devel-opment of drug addiction (50).

In summary, using a proteomics approach we specificallyanalyzed a PSD-enriched protein fraction from mouse hip-pocampus. This database along with other hippocampal PSDdatabases should be useful for further functional studies re-lated to learning and memory. Using differential isotopic la-beling we identified the components of the endocytic machin-ery that are substantially altered by morphine administration.These results suggest a critical involvement of the endocyticmachinery in the modulatory effects of morphine at the PSDand provide further insight into mechanisms underlying syn-aptic changes elicited by drugs of abuse.




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Acknowledgments—We thank Drs. Robert Blitzer and IvoneGomes for critical reading of the manuscript and members of the Devilaboratory for discussion. We also thank Dr. Victor Friedrich for helpwith electron microscopy.

* This work was supported by National Institutes of Health GrantsDA08863, NS26880, and DA19521 (to L. A. D.) and CA88325 (toR. W.). Electron microscopy was performed at the Mount Sinai Schoolof Medicine Microscopy Shared Resource Facility, which is supportedwith funding from NCI, National Institutes of Health Shared ResourcesGrant 5R24 CA095823-04, National Science Foundation Major Re-search Instrumentation Grant DBI-9724504, and National Institutes ofHealth Shared Instrumentation Grant 1 S10 RR0 9145-01. The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

¶ To whom correspondence should be addressed: Dept. of Phar-macology and Biological Chemistry, Mount Sinai School of Medicine,One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-8345;Fax: 212-996-7214; E-mail: [email protected].

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