Synthetic Rat V1a Vasopressin Receptor Fragments Interfere with Vasopressin Binding via Specific Interaction with the Receptor*

(Received for publication, April 16, 1997, and in revised form, June 4, 1997)

Christiane Mendre Dagger §, Marie Noëlle Dufour , Sylvie Le Roux Dagger , René Seyer , Laurent Guillou , Bernard Calas par and Gilles Guillon Dagger

From Dagger  INSERM U469 and  CNRS UPR 9023, Centre de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier cedex 5 and par  CNRS UPR 9008, Centre de Recherches de Biochimie Macromoléculaire du CNRS, Route de Mende, BP 5051, 34033 Montpellier cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a vasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V1a receptor. In contrast, the natural N-terminal part of the V1a vasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a receptor and synthetic V1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (Ki(app) for the peptide: 3.7, 14.6, and 64.5 µM for displacing [3H]vasopressin from rat V1a, V1b, and V2 receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [3H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1a vasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.


INTRODUCTION

Vasopressin (AVP),1 a small polypeptidic neurohypophysial hormone, exerts different biological effects in mammals. At the periphery, its major physiological role is played in regulating water and solute excretion by the kidney. This hormone is also involved in blood pressure control, platelet aggregation, corticotropin and aldosterone secretion (by the adenohypophysis and the adrenals, respectively), hepatic glycogenolysis, and uterine motility (for review, see Refs. 1 and 2). In the central nervous system, AVP is also involved in interneuronal communication (3).

These distinct biological functions are mediated, in mammals, by at least three distinct receptor subtypes: V2, V1a, and V1b. V2 receptors, involved in the antidiuretic response, are positively coupled to adenylyl cyclase (4). V1a and V1b receptors, involved in multiple peripheral responses and in corticotropin release, stimulate phospholipase C and activate protein kinase C (5, 6). Cloning the different subtypes of receptors (7-9) confirmed that these peptidic receptors belong to the family of the G-protein-coupled receptors.

Small amounts of vasopressin receptors in natural tissues and difficulties in solubilizing and purifying vasopressin receptors (10) led to restricted information on the topology of the hormonal binding domain of AVP receptors. However, molecular biology and biochemical approaches have recently headed the way to characterize the vasopressin receptor binding domain. Using a tritium-labeled photoreactive vasopressin agonist, Kojro et al. (11) demonstrated that residues Arg106 and Thr102 present in the second loop of the bovine kidney V2 receptor are involved in AVP binding. Chini et al. (12) showed that replacement of Tyr115 by an alanine, in the first loop of the human V1a vasopressin receptor, greatly affects its ligand selectivity toward a series of vasopressin analogues. More recently, three-dimensional computer modeling of rat V1a vasopressin receptor structure, combined with directed site-mutagenesis experiments, has indicated that the transmembrane domains of the receptor are also involved in the vasopressin binding site (13). However, the precise location of vasopressin binding to the three receptor isoforms remains incompletely characterized.

Another approach to the study of hormone-receptor interactions involves the use of small synthetic peptides mimicking the sequence of the supposed active region of the receptor. This approach allows the use of pure and well characterized fragments, available in large amounts, to determine their possible interaction with the specific ligand or with the receptor itself. This approach has been successfully used in the case of the thyrotropin-stimulating hormone receptor and luteotropin human choriogonadotropin receptor, where synthetic peptides mimicking the N-terminal extracellular sequence of the receptor were able to bind the hormone (14, 15). Similarly, peptide fragments corresponding to the N-terminal segment of the follitropin receptor were shown able to bind this hormone (16). More recently, Howl and collaborators (17) also described the inhibitory properties of some synthetic peptides mimicking neurohypophysial hormone receptor. A 20-amino acid synthetic peptide derived from the tumor necrosis factor receptor inhibited the binding and the cytolytic activity of the corresponding recombinant human hormone (18). This approach has also been successfully used in characterizing hormonal receptor-G-protein interactions, such as synthetic peptides mimicking the third intracellular loop of the 5HT1a receptor which prevent hormonal adenylyl cyclase inhibition (19), and also the interactions between other classes of proteins like actin-tropomyosin and calponin (20).

To further delineate the vasopressin binding site of the V1a vasopressin receptor, we elected to follow an approach similar to that described above. Thus, we synthesized peptides corresponding to extracellular regions of the rat V1a vasopressin receptor (N-terminal sequence and hydrophilic extracellular loops) and examined their ability to alter specific vasopressin binding to V1a vasopressin receptor and to modify vasopressin-stimulated phospholipase C activity. Our data indicate that some synthetic peptides and particularly one fragment of the second extracellular loop with an additional cysteine residue exhibited antagonistic properties. We studied the mechanisms involved in such inhibition.


MATERIALS AND METHODS

Chemicals

Tritiated [Arg8]vasopressin (60 Ci/mmol) ([3H]AVP)and myo-[3H]inositol (10-20 Ci/mmol) were obtained from NEN Life Science Products; OH-LVA, a specific V1a vasopressin antagonist (2000 Ci/mol), and Sar-AngII, an angiotensin II antagonist (2000 Ci/mmol), were radioiodinated using the IODO-GEN technique as described previously (21, 22). [Arg8]Vasopressin was obtained from Bachem. The peptides Nt-(50-79) (deriving from the N-terminal part of the bovine endothelin A receptor) and GLP (a glucagon-like-peptide) were synthesized and characterized in the laboratory by solid phase peptide synthesis as described previously (23, 24). Dowex AG1-X8 (100-200-mesh), chloride form, was obtained from Fluka. Affinity chromatography was performed with Affi-GelR 10 gel from Bio-Rad. All other chemicals were of A-grade purity.

Synthesis and Purification of Receptor Fragments

All peptides were obtained by solid phase peptide synthesis, using two different procedures. The fluoromethoxycarbonyl strategy consisted in using a continuous flow procedure in an automated solid-phase synthesizer (PepSynthesizer 9050, from Perseptive Biosystems, Millipore) (25). The resin was Fmoc-Amino-Acid-PAL-PEG-PSR from Perseptive Biosystems, Millipore (1 g of 0.2 mmol NH2/g). We used free alpha CO2H amino acids (0.6 mmol) with temporary protection on the alpha NH2 and the side-chain protections as follows: pentamethylchromanesulfonyl for Arg; trityl for Cys, Asn, and Gln; tertiobutyloxycarbonyl for Lys; tertiobutyl ether for Ser, Thr, and Tyr; tertiobutyl ester for Asp and Glu. The coupling agent used was 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (0.6 mmol) and diisopropylethylamine as base (1.2 mmol). After the completion of the synthesis, the peptides were deprotected and cleaved from the support using K reagent (CF3COOH/phenol/water/thioanisole/ethanedithiol, 85/5/2.5/2.5, v/v) for 0.5-4 h, depending on the presence or not of Arg(pentamethylchroman sulfonyl) in the sequence. The tertiobutyloxycarbonyl strategy was carried out using either an automated solid-phase synthesizer (Applied Biosystems 430A) or a manual device as described previously (26). The resin used was a p-methylbenzhydrylamine polystyrene from Bachem (1 g of 1.1 mmol NH2/g for automatic synthesis, and 3 g of 0.6 mmol NH2/g of titratable amine for manual synthesis). We used alpha CO2H amino acids (2.2 and 3.6 mmol for automatic and manual synthesis, respectively) with the following side-chain protections: p-toluenesulfonyl for Arg; p-methylbenzyl for Cys, benzyl for Ser and Thr, cyclohexyl for Glu and Asp, o,o-dichlorobenzyl for Tyr, and o-chlorobenzyloxycarbonyl for Lys with (benzotriazolyloxy)tripyrrolidinophosphonium hexafluorophosphate as coupling agent (2.2 or 3.6 mmol for automatic or manual synthesis, respectively) (27) and diisopropylethylamine as base (4.4 or 7.2 mmol for automatic or manual synthesis, respectively). The coupling efficiency was usually evaluated by the colorimetric Kaiser test. In this strategy, the deprotection was performed by CF3COOH/CH2Cl2/ethanedithiol (50/47/3, v/v) for 2 and 28 min, and finally 60 min in HF/anisole/dimethylsulfide (9/0.8/0.2, v/v).

Peptides were purified by reverse phase HPLC. Analytical HPLC was performed using a C18 cartridge and linear CH3CN/H2O/0.1% CF3COOH (v/v) gradients as described previously (28). Preparative runs were performed using linear gradients of CH3CN in H2O, acidified (pH 2-3) by 0.1% CF3COOH. The purified peptide structure was checked via amino acid analysis, electrospray ionization mass spectrometry (ESI-MS, VG FISONS, TRIO 2000) as described previously (29) or by fast atom bombardment (FAB-MS, JEOL, SX 102). For the ESI-MS method, determination of the peptide true molecular weight from the raw m/z data was performed using VG TRIO 2000 mass spectrometer with Maxent software (29). For the FAB-MS, determination of the true molecular weight was derived from the (MH+) peak of the fragmentation profile. Peptide quantification was performed by weighing, assuming CF3COOH as counter-ion in the lyophilized compounds, but without considering any hydration of the peptides.

Rapid Thiol Blocking Procedure

The e2-(194-218)C peptide was treated with iodoacetamide, as described previously (30), to block its cysteinyl residues. This technique was preferred to N-ethylmaleimide blockade, since it introduced a reduced steric modification with no additional charge on the native peptide.

Plasma Membrane Preparations

Animals used in this study were female Wistar rats (160-180 g body weight). Purified plasma membranes from liver were prepared according to the procedure of Neville (31) up to step 11. They were stored in liquid nitrogen. Crude plasma membranes from the inner kidney medulla or from the adenohypophysis were prepared according to Butlen (4) and Jard (6), respectively. Briefly, the tissues were homogenized at 4 °C in an isotonic buffer containing 250 mM sucrose, 5 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 1 mM EDTA, 0.1 mM phenymethylsulfonyl fluoride, using a glass/glass Potter-Elvehjem. The extracts were then centrifuged at 4 °C for 15 min at 3500 rpm. The supernatants were discarded and pellets resuspended in a large volume of hypotonic buffer, the composition of which was similar to the one described above, but without sucrose. After a 20-min incubation at 0 °C, extracts were centrifuged under the same conditions. Resulting pellets were resuspended in the hypotonic medium and centrifuged again. These pellets were collected, resuspended in hypotonic medium (1 mg of protein/ml), and used immediately for binding experiments.

Cell Culture

WRK1 cells, a rat mammary tumor cell line, were cultured as described previously (32). Briefly, cells were plated at a density of 105 cells/dish in a modified minimum essential medium, containing 5% calf serum, 2% rat serum, 290 mg/ml glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. They were cultured at 37 °C in a humidified atmosphere (5% CO2, 95% air). Two days after plating, the culture medium was removed and replaced by a fresh one containing 1 µCi/ml myo-[3H]inositol and cells were used 2 days thereafter.

Binding Experiments

[3H]AVP binding to plasma membrane preparations was performed as described previously (4, 6, 33). Briefly, kidney, adenohypophysis or liver membrane preparations (30-60 µg of protein/assay) were incubated 1 h at 37 °C in 200 µl of a buffer containing 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 1 mg/ml bovine serum albumin, 0.1 mM phenymethylsulfonyl fluoride, 1 nM [3H]AVP, and increasing amounts of vasopressin receptor fragments (total binding determinations). An excess of unlabeled AVP (1 µM) was also added in the incubation medium to determine the nonspecific binding. The reaction was initiated by adding the membranes, stopped by addition of 4 ml of cold washing solution (10 mM Tris-HCl, pH 7.4, 3 mM MgCl2), and immediately filtered onto Whatman glass fiber (0.8-1.2 µm) previously presoaked 2 h with 10 mg/ml BSA. The filters were then rinsed three times with 4 ml of the washing solution and the remaining radioactivity, measured by liquid scintillation spectrometry. The specific binding was calculated as the difference between the total and nonspecific values. 125I-OH-LVA binding was performed as described previously (21). Briefly, 1-2 µg of rat liver membranes were incubated 1 h at 37 °C in 200 µl of the same incubation medium as the one described above. [3H]AVP was replaced with a single concentration of 125I-OH-LVA (concentration-displacement experiments) or increasing amounts of 125I-OH-LVA (concentration-dependent binding experiments). The concentration of 125I-OH-LVA used in the displacement experiments was around 20 pM, a value corresponding to the Kd of this labeled analogue for the rat liver V1a vasopressin receptor (21). Binding experiments were carried out as described for [3H]AVP, except that radioactivity was measured using a gamma  counter.

To verify if our binding assay conditions outlined the kinetic equilibrium conditions between the synthetic peptides and the hormonal receptor, we tested the influence of a preincubation between the synthetic peptides and the plasma membranes, prior addition of 125I-OH-LVA. As shown in Fig. 1A, a 30-min preincubation at 37 °C did not modify the ability of e2-(194-218)C to inhibit 125I-OH-LVA specific binding. These results validate the experimental protocol chosen for measuring the interactions between the synthetic peptides and the hormonal receptor. To further characterize such interactions, we also calculated the Hill coefficient for the e2-(194-218)C peptide, as described previously (33). As illustrated on Fig. 1B, the Hill coefficient was found independent from the preincubation conditions. Its value (1.14 ± 0.07, n = 7) was near to unit, suggesting a single site binding interaction. Similar results were also obtained with the same synthetic peptide on rat liver plasma membrane using [3H]AVP as radioligand (Hill coefficient = 1.11 ± 0.09, n = 5) (data not shown).


Fig. 1. Influence of preincubation on the interaction between V1a vasopressin receptor and e2-(194-218)C peptide. Panel A, 1.5 µg of rat plasma membrane were preincubated 30 min at 37 °C with increasing amounts of e2-(194-218)C synthetic peptide (bullet ) or vehicle (control). Then, either 20 pM 125I-OH-LVA (total binding) or 20 pM labeled ligand plus 1 µM unlabeled AVP (nonspecific binding) were added in the incubation medium and the reactions allowed to proceed for another 60-min period at the same temperature. In another set of experiment (open circle ), rat liver membranes were preincubated 30 min without synthetic peptide. Then, increasing amounts of e2-(194-218)C peptide were added together with 20 pM 125I-OH-LVA (total binding) or 20 pM 125I-OH-LVA plus 1 µM unlabeled AVP and incubation proceeded for 60 additional min. Radioactivity found associated to plasma membrane was determined by filtration as described under "Materials and Methods." Specific binding were calculated in each condition (total-nonspecific binding) and expressed as percent of control specific binding (100% = 2900 ± 150 dpm/assay for membrane preincubated with or without e2-(194-218)C peptide). Results are the mean of triplicate determinations from a single experiment representative of three. Panel B, data from panel A were plotted as described previously (33) for the determination of the Hill coefficient. Bo, control specific binding; B, specific binding measured in the presence of a given concentration (I) of e2-(194-218)C peptide; H, concentration of 125I-OH-LVA used in the assay; Kd, dissociation constant of the rat liver vasopressin receptor for 125I-OH-LVA; [I], concentration of synthetic peptide.
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125I-Sar-AngII Binding to Rat Liver Membrane Preparation

125I-Sar-AngII binding was performed on rat liver membranes as described for 125I-OH-LVA. The concentration of labeled hormone used in this assay was around 0.2 nM, a value similar to the Kd of this analogue for rat liver AngII receptor (22).

Inositol Phosphate Accumulation Measurements

As described previously (34), myo-[3H]inositol-prelabeled WRK1 cells were incubated for 45 min, at 37 °C in a culture medium deprived of myo-[3H]inositol and sera, washed twice with 1.5 ml of phosphate-buffered saline (PBS) and then preincubated 15 min at 37 °C with 0.7 ml of PBS supplemented with: 1 g/liter glucose, 10 mM LiCl, 1 mg/ml BSA, and the vasopressin receptor fragments tested. AVP (1 nM) was then added to the incubation medium for an additional 6-min period. Perchloric acid (5% final concentration) together with 0.1 ml of BSA (20 mg/ml) were added to the incubation medium to stop the reaction. Cells were scraped and cellular extracts centrifuged. Labeled mono-, bis-, and triphosphate inositol present in the supernatant were separated from inositol and glycerophosphoinositides using Dowex AG1-X8 columns and measured by liquid scintillation spectrometry as described previously (34).

Data Analysis

Data presented are the mean of triplicate determinations. The standard error (S.E.) associated to each experimental value never exceeded 10% both for binding and inositol phosphate accumulation measurements. The number of distinct experiments performed (n) is indicated in parentheses. Determination of the apparent dissociation constant (Ki(app)) for the vasopressin receptor fragments was calculated as described previously (33), assuming a direct competition between the vasopressin receptor fragment and labeled AVP or OH-LVA molecules for the vasopressin receptor binding sites. Briefly, the concentration of vasopressin fragment leading to half-maximal specific binding inhibition of labeled hormone (ED50) was determined by concentration-displacement experiments and its Ki(app), calculated according to the Cheng-Prusoff equation: Ki(app) = ED50 × Kd/(Kd + [H*]), where Kd is the dissociation constant of the labeled ligand used for the vasopressin receptor considered and [H*] the concentration of the labeled hormone used in the assay.


RESULTS

Sequences and Purity of V1a Vasopressin Receptor Fragments

We prepared synthetic fragments of the rat V1a vasopressin receptor from the primary sequence published (8). These peptides (from 13 to 51 residues) mainly corresponded to hydrophilic extracellular sequences of the rat V1a receptor. The C termini of the synthetic peptides, for which length did not exceed 26 residues, were amidated to suppress the carboxyl negative charge not present in the corresponding domain of the native protein. The putative importance of cysteine residues in the receptor binding domain (35), led us to synthesize "modified peptides." They were either cysteinylated (addition of a C-terminal extra-cysteinyl residue) or alkylated by iodoacetamide (blockade of cysteinyl residues).

The sequences and the abbreviations of the peptides are summarized in Table I. Peptide purity was determined by analytical HPLC and evaluated to be higher than 98%, as can be shown for e2-(194-218)C peptide (Fig. 2, inset). Moreover, amino acid analysis of each peptide is in good agreement with its sequence (data not shown). Finally, the mass of each peptide was confirmed using mass spectrometry. As shown in Table II, the experimental mass values found for each peptide corresponded to the calculated mass. Fig. 2 illustrates a typical result of mass spectrometry for e2-(194-218)C synthetic fragment.

Table I. Positions and sequences of natural and modified synthetic fragments of rat V1a vasopressin receptor


   Position             Abbreviations                                       Sequences


Fig. 2. Purity and mass measurement of e2-(194-218)C peptide. The deconvoluted electrospray mass spectrum of e2-(194-218)C peptide is illustrated. The calculated mass corresponding to the principal peak was 3002 Da. The inset illustrates the reverse-phase HPLC profile of the same synthetic peptide performed on a C18 column, monitored at 214 nm, using a 0-40% acetonitrile gradient in 0.1% trifluoroacetic acid.
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Table II. Mass spectrometry characterization of synthetic peptides

The structure of purified synthetic peptides was assessed by ESI-MS or FAB-MS. Experimental mass values were determined as described under "Materials and Methods."

Abbreviations Mass spectrometry data
Theoretical mass value Experimental mass value
ESI-MS FAB-MS

Nt-(1-51) 5434 5432
e1-(111-124) 1827 1828
e3-(319-335) 2111 2112
e2-(206-218) 1595 1596
e2-(205-218) 1697 1698
e2-(194-218) 2899 2895
e2-(194-218)S 2883 2885
e2-(194-218)C 3002 3004
e2-(203-218)C 2043 2045
e2-(201-218)C 2272 2274
e2-(194-218)C(cam)2 3118 3117

HPLC control experiments performed on the synthetic fragment e2-(194-218)C, containing cysteinyl residues, showed that it was not able to dimerize or to be cyclized (data not shown).

Influence of V1a Vasopressin Receptor Fragments on the Iodinated OH-LVA Specific Binding to Rat V1a Vasopressin Receptor

Fig. 3 summarizes the effects of some receptor fragments on the specific binding of 125I-OH-LVA on rat liver plasma membranes, a preparation that contained only the V1a vasopressin receptor subtype (33). Short fragments of the first loop (e1-(111-124)), of the second loops (e2-(205-218), e2-(206-218), e2-(194-218)), and of the third loop (e3-(319-335)) inhibited, in a concentration-dependent fashion, the specific binding of 125I-OH-LVA with an apparent inhibition constant ranging between 7 and 61 µM (see Table III). Finally, the N-terminal part of the V1a vasopressin receptor, Nt-(1-51) peptide, had no effect even up to 300 µM. From these results, it appears that among the V1a vasopressin receptor fragments tested, the peptides corresponding to the second extracellular loops were the most active. More interestingly, their size and cysteine composition seemed to represent crucial parameters for their activities. Thus, increasing the size of peptide e2-(205-218) resulted in peptide e2-(194-218) with a reduced activity (see Table III). The importance of the cysteine 205 residue in e2-(194-218) is further evidenced in Fig. 4. Its replacement by a serine in e2-(194-218)S reduced by at least 10-fold the peptide activity. Moreover, peptide e2-(205-218), which contained only one N-terminal cysteine more than e2-(206-218), was found more potent (see Table III).


Fig. 3. Influence of extracellular fragments of rat V1a vasopressin receptor on 125I-OH-LVA specific binding. Rat liver membranes (1-2 µg of protein/assay) were incubated with 20 pM 125I-OH-LVA (total binding) or with the same amount of labeled hormone plus 1 µM unlabeled AVP (nonspecific binding) in the absence (control) or in the presence of increasing amounts of rat V1a vasopressin receptor fragments (Nt-(1-51) (triangle ), e2-(194-218) (square ), e3-(319-335) (open circle ), e1-(111-124) (black-square), e2-(206-218) (black-triangle), and e2-(205-218) (bullet )). Specific binding were calculated in each condition (total binding - nonspecific binding) and expressed as percent of control specific binding (100% = 3500 ± 200 cpm). Results were the mean of triplicate determinations from a single experiment representative of three.
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Table III. Biological properties of natural and modified synthetic fragments of rat V1a vasopressin receptor

The ability of synthetic fragments of V1a vasopressin receptor to inhibit [3H]AVP or 125I-OH-LVA binding to rat liver membranes was determined as described in legend of Figs. 3 and 5. The apparent inhibition constant of each peptide was calculated as indicated under "Materials and Methods." The antagonistic properties of each synthetic peptide was determined as described in legend of Fig. 9. Values in the table corresponded to the inhibition of vasopressin-stimulated inositol phosphate accumulation induced by synthetic peptides. Results were expressed as: 100 - (100 × inositol phosphate accumulated in the presence of 1 nM AVP plus 30 µM peptide over basal/inositol phosphate accumulated in the presence of 1 nM AVP over basal). Results were the mean ± S.E. of the number of distinct experiments indicated in parentheses (undetectable = no significant inhibition observed for 10-4 M peptide). ND, not determined.

Abbreviations Apparent inhibition constant of 125I-OH-LVA binding (Ki(app)) Apparent inhibition constant of [3H]AVP binding (Ki(app)) Inhibition of AVP-stimulated inositol phosphate accumulation induced by 30 µM peptide

µM µM %
Nt-(1-51) Undetectable (n = 3) ND Undetectable (n = 2)
e1-(111-124) 27  ± 6 (n = 4) ND 25 (n = 1)
e3-(319-335) 61  ± 22 (n = 4) ND ND
e2-(206-218) 22  ± 3 (n = 3) ND ND
e2-(205-218) 7  ± 2 (n = 3) 17.0 (n = 2) 37  ± 12 (n = 3)
e2-(194-218) 33  ± 6 (n = 5) 56  ± 7 (n = 3) 9  ± 2 (n = 3)
e2-(194-218)S >300 (n = 2) ND ND
e2-(194-218)C 0.55  ± 0.15 (n = 5) 3.7  ± 1.4 (n = 5) 90 ± 7 (n = 3) for 10 µM peptide
e2-(203-218)C >100 (n = 3) ND ND
e2-(201-218)C 65  ± 21 (n = 3) ND ND
e2-(194-218)C(cam)2 >80 (n = 1) ND ND


Fig. 4. Influence of modified extracellular fragments of rat V1a vasopressin receptor on 125I-OH-LVA specific binding. Rat liver membranes were incubated as described in legend of Fig. 2 without (control) or with increasing amounts of modified peptides from the second extracellular loop of rat V1a vasopressin receptor: e2-(194-218)C (black-square), e2-(194-218)C(cam)2 (bullet ), e2-(201-218)C (black-triangle), e2-(203-218)C (triangle ), e2-(194-218) (square ), e2-(194-218)S (open circle ). Specific binding were calculated and expressed as % of control as described in Fig. 1. Results were the mean of triplicate determinations from a single experiment representative of three.
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Influence of Cysteinyl V1a Vasopressin Receptor Fragments on the Iodinated OH-LVA Specific Binding to Rat V1a Vasopressin Receptor

As the e2-(205-218) peptide was the most potent in inhibiting iodinated OH-LVA specific binding and as its activity was due in part to its N-terminal cysteine, we synthesized new peptides differing by their size or cysteine composition. The sequences of these new fragments are illustrated in Table I. The most striking finding is illustrated in Fig. 4. The elongation of e2-(194-218) by a single cysteine residue in the C-terminal position (e2-(194-218)C) increased its activity by 60-fold (Ki(app) of 0.55 µM) (Table III). The importance of the two cysteines of e2-(194-218)C peptide is further illustrated in Fig. 4. e2-(194-218)C(cam)2, deriving from e2-(194-218)C by iodoacetamide treatment which blocked its cysteinyl residues, exhibited a reduced activity (Ki(app) larger than 80 µM). Reducing its sequence on its N-terminal part led to e2-(201-218)C and e2-(203-218)C, which were found to be 120- and at least 200-fold less active than e2-(194-218)C despite a similar cysteinyl residue composition (Table III).

Influence of Active V1a Vasopressin Receptor Fragments on the [3H]AVP Specific Binding to Rat V1a Vasopressin Receptor

As the agonist and antagonist binding sites are probably distinct, we compared the activity of some of the most active vasopressin receptor fragments on the specific binding of 125I-OH-LVA (a vasopressin linear antagonist) and [3H]AVP (the natural agonist) to the V1a vasopressin receptor. e2-(194-218), e2-(194-218)C, and e2-(205-218) peptides were able to fully inhibit the specific [3H]AVP binding to rat liver membranes in a concentration-dependent manner (Fig. 5). The rank order of potency to inhibit [3H]AVP binding was found to be similar to that observed for the inhibition of 125I-OH-LVA. However, the e2-(194-218)C peptide was found to be more active in displacing the iodinated OH-LVA than the tritiated AVP (Ki(app) = 0.55 and 3.7 µM, respectively) (Table III). In contrast, e2-(205-218) and e2-(194-218) peptides exhibited a similar efficiency in inhibition of both antagonist and agonist specific binding (Table III).


Fig. 5. Influence of extracellular fragments of rat V1a receptor on [3H]AVP specific binding. Rat liver membranes (30-40 µg of protein/assay) were incubated 60 min at 37 °C with 1 nM [3H]AVP (total binding), or the same amount of labeled AVP plus 1 µM unlabeled AVP (nonspecific binding) in the absence (control) or presence of increasing amounts of V1a vasopressin receptor fragments (e2-(194-218) (black-square), e2-(205-218) (bullet ), and e2-(194-218)C (black-triangle)). Specific binding were calculated and expressed as percent of control values (100% = 7500 ± 600 dpm). Results were the mean of triplicate determinations from a single experiment representative of three.
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Selectivity of the e2-(194-218)C Fragment on the [3H]AVP Specific Binding to Various Vasopressin Receptor Subtypes

To further test the specificity of the e2-(194-218)C peptide, we tested its ability to inhibit specific hormone binding on the two other vasopressin receptor subtypes, earlier characterized: V1b present in rat adenohypophysis (6) and V2 present in rat kidney inner medulla (4). We also compared these values to those obtained with the rat liver V1a vasopressin receptor. For these studies, we used [3H]AVP as radioligand, since this hormone exhibited the same affinity for all receptor subtypes studied (2, 6). As illustrated in Fig. 6, the e2-(194-218)C peptide was found to be more active to inhibit [3H]AVP binding to the V1a vasopressin receptor than to the other subtypes. However, it entirely suppressed the specific binding to the V1b receptor, but with a lower potency (Ki(app) = 3.7 ± 1.4, n = 3 and 14.6 ± 4.5 µM, n = 4 for V1a and V1b receptors, respectively). It also weakly inhibited the [3H]AVP binding to the V2 vasopressin receptor (Ki(app) = 64.5 ± 3.5 µM, n = 4).


Fig. 6. Influence of e2-(194-218)C fragment on [3H]AVP specific binding to distinct rat vasopressin receptor subtypes. Rat liver membranes (black-square), rat kidney membranes (black-triangle), or rat adenohypophysis membranes (bullet ) (30-50 µg of protein/assay) were incubated 60 min at 37 °C with 1 nM [3H]AVP in the absence (control) or in the presence of increasing amounts of e2-(194-218)C fragments of rat V1a vasopressin receptor. Specific binding was calculated as described in Fig. 5. Results, expressed as percent of corresponding control values, are the mean of triplicate determinations from a single experiment representative of three (100% = 7500 ± 600, 5200 ± 400, and 490 ± 70 dpm for rat liver, rat kidney, and rat adenohypophysis membrane preparations, respectively).
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Nature of the Interaction between the e2-(194-218)C and the V1a Vasopressin Receptor

To further study the mechanisms by which e2-(194-218)C peptide inhibited the hormone specific binding, we analyzed the effects of this peptide on the binding parameters of 125I-OH-LVA to rat liver membranes. As shown in Fig. 7, and as described previously (21), 125I-OH-LVA interacts with a single class of vasopressin receptors, present in rat liver membranes. The addition of e2-(194-218)C in the incubation medium drastically reduced the specific binding whatever the concentration of iodinated antagonist used. Scatchard representations of dose-dependent binding experiments indicated that e2-(194-218)C affected both the affinity of 125I-OH-LVA for the V1a vasopressin receptor and its maximal specific binding capacity. As illustrated in Table IV, when membranes were incubated with 3.16 µM e2-(194-218)C peptide, the apparent affinity was reduced by 2.3-fold. The maximal binding capacity (Bmax) was slightly reduced, but this effect was not statistically significant. Increasing the amounts of synthetic peptide in the incubation medium up to 10 µM greatly increased the Kd by 7-fold and reduced the Bmax significantly (by 2-fold). Such results indicate that e2-(194-218)C peptide inhibits 125I-OH-LVA in an uncompetitive fashion, which suggests interaction between the receptor and the peptide. We also tested the reversible nature of the interaction between e2-(194-218)C fragment and the V1a vasopressin receptor. For this purpose, rat liver membranes were first preincubated with or without a concentration of synthetic peptide allowing an almost maximal inhibitory effect and washed to eliminate the synthetic peptide. The kinetic binding parameters of the V1a vasopressin receptor were then measured in control and preincubated membranes. As illustrated in Fig. 8A, the inhibitory effect of e2-(194-218)C peptide still persisted even after washing the membranes previously incubated with the synthetic receptor fragment. However, at variance with membrane directly incubated with the synthetic receptor fragment, only the effect on the maximal binding capacity was observed. Fig. 8B also showed that membranes preincubated with e2-(194-218)C peptide and further washed were still sensitive to the synthetic receptor fragment. A new addition of e2-(194-218)C peptide always strongly reduced the remaining specific binding of 125I-OH-LVA. Altogether, these results indicate that the interactions between e2-(194-218)C peptide and the V1a vasopressin receptor are partly irreversible.


Fig. 7. Influence of e2-(194-218)C fragment on the affinity and the maximal binding capacity of rat liver vasopressin receptor for 125I-OH-LVA. Rat liver membranes (1-2 µg of protein/assay) were incubated 60 min at 37 °C with increasing amounts of 125I-OH-LVA in the absence (total binding) or in the presence of 1 µM unlabeled AVP (nonspecific binding) under three different conditions: in the absence (open circle ) or in the presence of 3.16 (black-square) or 10 µM (bullet ) of e2-(194-218)C fragment. Specific radioactivity associated to membrane (Bound) was calculated in each condition and plotted against the concentration of 125I-OH-LVA (Free) (panel A) or against the ratio (Bound/Free, panel B). Results are the mean of triplicate determinations from a single experiment representative of three.
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Table IV. Influence of e2-(194-218)C fragment on the binding kinetical parameters of 125I-OH-LVA for rat liver V1a vasopressin receptor

Data illustrated in this table are the mean ± S.E. of three distinct experiments performed as described in legend of Fig. 7. *, statistically different from corresponding control values (p < 0.05).

Assay conditions Kd Bmax

pM pmol 125I-OH-LVA specifically bound·mg-1 protein
Control membrane 18.2  ± 6.5 1.54  ± 0.6
Membrane + 3.16 µM e2-(194-218)C peptide 42.2*  ± 5.2 1.32  ± 0.20
Control membrane 17.2  ± 1.3 1.39  ± 0.10
Membrane + 10 µM e2-(194-218)C peptide 123.7*  ± 30.0 0.74*  ± 0.27


Fig. 8. Partial irreversibility of e2-(194-218)C effect on rat liver vasopressin receptor. Rat liver membranes (40 µg protein/ml) were preincubated 30 min at 37 °C with (bullet ) or without (open circle , square  = control) 3.16 µM e2-(194-218)C peptide. Reaction was stopped by diluting 6-fold the incubation medium with an ice-cold buffer containing 50 mM Tris-HCl, 7.4, and 1 mM MgCl2. Membranes were then quickly centrifuged for 15 min at 14,000 × g at 0 °C. The supernatants were discarded and the pellets resuspended in the ice-cold buffer to a protein concentration of 20-30 µg/ml. Panel A, control membranes (open circle , square ) were further incubated 60 min at 37 °C with increasing amounts of free 125I-OH-LVA in the presence (square ) or in the absence (open circle ) of 3.16 µM e2-(194-218)C peptide. Specific binding (Bound) was calculated in each condition as the difference between total and nonspecific binding and these values plotted against the ratio (Bound/Free). Similarly, membranes preincubated with 3.16 µM e2-(194-218)C peptide (bullet ) were further incubated 60 min at 37 °C with increasing amounts of 125I-OH-LVA in the absence of synthetic peptide. Specific binding (Bound) were calculated and plotted against the ratio (Bound/Free). Panel B, in this set of experiments, plasma membranes were preincubated 30 min at 37 °C with or without 3.16 µM e2-(194-218)C fragment and further washed as described above. Both two sets of membranes were further incubated 60 min at 37 °C in the presence of 20 pM 125I-OH-LVA with or without 3.16 µM e2-(194-218)C fragment. Specific binding was calculated in each condition. Results are the mean of triplicate determinations from a single experiment representative of two.
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Influence of V1a Vasopressin Receptor Fragments on Vasopressin-stimulated Inositol Phosphate Accumulation

As the V1a vasopressin receptor subtype is positively coupled to phospholipase C activation, we tested the activity of some of the most active V1a vasopressin receptor fragments characterized above. Such studies were performed on WRK1 cells, which expressed, as rat liver membranes only, a V1a vasopressin receptor tightly coupled to phospholipase C (32, 34). As illustrated in Fig. 9, e2-(194-218), e2-(194-218)C, and e2-(205-218) peptides up to 300 µM did not stimulate basal inositol phosphate accumulation. However, they were able to fully inhibit in a concentration-dependent manner the vasopressin-stimulated inositol phosphate accumulation. As expected, e2-(194-218)C peptide was the most potent in inhibiting the vasopressin effect (Ki(app) = 0.44 ± 0.07 µM, 3 distinct experiments). The two other peptides, e2-(205-218) and e2-(194-218), were less active (Ki(app) = 23 and 84 µM, respectively). This rank order of potency corresponded to those found for binding studies (Table III).


Fig. 9. Influence of extracellular fragments of rat V1a vasopressin receptor on AVP-stimulated inositol phosphate accumulation in WRK1 cells. myo-[3H]Inositol-prelabeled WRK1 cells (0.3-0.5 × 106 cells/dish) were preincubated 15 min at 37 °C in PBS medium supplemented with 10 mM LiCl (see "Materials and Methods") in the absence or presence of increasing amounts of rat V1a vasopressin receptor fragments (e2-(194-218) (square , black-square); e2-(205-218) (open circle , bullet ); e2-(194-218)C (triangle , black-triangle)). AVP (1 nM final concentration) (black-square, bullet , black-triangle) or vehicle (square , open circle , triangle ) were then added to the medium and the incubation allowed to proceed for an additional 6-min period. Reaction was stopped by adding 5% (v/v) of perchloric acid and the total amount of inositol phosphates that accumulated determined as described under "Materials and Methods." Results expressed as dpm/dish are the mean of triplicate determinations from a single experiment representative of three.
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Specificity of V1a Vasopressin Receptor Fragment Activity

To further demonstrate that peptide e2-(194-218)C specifically inhibited the binding of 125I-OH-LVA to V1a vasopressin receptor, we performed the following control experiments. First, we tested the ability of a 30-amino acid peptide derived from the N-terminal part of the endothelin A receptor (peptide Nt-(50-79)) to inhibit specific vasopressin binding. This peptide, containing a cysteinyl residue and a size similar to e2-(194-218)C fragment with no sequence homology did not alter the binding of 125I-OH-LVA on rat liver membranes, even tested at 30 µM. Under the same experimental conditions, e2-(194-218)C peptide almost completely inhibited the 125I-OH-LVA binding (Fig. 10A). Second, we also tested the influence of GLP, known to exhibit amphiphilic properties due to its helicoidal structure (36), to exclude any nonspecific putative detergent properties of the hormonal receptor fragments. As illustrated on Fig. 10A, GLP did not inhibit 125I-OH-LVA binding at a maximal dose of 30 µM. Third, we checked that e2-(194-218)C peptide was not able to alter the binding of 125I-Sar-AngII to the angiotensin II receptor also present in rat liver membranes. As seen on Fig. 10B, under experimental conditions where 125I-OH-LVA specific binding was completely suppressed (30 µM e2-(194-218)C), no significant modification of 125I-Sar-AngII was observed in the same plasma membrane sample. Fourth, to verify that there is no direct interaction between the e2-(194-218)C peptide and the labeled hormone, we compared the elution profiles of [3H]AVP either on an Affi-GelR column where e2-(194-218)C peptide was covalently fixed by its N-terminal part or on a control Affi-GelR column. Whatever the column used, the two elution profiles were identical. All the radioactivity was recovered in a single symmetrical peak coeluting with blue dextran, a marker of the column void volume (data not shown). Control experiments that validate this negative experiment have been previously published by Pradelles and collaborators. They demonstrated that the well known protein/protein interactions between AVP and neurophysin still persist even if neurophysin was covalently linked to the Sepharose column (37). This property was routinely used in the laboratory to purify tritiated vasopressin.


Fig. 10. Specificity of e2-(194-218)C peptide for binding to V1a vasopressin receptor. Rat liver membranes (1-2 µg of protein/assay) were incubated for 1 h at 37 °C as described under "Materials and Methods" with 20 pM 125I-OH-LVA (panel A) or 0.2 nM 125I-Sar-AngII (panel B) with or without (control) various synthetic peptides: 30 µM e2-(194-218)C, 30 µM GLP, 30 µM Nt-(50-79). Nonspecific binding were measured in each condition by adding either 1 µM unlabeled AVP (panel A) or 1 µM unlabeled AngII (panel B). Specific binding, calculated as the difference between total and corresponding nonspecific values, were expressed as % of control values and are the mean ± S.E. of three distinct experiments each performed in triplicate. (100% = 1270 ± 60 and 4400 ± 380 cpm/assay for panels A and B, respectively.)
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DISCUSSION

Vasopressin receptors belong to the G-protein-coupled receptor family exhibiting an N-terminal part, seven transmembrane domains connected by three extracellular loops and three intracellular loops, and possessing a C-terminal intracellular tail (38). Only recently, some data have helped to identify the receptor functional domain underlying binding of vasopressin. As was earlier established for the many G-protein-coupled receptors, some specific regions of transmembrane helices of the V1a vasopressin receptor are responsible for vasopressin anchoring (39, 40). However, additional studies have also demonstrated that the external loops of V1a vasopressin receptors may also play a role in these mechanisms. Mutation of one amino acid of the first extracellular loop of the V1a vasopressin receptor deeply affects its selectivity for two series of vasopressin analogues (12), photoaffinity labeling of V2 vasopressin receptor pointed to the second extracellular loop as a potential binding site for the hormone (11), and a peptide strategy suggested that the first extracellular loop of the V1a vasopressin receptor may represent a putative binding site for radioligands (17).

To further characterize the interaction between the vasopressin and the extracellular loops of the V1a vasopressin receptor, we synthesized a series of peptides mimicking these hydrophilic loops and tested their abilities to interfere with hormonal binding. Results presented in this study indicate that the peptide mimicking the N-terminal part of the V1a receptor is unable to alter specific agonist or antagonist binding to this receptor. This suggests either that this receptor domain is not concerned in binding or the structural determinants such as glycosylation or three-dimensional organization are lacking for this longer peptide. In contrast, fragments of V1a receptor exhibiting sequences similar to those of the external loops one, two, and three are active. They inhibit in a concentration-dependent manner the specific [3H]AVP and 125I-OH-LVA binding to the rat V1a vasopressin receptor with Ki(app) ranging between 10 and 60 µM (Table III).

To study structure-activity relationships of the most active peptide found, e2-(205-218), we increased its size and modified its cysteinyl composition by adding a C-terminal cysteine residue. These modifications were based upon the following arguments. 1) Hydropathy profile of the primary receptor sequence indicated that the second extracellular loop, determined by hydrophilic amino acid composition, is larger than the e2-(205-218) sequence. 2) Cysteinyl residues of rat V2 and, to a lesser extent, those of rat V1a vasopressin receptors play an important role in AVP binding (35). 3) Most of our active fragments exhibited a cysteinyl residue. 4) Recent representations of the vasopressin receptor favored the existence of a disulfide bridge between the cysteinyl residues of the extracellular loops one and two (13). Results obtained indicate that increasing the size of the most active peptide, e2-(205-218), leads to e2-(194-218), 5-fold less active on 125I-OH-LVA binding assays on rat liver membranes. In contrast, increasing the size of the inactive modified e2-(203-218)C peptide leads to e2-(201-218)C and e2-(194-218)C peptides with enhanced activity. Addition of 2 or 9 amino acids to the N-terminal part of the inactive fragment, e2-(203-218)C, led to peptides active at the 50 µM and 1 µM concentration range, respectively. Moreover, the presence of cysteine residues in the synthetic fragments also greatly improve their activities; e2-(194-218)C peptide, which differs from e2-(194-218) peptide by only one cysteinyl residue in the C-terminal part, is 60-fold more active. On the opposite, blocking its cysteinyl residues leads to a nearly inactive fragment. All together, these data suggest that the size and the cysteinyl composition of the most active peptide, e2-(194-218)C, constitute crucial parameters for its activity.

At least two reasons may explain why e2-(194-218)C peptide inhibits specific vasopressin binding. This receptor fragment may interact either with the receptor itself or with the hormone. In both cases, such interactions prevent the hormonal binding to its specific receptor. Experimental data presented in this study favor the first hypothesis for the following reasons. 1) No direct interaction between AVP and e2-(194-218)C peptide could be observed by an affinity chromatography approach. [3H]AVP was not retained on an Affi-Gel column on which e2-(194-218)C peptide was covalently linked, as neurophysin was retained, using the same experimental approach (37). 2) A direct interaction between [3H]AVP and e2-(194-218)C peptide would lead to identical inhibitory effects on the three vasopressin receptor subtypes, since they possessed the same affinity for [3H]AVP (Kd = 3.5 ± 0.2, 1.0 ± 0.2, and 1.5 ± 0.3 nM, for V1a, V1b, and V2 vasopressin receptors present on rat liver, rat pituitary, and rat kidney membranes, respectively). Data presented in Fig. 6 clearly indicate that it is not the case. e2-(194-218)C peptide preferentially inhibited the binding of [3H]AVP on the V1a vasopressin receptor and is 4- and 17-fold less active on the V1b and the V2 vasopressin receptors, respectively. 3) The inhibitory effects of e2-(194-218)C peptide on specific 125I-OH-LVA binding were partially irreversible. They still persist even if rat liver plasma membranes preincubated with the synthetic receptor fragment were washed before the hormonal binding assay (Fig. 8).

The effects we observed with the synthetic peptides mimicking the rat V1a vasopressin receptor on vasopressin binding are specific and could not be explained by their putative detergent properties or cysteinyl composition. Indeed, we demonstrated that 30 µM e2-(194-218)C peptide, which completely inhibited 125I-OH-LVA binding on rat liver plasma membranes, did not affect, under the same experimental conditions, the specific binding of 125I-Sar-AngII (Fig. 10B). Similarly, we tested the effects of unrelated V1a vasopressin receptor peptides exhibiting either cysteine residue (Nt-(50-79), a fragment of the endothelin A receptor) or an helicoidal structure with potential detergent properties (GLP, glucagon-like peptide) on 125I-OH-LVA binding. As observed on Fig. 10A, neither Nt-(50-79) nor GLP peptide inhibited specific 125I-OH-LVA binding to rat liver membranes even tested at 30 µM.

The mechanisms by which e2-(194-218)C peptide specifically inhibits hormonal binding to rat V1a vasopressin receptor are probably complex, since we observed both an irreversible loss of the maximal binding capacity and a reversible alteration of the affinity of the vasopressin receptor for its ligand. The substantial loss of specific binding site observed for high concentrations of e2-(194-218)C peptide (Table IV) is probably due to covalent interactions between the receptor itself and the synthetic peptide via cysteinyl residues since 1) both the V1a vasopressin receptor and the e2-(194-218)C peptide exhibited cysteinyl residues (Ref. 35 and Table I), 2) replacement of the Cys205 residue of the natural receptor fragment e2-(194-218) by a serine led to a compound (e2-(194-218)S) more than 10-fold less active, 3) blockade of the two cysteinyl residues of e2-(194-218)C peptide led to peptide e2-(194-218)C(cam2) exhibiting a Ki of at least 150-fold higher, and 4) the effect of e2-(194-218)C peptide on 125I-OH-LVA binding to rat liver membranes is partly irreversible (Fig. 8). The modulation of the affinity of the V1a vasopressin receptor for its ligand induced by e2-(194-218)C fragment also probably involved non-covalent protein-protein interactions between the synthetic peptide and the receptor, since these effects were reversible (Fig. 8). The fact that synthetic peptides devoid of any cysteinyl residue like e2-(206-218) were active reinforces this assumption (see Table III). Probably, we have synthesized a cysteinyl site-directed peptide able to interact with the V1a vasopressin receptor and as a consequence to block its ability to recognize its natural hormone and to transduce intracellular second messenger generation. However, as shown in Fig. 10, the cysteine is not sufficient to confer activity to a peptide, since a fragment of endothelin receptor of size similar to e2-(194-218)C contains cysteine and yet did not inhibit 125I-OH-LVA binding. This indicates that sequence-specific interactions are involved in the biological activity of the peptide studied. Such results would imply that synthetic peptides mimicking some specific regions of the extracellular domains of the V1a vasopressin receptor are able to interact with unidentified part of the V1a vasopressin receptor and block its activity. This suggests that intramolecular protein-protein interactions may represent a crucial parameter for vasopressin receptor activity. Such a hypothesis was already verified by Ridge et al. (41), who showed that co-expression of two or three complementary fragments of rhodopsin in COS cells led to the expression of a protein that reproduced the spectral properties of native rhodopsin. Alternatively, our data would be also consistent with the proposed importance of intermolecular protein-protein interactions between two receptors domains of distinct proteins. Such specific interactions may explain the formation of hormonal receptor dimers as described previously for the muscarinic, the beta -adrenergic and the glucagon receptors (42, 43). Similarly, the V1a vasopressin receptor photolabeling, in rat liver membranes showed, by SDS-PAGE autoradiography, higher molecular weight structures suggesting the possibility of dimer formation (44). More interestingly, they may represent a new mechanism to regulate their activities (45). Thus, Bouvier and collaborators confirmed that beta 2-adrenergic receptor may dimerize. They also demonstrate that a peptide mimicking the transmembrane domain VI of this receptor prevents dimerization and also inhibits beta -adrenergic stimulation of adenylyl cyclase activity (46). Maggio et al. (47) also demonstrated that co-expression in COS cells of two muscarinic/adrenergic receptor chimera, individually devoid of any binding activity, led to specific active muscarinic and adrenergic binding sites.

In conclusion, the specific interaction evidenced in the paper between synthetic peptide mimicking the sequence of the V1a vasopressin receptor and the receptor itself strongly suggests the importance of intra- or intermolecular interactions between two receptor molecules and provides a new molecular basis to study their regulations.


FOOTNOTES

*   This work was supported by CNRS and INSERM, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 33-4-6714-2984; Fax: 33-4-6754-2432.
1   The abbreviations used are: AVP, vasopressin; GLP, glucagon-like peptide-(17-37) amide; OH-LVA, hydroxyl linear vasopressin antagonist; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Nt, N-terminal peptide; Sar-AngII, sarcosin 1-angiotensin II; ESI-MS, electrospray ionization mass spectrometry; FAB-MS, fast atom bombardment mass spectroscopy.

ACKNOWLEDGEMENTS

We thank M. Passama and D. Bellenoue for drawings, M. C. Maraval and M. Chalier for English editing, Dr. J. Marie for 125I-Sar-AngII, and Dr. M. Bouvier, Dr. C. Chevillard, and Dr. R. Pascal for fruitful discussions.


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