Purification and Identification of G Protein- coupled Receptor Protein Complexes under Native Conditions

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Receptor Protein Complexes under Native Conditions

Avais Daulat, Pascal Maurice, Carine Froment, Jean-Luc Guillaume, Cedric

Broussard, Bernard Monsarrat, Philippe Delagrange, Ralf Jockers

To cite this version:

Avais Daulat, Pascal Maurice, Carine Froment, Jean-Luc Guillaume, Cedric Broussard, et al..

Purifi-cation and IdentifiPurifi-cation of G Protein- coupled Receptor Protein Complexes under Native Conditions.

Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2007.

�hal-02348074�

Purification and Identification of G

Protein-coupled Receptor Protein Complexes under

Native Conditions*

□

S

Avais M. Daulatद

储**, Pascal Maurice‡§¶储, Carine Froment‡‡,

Jean-Luc Guillaumeद

储, Ce´dric Broussard‡§¶储, Bernard Monsarrat‡‡§§,

Philippe Delagrange¶¶, and Ralf Jockers‡§¶

储 储储

G protein-coupled receptors (GPCRs) constitute the larg-est family of membrane receptors and are of major thera-peutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not sat-isfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as “bait.” We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limi-tations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT1and MT2melatonin receptors were chosen as model GPCRs. 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. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of Gi pro-teins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT1- and MT2-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associ-ated proteins under native conditions in quantities suitable for mass spectrometry analysis. Molecular & Cellular Proteomics 6:835– 844, 2007.

With more than 800 members, GPCRs1_{constitute the}

larg-est family of membrane receptors (1, 2). They respond to a wide variety of extracellular stimuli and are targeted by about

half of the drugs prescribed for human diseases (3). GPCRs are key controllers of physiological processes such as neuro-transmission, cellular metabolism, secretion, cell differentia-tion, and growth. The prototypic topology of GPCRs consists of an extracellular amino-terminal segment, a hydrophobic core of seven transmembrane (7TM) ␣-helices that interact together to form a three-dimensional barrel within the plasma membrane, 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 of three 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 protein complexes from tissues (5). Subsequently based on the pio-neering work of Husi et al. (6), more systematic proteomics analysis of GPCR-associated protein complexes was con-ducted using receptor-specific antibodies (7). However, the general application of these approaches was limited by the availability of adequate tools (labeled ligands, antibodies, etc.) for each GPCR. Later on, isolated intracellular domains were widely used to identify GPCR-associated proteins either as bait 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 5HT_2c receptor expressed as GST fusion protein, more than 15 proteins have been identified (12). Furthermore isolated proteprotein in-teraction motifs such as PDZ domain recognition motifs of GPCRs have been successfully used to identify interacting partners of the PDZ domain recognition motifs of the 5HT2a,

5HT2c, and 5HT4receptors (13, 14).

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

From the ‡Department of Cell Biology, Institut Cochin, §INSERM U567, ¶CNRS UMR 8104, and储Faculte´ de Me´decine Rene´ Descartes, UMR-S 8104, Universite´ Paris Descartes, Paris F-75014, France, ‡‡Institut de Biologie Structurale et de Pharmacologie, CNRS UMR 5089, Toulouse 31076, France, and ¶¶Institut de Recherches SER-VIER, Suresnes 92150, France

Received, August 7, 2006, and in revised form, November 24, 2006 Published, MCP Papers in Press, January 9, 2007, DOI 10.1074/ mcp.M600298-MCP200

1_{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; [125_{I]MLT, 2-[}125_{I]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

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the C-tail, and receptor oligomerization), 2) do not provide an adequate membrane environment, and 3) do not allow the recruitment of protein complexes upon agonist activation. Not surprisingly, well known interaction partners of GPCRs such as heterotrimeric G proteins are difficult to identify using these techniques.

Recently a tandem affinity purification (TAP) method has been described (15). Importantly this method overcomes the aforementioned limitations and can be potentially applied to any protein. The full-length protein of interest can be ex-pressed in mammalian cells where subcellular localization and post-translational modifications are conserved. In addition, the recruitment of protein complexes may be induced by treatment 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, the TAP method relies on the presence of a TAP tag composed of two IgG binding domains, a tobacco etch virus (TEV) protease cleavage site, and a calmodulin binding domain (16). The TAP method has been successfully used for high throughput iden-tification of soluble proteins engaged in interacting complexes in yeast and mammalian cells (17, 18). However, purification of membrane protein complexes in general and of GPCR-associated complexes in particular has been unsuccessful.

In the present study, we developed a modified TAP method suitable for the purification of GPCR-associated complexes. The human MT₁and MT₂melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the TAP tag at 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 scale experiments and subsequently identified by nano-LC-nano-ESI MS/MS.

EXPERIMENTAL PROCEDURES

Receptor Constructs—MT1-Rluc and MT2-Rluc constructs have

been described elsewhere (19). To obtain the MT1-TAP and MT2-TAP

constructs, the TAP tag cassette from the pcDNA3-CMV-TAP plas-mid (a gift from Nicolas Goardon, Institut Cochin, Paris, France) was fused in frame to the 3⬘-end of the MT1and MT2coding region.

Cell Culture and Transfection—HEK 293 cells were grown and

transfected 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 saturat-TAP-express-ing concentration (500 pM) of 2-[125_{I]iodomelatonin ([}125_{I]MLT), and}

[125_{I]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姞96V, 0.5% digitonin, 0.5% Nonidet P-40 (all from Sigma) and 0.5% dodecylmaltoside (Roche Applied Science) were used for overnight solubilization at 4 °C in solubilization buffer (75 mM

Tris, 2 mMEDTA, 5 mMMgCl2, 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. The soluble fraction was separated from the insoluble fraction by centrif-ugation at 40000⫻ g. The insoluble fraction (pellet) was resuspended in the same buffer, and luciferase activity was measured in the soluble and resuspended insoluble fraction by adding coelenterazine h (In-terchim, Montluc¸on, 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 the percentage of luciferase activity in the supernatant over total lucifer-ase activity (pellet ⫹ supernatant). Use of Nonidet P-40 was not compatible with the luciferase activity assay.

Immunofluorescence Microscopy—HEK 293 cells were grown on

sterile 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-MT1or anti-MT2antibodies for 1 h at room

temperature. Coverslips were washed three times with PBS and incubated with a FITC-coupled secondary antibody at 1:1000 dilution in PBS, 3% BSA (Jackson ImmunoResearch Laboratories) for 30 min at room temperature. Coverslips were mounted and analyzed by confocal laser microscopy (Leica TCS SP2 AOBS).

SDS-PAGE and Western Blotting—Whole cells (ERK activation) or

crude membranes (receptor detection) were denatured overnight at room temperature in SDS-PAGE loading buffer (62.5 mMTris/HCl, pH 6.8, 5% SDS, 10% glycerol, 0.5% bromphenol blue). Proteins were separated 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 (Roche Applied Science), 1 mMorthovanadate, and 2 mMNaF. For large scale experiments, crude membranes were prepared from⬃1 ⫻ 109_HEK

293 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 for 30 min and incubated for 4 h with 400 ␮l of rabbit IgG-Agarose (Sigma). The resin was washed three times with 1 ml of solubilization buffer, resuspended in 500 ␮l of the same buffer, and incubated overnight with 100 units of TEV protease (Invitrogen). The supernatant was collected, mixed with 500␮l of calmodulin buffer (75 mMTris, 5 mMMgCl2, 50 mMCaCl2, and 0.5% digitonin or 0.25% Brij96V, pH

8.0) and incubated for 2 h with 100␮l of calmodulin beads (Strat-agene, La Jolla, CA). Beads were washed five times with 1 ml of calmodulin buffer, and retained proteins were eluted with SDS-PAGE loading buffer.

Mass Spectrometry and Protein Identification—Coomassie

Blue-stained or silver-Blue-stained (23) bands were excised and subjected to in-gel tryptic digestion using modified porcine trypsin (Promega, Lyon, France) as described previously (24). The tryptic digest was analyzed by on-line capillary HPLC (Dionex/LC Packings) coupled to a nanospray Qq-TOF mass spectrometer (QSTAR Pulsar XL, Applied Biosystems, Foster City, CA). Peptides were separated on a 75-␮m inner diameter⫻ 15-cm C18PepMap

TM_{column after loading onto a}

300-␮m inner diameter ⫻ 5-mm PepMap C18precolumn (Dionex/LC

Packings). The flow rate was set at 200 nl/min. Peptides were eluted using a 0 –50% linear gradient of solvent B in 50 min (solvent A was 0.2% formic acid in 5% acetonitrile, and solvent B was 0.2% formic acid in 90% acetonitrile). The mass spectrometer was operated in positive ion mode at a 2.1- kV needle voltage. MS and MS/MS data were continuously acquired in an information-dependent acquisition mode consisting of a 10-s cycle time. Within each cycle, a MS

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spectrum was accumulated for 1 s over the range m/z 300 –2000 followed by three MS/MS acquisitions of 3 s each on the three most abundant ions in the MS spectrum. A dynamic exclusion duration was used 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 the charge state and mass value of the precursor ions. Peak lists of MS/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⫹, and 4⫹; MS/MS data were centroided and deisotoped with a threshold at 0% of the base peak; MS/MS spectra with less than five peaks were rejected; 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/MS data were searched against a human sequence in-house database, a compilation of UniProt Swiss-Prot and UniProt TrEMBL databases (72,049 entries, versions 49.1 and 32.1, respectively) using the

Mas-c

d

f

e

c

d

f

e

MT

₁

-TAP

MT

₂

-TAP

MT

₂

MT

₁

Anti-MT

₂

Anti-MT

₁

g

MT

₁

-TAP

MT

₂

-TAP

MT

₂

MT

₁

kDa

_MT2 -TAP NT

97

66

56

42

MT 2 NT

97

66

56

42

kDa

Anti-MT

₂

Anti-MT

₂ MT₂-TAP

kDa

97

66

56

42

MT 1 -TAP NT

a

b

Anti-MT

₁ NS MT₂ MT₁ FIG. 1. Functional expression of

MT1-TAP and MT2-TAP in stably

transfected HEK 293 cells. a and b,

crude membranes were prepared from HEK 293 cells stably transfected with the indicated receptors. Denatured samples were separated by SDS-PAGE and ana-lyzed by Western blot using anti-MT1(a)

or anti-MT2 (b) antibodies. Subcellular

localization of MT1(c), MT1-TAP (d), MT2

(e), or MT2-TAP (f) in stably transfected

HEK 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) ERK activation in stable clones expressing MT1, MT1-TAP, MT2, or MT2-TAP. No

activation of the ERK pathway by mela-tonin was observed in non-transfected HEK 293 cells (not shown). NS, nonspe-cific; P-ERK, phospho-ERK.

GPCR Protein Complexes

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cot姞 search engine (version 2.1.04). Up to two trypsin missed cleav-ages were allowed, and the mass tolerance for peptide and MS/MS fragment 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 the Mascot significance threshold of 36 (p⬍ 0.05). For the protein with a score close to the threshold value, the identification was confirmed by manual interpretation of corresponding MS/MS data. To evaluate the false-positive rate in these large scale experiments, we repeated the searches using identical search parameters and validation criteria against a random database made of the same compilation in which the 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 II

for a detailed list of identified peptides.

RESULTS AND DISCUSSION

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

The human MT1and MT2melatonin receptors were tagged

with the TAP tag at their C-tails, and corresponding stable clones were established in HEK 293 cells. HEK 293 cells are

of human origin, are extremely well characterized in terms of GPCR signal transduction, can be produced in quantities that are compatible with proteomics analysis, and do not express significant amounts of endogenous melatonin receptors. Western blot analysis with receptor-specific antibodies re-vealed immunoreactive bands at the expected molecular size of 85 kDa in an HEK-MT₁-TAP clone expressing 1.0 ⫾ 0.2 pmol of MT₁-TAP/mg of protein (n⫽ 3) and in an HEK-MT₂ -TAP clone expressing 340 ⫾ 41 fmol of MT₂-TAP/mg of protein (n⫽ 3) (Fig. 1, a and b). The affinity constants (Kd) of the melatonin receptor agonist [125_{I]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 of receptors at the cell surface was assessed by fluorescence microscopy using receptor-specific antibodies (Fig. 1, c–f). TAP-tagged and wild-type receptors were all expressed at the plasma membrane of stably transfected HEK 293 clones. Incubation of HEK-MT₁-TAP and HEK-MT₂-TAP clones with melatonin led to the expected transient increase

0 25 50 75 100 0 0.25 0.50 0.75 1.00 0 25 50 75 100 Detergent conc. (%) 0 4 8 12 16 0 25 50 75 100 Time (h) 12 16 0 4 8 0 25 50 75 100 Time (h) S o lu b iliz at io n (% o f to ta l) S o lu b iliz at io n (% o f to ta l) 0 0.25 0.50 0.75 1.00 Detergent conc. (%) MT2 MT1 MT2 MT1 MT2 MT1

FIG. 2. Detergent selection and

opti-mization of solubilization conditions of melatonin receptors. a and b,

mem-brane preparations of HEK 293 cells that stably express MT1-Rluc or MT2-Rluc

were incubated either for 15 h with in-creasing detergent concentrations at 4 °C (a) or for the indicated times in the presence of 0.5% CHAPS (●), digitonin (E), or dodecylmaltoside (䡺) or 0.25% Brij96V (f) (b). Luciferase activity was measured in the soluble and insoluble fraction to determine the percentage of solubilization. The use of Nonidet P-40 was not compatible with the luciferase activity assay. c, membrane prepara-tions of HEK 293 cells that stably ex-press MT1-TAP or MT2-TAP were

la-beled with [125_{I]MLT, incubated}

overnight at 4 °C in the presence of a 0.5% concentration of the indicated de-tergents with the exception of Brij96V, which was used at 0.25%. The amount of radioactivity in the soluble fraction was determined (black bars), [125

I]MLT-labeled MT1-TAP and MT2-TAP were

re-covered on IgG-coated beads, and the amount of recovered radioactivity was determined (white bars). The amount of [125_{I]MLT binding to non-transfected}

HEK 293 cells was negligible.

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of ERK1/2 phosphorylation, which was comparable to that obtained for wild-type receptors (Fig. 1g). Altogether these data demonstrate that the addition of the TAP tag does not affect the subcellular localization and the functionality of the melatonin receptor.

Optimization of Receptor Solubilization and Purification—

The crucial step for successful purification of GPCRs and associated proteins consists of mild but efficient solubilization of 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 MT1and MT2receptors (19). These cells were

incu-bated overnight with varying concentrations of detergents, and the solubilization yield was determined by measuring luciferase activity in the soluble and non-soluble fractions (Fig. 2a). The amount of solubilized receptor increased as a func-tion of the detergent concentrafunc-tion and reached maxima at

50% (CHAPS), 65% (Brij96V), 70% (digitonin), or 80% (dode-cylmaltoside). Data obtained with Nonidet P-40 could not be analyzed in this assay as Nonidet P-40 inhibits the luciferase activity. Comparable results were obtained for MT1-Rluc and

MT2-Rluc. Minimal detergent concentrations, which gave

max-imal receptor solubilization, were used to study the solubiliza-tion kinetics (Fig. 2b). Both receptors solubilized progressively with 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-TAP

and MT2-TAP, their ability to bind [125I]MLT was used.

Mem-branes prepared from HEK-MT1-TAP and HEK-MT2-TAP cells

were labeled with [125_{I]MLT. The receptors were solubilized}

and immobilized on IgG-coated beads via the IgG binding modules of the TAP tag (Fig. 2c). Digitonin and Brij96V were chosen for further experiments as 40 –50% of [125_I]MLT

bind-MT

₁

-TAP

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MT1

-

+ -

+ -

+

Cells mb Solubilization Supernatant Pellet

-

+ -

+ -

+

G

_iα

G

_iα IgG column TEV Eluate CAM column Unretained Eluate CAM column Eluate MT₂

-

+

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_iα CAM column Eluate MT₂

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_iα

FIG. 3. Purification of MT1-TAP and

MT2-TAP and associated Giproteins.

a and b, monitoring of the purification

of the digitonin-solubilized (white bars) or Brij96V-solubilized (black bars)

[125_{I]MLT-labeled MT}

1-TAP (a) and MT2

-TAP (b) at each step of the -TAP protocol. The purification yield was expressed as percentage of total membrane [125_I]MLT

binding sites. CAM, eluate from calmod-ulin column; IgG, bound to IgG-coated column; Sol, solubilized fraction; TEV, elution with TEV protease. c, monitoring of the co-purification of receptor-associ-ated Gi␣ proteins by Western blot from

melatonin-stimulated cells (15 min, 1 ␮M). mb, crude membrane fraction.

GPCR Protein Complexes

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ing sites were routinely retained on IgG beads under these conditions. Using these optimized solubilization conditions, the entire TAP procedure was carried out. The purification of functional receptors was monitored at each step with the [125_{I]MLT binding assay. The overall yield of [}125_{I]MLT-labeled}

receptors varied from 27⫾ 3 (digitonin) to 15 ⫾ 2% (Brij96V) and from 33⫾ 2 (digitonin) to 25 ⫾ 5% (Brij96V) (n ⬎ 5) for MT1and MT2, respectively (Fig. 3, a and b). The

co-purifica-tion of associated proteins upon melatonin stimulaco-purifica-tion was evaluated by the presence of the ␣ subunit of the heterotri-meric Giprotein (Gi␣) (20, 21). Whereas Gi␣ was readily

de-tected throughout the purification of digitonin-solubilized MT1-TAP and MT2-TAP (Fig. 3c), Gi␣ was rarely co-purified

when using Brij96V (not shown) because this detergent ap-parently destabilized the receptor/G protein interaction. The integrity of the heterotrimeric G protein was further confirmed by the presence of the G␤ subunit at the final purification step in the presence of digitonin (not shown), which was used for further experiments.

Purification and Identification of MT1- and MT2-associated Proteins

The ultimate aim of the TAP tag procedure is the purification of sufficient amounts of receptor to identify associated pro-teins by mass spectrometry analysis. To reach this goal, crude membranes of ⬃1 ⫻ 109 _HEK-MT

1-TAP, HEK-MT2

-TAP, and non-transfected HEK 293 cells were prepared, and the digitonin-solubilized fraction was submitted to the TAP procedure. Eluates were separated by one-dimensional gel electrophoresis, and depending on the receptor expression levels, proteins were detected either by Coomassie Blue (MT1-TAP,⬃1 pmol/mg) or by silver staining (MT2-TAP,⬃0.3

pmol/mg) (Fig. 4, a and b). Whereas only a few bands were

visible in non-transfected (NT) HEK 293 cells, several specific protein bands were reproducibly present in MT1-TAP- and

MT2-TAP-expressing cells (n⫽ 4). Lanes were systematically

excised and digested with trypsin, and the resulting peptides were analyzed by nano-LC-nano-ESI MS/MS and identified with 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 the MT1-TAP or MT2-TAP lane but absent from the NT lane were

considered to be specifically associated to MT1 or MT2,

respectively (Tables I and II). Consistently both bait proteins (MT1and MT2) were identified in the corresponding lanes.

Each receptor was identified in two regions, at 45 and 90 kDa, corresponding to the well documented monomeric and SDS-resistant dimeric form of the receptors, respectively (26). This indicates that both receptors reached their fully functional quaternary structure.

Importantly the presence of heterotrimeric G proteins in MT1- and MT2-associated complexes was confirmed by mass

spectrometry. Consistent with the known coupling of melato-nin receptors to Giproteins, all three Gi␣ isoforms (specific

peptides for Gi␣1–3) and two different G␤ isoforms (specific

peptides for G␤1,4) were identified. Co-purification of G

pro-teins and receptors validates our method and provides a major advantage compared with other currently available pro-tein-protein interaction assays where the G protein/receptor interaction is generally lost. These results clearly demonstrate that our procedure can identify functionally relevant GPCR-interacting proteins that associate only with intact receptors expressed in the natural membrane environment.

Several previously unknown melatonin receptor-associated proteins were identified. Interestingly these proteins localized to different subcellular compartments (cytosol, plasma mem-brane, and different intracellular membrane compartments such 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 of GPCRs (Tables I and II). The function of remaining proteins, classified as “others,” are unknown or appear not to relate directly to known GPCR function. This is the case for the MT₁-specific heterogeneous nuclear ribonucleoprotein A0 that is suspected to participate in mRNA maturation (27) and is a major substrate for MAPK-activated protein kinase 2, which is itself activated upon GPCR-promoted p38 MAPK stimulation (28).

Filamin A and insulin receptor substrate 4 (IRS4) were iden-tified as common members of MT1- and MT2-associated

complexes. Consistent with these findings, filamin A has been shown previously to interact with several other members of the 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-MT

1

-TAP, HEK-MT2-TAP, and non-transfected HEK 293 cells were

sub-mitted to the TAP protocol. Eluates were separated by gel electrophoresis, and proteins were detected by Coomassie Blue staining (a) or silver staining (b).

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

We were also able to identify several MT1-specific signaling

proteins such as Rac1, RAP-1A, and the 2 ⬘,3⬘-cyclic-nucleo-tide 3⬘-phosphodiesterase and the protein elongation factor 1-␥ (eEF-1B␥). Interestingly the small GTPases Rac1 and RAP-1 have been shown to function downstream of 5HT4

receptors and the cAMP guanine nucleotide exchange factor Epac1 (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 both second 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 signaling by NF␬B activation through regulation of Rho GTPases, its specific role in GPCR signaling is currently unknown (40). Several serine/threonine phosphatases participate in the de-phosphorylation of activated GPCRs (41). Phosphatases of the PP2A and PP2B subfamilies have been reported to target GPCRs, whereas PP2C subfamily members have been shown to dephosphorylate the metabotropic glutamate receptor 3 (42). It will be interesting to determine whether PP2C␥ partic-ipates in MT2dephosphorylation.

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

com-TABLEI

MT1-associated proteins

Trypsin-digested protein bands were analyzed by nano-LC-nano-ESI MS/MS, and proteins were identified with Mascot software in Swiss-Prot and TrEMBL databases. Comparison of proteins identified in eluates from MT1-TAP-expressing cells with those of non-transfected

HEK 293 cells led to the identification of MT1-specific proteins. Data represent the common results of four experiments.

Swiss-Prot/TrEMBL

accession no. Protein identity Score Coverage

Number of unique peptides Theoretical molecular mass Also identified in MT2 Subcellular localization % Da Bait protein

P48039 Melatonin receptor type 1A (MT1) 353 24 9 39,349 ⫹ Plasma membrane

Signal transduction

P62873 Guanine nucleotide-binding protein␤ subunit 1

405 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 G_i␣3 639 46 16 40,375 ⫹ Plasma membrane P63096 Guanine nucleotide-binding protein G_i␣1 566 46 14 40,204 ⫹ Plasma membrane P04899 Guanine nucleotide-binding protein G_i␣2 409 34 11 40,294 ⫹ Plasma membrane P09543 2⬘,cyclic-nucleotide

3⬘-phosphodiesterase

97 7 3 47,549 ⫺ Plasma membrane

P62834 Ras-related protein RAP-1A 93 11 2 20,974 ⫺ Plasma membrane

P63000 p21-Rac1 82 8 2 21,436 ⫺ Membrane

P26641 Elongation factor 1-␥ 197 11 5 49,956 ⫺ Cytoplasm

O14654 Insulin receptor substrate 4 146 4 4 133,685 ⫹ Cytoplasm

Q60FE5 Filamin A 94 1 3 278,053 ⫹ Cytoplasm

Biosynthesis

P27824 Calnexin precursor 837 30 20 67,526 ⫹ ER membrane

P11021 78-kDa glucose-regulated protein 511 25 16 72,288 ⫹ ER lumen

P27797 Calreticulin 189 12 7 48,112 ⫺ ER lumen

Q15084 Protein-disulfide isomerase A6 155 7 3 48,091 ⫹ ER lumen

Traffic

O95292 Vesicle-associated membrane protein-associated protein B/C

83 15 4 27,080 ⫺ Membrane

P61026 Ras-related protein Rab-10 121 14 3 22,527 ⫺ Endosome

Others

O00264 Membrane-associated progesterone receptor component 1

191 24 6 21,527 ⫺ Plasma membrane

Q96AG4 Leucine-rich repeat-containing protein 59 312 36 9 34,909 ⫺ Intracellular membrane

P57088 Transmembrane protein 33 102 12 3 27,960 ⫺ Membrane

Q13151 Heterogeneous nuclear ribonucleoprotein A0

377 28 7 30,822 ⫺ Cytoplasm

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

Apart from heterotrimeric Gi proteins, not much is known

about the repertoire of melatonin receptor interaction part-ners. This makes it difficult to estimate the proportion of known interaction partners that are covered by our data set. However, a rough estimation can be made with the assump-tion that many interacassump-tion partners are likely to interact at least with several other GPCRs. For MT1 and MT2 35 and 45%,

respectively, of the identified proteins have been shown to interact with other GPCRs. An additional 25 and 20% of the MT1and MT2interactors, respectively, have been associated

with GPCR function (signaling, trafficking, etc.). Thus the overall 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 in receptor desensitization, internalization, and signaling (44). This interaction is known to be agonist-stimulated and tran-sient. The absence of ␤-arrestin in our data set suggests that this transient interaction may need to be stabilized for instance by chemically cross-linking the partners prior to purification.

Purification of protein complexes from intact mammalian cells is one of the advantages of the TAP technique. This also implies that the components of the interacting complexes depend on the chosen cellular context. As we selected HEK 293 fibroblasts for our study we expected to identify rather ubiquitously expressed interaction partners than cell type-specific (i.e. neuron-type-specific) partners. The repertoire of pro-teins identified for both melatonin receptors confirmed this prediction. The next step will be to identify cell type-specific interaction 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 time the purification of GPCR-associated proteins under native

TABLEII

MT2-associated proteins

Trypsin-digested protein bands were analyzed by nano-LC-nano-ESI MS/MS, and proteins were identified with Mascot software in Swiss-Prot and TrEMBL databases. Comparison of proteins identified in eluates from MT2-TAP-expressing cells with those of non-transfected

HEK 293 cells led to the identification of MT2-specific proteins. Data represent the common results of four experiments.

Swiss-Prot/TrEMBL

accession no. Protein identity Score Coverage

Number of unique peptides Theoretical molecular mass Also identified in MT1 Subcellular localization % Da Bait protein

P49286 Melatonin receptor type 1B (MT2) 191 10 4 40,162 Plasma membrane

Signal transduction

P62879 Guanine nucleotide-binding protein␤ subunit 1

929 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 G_i␣1 905 60 20 40,204 ⫹ Plasma membrane P08754 Guanine nucleotide-binding protein G_i␣3 899 59 19 40,375 ⫹ Plasma membrane P04899 Guanine nucleotide-binding protein G_i␣2 775 64 20 40,294 ⫹ Plasma membrane

O14654 Insulin receptor substrate 4 546 12 16 133,685 ⫹ Cytoplasm

Q60FE5 Filamin A 369 6 14 278,053 ⫹ Cytoplasm

O60716 Catenin␦1 126 6 4 108,103 ⫺ Cytoplasm

O15355 Protein phosphatase 2C isoform␥ 119 5 3 59,235 ⫺ Cytoplasm

Biosynthesis

P27824 Calnexin 747 28 18 67,526 ⫹ ER membrane

P11021 78-kDa glucose-regulated protein 641 28 16 72,288 ⫹ ER lumen

Q15084 Protein-disulfide isomerase A6 235 16 4 48,091 ⫹ ER lumen

P27348 14-3-3 protein␪ 96 8 2 27,747 ⫺ Cytoplasm

Traffic

Q5T201 Coatomer protein complex,␣ subunit 547 15 20 138,258 ⫺ ER/Golgi

Q96QK1 Vacuolar sorting protein 35 377 15 11 91,649 ⫺ Membrane-associated

protein

P42356 Phosphatidylinositol 4-kinase␣ 134 3 4 231,143 ⫺ Intracellular membrane Others

Q9NQX7 Integral membrane protein 2C 80 12 2 30,224 ⫺ Membrane

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conditions in quantities suitable for mass spectrometry anal-ysis. Interaction partners from different cellular compartments recognizing extra- and intracellular receptor domains of mo-nomeric and/or dimeric receptor species were identified. This presents a major methodological advance in the identification of GPCR-associated protein complexes. In addition, this method is relatively fast, generates a low number of nonspe-cific proteins, and needs no confirmation of the interaction by co-immunoprecipitation experiments. Similar results were ob-tained between MT₁and MT2 in terms of solubilization

effi-ciency, complex stability, and purification yields with this pro-tocol, suggesting a more general application. The increasing number of TAP tag variants, including the split TAP tag (16), demonstrates the flexibility of this method and allows the possibility of rapid optimization for any GPCR homo- and heterodimer.

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 and anti-melatonin receptor antibodies, respectively. We thank Patty Chen for comments on the manuscript, Dr. Luc Camoin for expert advice, 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 must therefore be hereby marked “advertisement” in accordance with 18 U.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 Ge´nopole Toulouse Midi-Pyre´ne´es and Re´gion Midi Pyre´ne´es.

储储 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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