Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs

Article abstract of DOI:10.1016/j.bmcl.2005.10.061

A ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates.

Full text of DOI:10.1016/j.bmcl.2005.10.061

Bioorganic & Medicinal Chemistry Letters 16 (2006) 521–524
Design and synthesis of cyclic and linear peptide-agarose tools
for baiting interacting protein partners of GPCRs
a
a
b
a
´
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´ ´ ´
Sebastien Granier, Frederic Jean-Alphonse, Helene Demene, Gilles Guillon,
Robert Pascalc,* and Christiane Mendrea,*
aUMR 5203 CNRS, U 661 INSERM, Universite´ Montpellier 1, Universite´ Montpellier 2, Institut de Ge´nomique Fonctionnelle,
141 rue de la Cardonille, 34094 Montpellier Cedex, France
bUMR 5048 CNRS-UM1/UMR 554 INSERM-UM1, Centre de Biochimie Structurale, 29 rue de Navacelles,
34060 Montpellier Cedex, France
cUMR 5073, CNRS-Universite´ Montpellier 2, CC017, Place E. Bataillon, 34095 Montpellier Cedex, France
Received 25 July 2005; revised 18 October 2005; accepted 19 October 2005
Available online 11 November 2005
Abstract—A ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to
identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor
family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present
in cellular lysates.
Ó 2005 Elsevier Ltd. All rights reserved.
Cell surface receptors recognizing and responding to
extracellular stimuli are the key components required
for the communication between cells. G-protein-coupled
receptors (GPCRs) mediate signals from a number of
endogenous compounds, such as hormones, neurotrans-
mitters, autocrine and paracrine factors, as well as
having light, odorant, and taste sensing functions.1
Therefore, GPCRs are the target for a large number of
drugs and the subject of intensive research. Although
remarkably diverse in sequence and function, all GPCRs
share a highly conserved topological arrangement of a
seven-transmembrane helical core domain joined by
three intracellular loops, three extracellular loops, extra-
cellular N-terminal, and intracellular C-terminal do-
mains. Upon ligand binding, these receptors act as
switches from inactive to active conformations able to
bind and activate their intracellular partners such as
the well-known G-proteins. Numerous studies have
pointed out the importance of intracellular loops in
G-protein coupling1 or more generally coupling with
protein partners.2 More recently, new concepts are
emerging concerning the identification of multiple mem-
brane-associated proteins that not only govern the intra-
cellular sequestration and/or transport of GPCR but
also modulate quite dramatically GPCR ligand specific-
ity subsequent to the cell surface delivery.3
In the vasopressin family, the V2 receptor subtypeÕs i3
loop is the most fundamental determinant responsible
for Gs-protein interaction and further vasopressin anti-
diuretic action.4 This loop is also important for receptor
localization at the endoplasmic reticulum (ER).5 More-
over, the C-terminal tail of the V2 receptor is involved
in receptor trafficking.6 All these elements suggest the
existence of receptor complexes involving other proteins
than Gs and able to regulate new signalization or traf-
ficking pathways.
Then, we decided to develop baits including the i3
intracellular loop and the C-terminal tail from the V2
receptor to identify new partners for this receptor.
Glutathione seryl transferase (GST) pull-down assays or
immunoprecipitation experiments have been used to
identify new GPCR binding partners by proteomic
approaches.7 An alternative would be to use peptides
covalently linked to resins. Chemical modifications in
the bait sequence (e.g., cyclization) may be compatible
with this strategy. Chemical synthesis of peptide affinity
Keywords: G-protein-coupled receptors; Intracellular loop; Peptide
synthesis; Ligation; Proteomics.
*
Corresponding authors. Tel.: +33 4 6714 2984; fax: +33 467 54 24 32
(C.M.); fax: +33 67 63 10 46 (R.P.); e-mail addresses:
rpascal@univ-montp2.fr; Christiane.Mendre@igf.cnrs.fr
4
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.061

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S. Granier et al. / Bioorg. Med. Chem. Lett. 16 (2006) 521–524
supports can provide large amounts of bait allowing
an easier detection and identification of interacting
proteins. Moreover, a covalent anchorage of the
bait to the resin would avoid its elution with protein
partners as it is the case in GST pull-down or
immunoprecipitations.
Fmoc-Lys
1) SOCl2 / MeOH
2) Boc-Ahx-OH, BOP, HOBt, DIEA in CH2Cl2
Boc Ahx
Fmoc Lys
OMe
3) TFA
4) Boc-Ser(tBu)-OH, BOP, HOBt, DIEA in CH2Cl2
The structure of the affinity chromatography support (i3
cyc bait) used to identify proteins that could interact
with the i3 intracellular loop of V2 receptor is shown
in Scheme 1. It is derived from the previously synthe-
sized i3 cyc peptide8 in which the Gly-1 residue of the
cyclization linker is substituted for a lysine residue an-
chored to a solid support via its side chain. Any alter-
ation of the i3 cyc peptide structure upon anchoring
was intended to be restrained by the use of a covalent
anchor selectively located on the cyclization linker and
by the introduction of an aminohexanoic acid (Ahx) res-
idue on a lysine side chain that increases the distance
from the support. The affinity support bound peptide
(i3 cyc bait) was prepared using solid-phase peptide syn-
thesis followed by a double ligation strategy to cyclize a
linear precursor in solution and then to anchor it to the
affinity support. Supports for control experiments con-
tained either the bare tripeptide handle (control) or a
peptide derived from the C-terminal sequence of V2
receptor (Cter).
tBu
Boc Ser Ahx
Fmoc Lys
OMe
5) NaOH, in 0.8 M CaCl2 in iPrOH/H2O 7:3
tBu
Boc Ser Ahx
1
Fmoc Lys
OH
Scheme 2. Synthesis of the side-chain-derivatized lysine building block
1.
iPrOH/H2O 7:3, a reagent that is known to preserve the
base-labile Fmoc group.9
The i3 cyc affinity resin was prepared as follows (Scheme
3). A resin bearing the Val-3–Cys-51 sequence of the
peptide was synthesized as previously described for the
preparation of i3 cyc peptide.8 Fmoc-D-Gln and then
the tripeptide building block 1 were coupled at the N-
terminus of the protected peptide anchored on the solid
phase, which was finally acylated with bromoacetic
anhydride. The cyclic peptide 2 was obtained by acidol-
ysis of the resin and cyclization by thioether ligation10 in
a phosphate buffer (Supplementary data) and then puri-
fied by HPLC. The N-terminal serine residue was then
The side-chain-derivatized lysine building block 1 was
synthesized as shown in Scheme 2 (experimental informa-
tion in Supplementary data). It was prepared in solution
using BOP (benzotriazol-1-yloxy-tris(dimethylamino)-
phosphonium hexafluorophosphate) coupling reagent
from Fmoc-Lys after conversion into methyl ester. At
the end of the synthesis, the methyl ester was selectively
cleaved using NaOH (1.2 equiv) in 0.8 M CaCl2 in
Lys1
O
O
Gly1
Ahx
ACS
N
O
O
H
H H
H
N
HN
N
X = S or S=O
H
H
N
NH2
C
C
O
O
ACS: Affinity chromatography
Cys51
NH2
Cys51
NH2
S
X
support
H2N
HN
H2N
O
D-Gln2
HN
O
D-Gln2
HN
HN
O
O
O
O
Q225
L274
T
V
L
V
L
T
V
L
T
M272
Nle 49
Nle49
CO2H
M 266
I
R
I
R
I
R
F
V
F
V
F
V
ER
ER
TK
ER
TK
ER
TK
A
43
43
Nle
I
A
I
A
I
H
H
Nle
H
A
A
A
S
V
H
A
A
S
L
V
P
G
A
A
S
V
H
A
A
A
A
S
V
H
A
S
L
S
L
V
P
G
V
P
G
RG
RG
RG
R R
R
R
R
R
R
R
T
R
R
R
R
R
T
T
R
R
P
G
G
P
G
G
P
G
G
G
G
G
S
A
S
E
S
A
S
E
S
A
S
E
PS
PS
ER
PS
ER
i3 r-V2-R
i3 cyc
i3 cyc bait
O
O
O
O
ACS
Ahx
Fmoc-Lys-OH
ACS
Ahx RGRTPPSLGPQDESSTTASSSLAKDTSS-OH
HN
N
HN N
Control bait
Cter bait
Scheme 1. Structures of the i3 cyc peptide and of the i3 cyc bait (anchored to an affinity chromatography support, ACS) derived from the sequence of
vasopressin V2 receptor third intracellular loop (i3 r-V2R). Structures of the control affinity resins Control bait and Cter bait derived from the
linkage handle and of the C-terminal part of the h-V2 receptor.

S. Granier et al. / Bioorg. Med. Chem. Lett. 16 (2006) 521–524
523
Boc-Ser(tBu)-Ahx
Ser-Ahx
Br
Fmoc
–Cys–NH– Rink–TG
D-Gln
Lys
Br
–Cys–NH– Rink–TG
D-Gln
i3
Lys
Cys NH2
i3
i3
Fmoc SPPS
t-
t-
O
O
-
-
-
-
Cleavage
side-chain deprotection
=
i3
VLIFR EIHAS LVPGP SERAG RRRRG RRTGS PSEGA HVSAA NleAKTV RNleT
Cyclization
O
ACS
HN NH2
O
O
O
ACS
Ahx
Ahx
O
Ser Ahx
HN
N
i3 cyc bait
3
2
Scheme 3. Structure and synthesis of the i3 cyc peptide-derivatized affinity chromatography support. Residues Ala-11–Ser-12, Val-39–Ser-40, and
Lys-45–Thr-46 were introduced as Fmoc-protected pseudoproline dipeptides (wPro).12 Side-chain protecting groups used: tBu: tert-butyl (Ser, Thr,
Glu), Pbf: 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Arg), Trt: trityl (His, Cys), Boc: (Lys). Supports: TentagelÒ equipped with Rink
amide linker (Rink-TG), affinity chromatography support (ACS).
oxidized11 into aldehyde 3 with NaIO4 in acetonitrile/
water 1:1 (acetate buffer, pH 5.5). The completion of
the oxidation reaction was controlled by MS (MALDI)
(Supplementary data, Fig. S2), which showed the
presence of the expected aldehyde 3 (60%) calculated
(average) for C246H419N87O70S [M+H+] 5747.6, found
5751.8. A significant part (40%) of the product displayed
a mass of 5767.8 (+16), which is not unexpected since
the sulfur atom of Cys residue in the cyclization linker
is likely to be easily oxidized into the corresponding sulf-
oxide. This modification in the non-peptidic part of i3
cyc loop being unlikely to modify the structure, the
solution was directly used for the derivatization of the
Affi-GelÒ Hz hydrazide resin (BIO-RAD) using the pro-
cedure recommended by the supplier. The progress of
coupling to the resin was checked by HPLC analysis
of the supernatant (Fig. 1). As the reaction proceeded
(4 days), the concentration of aldehyde 3 was observed
to decrease. This analytical procedure also allowed us
to check that aldehyde 3 was not damaged in the medi-
um since side products were observed neither by HPLC
nor by MALDI-MS.
The control affinity resin bearing the linkage agent de-
rived from the side-chain-derivatized lysine building
1
1) TFA
H
Ser Ahx
Fmoc Lys
OH
2) NaIO4
O
Ahx
O
Fmoc Lys
OH
O
3)
ACS
HN NH2
O
O
Ahx
Fmoc-Lys-OH
ACS
HN N
Figure 1. Grafting of the N-terminal aldehyde peptide 3 on the
hydrazide resin. Evolution of the aldehyde HPLC peak in the
supernatant (retention time 15.7 min, column LiChrospher 100 RP-
18, buffer A 0.1% aqueous TFA, B 0.1% TFA in CH3CN, linear
gradient 20–60% buffer B over 30 min, and flow 2 mL/min).
Control
Scheme 4. Structure and synthesis of the control affinity chromato-
graphy support.

524
S. Granier et al. / Bioorg. Med. Chem. Lett. 16 (2006) 521–524
sis of tools for the identification of protein partners
using rather short peptide sequences easily accessible
by chemical synthesis, but involving cyclic structures de-
signed to mimic the peptidic segment mobility restriction
present in the native protein.
Acknowledgments
We thank Emmanuelle Demey for synthetic peptides
mass spectrometry and the European Community for
financial support (Grant: LSHB-CT-200-503337).
Supplementary data
Figure 2. 2D-electrophoresis of proteins interacting with i3 cyc bait or
control bait from Triton X-100 solubilized HEK lysate proteins.
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmcl.2005.10.061.
block 1 only was synthesized using a similar procedure
(Scheme 4).
The Cter affinity resin was prepared from the Cter pep-
tide containing an N-terminal Ser residue, which had
been previously synthesized by solid-phase peptide syn-
thesis using Fmoc chemistry (Supplementary data). The
Ser residue was then oxidized and the N-terminal alde-
hyde peptide Cter was grafted to the affinity gel.
References and notes
1. Bockaert, J. Curr. Opin. Neurobiol. 1991, 1, 32.
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J. 2002, 21, 2332.
8. Granier, S.; Terrillon, S.; Pascal, R.; Demene, H.; Bouvier,
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Tetrahedron Lett. 1998, 39, 4929.
The proteomic analysis to detect proteins interacting
with peptide baits was performed as follows. Solubilized
Triton X-100 HEK cell lysates were incubated with the
different resins overnight at 4 °C. The resins were then
rinsed and interacting proteins were eluted with 2D buff-
er to proceed to 2D electrophoresis (Fig. 2). The pres-
ence of proteins interacting specifically with the i3 cyc
peptide but not with the C-terminal peptide potentially
allows their identification by mass spectrometry using
customary proteomic analysis procedures. The selection
and identification process will be published elsewhere.
´
´ ´
11. Kawakami, T.; Akaji, K.; Aimoto, S. Org. Lett. 2001, 3,
1403.
12. (a) Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.;
Wo¨hr, T. Pept. Res. 1995, 8, 145; (b) Wo¨hr, T.; Wahl, F.;
Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.; Mutter, M.
J. Am. Chem. Soc. 1996, 118, 9218.
The strategy used here to anchor peptides on affinity res-
in bearing hydrazide groups has been shown to be com-
patible with three different peptides and orthogonal with
the ligation procedure involving cysteine alkylation by a
bromoacetyl group. It may be widely used in the synthe-