direct identification of human oxytocin receptor-binding domains

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: [email protected]

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,


4,Orn

8,Tyr(3I)-NH2

9]vasotocin ; [

125I]-OTA,


4,Orn

8,Tyr(3

125I)NH2

9]vasotocin ; [I]-ZOTA,


4,Orn

8,Phe(3I,4N3)NH2

9]vasotocin; [

125I]-ZOTA,


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


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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REFERENCES

1. Gainer, H., and Wray, S. (1994) in: The Physiology of Reproduction. Knobil E, Neill JD

eds, 2nd edition, pp. 1099-1129, Raven Press, New York

2 . Fuchs, A-R. (1985) in: Oxytocin. Clinical and laboratory Studies. Amico JA, Robinson

AG eds, pp. 207-235, Excerpta Medica, Amsterdam

3. Soloff, M.S., Alexandrova, M., and Fernstrom, MJ. (1979) Science 204:1313-1315

4. Marc, S., Leiber, D., and Harbon, S. (1986) FEBS Lett. 201, 9-14

5. Strakova, Z., and Soloff, M.S. (1997) Am. J. Physiol. 272, E870-E876

6. Kimura T., Tanizawa O., Mori K., Brownstein M.J., and Okayama H. (1992) Nature 356,

526-529

7. Gorbulev V., Buchner H., Akhundova A., and Fahrenholz F. (1993) Eur. J. Biochem. 215,

1-7

8. Rozen F., Russo C., Banville D., and Zingg H.H. (1995) Proc. Natl. Acad. Sci. USA. 92,

200-204

9. Bathgate R., Rust W., Balvers M., Hartung S., Morley S., and Ivell R. (1995) DNA Cell

Biol. 14, 1037-1048

10. Andersson, K.E., Forman, A., and Ulmstem, U. (1983) Clin. Obstet. Gynecol. 26, 56-77.

11.Williams, P.D., Bock, M.J., Evans, B.E. Freidinger, and R.M. Pettibone, D.J. (1998) Adv.

Exp. Med. Biol. 449, 473-479

12. Goodwin, T.M., and Zograbyan, A. (1998) Clin. Perinatol. 25, 859-871

13. Chan, W.Y., Wo, N.C., Stoev, S.T., Cheng, L.L. Manning, M. (2000) Exp. Physiol. 85,

7S-18S

14. Valenzuela, G.J., Sanchez-Ramos, L., Romero, R., Silver, H.M., Koltun, W.D., Millar,

L., Hobbins, J., Rayburn, W., Shangold, G., Wang, J., Smith, J., and Creasy, G.W.

(2000) Am. J. Obstet. Gynecol. 182, 1184-1190

15. Goodwin, T.M., Valenzuela, G., Silver, H., Hayashi, R., Creasy, G.V., and Lane, R.

(1996) Am. J. Perinatal. 13, 143-146

16. Yarwood, N.J., Wheatley, M. (1995) Adv. Exp. Med. Biol. 395, 343-344


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

17. Chini, B., Mouillac, B., Balestre, M.N., Trumpp-Kallmeyer, S., Hoflack, J., Hibert, M.,

Andriolo, M., Pupier, S., Jard, S., and Barberis, C. (1996) FEBS Lett. 397, 201-206

18. Postina, R., Kojro, E., and Fahrenholz, F. (1996) J. Biol. Chem. 271, 31593-31601

19. Fahrenholz, F., Hackenberg, M., and Muller, M. (1988) Eur. J. Biochem. 174, 81-85

20. Muller, M., Soloff, M.S., and Fahrenholz, F. (1989) FEBS Lett. 242, 333-336

21. Kojro, E., Hackenberg, M., Zsigo J., and Fahrenholz F. (1991) J. Biol. Chem. 266,

21416-21421

22. Hinko A., Soloff M.S., and Potier M. (1992) Endocrinology 130, 3554-3559

23. Elands J., Barberis C., Jard S., Tribollet E., Dreifuss J.J., Bankowski K., Manning M.,

and Sawyer W.H. (1987) Eur. J. Pharmacol. 147 197-207

24. Carnazzi, E., Aumelas, A., Phalipou, S., Mouillac, B., Guillon, G., Barberis, C., and

Seyer, R. (1997) Eur. J. Biochem. 247, 906-913

25. Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahé, E., Jard, S., Barberis, C., and

Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544

26. Phalipou, S., Seyer, R.,Cotte, N., Breton, C., Barberis, C., Hibert, M., and Mouillac, B.

(1999) J. Biol. Chem. 274, 23316-23327

27. Mouillac, B., Chini, B., Balestre, M.N., Elands, J., Trumpp-Kallmeyer, S., Hoflack, J.,

Hibert, M., Jard, S., and Barberis, C. (1995) J. Biol. Chem. 270, 25771-25777

28. Chini, B., Mouillac, B., Ala, Y., Balestre, M.N., Trumpp-Kallmeyer, S., Hoflack, J.,

Elands, J., Hibert, M., Manning, M., Jard, S., and Barberis, C. (1995) EMBO J. 14,

2176- 2182

29. Cotte, N., Balestre, M.N., Aumelas, A., Mahe, E., Phalipou, S., Morin, D., Hibert, M.,

Durroux, T., Barberis, C., and Mouillac, B. (2000) Eur. J. Biochem. 267, 1-12

30. Barberis, C., Mouillac, B., and Durroux, T. (1998) J. Endocrinol. 156, 223-229

31. Thibonnier, M., Auzan, C., Madhun, Z., Wilkins, P., Berti-Mattera, L., and Clauser, E.

(1994) J. Biol. Chem. 269, 3304-3310

32. Kassis, S., Henneberry, R. C., and Fishman, P. H. (1984) J. Biol. Chem. 259, 4910- 4916

33. Park, C., Chamberlin, M. E., Pan, C. J., and Chou, J. Y. (1996) Biochemistry 35, 9807-

9814


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

34. Munson, P.J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239

35. Laemmli, U.K. (1970) Nature 227, 680-685

36. Schägger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368-379

37. Schall, T.J., Lewis, M., Koller, K.J., Lee, A., Rice, G.C., Wong, G.H.W., Gatanaga, T.,

Granger, G.A., Lentz, R., Raab, H., Kohr, W.J., and Goeddel, D.V. (1990) Cell 61, 361-370

38. Kimura, T., Makino, Y., Bathgate, R., Ivell, R., Nobunaga, T., Kubota, Y., Kumazawa,

I., Saji, F., Murata, Y., Nishiara, T., Hashimoto, M., and Kinoshita, M. (1997) Mol.

Hum. Reprod. 11, 957-963

39. Ji, T.H., Grossmann, M., and Ji I. (1998) J. Biol. Chem. 273, 17299-17302

40. Thibonnier, M., Conarty, D.M., Preston, J.A., Wilkins, P.L., Berti-Mattera, L.N., and

Mattera, R. (1998) Adv. Exp. Med. Biol. 449, 251-276

41. Kojro, E., Eich, P., Gimpl, G., and Fahrenholz, F. (1993) Biochemistry 32, 13537-

13544

42. Kojro, E., Postina, R., Gilbert, S., Bender, F., Krause, G., and Fahrenholz, F. (1999) Eur.

J. Biochem. 266, 538-548

43. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox, B.A., Le

Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M., and Miyano, M.

(2000) Science 289, 739-745

44. Burley, S.K., and Petsko, G.A. (1988) Adv. Prot. Chem. 39, 125-189.

45. Barberis, C., Balestre, M-N., Jard, S., Tribollet, E., Arsenejevic, Y., Dreifuss, J.J.,

Bankowski, K., Manning, M., Chan, W.Y., Schlosser, S.S., Holsboer, F., and Elands, J.

(1995) Neuroendocrinology 62, 135-146

46. Strader, C.D., Sigal, I.S., Candelore, M.R., Rands, E., Hill, W.S., and Dixon, R.A.F.

(1988) J. Biol. Chem. 263, 10267-10271

47. Schertler, G.F.X., and Hargrave, P.A. (1995) Proc. Natl. Acad. Sci. USA 92, 11578-

11582


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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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A B C

Figure 2


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1 2 3 4

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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:

Alerts:

When a correction for this article is posted•

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to choose from all of JBC's e-mail alertsClick here


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direct identification of human oxytocin receptor-binding domains

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