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Direct identification of human oxytocin receptor-binding domains using a
photoactivatable cyclic peptide antagonist: comparison with the human V1a
vasopressin receptor.
Christophe Breton‡§, Hichem Chellil‡, Majida Kabbaj-Benmansour‡, Eric Carnazzi¶||, René
Seyer¶, Sylvie Phalipou‡, Denis Morin‡, Thierry Durroux‡, Hans Zingg**, Claude Barberis‡
and Bernard Mouillac‡‡‡.
From ‡U469 INSERM and ¶UPR 9023 CNRS, CCIPE, 141 rue de la Cardonille, 34094
Montpellier cedex 5, France; ∗∗ Laboratory of Molecular Endocrinology, McGill University
Health Centre, Montreal, Quebec, Canada H3A 1A1.
§Present address: Laboratoire d’Endocrinologie des Annélides, UPRESA CNRS 8017, SN3,
Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France.
||Present address: Laboratoire des Aminoacides, Peptides et Protéines, UMOLECULAR
MASS5810 CNRS, Faculté de Pharmacie, 15 avenue Charles Flahaut , 34060 Montpellier,
France.
‡‡To whom correspondence should be addressed.
tel: (33) 4 67 14 29 22
fax: (33) 4 67 54 24 32
e-mail: mouillac@u469.montp.inserm.fr
Running title: Antagonist binding to oxytocin and vasopressin receptors
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 3, 2001 as Manuscript M102073200 by guest on M
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SUMMARY
Understanding of the molecular determinants responsible for antagonist binding to the
oxytocin receptor should provide important insights that facilitate rational design of potential
therapeutic agents for the treatment of preterm labor. To study ligand-receptor interactions, we
used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, termed
[125
I]-ZOTA. This ligand had an equivalent high affinity for human oxytocin and V1a
vasopressin receptors expressed in CHO cells. Taking advantage of this dual specificity, we
conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and
V1a receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at
85-90 kDa and 46 kDa, respectively. To identify contact sites between the antagonist and the
receptors, the labeled 70-75 kDa and the 46 kDa proteins were cleaved with CNBr and
digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed to
identify a covalently labeled region in the oxytocin receptor transmembrane domain III,
consisting of the residues Leu114
-Val115
-Lys116
. Analysis of contact sites in the V1a receptor
led to the identification of the homologous region, consisting of the residues Val126
-Val127
-
Lys128
. Binding domains were confirmed by mutation of several CNBr cleavage sites in the
oxytocin receptor and of one Lys-C cleavage site in the V1a receptor. The results are in
agreement with previous experimental data and three-dimensional models of agonist and
antagonist binding to members of the oxytocin/vasopressin receptor family.
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INTRODUCTION
Oxytocin (OT)1, a neurohypophyseal nonapeptide, is among the strongest uterotonic agents
known to date (1,2). This hormone acts on the myometrium through specific OT receptors
(OTR) that show a dramatic increase in their expression pattern immediately before
parturition (3). In mammals, at term, OT-induced contractility of myometrial smooth muscle
cells is triggered through an increase in the intracellular calcium level (4). This calcium
signalling pathway is dependent on coupling OTR to both the Gq/11 and Gi proteins (5). The
OTR cDNA of several mammalian species has been cloned (6-9) and the deduced amino-acid
sequence confirmed that the receptor belongs to the large family of seven-helix
transmembrane G protein-coupled receptors (GPCR). OT is widely used in obstetrical practice
to promote labor and delivery (10). On the other hand, blockade of the OTR may provide a
unique approach for treatment of preterm labor by prolonging uterine quiescence (11-13).
Therefore, much effort has been focused on the design and development of OT antagonists as
potential tocolytic drugs. The utility of such OT antagonists has been demonstrated clinically
by using intravenously administered 1-deamino[D-Tyr(Et)2,Thr
4,Orn
8]vasotocin (Atosiban).
This peptide antagonist has been shown to inhibit uterine contractions in women with
threatened and established preterm labor (14, 15). However, the structure/function
relationships of the functional domains of the OTR and the topography of the ligand binding
sites are still poorly investigated. Such knowledge should provide valuable information on the
structural requirements for antagonist binding, and should be helpul for the rational design of
potential therapeutic agents and for a better understanding of the molecular mechanisms
leading to receptor inactivation. Agonist binding sites of the OTR have been investigated
previously by site-directed mutagenesis and three-dimensional (3D) molecular modeling (16-
18). In parallel, a major contribution to peptide antagonist binding affinity by the upper part of
transmembrane domain (TMD) VII has been demonstrated (18).
The photoaffinity labeling technique is an essential complement to modeling and mutagenesis
approaches and allows unambiguous direct determination of the ligand/receptor contact
regions. So far, radiolabeled OT analogues containing a photoactivatable group at the side
chain of residue 8 have been used to photolabel the OTR naturally expressed in the rat
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mammary gland, the guinea pig uterus, the rabbit amnion or transfected in COS cells
(recombinant porcine receptor). These studies allowed to identify the receptor as a
glycoprotein with an apparent molecular mass between 65 and 80 kDa (18-22). However, the
precise localization of the covalent attachment of these ligands to the receptor has not been
investigated.
The present study has been performed to localize accurately peptide antagonist-binding
domains of the human OTR, using a photoaffinity labeling approach. Based on the structure
of the previously reported potent and high-affinity cyclic peptide antagonist
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Tyr(3
125I)-NH2
9]vasotocin ([
125I]-OTA) (23), we have designed,
synthesized and characterized a new radiolabeled photosensitive antagonist containing an
azido group on the amino-acid at position 9, termed [125
I]-ZOTA (manuscript submitted for
publication). As this novel radioligand displays an equivalent high affinity for the human
OTR and the structurally related human V1a vasopressin (AVP) receptor, we decided to
conduct a comparative photolabeling study on both receptors. Because we already
accumulated a large amount of data on linear and cyclic peptide antagonist-binding sites of the
V1a receptor from direct receptor photoaffinity labeling experiments (24-26) as well as from
site-directed mutagenesis and 3D molecular modeling studies (27-29, and for review 30), we
therefore hypothesized that this dual comparative investigation should further increase our
understanding of OTR/antagonist interactions. We describe here the photolabeling of the
human OTR and the human V1a AVP receptor with [125
I]-ZOTA and their proteolytic
fragmentation using CNBr and Lys-C and Arg-C endoproteinases. Compilation of all the data
shows that covalent attachment is restricted to three residues, Leu114
-Val115
-Lys116
, in the
upper part of the OTR TMD III. Interestingly, the photolabeled region is equivalent in the V1a
receptor.
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FOOTNOTE
1The abbreviations used are:
OT, oxytocin; OTR, oxytocin receptor; GPCR, G protein-coupled receptor; 3D, three-
dimensional; TMD, transmembrane domain; AVP, arginine-vasopressin; [I]-OTA,
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Tyr(3I)-NH2
9]vasotocin ; [
125I]-OTA,
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Tyr(3
125I)NH2
9]vasotocin ; [I]-ZOTA,
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Phe(3I,4N3)NH2
9]vasotocin; [
125I]-ZOTA,
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Phe(3
125I,4N3)NH2
9]vasotocin ; CHO, Chinese hamster ovary;
PBS, phosphate-buffered saline; BSA, bovine serum albumin; IPs, inositol phosphates; SDS-
PAGE, sodiumdodecylsulfate-polyacrylamide gel electrophoresis; CNBr, cyanogen bromide;
DMSO, dimethylsulfoxide; kDa, kiloDalton.
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EXPERIMENTAL PROCEDURES
Radioiodination and azidation of the antagonist probe.
The detailed procedure of radioiodination and azidation leading to [125
I]-ZOTA has been
described elsewhere (manuscript submitted for publication). Briefly, a strategy consisting in
the solid-phase synthesis of the precursor nonapeptide
d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Phe(4NH2)-NH2
9]vasotocin and its subsequent radioiodination
and complete azidation has been designed. The HPLC-purified precursor (10-3
M) was first
iodinated with Na125
I (Amersham, 1 mCi) in a phosphate buffer (pH 6.8) using Iodo-Gen
(Pierce) as oxidant. The monoiodinated peptide d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Phe(3
125I,
4NH2)-NH29]vasotocin was purified by HPLC (Waters C18 µBondapak column). Then,
diazotization and azidation of this compound was done with an excess of NaNO2 (10-3
M) in
HCl (12 M) at 0 °C in dim light for 1 h before addition of NaN3 (10-3
M, progressively let to
room temperature), yielding the final product d(CH2)5[Tyr(Me)2,Thr
4,Orn
8,Phe(3
125I, 4N3)-
NH29]vasotocin ([
125I]-ZOTA). The [
125I]-ZOTA was purified by HPLC and its specific
activity was 2200 Ci/mmol. The corresponding non-radiolabeled iodinated peptide, termed
[I]-ZOTA, was also synthesized and purified.
Cell culture, receptor expression and membrane preparation.
The human V1a and OTR cDNA clones were generous gifts of Drs. Thibonnier (31) and
Kimura (6), respectively. The procedure of CHO cell transfection as well as stable cell line
establishment have been previously described (25, 26). CHO cells stably expressing either the
human OT, V1a, V2 or V1b receptor were cultured in Petri dishes in Dulbecco’s modified
Eagle’s medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal calf serum, 4
mM L-glutamine, 500 units/ml penicillin and streptomycin each, and 0.25 µg/ml amphotericin
B in an environment containing 95% air and 5% CO2 at 37 °C. Depending on the experiment
to be conducted, cells were treated overnight with 5 mM sodium butyrate to increase receptor
expression (32, 33). As already published, this treatment does not modify the pharmacological
properties of the receptors (17, 25, 26). Membranes were prepared as already described (27).
In short, cells were washed twice in PBS without Ca2+ and Mg2+, Polytron-homogenized in
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lysis buffer (15 mM Tris-HCl pH 7.4; 2 mM MgCl2; 0.3 mM EDTA) and centrifuged at
800xg for 5 min at 4 °C. Supernatants were recovered and centrifuged at 44,000xg for 20 min
at 4 °C. Pellets were washed in Buffer A (50 mM Tris-HCl pH 7.4; 5 mM MgCl2) and
centrifuged at 44,000xg for 20 min at 4 °C. Membranes were suspended in a small volume of
Buffer A and protein content was determined by the Bradford method (BioRad) using bovine
serum albumin (BSA) as the standard. Aliquots of membranes were used immediately for
binding assays and photolabeling experiments or stored at –80 °C.
Radioligand binding assays.
Binding assays were performed at 30 °C using [125
I]-OTA, [125
I]-ZOTA or [3H]-AVP as the
radioligands and 1-3 µg (for assays with [125
I]-OTA and [125
I]-ZOTA) or 10-15 µg (for assays
with [3H]-AVP) of membrane proteins in standard radioligand saturation and competition
binding assays as previously described (25-29). Briefly, membranes were incubated in Buffer
A supplemented with 1 mg/ml BSA and with radiolabeled and displacing peptides for 30 min
(with [3H]-AVP) or 60 min (with [
125I]-OTA and [
125I]-ZOTA). Affinities (Kd) for [
125I]-OTA
and [125
I]-ZOTA (concentrations from 20 to 2000 pM), as well as for [3H]AVP
(concentrations from 0.1 to 20 nM) were directly determined in saturation experiments.
Affinities (Ki) for the unlabeled ligand [I]-ZOTA were determined by competition
experiments using [3H]-AVP (≈ 1-2 nM) as the radioligand. The concentration of the
unlabeled ligands varied from 1 pM to 10 µM. In saturation and competition experiments,
depending on the radiolabeled peptide, non-specific binding was determined by adding
unlabeled AVP (10 µM), [I]-OTA (400 nM) or [I]-ZOTA (400 nM). Bound and free
radioactivity were separated by filtration over Whatman GF/C filters pre-soaked in a 10
mg/ml BSA solution for 3-4 hours. The ligand binding data were analyzed by nonlinear least-
squares regression using the computer program Ligand (34).
Inositol phosphate assays.
The antagonist activity of [I]-ZOTA was determined by measuring inhibition of OT-
stimulated inositol phosphate (IP) accumulation as previously described (25,26). Briefly,
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CHO cells expressing the human OT receptor were plated and grown in 6-well dishes for 48 h
in DMEM-supplemented medium and then labeled for 24 h with myo-[2-3H]inositol (10-20
Ci/mmol; Dupont-NEN) at a final concentration of 1 µCi/ml in a serum-free, inositol-free
medium (Life Technologies, Inc.). Cells were washed twice with PBS medium, equilibrated at
37 °C in PBS for 1 h and then incubated for 10 min in PBS supplemented with 10 mM LiCl,
in the presence or absence of increasing concentrations of [I]-ZOTA (from 10-12
to 10-6
M).
CHO cells were stimulated for 15 minutes with 10-8
M OT (a concentration close to the Kact
value determined in CHO cells). The reaction was stopped by adding ice-cold perchloric acid.
After neutralizing the samples, total IPs were extracted and purified by anion-exchange
chromatography (Dowex AG1x8 column, formate form, 200-400 mesh; Bio-Rad). For each
sample, a fraction containing total IPs was collected and counted. Kinact constants were
calculated as Kinact = IC50 / (1 + [OT] / Kact) in which IC50 is the concentration of antagonist
leading to 50% inhibition, [OT] was kept at 10 nM, and Kact is the concentration of OT
inducing half maximum accumulation of IPs (Kact = 12.9 nM in CHO cells expressing the
wild-type human OT receptor (17)).
Photoaffinity labeling of the receptors with [125I]-ZOTA.
Photoaffinity labeling experiments were carried out as previously described with the V1a
receptor photoactivatable antagonists (25, 26). Shortly, membranes expressing OTR or V1a
(500 µg) were resuspended in 4 ml of binding Buffer A containing BSA (0.5 mg/ml) in Corex
glass tubes. Then, [125
I]-ZOTA (≈ 1-2 nM) was added to the membranes and samples
incubated at 30 °C for 1 or 3 h in the dark with or without OT or AVP (10 µM) to determine
specific labeling. Membranes were separated from unbound ligand by washing with Buffer A
without BSA and two subsequent centrifugation steps (20 min, 44,000xg, 4 °C). The final
pellet was resuspended in 1 ml of Buffer A and photolyzed with ultraviolet light (254 nm) for
1 to 5 min on ice. After photolysis, membranes were washed twice (1 ml of Buffer A) and
finally resuspended in Laemmli buffer (35). Samples were subjected to SDS-polyacrylamide
gel electrophoresis (PAGE) using 1 mm thick 12% acrylamide gels. Gels were fixed in glacial
acetic acid: methanol: DMSO: water (16: 40: 2: 42), dried and exposed to Kodak XAR-5
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films at –80 °C. The assessment of covalent binding yield was performed after gel drying.
Gels were cut into slices and the radioactive content in each slice was determined. Covalent
binding was determined by calculating the amount (in pmols) of labeled receptors as a
percentage of the total number of receptors expressed in the membrane preparation.
Electroelution of the photolabeled OT and V1a receptors.
Photolabeled membranes were first subjected to SDS-PAGE using 12% acrylamide gels, as
described above. After electrophoresis, the labeled bands were excised from the preparative
gel and the human OTR and V1a were electroeluted using an electroeluter model 422 (Bio-
Rad) in Tris-Glycine running buffer (25 mM Tris; 182 mM glycine pH 8.3; 0.1% SDS).
Samples containing electroeluted OTR and V1a were washed and concentrated using
Microcon-50 or Microcon-30, respectively (Amicon). Fragmentation with CNBr and Lys-
C/Arg-C endoprotease digestions were performed using these concentrated samples.
Deglycosylation of the photolabeled OT receptors with N-glycosidase F.
The photolabeled CHO membranes (100 µg) or electroeluted OTR were resuspended in
deglycosylation buffer (100 mM Na2HPO4/NaH2PO4 pH 8.0; 10 mM EDTA; 1% digitonin;
1% 2-mercaptoethanol; 5 µg/ml leupeptin; 0.1% SDS) and digested with 2 units of N-
glycosidase F (Roche Molecular Biochemicals) for 2 days at 37 °C. Deglycosylated receptors
were analysed by SDS-PAGE using 12% gels.
Chemical and enzymatic cleavage of the photolabeled OT and V1a receptors.
The electroeluted receptors were first subjected to single digestion with CNBr or Lys-C/Arg-C
endoproteinase. CNBr, Lys-C and Arg-C cleave proteins specifically at the carboxy terminus
of methionine, lysine and arginine residues, respectively. Double digestions were also
performed (first digestion with Arg-C followed by a second digest with CNBr). CNBr
cleavage of the electroeluted OTR and V1a was carried out in 100 µl of 70% (v/v) formic acid
containing a few crystals of CNBr. The mixture was incubated in the dark for 24 h at room
temperature under argon and was then stopped by adding 500 µl of water. Sample volume was
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reduced under vacuum and formic acid was removed by solvent exchange with water.
Endoproteinase Lys-C (sequencing grade from Lysobacter enzymogenes, Roche Molecular
Biochemicals) was used at 0.2 µg/assay in a final volume of 50 µl. The digestion was
performed in 25 mM Tris-HCl (pH 8.5); 1 mM EDTA; 0.1% SDS at 37 °C for 16-24 h and
stopped by addition of Laemmli buffer. Endoproteinase Arg-C (sequencing grade from
Clostridium histolyticum, Roche Molecular Biochemicals) was used at 0.5 µg/assay for 24 h
at 37 °C in a final volume of 50 µl 0.1 M Tris-HCl (pH 7.6); 10 mM CaCl2. To perform
double fragmentations, the Arg-C digestion reaction was dried under vacuum. The pellet was
resuspended in 100 µl 70% (v/v) formic acid and CNBr cleavage was performed as indicated
above. Results of cleavage were analyzed by a Tricine discontinuous SDS-PAGE system (10-
16.5% acrylamide gels), applied to the separation of small molecular mass species (36). Gels
were fixed in glacial acetic acid:methanol:DMSO:water (10:50:2:38), dried and exposed to
Kodak XAR-5 films at –80 °C. The molecular masses of radiolabeled receptor fragments were
determined using a range of low molecular mass markers (RainbowTM
coloured protein,
Amersham Pharmacia Biotech).
Site-directed mutagenesis of the human OT and V1a receptors.
Point mutations M78A, M123A, M296A, M315A or M330A were introduced in the human
OTR cDNA sequence using the QuickChange site-directed mutagenesis kit (Stratagene). The
replacement of methionine residues corresponding to potential CNBr cleavage sites by alanine
residues was directly performed on the eukaryotic expression vector pCMV (37) and verified
by direct dideoxynucleotide sequencing (T7 SequencingTM
kit, Amersham Pharmacia
Biotech). The method used for the construction of the human V1a receptor mutant K128A has
been already reported elsewhere (26, 29). All the mutant receptors were transiently expressed
in CHO cells by electroporation as published earlier (27). For all mutated receptors, cell
membrane preparations, receptor photolabeling and fragmentation experiments were
conducted as described above.
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RESULTS
Binding and antagonistic properties of the new photoactivatable ligand.
Pharmacological and functional properties of the wild-type human OTR and AVP V1a
receptor stably expressed in CHO cells have been previously published (17, 26, 27). The
affinity of [125
I]-ZOTA, which differs from the OT antagonist [125
I]-OTA (23) only by
replacement of hydroxyl group on Tyr9 with a photosensitive azido group (see Figure 1), was
first directly determined for the human OTR by saturation binding experiments. As reported
in Table I, the radioiodinated peptide exhibited a high affinity for OTR in the range to that
measured for [125
I]-OTA (Kd = 0.25 nM versus 0.18 nM). Like [125
I]-OTA (see Table I), the
photoactivatable peptide is indeed nonselective for the OTR, displaying a very similar Kd
binding value for the human V1a AVP receptor (0.45 nM). By doing competition binding
experiments using the [3H]-AVP as the radioligand, the affinity (Ki) of the [I]-ZOTA was also
calculated for the four human AVP/OT receptor subtypes expressed in CHO cells. The results
obtained are reported in Table I and confirmed lack of selectivity between the OTR and the
V1a. In CHO cells expressing the human OTR, the photoactivatable peptide [I]-ZOTA
inhibited the IP accumulation induced by 10 nM OT in a concentration-dependent manner
(data not shown). The Kinact calculated from experimental IC50 values was 0.15 ± 0.02 nM (n
= 4), a value in agreement with the Kd determined in binding experiments. Moreover, no
partial agonistic activity of [I]-ZOTA was detected. In conclusion, the photosensitive cyclic
peptide [125
I]-ZOTA is a potent antagonist for the human OTR, displays equivalent high
affinity for human OT and V1a receptors, and thus constitutes a valuable tool to map peptide-
binding domains of both receptors.
Photoaffinity labeling of the human OT and V1a receptors.
Photoaffinity labeling of the human wild-type OT and V1a receptors was directly performed
on CHO cell membrane preparations. First, after incubating the photoactivatable ligand [125
I]-
ZOTA with the membranes, different UV irradiation times were compared to determine an
optimized covalent binding yield. As shown in Fig. 2A, a 254 nm irradiation of the sample for
1 min (lane 2) led to a strong labeling of the receptor and a covalent binding yield, measured
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as the fraction of specifically bound radioactivity recovered in the labeled bands, reaching �
7% (multiple determinations). For longer irradiation times (lanes 3-5), the covalent binding
yield decreased. In all cases, [125
I]-ZOTA covalently labeled a major protein band migrating at
approximately 70-75 kDa (Fig. 2A and C, lane3). An additional faint band at around 42 kDa
that might represent an immature or a non-glycosylated receptor form was sometimes
observable. No photolabeling was detected with non-irradiated membranes (lane 1) or in the
presence of a saturating 10 µM OT concentration (Fig. 2A, lane 6 and 2C, lane 4), indicating
that photolabeling of the protein bands was specific for the OTR.
To determine whether the photolabeled protein at 70-75 kDa could correspond to the native
glycosylated state of the receptor expressed in the CHO cell system, the membranes were
treated with N-glycosidase F before SDS-PAGE. As seen in Fig. 2B, this enzymatic treatment
converted the unique 70-75 kDa protein band (lane 1) into two bands of approximately 48
kDa and 38 kDa (lane 2), confirming that the receptor contained carbohydrates. As shown in
Fig. 3, the human OTR contains in its extracellular N-terminal domain three potential N-
glycosylation sites, located at Asn8, Asn
15 and Asn
26 (6, 38). The molecular mass of the
smaller band migrating at 38 kDa (including the antagonist) is close to the theoretical mass of
the receptor core deduced from the cDNA sequence (42.8 kDa). As reported for the guinea pig
uterine OTR (21), the deglycosylation treatment led to a reaction product with molecular mass
approximately 50% of the mass of the native receptor, which likely represents a final
digestion product. The nature of the band obtained at 42 kDa upon photolysis remains
unknown. It is interesting to note that deglycosylation of the photolabeled guinea pig receptor
resulted in a protein band migrating at 38 kDa, as well (21). Taking into account insensitivity
of the human OTR to the plasma membrane metalloproteinase proteolysis (18), the upper
band at 48 kDa likely represents a form of partial receptor deglycosylation rather than a
proteolytic receptor fragment. Indeed, when the human OTR was transiently expressed in
CHO cells or when the 70-75 kDa band was electroeluted from preparative SDS-PAGE,
deglycosylation of the photolabeled materials always gave rise to both 38 and 48 kDa species
(not shown). Taken together, the N-glycosidase F effect on the receptor protein and the lack of
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endoglycosidase H action (data not shown) suggest that the mature OT receptor is a
glycoprotein that contains a high percentage of asparagine-linked oligosaccharides.
Using the same experimental conditions (incubation for 1 h at 30 ˚C, then 1 min UV
irradiation), CHO cell membranes expressing the human AVP V1a receptor were photolabeled
with [125
I]-ZOTA. Two broad bands at 85-90 kDa and 46 kDa were covalently labeled (Fig.
2C, lane 1). Equivalent results were obtained in earlier photolabeling studies, using two V1a-
selective photoactivatable peptide antagonists (25, 26). The photolabeling of both 85-90 kDa
and 46 kDa protein bands was receptor-specific, because labeling could be completely
suppressed by adding 10 µM AVP (Fig. 2C, lane 2). The covalent binding efficiency of the
probe to the V1a receptor, calculated as the specifically bound radioactivity recovered in the
two labeled bands, was estimated at 3% (multiple determinations). As demonstrated in two
previous studies (25, 26), the 46 kDa photolabeled band originated from a proteolytic
cleavage of the entire receptor migrating at an apparent molecular mass 85-90 kDa. Indeed,
the relative abundance of the 46 kDa species (�50% in lane 1, Fig. 2C) could be significantly
reduced when incubation with [125
I]-ZOTA was done in the presence of ZnCl2 and protease
inhibitors for 1 h at 4 ˚C (data not shown). As illustrated in Fig. 4, the sequence Phe103
-Gln108
in the V1a receptor corresponds to a potential cleavage site for a metalloproteinase present in
the membrane preparation. This enzyme has been demonstrated to digest the bovine renal V2
receptor between Gln92
and Val93
upon ligand binding (18). As demonstrated previously with
two radioiodinated photoactivatable linear peptide antagonists (25, 26), we found here that the
human V1a is sensitive to metalloproteinase proteolysis as well during incubation with the
cyclic peptide antagonist [125
I]-ZOTA. Interestingly, while the proteolytic cleavage site of the
metalloproteinase is conserved in the human OTR (sequence Phe91
-Gln96
), this receptor is
resistant to the proteolysis (even with a longer 3 h incubation time, data not shown),
indicating that other residues, domains or specific conformations are necessary for an efficient
enzymatic action.
Fragmentation of the human OT and V1a photolabeled receptors and identification of the
covalently-labeled regions.
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In order to identify antagonist-binding domains covalently-bound to the photoactivatable
cyclic peptide [125
I]-ZOTA, photolabeled bands were excised from preparative SDS-PAGE
gels, electroeluted and subjected to chemical cleavage with CNBr or enzymatic digestion with
Lys-C and Arg-C endoproteinases. Based on our present knowledge on the localization of
ligand-binding sites on GPCR (see for review, 39), we have systematically excluded
fragments corresponding to putative intracellular portions of the receptors as potential
domains covalently-bound to the ligand. For the human OTR, the unique photolabeled 70-75
kDa band was used to perform the different fragmentation reactions (see Fig. 3 for the
localization of CNBr, Lys-C and Arg-C cleavage sites in the OTR sequence). The
photoactivatable peptide itself is protected from the different degradations as it does not
possess Met, Lys or Arg residues.
The experimental OTR fragmentation patterns deriving from CNBr, Lys-C, Arg-C or Arg-C +
CNBr digestions are presented in Fig. 5. For a better understanding of the results, a theoretical
fragmentation map of the OTR is shown in Fig. 6. CNBr cleavage of the photolabeled 70-75
kDa OTR yielded 3 small radiolabeled bands migrating at molecular mass ≈ 4.5, 5.5 and 6.5
kDa (Fig. 5A, lane 2). This result restricted the site of covalent attachment to only one
receptor domain, Lys79
-Met123
. Digestion of the photolabeled OTR with Lys-C endoproteinase
yielded 4 fragments at molecular mass ≈ 5, 8, 11 and 16 kDa (Fig. 5B, lane 2). Because the
smallest fragment migrated at ≈ 5 kDa, only 2 peptide sequences, His80
-Lys116
and Met276
-
Lys306
could be covalently-bound to [125
I]-ZOTA. Fragment Ala199
-Lys226
(3.1 kDa)
encompassing TMD V was eliminated as a potential candidate because of the absence of
CNBr cleavage sites in this region. Digestion of the photolabeled 70-75 kDa OTR with Arg-C
endoproteinase produced 4 labeled bands at molecular mass ≈ 3.4, 4, 7.8, and 10 kDa (Fig. 5C,
lane 2). Several fragments could account for this result, such as Met1-Arg
27, Val
41-Arg
65,
Leu114
-Arg137
, or Leu155
-Arg178
. Considering the cleavage results with CNBr or Lys-C
endoproteinase, there was only one reasonable candidate of ≈ 3.4 kDa, spanning from Lys114
to Arg137
and including the entire TMD III. Successive treatment of the 70-75 kDa
photolabeled receptor with Arg-C and CNBr (Fig.5D, lane 3) generated a new fragment of
molecular mass ≈ 3 kDa slightly smaller than the 3.4 kDa obtained with Arg-C alone (panel D,
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lane 2) or the 4.5 kDa produced with CNBr alone (panel A, lane 2). Among the predicted
fragments resulting from a digest with Arg-C, there is only one that fulfills the criteria of
having a molecular mass ≈ 3 kDa and possessing an internal CNBr cleavage site, namely the
fragment Leu114
-Met123
. Compilation of the data highlighted an overlap of the different
fragmentations in the upper part of TMD III (see Fig. 6) and indicated that covalent binding of
[125
I]-ZOTA is restricted to the 3 amino-acid residues Leu114
-Val115
-Lys116
(see Fig. 3).
We next analysed the proteolytic fragmentation patterns of the human V1a receptor (see Fig.
7), and the theoretical digestion map of this receptor is shown again in Fig. 6. In two previous
studies (25, 26), we had demonstrated that the 46 kDa photolabeled band corresponding to a
truncated glycosylated V1a receptor from Val105
to the C-terminus end, derives from the entire
85-90 kDa photolabeled receptor and contains all the covalently-bound radioactivity.
Therefore, the proteolytic fragmentation of the V1a receptor was only conducted using the
material recovered in the 46 kDa protein (see Fig. 4 for the localization of CNBr, Lys-C and
Arg-C cleavage sites in the V1a sequence). CNBr cleavage of the photoaffinity-labeled 46 kDa
species yielded 3 labeled fragments migrating at molecular mass of ≈ 4, 5.5 and at 7.5 kDa
(Fig. 7A, lane 2). Apart from the additional 4 kDa band, the CNBr cleavage pattern was
similar to the one previously described using the photoactivatable linear peptide antagonist
[125
I]-[Lys(3N3Phpa)8]HO-LVA (26). Based on their predicted size, there are several receptor
regions that could correspond to the labeled CNBr cleavage fragments observed: Cys110
-
Met135
, Ile171
-Met191
, Thr293
-Met312
and Ser320
-Met349
. Digestion of the radioiodinated
antagonist-bound 46 kDa photolabeled species with Lys-C endoproteinase yielded three
labeled fragment of molecular mass ≈ 5, 8 and 14 kDa (Fig. 7B, lane 2), a result equivalent to
that determined with [125
I]-[Lys(3N3Phpa)8]HO-LVA (26). Considering that CNBr cleavage
and Lys-C protease digestion of the V1a receptor are quite similar when the photolabeling is
performed either with [125
I]-ZOTA or [125
I]-[Lys(3N3Phpa)8]HO-LVA, the Val
105-Lys
128
sequence likely corresponds to the 5 kDa labeled band produced upon Lys-C fragmentation
(the N-terminus of this fragment is defined by the metalloproteinase cleavage site, Val105
).
Arg-C endoproteinase digestion of the photolabeled 46 kDa receptor yielded three labeled
fragments at molecular mass ≈ 4, 6.5 and 10 kDa (Fig. 7C, lane 2). Only two fragments could
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account for a labeled band with an electrophoretic migration at 4 kDa: Val105
-Arg116
and
Val126
-Arg149
. Although the Val126
-Arg149
fragment is the best candidate, labeling of both
regions could be in agreement with results obtained from the CNBr cleavage and the Lys-C
digestion. In order to confirm this localization, successive treatment of the 46 kDa
photolabeled receptor with ArgC and CNBr was done and generated a new fragment at
molecular mass ≈ 3 kDa (Fig. 7, lane 2) slightly smaller than the 4 kDa bands obtained with
CNB or Arg-C alone. The labeled receptor fragment at ≈ 3 kDa corresponds most likely to the
Val126
-Met135
sequence. The identification of this fragment as the site of covalent binding for
[125
I]-ZOTA would be entirely consistent with the results of CNBr, Lys-C and ArgC cleavages
described above. Compilation of all the fragmentation patterns (see Fig. 6) highlighted that
covalent attachment of [125
I]-ZOTA to the human V1a receptor might also be restricted to the
three amino-acids Val126
-Val127
-Lys128
in the upper part of the TMD III (see Fig. 4), which are
homologous to Leu114
-Val115
-Lys116
in the OTR.
Site-directed mutagenesis of the human OT and V1a receptors.
To confirm further the localization of the covalent binding of [125
I]-ZOTA in the TMD III of
the human OTR, we next carried out site-directed mutagenesis of the potential CNBr cleavage
sites in this region. Thus, M78A and M123A mutant receptors were first constructed (see Fig.
3 for localization of these Met residues). Moreover, to eliminate the possibility that
photolabeling might have occured elsewhere in the receptor, other potential CNBr cleavage
sites in TMD VI and VII were also mutated (M296A, M315A, M330A, see Fig. 3). All mutant
receptors were transiently expressed in CHO cells and photolabeled with [125
I]-ZOTA. CNBr
cleavage fragments obtained using radiolabeled mutant receptors were then compared to the
fragments obtained with the wild-type OTR. The fragmentation patterns of M296A, M315A,
M330A mutant receptors were equivalent to that of the wild type OTR (the three labeled
fragments at ≈ 4.5, 5.5 and 6.5 kDa were recovered, data not shown), confirming that regions
including TMD VI and VII were not photolabeled. By contrast, as illustred in Fig. 8, the CNBr
cleavage of both M78A and M123A mutant receptors only produced an unique major 6.5 kDa
radiolabeled band (lanes 4 and 6). The two smaller bands at 4.5 and 5.5 kDa, visualized in the
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wild-type OTR fragmentation (lane 2) were not present anymore, confirming that the
covalently-bound domain of the OTR with [125
I]-ZOTA is delimited by Met 78 and Met 123
residues.
To confirm, as well, our hypothesis regarding the localization of the covalent attachment site
of [125
I]-ZOTA to TMD III in the human V1a receptor, we used in this study a K128A mutant
V1a receptor (Lys128
constitutes a potential cleavage site for Lys-C protease, see Fig. 4) which
was constructed and described previously (26, 29). Following expression in CHO cells and
photolabeling of the mutated receptor, the corresponding 46 kDa radiolabeled fragment was
digested with Lys-C and the resulting fragmentation was compared to that obtained with the
wild-type receptor. As shown in Fig. 9, the 5 kDa band produced upon cleavage of the wild-
type receptor with Lys-C endoproteinase (lane 2) was no more visible when the mutant
K128A receptor was used instead (lane 4). The Lys-C fragmentation pattern yielded only the 8
and 14 kDa photolabeled fragments, confirming that covalent binding of [125
I]-ZOTA to the
human V1a receptor is located in TMD III. Taken together, these results demonstrate that upon
UV irradiation, the cyclic peptide antagonist [125
I]-ZOTA attaches covalently to both OTR
and V1a at an equivalent position in the upper part of TMD III.
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DISCUSSION
It is well established that OTR is a major therapeutic target in the control of labor, thus
analysis of the structure/function relationships of its functional domains, particularly the
ligand-binding pocket, is very important. However, relatively little information on the
characterization of OTR antagonist-binding sites is currently available. In one report, the
upper part of TMD VII has been demonstrated to participate in the binding of a [125
I]-OTA-
derived antagonist (18). To provide further information on structural requirements for
antagonist binding to the human OTR, we have undertaken a photoaffinity labeling study with
a novel photoactivatable cyclic peptide ligand. The structure of [I]-ZOTA or that of its
radiolabeled counterpart [125
I]-ZOTA, is based on that of [I]-OTA for which properties have
been described more than 10 years ago (23). We have first pharmacologically and functionally
characterized [125
I]-ZOTA (manuscript submitted for publication and the present paper), and
demonstrated that this OT antagonist combined high affinity, low nonspecific binding,
easiness and efficiency of radioiodination, and appreciable covalent binding yield. As [125
I]-
OTA (40), [125
I]-ZOTA behaved as a nonselective compound displaying equivalent high
affinity for both the human OTR and the human AVP V1a receptor. Taking advantage of this
lack of selectivity, we decided to conduct a comparative photoaffinity labeling study on both
receptors.
The photolabeled OTR migrated on SDS-PAGE as a unique glycosylated broad band with an
apparent molecular weight at 70-75 kDa. The size of the human OTR is consistent with the
molecular mass reported for OTRs isolated from rat mammary gland, guinea pig uterus or
rabbit amnion (19-22). Deglycosylation of the photolabeled OTR converted the 70-75 kDa
protein into two bands of molecular mass ≈ 48 and 38 kDa, the latter corresponding roughly to
the expected size of the peptidic core of the native receptor, as described for the guinea pig
OTR (21). By contrast, using the same photolabeling conditions (incubation for 1 h at 30 °C
followed by 1 min irradiation), the human V1a receptor was degradated during incubation with
the ligand, leading to two bands of molecular mass ≈ 85-90 kDa and 46 kDa. As described
previously, the 85-90 kDa molecular species corresponds to the glycosylated native state of
the receptor whereas the 46 kDa species represented a NH2-terminus truncated receptor. The
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latter results from a proteolytic cleavage of the 85-90 kDa band by an endogenous
metalloproteinase present in the CHO membrane preparation (25, 26). Equivalently,
photolabeling of bovine kidney membranes with a tritiated photoactivatable agonist
containing an arylazido group at the side chain of Lys8 gave rise to two AVP V2 receptor
bands, a glycosylated native receptor at ≈ 58 kDa and a NH2-terminus truncated form at 30
kDa (41, 42). In that case, the proteolytic cleavage occured between Gln 92 and Val 93, in the
6 amino-acid sequence FQVLPQ located in TMD II and conserved in all neurohypophyseal
hormone receptors, including the OTR. According to the authors, the proteolytic cleavage of
the V2 receptor would require a receptor conformational change dependent on the agonistic
properties of the ligand. We have presently shown that the human OTR is fully resistant to the
proteolysis after binding of the cyclic photoactivatable antagonist, a result previously shown
by others with a different photoactivatable antagonist (18, 42). By contrast, the
metalloproteinase cleavage occured in the human V1a receptor after incubation with [125
I]-
ZOTA and under the same experimental conditions. This finding is equivalent to that obtained
when incubating V1a-expressing membranes with two different photoactivatable linear peptide
antagonists (25, 26). Taken together, the present data suggest that cyclic as well as linear
peptide antagonists are able to induce a conformational change, leading to proteolytic
cleavage in the V1a receptor, whereas the OTR remains resistant.
Using CNBr chemical cleavage and Lys-C/Arg-C protease digestions of the photolabeled
human OTR and V1a, we demonstrated that [125
I]-ZOTA covalently bound to the upper part of
TMD III. This result has been confirmed using double fragmentation (CNBr followed by Arg-
C protease) experiments and site-directed mutagenesis of potential CNBR or Lys-C cleavage
sites in this OTR or V1a receptor region. The covalently-attached region of both receptors has
been restricted to 3 amino-acid residues only, Leu114
-Val115
-Lys116
in the OTR, corresponding
to Val126
-Val127
-Lys128
in the human V1a receptor (Fig. 3 and 4). Based on the high resolution
X-ray structure of bovine rhodopsin (43), these residues are predicted to be located at the top
of TM III. Interestingly, the Leu114
-Val115
-Lys116
motif in the OTR and the corresponding
Val126
-Val127
-Lys128
motif in the V1a are located in a position homologous to that of Glu113
in
rhodopsin, known for interacting with the retinal Schiff base. As illustrated in Fig. 10, this
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photoaffinity labeling study not only constituted the first direct identification of the human
OTR antagonist-binding sites determined so far, but also allowed us to localize a third
photolabeled region in the V1a receptor (25, 26). Indeed, the first extracellular loop and the
upper part of TMD VII of the V1a receptor were identified as contact regions for two
structurally related V1a-selective photoactivatable linear peptide antagonists, [125
I]-
[Lys(3N3Phpa)8]HO-LVA and [
125I]-3N3Phpa-LVA. The Lys
128 in the human V1a,
corresponding to Lys116
in the human OTR, is well conserved throughout the AVP/OT
receptor family (30). Moreover, this residue has been shown to play a pivotal role in the
binding of agonists (27, 29), two different classes of peptide antagonists such as linear
peptides (25, 26) or the cyclic peptide d(CH2)5[Tyr(Me)2]AVP (29) (see also Fig. 10), and
also the nonpeptide compound SR 49059 (29). In the present study, the presence of Lys128
residue in the tripeptide sequence covalently-attached to the ligand is very interesting. As for
V1a-selective linear and cyclic peptide antagonists as well as for SR 49059, this Lys residue
might be responsible for a direct interaction between the receptors and [125
I]-ZOTA. It is very
likely that the protonated amine group of Lys128
or Lys116
, depending on the receptor subtype,
could be involved in a classical pi-cation interaction with the electron cloud of the phenyl ring
of Phe9 of the ligand. This kind of interaction is often encountered in protein structures (44).
Alternatively, the ammonium group of Lys could form a hydrogen bond with a carbonyl
function of the peptide backbone. The predicted interaction between Lys128
and [125
I]-ZOTA
is in good agreement with 3D antagonists/V1a receptor models that we have constructed
previously for V1a-selective linear and cyclic peptide antagonists (25, 26, 29). Moreover in the
present study, we have performed the photolabeling of the K128A mutant V1a receptor using a
high concentration of [125
I]-ZOTA (around 2-3 nM) but we were not able to directly measure
the Kd of the radioligand in saturation studies. This suggests that its affinity was reduced and
also reinforces a putative role for this residue in the binding of the ligand. As a control, we
tried as well to directly measure the affinity for [3H]-AVP, but as already demonstrated in
COS cells (29), mutation of Lys128
of the human V1a receptor into an alanine significantly
decreased affinity for this radioligand. The affinity of AVP for the mutant K128A was finally
determined in competition studies using the V1a-selective antagonist [125
I]-HO-LVA (45) and
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Ki was 43 ± 6.8 nM (n=3). This value is in agreement with that measured in COS cells (29).
Altogether, these data magnify the role of Lys128
(Lys116
in OTR) and demonstrate again that
this residue participates to the overlap of agonist and antagonist binding sites in the human
V1a receptor binding pocket (29). Its role could be compared to that of Asp113
of cationic
neurotransmitter receptors, localized again in the upper part of TMD III. Substitution of this
residue dramatically affects the affinity of both ammonium group-containing agonists and
antagonists in the β-adrenergic receptor (46).
As depicted in Fig. 10, most of the interactions between linear or cyclic peptide antagonists
ligands with the V1a receptor (25-30), are located in a receptor central pocket surrounded by
the first extracellular loop and the upper parts of TMD II, TMD III, TMD VI and TMD VII.
The covalent attachment of [125
I]-ZOTA at the top of TMD III and the demonstration that the
upper part of TMD VII is involved in the binding of an [I]-OTA-derived antagonist (18), are
in agreement with such a model. Based on photoaffinity labeling results, 3D docking of two
linear peptide antagonists into the V1a receptor was equivalent to that of the natural hormone
AVP. When bound to the receptor, despite an open chain, they could adopt a pseudocyclic
conformation similar to that of the cyclic agonists. Moreover, based on site-directed
mutagenesis results, 3D docking of the cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP is
also equivalent to that of the hormone AVP. According to these models, AVP and its peptide
antagonists, at least in the V1a receptor, could enter this transmembrane central binding pocket
and interact with overlapping binding regions. As for AVP and the three peptide antagonists,
we hypothesize that [125
I]-ZOTA is also able to enter this central binding pocket and interact
with the V1a receptor in a similar way, while establishing its own network of molecular
interactions. Because i) [125
I]-ZOTA displays an equivalent affinity for the human OTR and
the human V1a receptor, ii) several residues involved in the binding of all classes of
antagonists are conserved throughout AVP/OT receptor family, and iii) OTR and V1a share a
significant sequence identity in the upper part of TMD II, TMD III, TMDVI and TMD VII as
well as in the first extracellular loop, we propose that binding sites for [125
I]-ZOTA in OTR
and V1a receptor could be equivalent. However, to allow a more complete definition of the
binding sites of the OTR for cyclic peptide antagonists such as [125
I]-ZOTA or Atosiban (14,
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15), and to generate meaningful information on receptor/antagonist interactions, it will be
necessary to perform other photolabeling studies.
In conclusion, covalent attachment of [125
I]-ZOTA to the upper part of TMD III of both OTR
and V1a receptor is fully consistent with our previous 3D antagonist/V1a receptor models.
Taken together, these findings suggest once again, as demonstrated previously, that agonist
and peptide antagonist binding pockets might be common to all receptor subtypes of the
OT/AVP family, and that specific residues can differentiate agonist versus antagonist binding
in these receptors (29). The delineation of a three amino-acid "contact domain" is a major step
"en route" to a more detailed mapping of the molecular interactions between OT-related
ligands and OTR. The use of other photoactivatable antagonist ligands as well as molecular
modeling of the OTR based on the recent 3D crystal structure of bovine rhodopsin (43),
should be very helpful in the future for rationalising the design of OT antagonists based on the
receptor structure.
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LEGEND TO FIGURES
Figure 1: Structure of the photoreactive antagonist [125I]-ZOTA.
The structure of [125
I]-ZOTA is compared to that of natural hormone OT and that of
antagonist [125
I]-OTA. The photosensitive compound only differs from its parent peptide
antagonist by substitution of an azido group (N3) to the hydroxyl group (OH) on Tyr9. As OT,
the two antagonists are cyclic nonapeptide ligands. Both antagonists can be radioiodinated at
the phenyl moiety of Tyr9.
Figure 2: Photoaffinity labeling of the human OTR with [125I]-ZOTA: comparison with
the human V1a receptor.
A, Photolabeling of the human OTR. Membrane proteins (500 µg) from CHO cells expressing
the OTR were incubated 1 hour with [125
I]-ZOTA and irradiated at 254 nm for 1 to 4 min
(lanes 2-5). To define non-specific photolabeling of the membranes, no irradiation was
performed (lane 1), or OT was added at a saturating concentration (10-5
M) during the
incubation step (lane 6). Irradiated and washed membranes were separated on a 12% SDS-
PAGE system. Equivalent amounts of proteins (25 µg) were loaded onto each well. B,
Deglycosylation of the photolabeled OTR. CHO cell membranes (100 µg) expressing the
receptor were photolabeled and solubilized in deglycosylation buffer in the presence (lane 2)
or not (lane 1) of 2 units of N-glycosidase F. Equivalent amounts of radioactivity (600,000
cpm) were loaded in each lane and proteins separated by SDS-PAGE using 12% gels. C,
Comparison of human OTR and V1a receptor photolabeling with [125
I]-ZOTA. CHO cell
membranes (500 µg) expressing the OTR (lane 3) or the V1a receptor (lane 1) were incubated
1 hour with the photoactivatable antagonist and irradiated for 1 min. Equivalent amounts of
photolabeled membranes (25 µg) were loaded in each lane before separation on SDS-PAGE.
Non-specific photolabeling of membranes was assessed by adding an excess of AVP (10-5
M)
(lane 2) or OT (10-5
M) (lane 4) during incubation with [125
I]-ZOTA. For each experiment
described in this figure, the gels were dried, exposed overnight at – 80 °C to Kodak XAR-5
films and autoradiograms are shown. Molecular mass markers are indicated (kDa) on the left.
Each panel in this figure is representative of three distinct experiments.
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Figure 3: Schematic representation of the human OTR.
The primary sequence of the receptor, deduced from the cloned cDNA (6) is shown, as well as
the predicted arrangement of the protein in the cell membrane. The position of the TMD was
predicted from GPCR hydrophobicity analysis and primary sequence comparison and
alignment. The three potential N-glycosylation sites are shown at Asn8, Asn
15 and Asn
26.
Shaded residues (Phe91
-Gln96
) indicate a consensus sequence of the cleavage site for a
metalloproteinase (41, 42). Predicted cleavage sites for CNBr (black diamonds), Lys-C (black
triangles) and Arg-C (black squares) are also indicated. The tripeptide fragment covalently-
labeled with the [125
I]-ZOTA antagonist is highlighted (residues Leu114
-Lys116
) at the top of
TMD III.
Figure 4: Schematic representation of the human V1a receptor.
The figure represents the primary human V1a receptor sequence deduced from the cloned
cDNA (31). The position of the TMD was predicted from GPCR hydrophobicity analysis and
primary sequence comparison and alignment. Three potential N-glycosylation sites are shown
at Asn14
, Asn27
and Asn196
. Shaded residues indicate a consensus sequence of the cleavage
site for a metalloproteinase (41, 42). Predicted cleavage sites for CNBr, Lys-C and Arg-C
endoproteinases are indicated as in the legend to Fig. 4. The tripeptide fragment covalently-
labeled with the [125
I]-ZOTA antagonist is highlighted (residues Val126
-Lys128
) at the top of
TMD III.
Figure 5: Single and double fragmentations of the human OTR with CNBr and Lys-
C/Arg-C endoproteinases.
CHO cell membranes (500 µg) expressing the OTR were incubated 1 h with the [125
I]-ZOTA
and irradiated for 1 min. Sample proteins were then separated on a preparative 12% gel and
the photolabeled 70-75 kDa species was electroeluted, washed and concentrated as described
in "Experimental Procedures". Equivalent amounts of electroeluted photolabeled OTR were
used in each digestion or chemical cleavage assays (15,000-25,000 cpm). The samples were
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then loaded on discontinuous 10-16.5% Tricine gels. A, CNBr chemical cleavage. The
electroeluted receptor was treated (lane 2) or not (lane 1) with CNBr for 24 h in the dark at
room temperature. B, Lys-C protease digestion. The 70-75 kDa species was treated (lane 2) or
not (lane 1) with the protease for 24 h at 37°C. C, Arg-C protease digestion. The electroeluted
receptor was treated (lane 2) or not (lane 1) with the enzyme for 24 h at 37°C. D, Arg-C
protease /CNBr double fragmentation. The radiolabeled OTR was treated with Arg-C protease
alone (lane 2) or in two steps with Arg-C protease followed by CNBr cleavage (lane 3). The
figure shows autoradiograms of dried gels exposed to Kodak XAR-5 films at – 80 °C for 48-
72 h. Molecular mass markers are indicated on the left in each panel. Each assay is
representative of at least three distinct experiments.
Figure 6 : Theoretical fragmentation maps of OTR and V1a receptor.
The theoretical fragmentation maps of OTR (A) and V1a receptor (B) using CNBr, Lys-C
endoproteinase or Arg-C endoproteinase are shown. TMDs are highlighted as solid black
lines. Cleavage sites for CNBr and enzymes are marked with black dots and the size of the
theoretical fragments are indicated. Only fragments possessing a molecular mass bigger than 1
kDa are shown. In each digestion, the best candidate fragment for covalent attachment of
[125
I]-ZOTA, based on experimental digestions, is shown as a solid black line. For the V1a
receptor, cleavage of the receptor with a metalloproteinase during incubation with [125
I]-
ZOTA is indicated by an arrow. The resulting radiolabeled 46 kDa protein used in the
different fragmentation reactions starts at Val105
. For a direct comparison between these
theoretical fragmentation maps and the experimental fragmentation results, molecular mass of
the photoactivatable antagonist [125
I]-ZOTA, covalently attached to the receptors, has to be
added (1.3 kDa).
Figure 7: Single and double fragmentations of the human V1a vasopressin receptor with
CNBr and Lys-C/Arg-C endoproteinases.
CHO cells membranes (500 µg) expressing the wild-type human V1a vasopressin receptor
were incubated 3 h (a condition that favors the preferential accumulation of the 46 kDa
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species) with the [125
I]-ZOTA and irradiated for 1 min. Membrane proteins were then
separated on a preparative 12% gel, and the photolabeled 46 kDa species was electroeluted,
washed and concentrated as described in "Experimental Procedures". Equivalent amounts of
electroeluted photolabeled V1a receptor was used in each digestion or chemical cleavage
assays (15,000-25,000 cpm). A, CNBr chemical cleavage. The electroeluted receptor was
treated (lane 2) or not (lane 1) with CNBr for 24 h in the dark at room temperature. B, Lys-C
protease digestion. The electroeluted receptor was treated (lane 2) or not (lane 1) with Lys-C
protease for 24 h at 37 °C. C, Arg-C protease digestion. The electroeluted receptor was treated
(lane 2) or not (lane 1) with Arg-C protease for 24 h at 37 °C. D, Arg-C protease /CNBr
fragmentation. The electroeluted V1a receptor was treated (lane 3) or not (lane 1) with Arg-C
protease alone, or subjected to a double fragmentation with Arg-C protease and CNBr (lane
2). The figure shows autoradiograms of dried gels exposed to Kodak XAR-5 films at –80 °C
for 48-72 h. Molecular mass markers are indicated on the left in each panel. Each assay is
representative of at least three distinct experiments.
Figure 8: Chemical CNBr fragmentation of photolabeled M78A and M123A mutant
OTR.
Photolabeling of CHO cell membranes (1 mg) expressing wild-type, mutant M78A or mutant
M123A OTR, as well as preparative 12% SDS-PAGE were performed as described in legend
to Figs. 2 and 5. Radioactive material at 70-75 kDa, corresponding to each receptor, was
electroeluted, washed and concentrated before being subjected to CNBr cleavage. Equivalent
amounts of photolabeled receptors were used in each chemical cleavage assay (20,000 cpm).
The radiolabeled samples, i.e. wild-type (lanes 1, 2), mutant M78A (lanes 3, 4) and mutant
M123A (lanes 5, 6) were treated (lanes 2, 4, 6) or not (lanes 1, 3, 5) with CNBr for 24 h in the
dark at room temperature. An autoradiogram of a discontinuous 10-16.5 % dried gel exposed
to Kodak XAR-5 film at –80 °C for 72 h is shown. Molecular mass markers are indicated on
the left. This fragmentation pattern is representative of three distinct experiments.
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Figure 9: Endoproteinase Lys-C fragmentation of photolabeled K128A mutant V1a
receptor.
Photolabeling of CHO cell membranes (1 mg) expressing wild-type or mutant K128A V1a
receptors was performed as described in legend to Figure 6. The corresponding 46 kDa
photolabeled species were then electroeluted from a preparative 12% SDS-PAGE and
subjected to Lys-C endoproteinase digestion. Equivalent amounts of photolabeled receptors
were used in each digestion assay (10,000 cpm). The radiolabeled samples, i.e. wild-type
(lanes 1, 2) or mutant K128A (lanes 3, 4) were treated (lanes 2, 4) or not (lanes 1, 3) with Lys-
C proteinase for 24 h at 37 °C. An autoradiogram of a discontinuous 10-16.5 % dried gel
exposed to Kodak XAR-5 film at –80 °C for 72 h is shown. Molecular mass markers are
indicated on the left. This fragmentation pattern is representative of three distinct experiments.
Figure 10: Schematic model of human OTR and V1a receptor binding pockets.
The arrangement of transmembrane domains of OTR and V1a receptors viewed from the
extracellular surface is based on the two-dimensional crystal projection map from of frog
rhodopsin (47). The TMD are represented as shaded cylinders, the first extracellular loop as a
solid black line between TMD II and III. Receptor regions that have been demonstrated to be
covalently attached to photoactivatable peptide antagonists are indicated with black triangles.
In the human V1a receptor, the top of TMD VII, the first extracellular loop and the top of TM
III have been photolabeled with [125
I]-3N3Phpa-LVA (25), [125
I]-[Lys(3N3Phpa)8]HO-LVA
(26) and [125
I]-ZOTA (this report), respectively. In the human OTR, only the top of TMD III
has been demonstrated so far to be covalently-attached to [125
I]-ZOTA (this report). Residues
or regions likely to be involved in peptide antagonist binding affinity, on the basis of site-
directed mutagenesis studies, are indicated with black spheres. In the human V1a receptor,
Gln108
(TMD II), Lys128
(TMD III), Gln185
(TMD IV) and Phe307
(TMD VI) play a role in the
binding of V1a-selective cyclic (29) and linear (25, 26) peptide antagonists. In the human
OTR, the top of TMD VII has been proposed to be a major contributor to high affinity binding
of a [125
I]-OTA-related cyclic peptide antagonist (18).
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Table I: Binding properties of the cyclic peptide antagonist [125I]-ZOTA and its non
radiolabeled analogue [I]-ZOTA for the OT/AVP family receptors.
----------------------------------------------------------------------------------------------------------------- (a) (a) (b) (c)
Receptors [125
I]-OTA [125
I]-ZOTA [3H]-AVP [I]-ZOTA
Kd (nM) Kd (nM) Kd (nM) Ki (nM) -----------------------------------------------------------------------------------------------------------------
OT 0.18±0.04 (7) 0.25±0.04 (3) 1.36±1.00 (3) 0.9±0.03 (3)
V1a 0.93±0.06 (3) 0.46±0.09 (3) 0.7±0.17 (3) 1.38±0.21 (3)
V1b n.d. n.d. 0.37±0.05 (3) 5665±1910 (3)
V2 n.d. n.d. 1.36±0.45 (3) 204±100 (3)
-----------------------------------------------------------------------------------------------------------------
(a) Affinities (Kd) of [125
I]-OTA and [125
I]-ZOTA for the human OTR and V1a were directly
determined in saturation experiments. (b) Affinities (Kd) of [3H]AVP for all four wild-type
receptor subtypes were directly determined in saturation experiments and have already been
published (25, 26). (c) Affinities (Ki) of the non radiolabeled [I]-ZOTA were determined in
competition assays by displacement of [3H]AVP. Saturation and competition assays are
described in the "Experimental Procedures" section. Data were analyzed by non linear least-
squares regression with the program Ligand. All values in this table are expressed as the mean
± S.E. calculated from at least three independent experiments, each performed in triplicate.
n.d. : not determined.
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AKNOWLEDGMENTS
Many thanks to M. Passama and L. Charvet for help in the illustrations. Special thanks to Drs.
Tuhinadri Sen and Jean-Philippe Pin for critical reading of the manuscript. This research was
supported by grants of INSERM and CNRS.
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CH21 CO Tyr(Me)2 Ile3 Thr4 Asn5 Cys6 Pro7 Orn8 NH CH9 CO NH2
S S
125IOH
CH2
[125I]-OTA
CH21 CO Tyr(Me)2 Ile3 Thr4 Asn5 Cys6 Pro7 Orn8 NH CH9 CO NH2
S S
N3
125I
CH2
[125I]-ZOTA
CH1 CO Tyr2 Ile3 Gln4 Asn5 Cys6 Pro7 Leu8 Gly9 NH2
S S
OT
NH2
Figure 1
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1 2 3 4 5 6
220
97.4
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202
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97.4
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30
A B C
Figure 2
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Figure 8
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Figure 9
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I
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Figure 10
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Bernard MouillacandSeyer, Sylvie Phalipou, Denis Morin, Thierry Durroux, Hans H. Zingg, Claude Barberis
Christophe Breton, Hichem Chellil, Majida Kabbaj-Benmansour, Eric Carnazzi, Renevasopressin receptor
photoactivatable cyclic peptide antagonist: Comparison with the human V1a Direct identification of human oxytocin receptor-binding domains using a
published online May 3, 2001J. Biol. Chem.
10.1074/jbc.M102073200Access the most updated version of this article at doi:
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