Purification and Identification of G Protein-coupled Receptor Protein Complexes underNative Conditions*□S

Avais M. Daulat‡§¶�**, Pascal Maurice‡§¶�, Carine Froment‡‡,Jean-Luc Guillaume‡§¶�, Cedric Broussard‡§¶�, Bernard Monsarrat‡‡§§,Philippe Delagrange¶¶, and Ralf Jockers‡§¶� ��

G protein-coupled receptors (GPCRs) constitute the larg-est family of membrane receptors and are of major thera-peutic importance. The identification of GPCR-associatedproteins is an important step toward a better understandingof these receptors. However, current methods are not sat-isfying as only isolated receptor domains (intracellularloops or carboxyl-terminal tails) can be used as “bait.” Wereport here a method based on tandem affinity purificationcoupled to mass spectrometry that overcomes these limi-tations as the entire receptor is used to identify proteincomplexes formed in living mammalian cells. The humanMT1 and MT2 melatonin receptors were chosen as modelGPCRs. Both receptors were tagged with the tandem affin-ity purification tag at their carboxyl-terminal tails and ex-pressed in human embryonic kidney 293 cells. Receptorsolubilization and purification conditions were optimized.The method was validated by the co-purification of Gi pro-teins, which are well known GPCR interaction partners butwhich are difficult to identify with current protein-proteininteraction assays. Several new and functionally relevantMT1- and MT2-associated proteins were identified; some ofthem were common to both receptors, and others werespecific for each subtype. Taken together, our protocolallowed for the first time the purification of GPCR-associ-ated proteins under native conditions in quantities suitablefor mass spectrometry analysis. Molecular & CellularProteomics 6:835–844, 2007.

With more than 800 members, GPCRs1 constitute the larg-est family of membrane receptors (1, 2). They respond to awide variety of extracellular stimuli and are targeted by about

half of the drugs prescribed for human diseases (3). GPCRsare key controllers of physiological processes such as neuro-transmission, cellular metabolism, secretion, cell differentia-tion, and growth. The prototypic topology of GPCRs consistsof an extracellular amino-terminal segment, a hydrophobiccore of seven transmembrane (7TM) �-helices that interacttogether to form a three-dimensional barrel within the plasmamembrane, and a cytosolic carboxyl-terminal tail (C-tail).Whereas amino acids within the extracellular and/or hydro-phobic 7TM core of the receptor are involved in ligand bind-ing, the intracellular domain of the receptor composed ofthree loops and the C-tail is important for signal transduction(4).

Several strategies have been used to identify GPCR-asso-ciated complexes. Early work used affinity columns with bio-tinylated ligands to purify somatostatin receptor-G proteincomplexes from tissues (5). Subsequently based on the pio-neering work of Husi et al. (6), more systematic proteomicsanalysis of GPCR-associated protein complexes was con-ducted using receptor-specific antibodies (7). However, thegeneral application of these approaches was limited by theavailability of adequate tools (labeled ligands, antibodies, etc.)for each GPCR. Later on, isolated intracellular domains werewidely used to identify GPCR-associated proteins either asbait in yeast two-hybrid screens or to generate affinity matri-ces for the purification of interacting proteins from cell ex-tracts (8–11). Using the entire C-tail of the 5HT2c receptorexpressed as GST fusion protein, more than 15 proteins havebeen identified (12). Furthermore isolated protein-protein in-teraction motifs such as PDZ domain recognition motifs ofGPCRs have been successfully used to identify interactingpartners of the PDZ domain recognition motifs of the 5HT2a,5HT2c, and 5HT4 receptors (13, 14).

Although several GPCR-interacting proteins could be iden-tified, these methods obviously have important limitations asisolated receptor subdomains 1) do not mimic the GPCRtopology (7TM domain, arrangement of intracellular loops and

From the ‡Department of Cell Biology, Institut Cochin, §INSERMU567, ¶CNRS UMR 8104, and �Faculte de Medecine Rene Descartes,UMR-S 8104, Universite Paris Descartes, Paris F-75014, France,‡‡Institut de Biologie Structurale et de Pharmacologie, CNRS UMR5089, Toulouse 31076, France, and ¶¶Institut de Recherches SER-VIER, Suresnes 92150, France

Received, August 7, 2006, and in revised form, November 24, 2006Published, MCP Papers in Press, January 9, 2007, DOI 10.1074/

mcp.M600298-MCP2001 The abbreviations used are: GPCR, G protein-coupled receptor;

7TM, seven transmembrane; C-tail, carboxyl-terminal tail; ERK, ex-tracellular signal-regulated kinase; MT, melatonin receptor; TAP, tan-

dem affinity purification; TEV, tobacco etch virus; HEK, human em-bryonic kidney; [125I]MLT, 2-[125I]iodomelatonin; NT, non-transfected;MAPK, mitogen-activated protein kinase; IRS, insulin receptor sub-strate; PP2, protein phosphatase 2; ER, endoplasmic reticulum.

Research

© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 6.5 835This paper is available on line at http://www.mcponline.org

the C-tail, and receptor oligomerization), 2) do not provide anadequate membrane environment, and 3) do not allow therecruitment of protein complexes upon agonist activation. Notsurprisingly, well known interaction partners of GPCRs suchas heterotrimeric G proteins are difficult to identify using thesetechniques.

Recently a tandem affinity purification (TAP) method hasbeen described (15). Importantly this method overcomes theaforementioned limitations and can be potentially applied toany protein. The full-length protein of interest can be ex-pressed in mammalian cells where subcellular localization andpost-translational modifications are conserved. In addition,the recruitment of protein complexes may be induced bytreatment of the cells with different hormonal or pharmaco-logical compounds. To perform a two-step affinity chroma-tography purification of complexes formed in intact cells, theTAP method relies on the presence of a TAP tag composed oftwo IgG binding domains, a tobacco etch virus (TEV) proteasecleavage site, and a calmodulin binding domain (16). The TAPmethod has been successfully used for high throughput iden-tification of soluble proteins engaged in interacting complexesin yeast and mammalian cells (17, 18). However, purificationof membrane protein complexes in general and of GPCR-associated complexes in particular has been unsuccessful.

In the present study, we developed a modified TAP methodsuitable for the purification of GPCR-associated complexes.The human MT1 and MT2 melatonin receptors were chosen asmodel GPCRs. Both receptors were tagged with the TAP tagat their C-tails, and corresponding stable clones were estab-lished in HEK 293 cells. Receptor solubilization and purifica-tion was optimized in small scale experiments, and receptor-associated complexes were purified from large scaleexperiments and subsequently identified by nano-LC-nano-ESI MS/MS.

EXPERIMENTAL PROCEDURES

Receptor Constructs—MT1-Rluc and MT2-Rluc constructs havebeen described elsewhere (19). To obtain the MT1-TAP and MT2-TAPconstructs, the TAP tag cassette from the pcDNA3-CMV-TAP plas-mid (a gift from Nicolas Goardon, Institut Cochin, Paris, France) wasfused in frame to the 3�-end of the MT1 and MT2 coding region.

Cell Culture and Transfection—HEK 293 cells were grown andtransfected as described elsewhere (19). Stable cell lines were se-lected with G418 (Invitrogen).

Crude Membrane Preparation, Radioligand Binding Assay, and Sol-ubilization—Crude membranes were prepared as described previ-ously (20, 21) from non-transfected, MT1-TAP-, or MT2-TAP-express-ing HEK 293 cells. Membranes were labeled with a saturatingconcentration (500 pM) of 2-[125I]iodomelatonin ([125I]MLT), and[125I]MLT binding sites were determined on crude membranes, solu-bilized extracts, or at different steps of the TAP procedure. 0.5%CHAPS, 0.25% Brij�96V, 0.5% digitonin, 0.5% Nonidet P-40 (all fromSigma) and 0.5% dodecylmaltoside (Roche Applied Science) wereused for overnight solubilization at 4 °C in solubilization buffer (75 mM

Tris, 2 mM EDTA, 5 mM MgCl2, pH 8.0).Luminescence Measurements—Crude membranes were prepared

from HEK 293 cell lines stably expressing MT1-Rluc or MT2-Rluc and

solubilized in solubilization buffer supplemented with increasing con-centrations of CHAPS, Brij96V, dodecylmaltoside, or digitonin. Thesoluble fraction was separated from the insoluble fraction by centrif-ugation at 40000 � g. The insoluble fraction (pellet) was resuspendedin the same buffer, and luciferase activity was measured in the solubleand resuspended insoluble fraction by adding coelenterazine h (In-terchim, Montlucon, France) at a final concentration of 5 �M. Read-ings were performed with a luminometer at 488 nm (FusionTM, Pack-ard Instrument Co.). Solubilization yields were defined as thepercentage of luciferase activity in the supernatant over total lucifer-ase activity (pellet � supernatant). Use of Nonidet P-40 was notcompatible with the luciferase activity assay.

Immunofluorescence Microscopy—HEK 293 cells were grown onsterile coverslips and fixed and permeabilized for 20 min in ethanol at�20 °C. After blocking in PBS, 3% BSA for 20 min, cells were incu-bated with polyclonal anti-MT1 or anti-MT2 antibodies for 1 h at roomtemperature. Coverslips were washed three times with PBS andincubated with a FITC-coupled secondary antibody at 1:1000 dilutionin PBS, 3% BSA (Jackson ImmunoResearch Laboratories) for 30 minat room temperature. Coverslips were mounted and analyzed byconfocal laser microscopy (Leica TCS SP2 AOBS).

SDS-PAGE and Western Blotting—Whole cells (ERK activation) orcrude membranes (receptor detection) were denatured overnight atroom temperature in SDS-PAGE loading buffer (62.5 mM Tris/HCl, pH6.8, 5% SDS, 10% glycerol, 0.5% bromphenol blue). Proteins wereseparated by 10% SDS-PAGE and transferred to nitrocellulose. Im-munoblot analysis was carried out with polyclonal anti-MT1, anti-MT2

(22), anti-phospho-ERK, or anti-ERK2 antibodies (Santa Cruz Bio-technology). Immunoreactivity was revealed using secondary anti-bodies coupled to horseradish peroxidase and the ECL reagent(Perbio).

Optimized TAP Tag Procedure—All purification steps were con-ducted at 4 °C in the presence of a protease inhibitor mixture (RocheApplied Science), 1 mM orthovanadate, and 2 mM NaF. For large scaleexperiments, crude membranes were prepared from �1 � 109 HEK293 cells and solubilized overnight in solubilization buffer with 0.5%digitonin or 0.25% Brij96V at a concentration of 2 mg of protein/ml.The supernatant was recovered after centrifugation at 40,000 � g for30 min and incubated for 4 h with 400 �l of rabbit IgG-Agarose(Sigma). The resin was washed three times with 1 ml of solubilizationbuffer, resuspended in 500 �l of the same buffer, and incubatedovernight with 100 units of TEV protease (Invitrogen). The supernatantwas collected, mixed with 500 �l of calmodulin buffer (75 mM Tris, 5mM MgCl2, 50 mM CaCl2, and 0.5% digitonin or 0.25% Brij96V, pH8.0) and incubated for 2 h with 100 �l of calmodulin beads (Strat-agene, La Jolla, CA). Beads were washed five times with 1 ml ofcalmodulin buffer, and retained proteins were eluted with SDS-PAGEloading buffer.

Mass Spectrometry and Protein Identification—Coomassie Blue-stained or silver-stained (23) bands were excised and subjected toin-gel tryptic digestion using modified porcine trypsin (Promega,Lyon, France) as described previously (24). The tryptic digest wasanalyzed by on-line capillary HPLC (Dionex/LC Packings) coupled toa nanospray Qq-TOF mass spectrometer (QSTAR Pulsar XL, AppliedBiosystems, Foster City, CA). Peptides were separated on a 75-�minner diameter � 15-cm C18 PepMapTM column after loading onto a300-�m inner diameter � 5-mm PepMap C18 precolumn (Dionex/LCPackings). The flow rate was set at 200 nl/min. Peptides were elutedusing a 0–50% linear gradient of solvent B in 50 min (solvent A was0.2% formic acid in 5% acetonitrile, and solvent B was 0.2% formicacid in 90% acetonitrile). The mass spectrometer was operated inpositive ion mode at a 2.1- kV needle voltage. MS and MS/MS datawere continuously acquired in an information-dependent acquisitionmode consisting of a 10-s cycle time. Within each cycle, a MS

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836 Molecular & Cellular Proteomics 6.5

spectrum was accumulated for 1 s over the range m/z 300–2000followed by three MS/MS acquisitions of 3 s each on the three mostabundant ions in the MS spectrum. A dynamic exclusion duration wasused to prevent repetitive selection of the same ions within 30 s.MS/MS data were acquired using a 3 m/z unit ion isolation window.Collision energies were automatically adjusted according to thecharge state and mass value of the precursor ions. Peak lists ofMS/MS spectra were created using Mascot.dll script (version 1.6b21)in Analyst QS software (version 1.1, Applied Biosystems). For gener-

ation of the peak lists, the default charge state was set to 2�, 3�, and4�; MS/MS data were centroided and deisotoped with a threshold at0% of the base peak; MS/MS spectra with less than five peaks wererejected; the precursor mass tolerance for grouping was set to 0.1;and the maximum number of cycles between groups and the mini-mum cycles per group were set to 60 and 1, respectively. MS/MSdata were searched against a human sequence in-house database, acompilation of UniProt Swiss-Prot and UniProt TrEMBL databases(72,049 entries, versions 49.1 and 32.1, respectively) using the Mas-

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FIG. 1. Functional expression ofMT1-TAP and MT2-TAP in stablytransfected HEK 293 cells. a and b,crude membranes were prepared fromHEK 293 cells stably transfected with theindicated receptors. Denatured sampleswere separated by SDS-PAGE and ana-lyzed by Western blot using anti-MT1 (a)or anti-MT2 (b) antibodies. Subcellularlocalization of MT1 (c), MT1-TAP (d), MT2

(e), or MT2-TAP (f) in stably transfectedHEK 293 cells was monitored by immu-nofluorescence using anti-MT1 (c and d)or anti-MT2 (e and f) antibodies. g, kinet-ics of melatonin-stimulated (100 nM) ERKactivation in stable clones expressingMT1, MT1-TAP, MT2, or MT2-TAP. Noactivation of the ERK pathway by mela-tonin was observed in non-transfectedHEK 293 cells (not shown). NS, nonspe-cific; P-ERK, phospho-ERK.

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Molecular & Cellular Proteomics 6.5 837

cot� search engine (version 2.1.04). Up to two trypsin missed cleav-ages were allowed, and the mass tolerance for peptide and MS/MSfragment ions was 0.5 Da. Cysteine carbamidomethylation and pro-pionamide and methionine oxidation were set as variable modifica-tions. Identification was considered positive if the protein was iden-tified with at least one peptide with an ion score greater than theMascot significance threshold of 36 (p � 0.05). For the protein with ascore close to the threshold value, the identification was confirmed bymanual interpretation of corresponding MS/MS data. To evaluate thefalse-positive rate in these large scale experiments, we repeated thesearches using identical search parameters and validation criteriaagainst a random database made of the same compilation in whichthe sequences have been reversed. These statistical analyses pro-vided respectively 2.1, 4.1, and 5.8% false-positive rate for MT1-TAP,MT2-TAP, and control experiments. See Supplemental Tables I and IIfor a detailed list of identified peptides.

RESULTS AND DISCUSSION

Functional Expression of MT1-TAP and MT2-TAP FusionProteins in HEK 293 Cells

The human MT1 and MT2 melatonin receptors were taggedwith the TAP tag at their C-tails, and corresponding stableclones were established in HEK 293 cells. HEK 293 cells are

of human origin, are extremely well characterized in terms ofGPCR signal transduction, can be produced in quantities thatare compatible with proteomics analysis, and do not expresssignificant amounts of endogenous melatonin receptors.Western blot analysis with receptor-specific antibodies re-vealed immunoreactive bands at the expected molecular sizeof 85 kDa in an HEK-MT1-TAP clone expressing 1.0 � 0.2pmol of MT1-TAP/mg of protein (n � 3) and in an HEK-MT2-TAP clone expressing 340 � 41 fmol of MT2-TAP/mg ofprotein (n � 3) (Fig. 1, a and b). The affinity constants (Kd) ofthe melatonin receptor agonist [125I]MLT were 85 � 53 (n �

3) and 267 � 78 pM (n � 3) for MT1-TAP and MT2-TAP,respectively, which are in agreement with previously re-ported values for wild-type receptors (25). Expression ofreceptors at the cell surface was assessed by fluorescencemicroscopy using receptor-specific antibodies (Fig. 1, c–f).TAP-tagged and wild-type receptors were all expressed atthe plasma membrane of stably transfected HEK 293clones. Incubation of HEK-MT1-TAP and HEK-MT2-TAPclones with melatonin led to the expected transient increase

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FIG. 2. Detergent selection and opti-mization of solubilization conditionsof melatonin receptors. a and b, mem-brane preparations of HEK 293 cells thatstably express MT1-Rluc or MT2-Rlucwere incubated either for 15 h with in-creasing detergent concentrations at4 °C (a) or for the indicated times in thepresence of 0.5% CHAPS (● ), digitonin(E), or dodecylmaltoside (�) or 0.25%Brij96V (f) (b). Luciferase activity wasmeasured in the soluble and insolublefraction to determine the percentage ofsolubilization. The use of Nonidet P-40was not compatible with the luciferaseactivity assay. c, membrane prepara-tions of HEK 293 cells that stably ex-press MT1-TAP or MT2-TAP were la-beled with [125I]MLT, incubatedovernight at 4 °C in the presence of a0.5% concentration of the indicated de-tergents with the exception of Brij96V,which was used at 0.25%. The amountof radioactivity in the soluble fractionwas determined (black bars), [125I]MLT-labeled MT1-TAP and MT2-TAP were re-covered on IgG-coated beads, and theamount of recovered radioactivity wasdetermined (white bars). The amount of[125I]MLT binding to non-transfectedHEK 293 cells was negligible.

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838 Molecular & Cellular Proteomics 6.5

of ERK1/2 phosphorylation, which was comparable to thatobtained for wild-type receptors (Fig. 1g). Altogether thesedata demonstrate that the addition of the TAP tag does notaffect the subcellular localization and the functionality of themelatonin receptor.

Optimization of Receptor Solubilization and Purification—The crucial step for successful purification of GPCRs andassociated proteins consists of mild but efficient solubilizationof cells to extract maximal amounts of intact membrane-bound complexes. To quantify the amount of solubilized MT1

and MT2, we used cells expressing Rluc (Renilla luciferase)-tagged MT1 and MT2 receptors (19). These cells were incu-bated overnight with varying concentrations of detergents,and the solubilization yield was determined by measuringluciferase activity in the soluble and non-soluble fractions (Fig.2a). The amount of solubilized receptor increased as a func-tion of the detergent concentration and reached maxima at

50% (CHAPS), 65% (Brij96V), 70% (digitonin), or 80% (dode-cylmaltoside). Data obtained with Nonidet P-40 could not beanalyzed in this assay as Nonidet P-40 inhibits the luciferaseactivity. Comparable results were obtained for MT1-Rluc andMT2-Rluc. Minimal detergent concentrations, which gave max-imal receptor solubilization, were used to study the solubiliza-tion kinetics (Fig. 2b). Both receptors solubilized progressivelywith time reaching a plateau after 15 h. For further experiments,receptors were solubilized with 0.5% CHAPS, digitonin, dode-cylmaltoside, or Nonidet P-40 or with 0.25% for Brij96V for 15 h.

To evaluate the topological integrity of solubilized MT1-TAPand MT2-TAP, their ability to bind [125I]MLT was used. Mem-branes prepared from HEK-MT1-TAP and HEK-MT2-TAP cellswere labeled with [125I]MLT. The receptors were solubilizedand immobilized on IgG-coated beads via the IgG bindingmodules of the TAP tag (Fig. 2c). Digitonin and Brij96V werechosen for further experiments as 40–50% of [125I]MLT bind-

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FIG. 3. Purification of MT1-TAP andMT2-TAP and associated Gi proteins.a and b, monitoring of the purificationof the digitonin-solubilized (white bars)or Brij96V-solubilized (black bars)[125I]MLT-labeled MT1-TAP (a) and MT2-TAP (b) at each step of the TAP protocol.The purification yield was expressed aspercentage of total membrane [125I]MLTbinding sites. CAM, eluate from calmod-ulin column; IgG, bound to IgG-coatedcolumn; Sol, solubilized fraction; TEV,elution with TEV protease. c, monitoringof the co-purification of receptor-associ-ated Gi� proteins by Western blot frommelatonin-stimulated cells (15 min, 1�M). mb, crude membrane fraction.

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Molecular & Cellular Proteomics 6.5 839

ing sites were routinely retained on IgG beads under theseconditions. Using these optimized solubilization conditions,the entire TAP procedure was carried out. The purification offunctional receptors was monitored at each step with the[125I]MLT binding assay. The overall yield of [125I]MLT-labeledreceptors varied from 27 � 3 (digitonin) to 15 � 2% (Brij96V)and from 33 � 2 (digitonin) to 25 � 5% (Brij96V) (n 5) forMT1 and MT2, respectively (Fig. 3, a and b). The co-purifica-tion of associated proteins upon melatonin stimulation wasevaluated by the presence of the � subunit of the heterotri-meric Gi protein (Gi�) (20, 21). Whereas Gi� was readily de-tected throughout the purification of digitonin-solubilizedMT1-TAP and MT2-TAP (Fig. 3c), Gi� was rarely co-purifiedwhen using Brij96V (not shown) because this detergent ap-parently destabilized the receptor/G protein interaction. Theintegrity of the heterotrimeric G protein was further confirmedby the presence of the G� subunit at the final purification stepin the presence of digitonin (not shown), which was used forfurther experiments.

Purification and Identification of MT1- and MT2-associatedProteins

The ultimate aim of the TAP tag procedure is the purificationof sufficient amounts of receptor to identify associated pro-teins by mass spectrometry analysis. To reach this goal,crude membranes of �1 � 109 HEK-MT1-TAP, HEK-MT2-TAP, and non-transfected HEK 293 cells were prepared, andthe digitonin-solubilized fraction was submitted to the TAPprocedure. Eluates were separated by one-dimensional gelelectrophoresis, and depending on the receptor expressionlevels, proteins were detected either by Coomassie Blue(MT1-TAP, �1 pmol/mg) or by silver staining (MT2-TAP, �0.3pmol/mg) (Fig. 4, a and b). Whereas only a few bands were

visible in non-transfected (NT) HEK 293 cells, several specificprotein bands were reproducibly present in MT1-TAP- andMT2-TAP-expressing cells (n � 4). Lanes were systematicallyexcised and digested with trypsin, and the resulting peptideswere analyzed by nano-LC-nano-ESI MS/MS and identifiedwith Mascot software in Swiss-Prot and TrEMBL databases.Several abundantly expressed proteins, mostly of mitochon-drial or ribosomal origin, were detected in all three lanes(NT, MT1-TAP, and MT2-TAP) and were classified as non-specific proteins. The proteins repeatedly present in theMT1-TAP or MT2-TAP lane but absent from the NT lane wereconsidered to be specifically associated to MT1 or MT2,respectively (Tables I and II). Consistently both bait proteins(MT1 and MT2) were identified in the corresponding lanes.Each receptor was identified in two regions, at 45 and 90kDa, corresponding to the well documented monomeric andSDS-resistant dimeric form of the receptors, respectively(26). This indicates that both receptors reached their fullyfunctional quaternary structure.

Importantly the presence of heterotrimeric G proteins inMT1- and MT2-associated complexes was confirmed by massspectrometry. Consistent with the known coupling of melato-nin receptors to Gi proteins, all three Gi� isoforms (specificpeptides for Gi�1–3) and two different G� isoforms (specificpeptides for G�1,4) were identified. Co-purification of G pro-teins and receptors validates our method and provides amajor advantage compared with other currently available pro-tein-protein interaction assays where the G protein/receptorinteraction is generally lost. These results clearly demonstratethat our procedure can identify functionally relevant GPCR-interacting proteins that associate only with intact receptorsexpressed in the natural membrane environment.

Several previously unknown melatonin receptor-associatedproteins were identified. Interestingly these proteins localizedto different subcellular compartments (cytosol, plasma mem-brane, and different intracellular membrane compartmentssuch as the endoplasmic reticulum). Melatonin receptor-as-sociated proteins could be divided into three functionally dis-tinct groups: proteins likely to be involved in receptor biosyn-thesis, intracellular trafficking, and signaling/regulation ofGPCRs (Tables I and II). The function of remaining proteins,classified as “others,” are unknown or appear not to relatedirectly to known GPCR function. This is the case for theMT1-specific heterogeneous nuclear ribonucleoprotein A0that is suspected to participate in mRNA maturation (27) andis a major substrate for MAPK-activated protein kinase 2,which is itself activated upon GPCR-promoted p38 MAPKstimulation (28).

Filamin A and insulin receptor substrate 4 (IRS4) were iden-tified as common members of MT1- and MT2-associatedcomplexes. Consistent with these findings, filamin A has beenshown previously to interact with several other members ofthe GPCR family, including dopamine D2/D3 (29, 30), calci-um-sensing (31, 32), and �-opioid receptors (33). The role of

FIG. 4. Large scale purification of the MT1-TAP (a) and MT2-TAP-associated (b) complex. Approximately 1 � 109 HEK-MT1-TAP, HEK-MT2-TAP, and non-transfected HEK 293 cells were sub-mitted to the TAP protocol. Eluates were separated by gelelectrophoresis, and proteins were detected by Coomassie Bluestaining (a) or silver staining (b).

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840 Molecular & Cellular Proteomics 6.5

IRS4 is less well documented. The involvement of IRS4 infibroblast growth factor receptor signaling (34) and interactionwith the protein phosphatase 4 have been described (35).

We were also able to identify several MT1-specific signalingproteins such as Rac1, RAP-1A, and the 2�,3�-cyclic-nucleo-tide 3�-phosphodiesterase and the protein elongation factor1-� (eEF-1B�). Interestingly the small GTPases Rac1 andRAP-1 have been shown to function downstream of 5HT4

receptors and the cAMP guanine nucleotide exchange factorEpac1 (36). The 2�,3�-cyclic-nucleotide 3�-phosphodiester-ase, belonging to the PDE3A family, is involved in the degra-dation of second messengers such as cAMP and cGMP (37).Activation of melatonin receptors is known to modulate bothsecond messengers (25). eEF-1B� and other elongation fac-tors have been reported to modulate GPCR function by direct

interaction with the receptor (38, 39).Catenin �1 (p120) and the protein phosphatase 2C�

(PP2C�) have been identified as MT2-specific signaling pro-teins. Whereas p120 is known to affect intracellular signalingby NF�B activation through regulation of Rho GTPases, itsspecific role in GPCR signaling is currently unknown (40).Several serine/threonine phosphatases participate in the de-phosphorylation of activated GPCRs (41). Phosphatases ofthe PP2A and PP2B subfamilies have been reported to targetGPCRs, whereas PP2C subfamily members have been shownto dephosphorylate the metabotropic glutamate receptor 3(42). It will be interesting to determine whether PP2C� partic-ipates in MT2 dephosphorylation.

Most of the identified proteins that are involved in receptorbiosynthesis are present in both receptor-associated com-

TABLE IMT1-associated proteins

Trypsin-digested protein bands were analyzed by nano-LC-nano-ESI MS/MS, and proteins were identified with Mascot software inSwiss-Prot and TrEMBL databases. Comparison of proteins identified in eluates from MT1-TAP-expressing cells with those of non-transfectedHEK 293 cells led to the identification of MT1-specific proteins. Data represent the common results of four experiments.

Swiss-Prot/TrEMBLaccession no.

Protein identity Score CoverageNumber

of uniquepeptides

Theoreticalmolecular

mass

Alsoidentified

in MT2

Subcellularlocalization

% Da

Bait proteinP48039 Melatonin receptor type 1A (MT1) 353 24 9 39,349 � Plasma membrane

Signal transductionP62873 Guanine nucleotide-binding protein �

subunit 1405 29 10 37,222 � Plasma membrane

Q9HAV0 Guanine nucleotide-binding protein �subunit 4

318 22 8 37,412 � Plasma membrane

P08754 Guanine nucleotide-binding protein Gi�3 639 46 16 40,375 � Plasma membraneP63096 Guanine nucleotide-binding protein Gi�1 566 46 14 40,204 � Plasma membraneP04899 Guanine nucleotide-binding protein Gi�2 409 34 11 40,294 � Plasma membraneP09543 2�,3�-cyclic-nucleotide 3�-

phosphodiesterase97 7 3 47,549 � Plasma membrane

P62834 Ras-related protein RAP-1A 93 11 2 20,974 � Plasma membraneP63000 p21-Rac1 82 8 2 21,436 � MembraneP26641 Elongation factor 1-� 197 11 5 49,956 � CytoplasmO14654 Insulin receptor substrate 4 146 4 4 133,685 � CytoplasmQ60FE5 Filamin A 94 1 3 278,053 � Cytoplasm

BiosynthesisP27824 Calnexin precursor 837 30 20 67,526 � ER membraneP11021 78-kDa glucose-regulated protein 511 25 16 72,288 � ER lumenP27797 Calreticulin 189 12 7 48,112 � ER lumenQ15084 Protein-disulfide isomerase A6 155 7 3 48,091 � ER lumen

TrafficO95292 Vesicle-associated membrane protein-

associated protein B/C83 15 4 27,080 � Membrane

P61026 Ras-related protein Rab-10 121 14 3 22,527 � EndosomeOthers

O00264 Membrane-associated progesteronereceptor component 1

191 24 6 21,527 � Plasma membrane

Q96AG4 Leucine-rich repeat-containing protein 59 312 36 9 34,909 � Intracellularmembrane

P57088 Transmembrane protein 33 102 12 3 27,960 � MembraneQ13151 Heterogeneous nuclear ribonucleoprotein

A0377 28 7 30,822 � Cytoplasm

GPCR Protein Complexes

Molecular & Cellular Proteomics 6.5 841

plexes. This is expected because all GPCRs are suspected tofollow the same biosynthetic pathway. Interestingly identifiedproteins interact with different distinct receptor domains in-cluding the cytoplasmic and the endoplasmic reticulum (ER)luminal receptor interface. In contrast, proteins involved intrafficking differ clearly between the two receptor subtypesindicating different trafficking behavior.

Apart from heterotrimeric Gi proteins, not much is knownabout the repertoire of melatonin receptor interaction part-ners. This makes it difficult to estimate the proportion ofknown interaction partners that are covered by our data set.However, a rough estimation can be made with the assump-tion that many interaction partners are likely to interact at leastwith several other GPCRs. For MT1 and MT2 35 and 45%,respectively, of the identified proteins have been shown tointeract with other GPCRs. An additional 25 and 20% of theMT1 and MT2 interactors, respectively, have been associatedwith GPCR function (signaling, trafficking, etc.). Thus theoverall rate of interaction partners that “make sense” repre-sents �60% for both receptors.

Did we miss any known/suspected interaction partners?Although not directly shown, functional studies clearly indi-

cate that melatonin receptors (43), like most other GPCRs,interact with �-arrestins, a family of proteins involved inreceptor desensitization, internalization, and signaling (44).This interaction is known to be agonist-stimulated and tran-sient. The absence of �-arrestin in our data set suggeststhat this transient interaction may need to be stabilized forinstance by chemically cross-linking the partners prior topurification.

Purification of protein complexes from intact mammaliancells is one of the advantages of the TAP technique. This alsoimplies that the components of the interacting complexesdepend on the chosen cellular context. As we selected HEK293 fibroblasts for our study we expected to identify ratherubiquitously expressed interaction partners than cell type-specific (i.e. neuron-specific) partners. The repertoire of pro-teins identified for both melatonin receptors confirmed thisprediction. The next step will be to identify cell type-specificinteraction partners by expressing TAP-tagged melatonin re-ceptors in neurons or endocrine cells that express endoge-nous receptors with well defined functions.

Taken together, our TAP protocol allowed for the first timethe purification of GPCR-associated proteins under native

TABLE IIMT2-associated proteins

Trypsin-digested protein bands were analyzed by nano-LC-nano-ESI MS/MS, and proteins were identified with Mascot software inSwiss-Prot and TrEMBL databases. Comparison of proteins identified in eluates from MT2-TAP-expressing cells with those of non-transfectedHEK 293 cells led to the identification of MT2-specific proteins. Data represent the common results of four experiments.

Swiss-Prot/TrEMBLaccession no.

Protein identity Score CoverageNumber

of uniquepeptides

Theoreticalmolecular

mass

Alsoidentified

in MT1

Subcellular localization

% Da

Bait proteinP49286 Melatonin receptor type 1B (MT2) 191 10 4 40,162 Plasma membrane

Signal transductionP62879 Guanine nucleotide-binding protein �

subunit 1929 52 15 37,222 � Plasma membrane

Q9HAV0 Guanine nucleotide-binding protein �subunit 4

511 29 10 37,412 � Plasma membrane

P63096 Guanine nucleotide-binding protein Gi�1 905 60 20 40,204 � Plasma membraneP08754 Guanine nucleotide-binding protein Gi�3 899 59 19 40,375 � Plasma membraneP04899 Guanine nucleotide-binding protein Gi�2 775 64 20 40,294 � Plasma membraneO14654 Insulin receptor substrate 4 546 12 16 133,685 � CytoplasmQ60FE5 Filamin A 369 6 14 278,053 � CytoplasmO60716 Catenin �1 126 6 4 108,103 � CytoplasmO15355 Protein phosphatase 2C isoform � 119 5 3 59,235 � Cytoplasm

BiosynthesisP27824 Calnexin 747 28 18 67,526 � ER membraneP11021 78-kDa glucose-regulated protein 641 28 16 72,288 � ER lumenQ15084 Protein-disulfide isomerase A6 235 16 4 48,091 � ER lumenP27348 14-3-3 protein � 96 8 2 27,747 � Cytoplasm

TrafficQ5T201 Coatomer protein complex, � subunit 547 15 20 138,258 � ER/GolgiQ96QK1 Vacuolar sorting protein 35 377 15 11 91,649 � Membrane-associated

proteinP42356 Phosphatidylinositol 4-kinase � 134 3 4 231,143 � Intracellular membrane

OthersQ9NQX7 Integral membrane protein 2C 80 12 2 30,224 � Membrane

GPCR Protein Complexes

842 Molecular & Cellular Proteomics 6.5

conditions in quantities suitable for mass spectrometry anal-ysis. Interaction partners from different cellular compartmentsrecognizing extra- and intracellular receptor domains of mo-nomeric and/or dimeric receptor species were identified. Thispresents a major methodological advance in the identificationof GPCR-associated protein complexes. In addition, thismethod is relatively fast, generates a low number of nonspe-cific proteins, and needs no confirmation of the interaction byco-immunoprecipitation experiments. Similar results were ob-tained between MT1 and MT2 in terms of solubilization effi-ciency, complex stability, and purification yields with this pro-tocol, suggesting a more general application. The increasingnumber of TAP tag variants, including the split TAP tag (16),demonstrates the flexibility of this method and allows thepossibility of rapid optimization for any GPCR homo- andheterodimer.

Acknowledgments—We are grateful to N. Goardon (Institut Cochin,Paris, France) and to Drs. G. Fraschini (Milan, Italy) and D. Angeloni(Pisa, Italy) for kindly providing the TAP tag expression cassette andanti-melatonin receptor antibodies, respectively. We thank PattyChen for comments on the manuscript, Dr. Luc Camoin for expertadvice, and Viola Baradari for help in the initial phase of the project.

* This work was supported in part by grants from SERVIER, IN-SERM, and CNRS. The costs of publication of this article were de-frayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

** Holds an EGIDE fellowship.§§ Supported by grants from Genopole Toulouse Midi-Pyrenees

and Region Midi Pyrenees.�� To whom correspondence should be addressed. Tel.: 331-40-

51-64-34; Fax: 331-40-51-64-30; E-mail: jockers@cochin.inserm.fr.

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