Research
Purification and Identification of G Protein-
coupled Receptor Protein Complexes under
Native Conditions*□ S
Avais M. Daulat‡§¶储**, Pascal Maurice‡§¶储, Carine Froment‡‡,
Jean-Luc Guillaume‡§¶储, Cédric 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
MT1 and MT2 melatonin 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
From the ‡Department of Cell Biology, Institut Cochin, §INSERM
U567, ¶CNRS UMR 8104, and 储Faculté de Médecine René Descartes,
UMR-S 8104, Université 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-
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
This paper is available on line at http://www.mcponline.org
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 5HT2c receptor
expressed as GST fusion protein, more than 15 proteins have
been identified (12). Furthermore isolated protein-protein 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 5HT4 receptors (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
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.
Molecular & Cellular Proteomics 6.5 835
GPCR Protein Complexes
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 MT1 and MT2 melatonin 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 MT1 and MT2 coding 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 saturating
concentration (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 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
836 Molecular & Cellular Proteomics 6.5
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, Montluçon, 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-MT1 or anti-MT2 antibodies 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 mM Tris/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 mM orthovanadate, and 2 mM NaF. 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 mM Tris, 5
mM MgCl2, 50 mM CaCl2, 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-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 C18 PepMapTM column after loading onto a
300-␮m inner diameter ⫻ 5-mm PepMap C18 precolumn (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
 GPCR Protein Complexes

a b

AP
P
1 -T A

2 -T

2
MT

NT
MT

MT
kDa

NT

NT
kDa kDa
97 97
97 MT2-TAP
MT1 66
66 NS 66
56 56
56
42 MT2 42
42
Anti-MT1 Anti-MT2 Anti-MT2

FIG. 1. Functional expression of

MT1-TAP and MT2-TAP in stably
transfected HEK 293 cells. a and b, MT1 MT1-TAP
crude membranes were prepared from
HEK 293 cells stably transfected with the
indicated receptors. Denatured samples c d
were separated by SDS-PAGE and ana-
lyzed by Western blot using anti-MT1 (a)
Anti-MT1
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)
Anti-MT2
or anti-MT2 (e and f) antibodies. g, kinet-
ics of melatonin-stimulated (100 nM) ERK
activation in stable clones expressing
e f
MT1, MT1-TAP, MT2, or MT2-TAP. No
activation of the ERK pathway by mela-
MT2 MT2-TAP
tonin was observed in non-transfected
HEK 293 cells (not shown). NS, nonspe-
cific; P-ERK, phospho-ERK.

MT1-TAP MT2-TAP

MT1 MT2

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-

Molecular & Cellular Proteomics 6.5 837

GPCR Protein Complexes

100 100

Solubilization
(% of total)
FIG. 2. Detergent selection and opti-
mization of solubilization conditions 75 75
of melatonin receptors. a and b, mem-
50 50
brane preparations of HEK 293 cells that
stably express MT1-Rluc or MT2-Rluc 25 25
were incubated either for 15 h with in- MT1 MT2
creasing detergent concentrations at 0 0
4 °C (a) or for the indicated times in the 0 0.25 0.50 0.75 1.00 0 0.25 0.50 0.75 1.00
Detergent conc. (%) Detergent conc. (%)
presence of 0.5% CHAPS (●), digitonin
(E), or dodecylmaltoside (䡺) or 0.25% 100 100
Brij96V (f) (b). Luciferase activity was

Solubilization
75 75

(% of total)
measured in the soluble and insoluble
fraction to determine the percentage of
50 50
solubilization. The use of Nonidet P-40
was not compatible with the luciferase 25 25
activity assay. c, membrane prepara- MT1 MT2
tions of HEK 293 cells that stably ex- 0 0
press MT1-TAP or MT2-TAP were la- 0 4 8 12 16 0 4 8 12 16
beled with [125I]MLT, incubated Time (h) Time (h)
overnight at 4 °C in the presence of a
0.5% concentration of the indicated de-
tergents with the exception of Brij96V, MT1 MT2
which was used at 0.25%. The amount
of radioactivity in the soluble fraction
was determined (black bars), [125I]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
[125I]MLT binding to non-transfected
HEK 293 cells was negligible.

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 MT1 and MT2 melatonin 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 [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 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

838 Molecular & Cellular Proteomics 6.5

 GPCR Protein Complexes

100

Purification yield (% of total)

a MT1-TAP
80
60
40
20

0
FIG. 3. Purification of MT1-TAP and Total Sol IgG TEV CAM
MT2-TAP and associated Gi proteins.
100
b

Purification yield (% of total)

a and b, monitoring of the purification
of the digitonin-solubilized (white bars) MT2-TAP
or Brij96V-solubilized (black bars) 80
[125I]MLT-labeled MT1-TAP (a) and MT2-
TAP (b) at each step of the TAP protocol.
The purification yield was expressed as
60
percentage of total membrane [125I]MLT
binding sites. CAM, eluate from calmod- 40
ulin column; IgG, bound to IgG-coated
column; Sol, solubilized fraction; TEV, 20
elution with TEV protease. c, monitoring
of the co-purification of receptor-associ- 0
ated Gi␣ proteins by Western blot from
melatonin-stimulated cells (15 min, 1
Total Sol IgG TEV CAM
␮M). mb, crude membrane fraction.
c Solubilization IgG column CAM column
Cells mb Supernatant Pellet TEV Eluate Unretained Eluate
MT1 - + - + - + - + - + - +
Giα
CAM column
Eluate
MT2 - +
Giα

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 MT1 and MT2 receptors (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 concentration 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 [125I]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 [125I]MLT bind-

Molecular & Cellular Proteomics 6.5 839

GPCR Protein Complexes
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 gel
electrophoresis, and proteins were detected by Coomassie Blue
staining (a) or silver staining (b).
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
[125I]MLT binding assay. The overall yield of [125I]MLT-labeled
receptors varied from 27 ⫾ 3 (digitonin) to 15 ⫾ 2% (Brij96V)
and from 33 ⫾ 2 (digitonin) to 25 ⫾ 5% (Brij96V) (n ⬎ 5) for
MT1 and MT2, respectively (Fig. 3, a and b). The co-purifica-
tion of associated proteins upon melatonin stimulation was
evaluated 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-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-MT1-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
840 Molecular & Cellular Proteomics 6.5
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
(MT1 and 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 Gi proteins, 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
 GPCR Protein Complexes

TABLE I
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.
Number Theoretical Also
Swiss-Prot/TrEMBL Subcellular
Protein identity Score Coverage of unique molecular identified
accession no. localization
peptides mass in MT2
% Da

Bait protein
P48039 Melatonin receptor type 1A (MT1) 353 24 9 39,349 ⫹ Plasma membrane
Signal transduction
P62873 Guanine nucleotide-binding protein ␤ 405 29 10 37,222 ⫹ Plasma membrane
subunit 1
Q9HAV0 Guanine nucleotide-binding protein ␤ 318 22 8 37,412 ⫹ Plasma membrane
subunit 4
P08754 Guanine nucleotide-binding protein Gi␣3 639 46 16 40,375 ⫹ Plasma membrane
P63096 Guanine nucleotide-binding protein Gi␣1 566 46 14 40,204 ⫹ Plasma membrane
P04899 Guanine nucleotide-binding protein Gi␣2 409 34 11 40,294 ⫹ Plasma membrane
P09543 2⬘,3⬘-cyclic-nucleotide 3⬘- 97 7 3 47,549 ⫺ Plasma membrane
phosphodiesterase
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- 83 15 4 27,080 ⫺ Membrane
associated protein B/C
P61026 Ras-related protein Rab-10 121 14 3 22,527 ⫺ Endosome
Others
O00264 Membrane-associated progesterone 191 24 6 21,527 ⫺ Plasma membrane
receptor component 1
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 377 28 7 30,822 ⫺ Cytoplasm
A0

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 MT2 dephosphorylation.
Most of the identified proteins that are involved in receptor
biosynthesis are present in both receptor-associated com-

Molecular & Cellular Proteomics 6.5 841

GPCR Protein Complexes

TABLE II
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.
Number Theoretical Also
Swiss-Prot/TrEMBL
Protein identity Score Coverage of unique molecular identified Subcellular localization
accession no.
peptides mass in MT1
% Da

Bait protein
P49286 Melatonin receptor type 1B (MT2) 191 10 4 40,162 Plasma membrane
Signal transduction
P62879 Guanine nucleotide-binding protein ␤ 929 52 15 37,222 ⫹ Plasma membrane
subunit 1
Q9HAV0 Guanine nucleotide-binding protein ␤ 511 29 10 37,412 ⫹ Plasma membrane
subunit 4
P63096 Guanine nucleotide-binding protein Gi␣1 905 60 20 40,204 ⫹ Plasma membrane
P08754 Guanine nucleotide-binding protein Gi␣3 899 59 19 40,375 ⫹ Plasma membrane
P04899 Guanine nucleotide-binding protein Gi␣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

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 interaction 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
MT1 and MT2 interactors, 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-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

842 Molecular & Cellular Proteomics 6.5


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 MT1 and 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 Génopole Toulouse Midi-Pyrénées
and Région Midi Pyrénées.
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.
REFERENCES
1. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., and Schioth, H. B. (2003)
The G-protein-coupled receptors in the human genome form five main
families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol.
Pharmacol. 63, 1256 –1272
2. Vassilatis, D. K., Hohmann, J. G., Zeng, H., Li, F., Ranchalis, J. E., Mortrud,
M. T., Brown, A., Rodriguez, S. S., Weller, J. R., Wright, A. C., Bergmann,
J. E., and Gaitanaris, G. A. (2003) The G protein-coupled receptor rep-
ertoires of human and mouse. Proc. Natl. Acad. Sci. U. S. A. 100,
4903– 4908
3. Nature Reviews Drug Discovery GPCR Questionnaire Participants (2004)
The state of GPCR research in 2004. Nat. Rev. Drug Discov. 3, 575,
577– 626
4. Gainetdinov, R. R., Premont, R. T., Bohn, L. M., Lefkowitz, R. J., and Caron,
M. G. (2004) Desensitization of G protein-coupled receptors and neuro-
nal functions. Annu. Rev. Neurosci. 27, 107–144
5. Brown, P. J., and Schonbrunn, A. (1993) Affinity purification of a soma-
tostatin receptor-G-protein complex demonstrates specificity in recep-
tor-G-protein coupling. J. Biol. Chem. 268, 6668 – 6676
6. Husi, H., Ward, M. A., Choudhary, J. S., Blackstock, W. P., and Grant,
S. G. N. (2000) Proteomic analysis of NMDA receptor-adhesion protein
signaling complexes. Nat. Neurosci.3, 661– 669
7. Farr, C. D., Gafken, P. R., Norbeck, A. D., Doneanu, C. E., Stapels, M. D.,
Barofsky, D. F., Minami, M., and Saugstad, J. A. (2004) Proteomic
analysis of native metabotropic glutamate receptor 5 protein complexes
reveals novel molecular constituents. J. Neurochem. 91, 438 – 450
8. Milligan, G., and White, J. H. (2001) Protein-protein interactions at G-
GPCR Protein Complexes
protein-coupled receptors. Trends Pharmacol. Sci. 22, 513–518
9. El Far, O., and Betz, H. (2002) G-protein-coupled receptors for neurotrans-
mitter amino acids: C-terminal tails, crowded signalosomes. Biochem. J.
2, 329 –336
10. Bockaert, J., Marin, P., Dumuis, A., and Fagni, L. (2003) The ‘magic tail’ of
G protein-coupled receptors: an anchorage for functional protein net-
works. FEBS Lett. 546, 65–72
11. Bockaert, J., Roussignol, G., Becamel, C., Gavarini, S., Joubert, L., Dumuis,
A., Fagni, L., and Marin, P. (2004) GPCR-interacting proteins (GIPs):
nature and functions. Biochem. Soc. Trans. 32, 851– 855
12. Becamel, C., Alonso, G., Geleotti, N., Demey, E., Jouin, P., Ullmer, C.,
Dumuis, A., Bockaert, J., and Marin, P. (2002) Synaptic multiprotein
complexes associated with 5-HT2C receptors: a proteomic approach.
EMBO J. 21, 2332–2342
13. Becamel, C., Gavarini, S., Chanrion, B., Alonso, G., Galeotti, N., Dumuis, A.,
Bockaert, J., and Marin, P. (2004) The serotonin 5-HT2A and 5-HT2C
receptors interact with specific sets of PDZ proteins. J. Biol. Chem. 279,
20257–20266
14. Joubert, L., Hanson, B., Barthet, G., Sebben, M., Claeysen, S., Hong, W.,
Marin, P., Dumuis, A., and Bockaert, J. (2004) New sorting nexin (SNX27)
and NHERF specifically interact with the 5-HT4a receptor splice variant:
roles in receptor targeting. J. Cell Sci. 117, 5367–5379
15. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B.
(1999) A generic protein purification method for protein complex char-
acterization and proteome exploration. Nat. Biotechnol. 17, 1030 –1032
16. Dziembowski, A., and Seraphin, B. (2004) Recent developments in the
analysis of protein complexes. FEBS Lett. 556, 1– 6
17. Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A.,
Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert,
C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak,
M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B.,
Leutwein, C., Heurtier, M. A., Copley, R. R., Edelmann, A., Querfurth, E.,
Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin,
B., Kuster, B., Neubauer, G., and Superti-Furga, G. (2002) Functional
organization of the yeast proteome by systematic analysis of protein
complexes. Nature 415, 141–147
18. Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P. O., Bergamini, G.,
Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., Hopf,
C., Huhse, B., Mangano, R., Michon, A. M., Schirle, M., Schlegl, J.,
Schwab, M., Stein, M. A., Bauer, A., Casari, G., Drewes, G., Gavin, A. C.,
Jackson, D. B., Joberty, G., Neubauer, G., Rick, J., Kuster, B., and
Superti-Furga, G. (2004) A physical and functional map of the human
TNF-␣/NF-␬B signal transduction pathway. Nat. Cell Biol. 6, 97–105
19. Ayoub, M. A., Couturier, C., Lucas-Meunier, E., Angers, S., Fossier, P.,
Bouvier, M., and Jockers, R. (2002) Monitoring of ligand-independent
dimerization and ligand-induced conformational changes of melatonin
receptors in living cells by bioluminescence resonance energy transfer.
J. Biol. Chem. 277, 21522–21528
20. Barrett, P., MacLean, A., and Morgan, P. J. (1994) Evidence for multiple
forms of melatonin receptor-G-protein complexes by solubilization and
gel electrophoresis. J. Neuroendocrinol. 6, 509 –515
21. Brydon, L., Roka, F., Petit, L., deCoppet, P., Tissot, M., Barrett, P., Morgan,
P. J., Nanoff, C., Strosberg, A. D., and Jockers, R. (1999) Dual signaling
of human Mel1a melatonin receptors via Gi2, Gi3, and Gq/11 proteins. Mol.
Endocrinol. 13, 2025–2038
22. Angeloni, D., Longhi, R., and Fraschini, E. (2000) Production and charac-
terization of antibodies directed against the human melatonin receptors
Mel-1a (Mt1) and Mel-1b (MT2). Eur. J. Histochem. 44, 199 –204
23. Rabilloud, T. (1999) Silver staining of 2-D electrophoresis gels. Methods
Mol. Biol. 112, 297–305
24. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectro-
metric sequencing of proteins silver-stained polyacrylamide gels. Anal.
Chem. 68, 850 – 858
25. Petit, L., Lacroix, I., deCoppet, P., Strosberg, A. D., and Jockers, R. (1999)
Differential signaling of human Mel1a and Mel1b melatonin receptors
through the cyclic guanosine 3⬘-5⬘-monophosphate pathway. Biochem.
Pharmacol. 58, 633– 639
26. Ayoub, M. A., Levoye, A., Delagrange, P., and Jockers, R. (2004) Prefer-
ential formation of MT1/MT2 melatonin receptor heterodimers with dis-
tinct ligand interaction properties compared with MT2 homodimers. Mol.
Pharmacol. 66, 312–321
Molecular & Cellular Proteomics 6.5 843
GPCR Protein Complexes
27. Krecic, A. M., and Swanson, M. S. (1999) hnRNP complexes: composition,
structure, and function. Curr. Opin. Cell Biol. 11, 363–371
28. Rousseau, S., Morrice, N., Peggie, M., Campbell, D. G., Gaestel, M., and
Cohen, P. (2002) Inhibition of SAPK2a/p38 prevents hnRNP A0 phos-
phorylation by MAPKAP-K2 and its interaction with cytokine mRNAs.
EMBO J. 21, 6505– 6514
29. Li, M., Bermak, J. C., Wang, Z. W., and Zhou, Q. Y. (2000) Modulation of
dopamine D2 receptor signaling by actin-binding protein (ABP-280). Mol.
Pharmacol. 57, 446 – 452
30. Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P., and Levenson, R. (2001)
Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via
interaction with filamin A. Proc. Natl. Acad. Sci. U. S. A. 98, 5258 –5263
31. Awata, H., Huang, C., Handlogten, M. E., and Miller, R. T. (2001) Interaction
of the calcium-sensing receptor and filamin, a potential scaffolding pro-
tein. J. Biol. Chem. 276, 34871–34879
32. Hjalm, G., MacLeod, R. J., Kifor, O., Chattopadhyay, N., and Brown, E. M.
(2001) Filamin-A binds to the carboxyl-terminal tail of the calcium-sens-
ing receptor, an interaction that participates in CaR-mediated activation
of mitogen-activated protein kinase. J. Biol. Chem. 276, 34880 –34887
33. Onoprishvili, I., Andria, M. L., Kramer, H. K., Ancevska-Taneva, N., Hiller,
J. M., and Simon, E. J. (2003) Interaction between the ␮ opioid receptor
and filamin A is involved in receptor regulation and trafficking. Mol.
Pharmacol. 64, 1092–1100
34. Hinsby, A. M., Olsen, J. V., and Mann, M. (2004) Tyrosine phosphopro-
teomics of fibroblast growth factor signaling: a role for insulin receptor
substrate-4. J. Biol. Chem. 279, 46438 – 46447
35. Mihindukulasuriya, K. A., Zhou, G., Qin, J., and Tan, T. H. (2004) Protein
phosphatase 4 interacts with and down-regulates insulin receptor sub-
strate 4 following tumor necrosis factor-alpha stimulation. J. Biol. Chem.
279, 46588 – 46594
36. Maillet, M., Robert, S. J., Cacquevel, M., Gastineau, M., Vivien, D., Berto-
844 Molecular & Cellular Proteomics 6.5
glio, J., Zugaza, J. L., Fischmeister, R., and Lezoualc’h, F. (2003)
Crosstalk between Rap1 and Rac regulates secretion of sAPP␣. Nat. Cell
Biol. 5, 633– 639
37. Lugnier, C. (2006) Cyclic nucleotide phosphodiesterase (PDE) superfamily:
a new target for the development of specific therapeutic agents. Phar-
macol. Ther. 109, 366 –398
38. McClatchy, D. B., Knudsen, C. R., Clark, B. F., Kahn, R. A., Hall, R. A., and
Levey, A. I. (2002) Novel interaction between the M4 muscarinic acetyl-
choline receptor and elongation factor 1A2. J. Biol. Chem. 277,
29268 –29274
39. Cho, D. I., Oak, M. H., Yang, H. J., Choi, H. K., Janssen, G. M., and Kim,
K. M. (2003) Direct and biochemical interaction between dopamine D3
receptor and elongation factor-1B␤␥. Life Sci. 73, 2991–3004
40. Perez-Moreno, M., Davis, M. A., Wong, E., Pasolli, H. A., Reynolds, A. B.,
and Fuchs, E. (2006) p120-catenin mediates inflammatory responses in
the skin. Cell 124, 631– 644
41. Shih, M. L., Lin, F. B., Scott, J. D., Wang, H. Y., and Malbon, C. C. (1999)
Dynamic complexes of ␤2-adrenergic receptors with protein kinases and
phosphatases and the role of gravin. J. Biol. Chem. 274, 1588 –1595
42. Flajolet, M., Rakhilin, S., Wang, H., Starkova, N., Nuangchamnong, N.,
Nairn, A. C., and Greengard, P. (2003) Protein phosphatase 2C binds
selectively to and dephosphorylates metabotropic glutamate receptor 3.
Proc. Natl. Acad. Sci. U. S. A. 100, 16006 –16011
43. Levoye, A., Dam, J., Ayoub, M. A., Guillaume, J. L., Couturier, C., Dela-
grange, P., and Jockers, R. (2006) The orphan GPR50 receptor specifi-
cally inhibits MT1 melatonin receptor function through heterodimeriza-
tion. EMBO J. 25, 3012–3023
44. Reiter, E., and Lefkowitz, R. J. (2006) GRKs and ␤-arrestins: roles in
receptor silencing, trafficking and signaling. Trends Endocrinol. Metab.
17, 159 –165