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• W2100492328 abstract "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, d(CH2)5 [Tyr(Me)2,Thr4,Orn8,Phe(3125I,4N3)-NH29]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V1a vasopressin receptors expressed in Chinese hamster ovary 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 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70–75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of 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. 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, d(CH2)5 [Tyr(Me)2,Thr4,Orn8,Phe(3125I,4N3)-NH29]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V1a vasopressin receptors expressed in Chinese hamster ovary 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 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70–75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of 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. oxytocin OT receptor three-dimensional transmembrane domain arginine-vasopressin d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(3I)-NH29]vasotocin d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(3125I)-NH29]vasotocin d(CH2)5[Tyr(Me)2,Thr4,Orn8,Phe(3I,4N3)-NH29]vasotocin d(CH2)5[Tyr(Me)2,Thr4,Orn8,Phe(3125I,4N3)-NH29]vasotocin Chinese hamster ovary bovine serum albumin inositol phosphate polyacrylamide gel electrophoresis high pressure liquid chromatography N-[2-hydroxy-1,1-bis- (hydroxymethyl)ethyl]glycine 4-hydroxyphenylpropionyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Lys(3-azidophenylpropionyl)8-NH2 3-azidophenylpropionyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-Tyr9-NH2 4-hydroxyphenylacetyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-NH2 Oxytocin (OT),1 a neurohypophyseal nonapeptide, is among the strongest uterotonic agents known to date (1Gainer H. Wray S. Knobil E. Neill J.D. The Physiology of Reproduction. 2nd Ed. Raven Press, New York1994: 1099-1129Google Scholar, 2Fuchs A.-R. Amico J.A. Robinson A.G. Oxytocin. Clinical and Laboratory Studies. Excerpta Medica, Amsterdam1985: 207-235Google Scholar). This hormone acts on the myometrium through specific OT receptors (OTRs) that show a dramatic increase in their expression pattern immediately before parturition (3Soloff M.S. Alexandrova M. Fernstrom M.J. Science. 1979; 204: 1313-1315Crossref PubMed Scopus (342) Google Scholar). In mammals at term, OT-induced contractility of myometrial smooth muscle cells is triggered through an increase in the intracellular calcium level (4Marc S. Leiber D. Harbon S. FEBS Lett. 1986; 201: 9-14Crossref PubMed Scopus (140) Google Scholar). This calcium signaling pathway is dependent on coupling the OTR to both the Gq/11 and Gi proteins (5Strakova Z. Soloff M.S. Am. J. Physiol. 1997; 272: E870-E876Crossref PubMed Google Scholar). The OTR cDNA of several mammalian species has been cloned (6Kimura T. Tanizawa O. Mori K. Brownstein M.J. Okayama H. Nature. 1992; 356: 526-529Crossref PubMed Scopus (560) Google Scholar, 7Gorbulev V. Buchner H. Akhundova A. Fahrenholz F. Eur. J. Biochem. 1993; 215: 1-7Crossref PubMed Scopus (117) Google Scholar, 8Rozen F. Russo C. Banville D. Zingg H.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 200-204Crossref PubMed Scopus (197) Google Scholar, 9Bathgate R. Rust W. Balvers M. Hartung S. Morley S. Ivell R. DNA Cell Biol. 1995; 14: 1037-1048Crossref PubMed Scopus (65) Google Scholar), and the deduced amino acid sequence confirmed that the receptor belongs to the large family of seven-helix transmembrane G protein-coupled receptors. OT is widely used in obstetrical practice to promote labor and delivery (10Andersson K.E. Forman A. Ulmstem U. Clin. Obstet. Gynecol. 1983; 26: 56-77Crossref PubMed Scopus (17) Google Scholar). On the other hand, blockade of the OTR may provide a unique approach for treatment of preterm labor by prolonging uterine quiescence (11Williams P.D. Bock M.J. Evans B.E. Freidinger R.M. Pettibone D.J. Adv. Exp. Med. Biol. 1998; 449: 473-479Crossref PubMed Scopus (29) Google Scholar, 12Goodwin T.M. Zograbyan A. Clin. Perinatol. 1998; 25: 859-871Abstract Full Text PDF PubMed Google Scholar, 13Chan W.Y. Wo N.C. Stoev S.T. Cheng L.L. Manning M. Exp. Physiol. 2000; 85S: 7S-18SCrossref Google Scholar). 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,Thr4,Orn8]vasotocin (Atosiban). This peptide antagonist has been shown to inhibit uterine contractions in women with threatened and established preterm labor (14Valenzuela 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. Creasy G.W. Am. J. Obstet. Gynecol. 2000; 182: 1184-1190Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 15Goodwin T.M. Valenzuela G. Silver H. Hayashi R. Creasy G.V. Lane R. Am. J. Perinatol. 1996; 13: 143-146Crossref PubMed Scopus (88) Google Scholar). 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 helpful 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 (16Yarwood N.J. Wheatley M. Adv. Exp. Med. Biol. 1995; 395: 343-344PubMed Google Scholar, 17Chini B. Mouillac B. Balestre M.N. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Andriolo M. Pupier S. Jard S. Barberis C. FEBS Lett. 1996; 397: 201-206Crossref PubMed Scopus (97) Google Scholar, 18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In parallel, a major contribution to peptide antagonist binding affinity by the upper part of transmembrane domain (TMD) VII has been demonstrated (18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).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 mammary gland, the guinea pig uterus, and the rabbit amnion or transfected in COS cells (recombinant porcine receptor). These studies allowed the identification of the receptor as a glycoprotein with an apparent molecular mass between 65 and 80 kDa (18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Fahrenholz F. Hackenberg M. Muller M. Eur. J. Biochem. 1988; 174: 81-85Crossref PubMed Scopus (21) Google Scholar, 20Muller M. Soloff M.S. Fahrenholz F. FEBS Lett. 1989; 242: 333-336Crossref PubMed Scopus (23) Google Scholar, 21Kojro E. Hackenberg M. Zsigo J. Fahrenholz F. J. Biol. Chem. 1991; 266: 21416-21421Abstract Full Text PDF PubMed Google Scholar, 22Hinko A. Soloff M.S. Potier M. Endocrinology. 1992; 130: 3554-3559Crossref PubMed Scopus (20) Google Scholar). 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 peptie 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,Thr4,Orn8,Tyr(3125I)-NH29]vasotocin (125I-OTA) (23Elands J. Barberis C. Jard S. Tribollet E. Dreifuss J.J. Bankowski K. Manning M. Sawyer W.H. Eur. J. Pharmacol. 1988; 147: 197-207Crossref PubMed Scopus (250) Google Scholar), we have designed, synthesized, and characterized a new radiolabeled photosensitive antagonist containing an azido group on the amino acid at position 9, termed125I-ZOTA 2E. Carnazzi, A. Aumelas, B. Mouillac, C. Breton, L. Guillou, C. Barberis, and R. Seyer, manuscript submitted for publication. 2E. Carnazzi, A. Aumelas, B. Mouillac, C. Breton, L. Guillou, C. Barberis, and R. Seyer, manuscript submitted for publication.. Because this novel radioligand displays an equivalent high affinity for the human OTR and the structurally related human V1aarginine-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 (24Carnazzi E. Aumelas A. Phalipou S. Mouillac B. Guillon G. Barberis C. Seyer R. Eur. J. Biochem. 1997; 247: 906-913Crossref PubMed Scopus (9) Google Scholar, 25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) as well as from site-directed mutagenesis and 3D molecular modeling studies (27–29; for review, see Ref. 30Barberis C. Mouillac B. Durroux T. J. Endocrinol. 1998; 156: 223-229Crossref PubMed Scopus (234) Google Scholar), 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 with125I-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.DISCUSSIONIt 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 an125I-OTA-derived antagonist (18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). 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 125I-ZOTA is based on that of I-OTA for which properties were described more than 10 years ago (23Elands J. Barberis C. Jard S. Tribollet E. Dreifuss J.J. Bankowski K. Manning M. Sawyer W.H. Eur. J. Pharmacol. 1988; 147: 197-207Crossref PubMed Scopus (250) Google Scholar). We have first pharmacologically and functionally characterized 125I-ZOTA2 (present study) and demonstrated that this OT antagonist combined high affinity, low nonspecific binding, easiness and efficiency of radioiodination, and appreciable covalent binding yield. Like 125I-OTA (40Thibonnier M. Conarty D.M. Preston J.A. Wilkins P.L. Berti-Mattera L.N. Mattera R. Adv. Exp. Med. Biol. 1998; 449: 251-276Crossref PubMed Scopus (125) Google Scholar),125I-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-polyacrylamide gels as a unique glycosylated broad band with an apparent molecular mass of 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 (19Fahrenholz F. Hackenberg M. Muller M. Eur. J. Biochem. 1988; 174: 81-85Crossref PubMed Scopus (21) Google Scholar, 20Muller M. Soloff M.S. Fahrenholz F. FEBS Lett. 1989; 242: 333-336Crossref PubMed Scopus (23) Google Scholar, 21Kojro E. Hackenberg M. Zsigo J. Fahrenholz F. J. Biol. Chem. 1991; 266: 21416-21421Abstract Full Text PDF PubMed Google Scholar, 22Hinko A. Soloff M.S. Potier M. Endocrinology. 1992; 130: 3554-3559Crossref PubMed Scopus (20) Google Scholar). Deglycosylation of the photolabeled OTR converted the 70–75-kDa protein into two bands of molecular masses of ≈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 (21Kojro E. Hackenberg M. Zsigo J. Fahrenholz F. J. Biol. Chem. 1991; 266: 21416-21421Abstract Full Text PDF PubMed Google Scholar). By contrast, using the same photolabeling conditions (incubation for 1 h at 30 °C followed by 1 min of irradiation), the human V1a receptor was degraded during incubation with the ligand, leading to two bands of molecular masses of ≈85–90 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 an NH2-terminal-truncated receptor. The latter results from a proteolytic cleavage of the 85–90-kDa band by an endogenous metalloproteinase present in the CHO membrane preparation (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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 an NH2-terminal-truncated form at 30 kDa (41Kojro E. Eich P. Gimpl G. Fahrenholz F. Biochemistry. 1993; 32: 13537-13544Crossref PubMed Scopus (81) Google Scholar, 42Kojro E. Postina R. Gilbert S. Bender F. Krause G. Fahrenholz F. Eur. J. Biochem. 1999; 266: 538-548Crossref PubMed Scopus (17) Google Scholar). In that case, the proteolytic cleavage occurred between Gln92 and Val93 in the six-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 (42Kojro E. Postina R. Gilbert S. Bender F. Krause G. Fahrenholz F. Eur. J. Biochem. 1999; 266: 538-548Crossref PubMed Scopus (17) Google Scholar). 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 (18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 42Kojro E. Postina R. Gilbert S. Bender F. Krause G. Fahrenholz F. Eur. J. Biochem. 1999; 266: 538-548Crossref PubMed Scopus (17) Google Scholar). By contrast, the metalloproteinase cleavage occurred in the human V1a receptor after incubation with125I-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 (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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 that125I-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 three amino acid residues only, Leu114-Val115-Lys116 in the OTR, which corresponds to Val126-Val127-Lys128 in the human V1a receptor (Figs. 3 and 4). Based on the high resolution x-ray structure of bovine rhodopsin (43Palczewski 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. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4991) Google Scholar), these residues are predicted to be located at the top of TMD 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 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 (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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,125I-[Lys(3N3Phpa)8]HO-LVA and125I-3N3Phpa-LVA. The Lys128 in the human V1a, corresponding to Lys116 in the human OTR, is well conserved throughout the AVP/OT receptor family (30Barberis C. Mouillac B. Durroux T. J. Endocrinol. 1998; 156: 223-229Crossref PubMed Scopus (234) Google Scholar). Moreover, this residue has been shown to play a pivotal role in the binding of agonists (27Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar), two different classes of peptide antagonists such as linear peptides (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) or the cyclic peptide d(CH2)5[Tyr(Me)2]AVP (29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar) (see also Fig. 10), and also the nonpeptide compound SR 49059 (29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar). 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 and125I-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 π-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 (44Burley S.K. Petsko G.A. Adv. Protein Chem. 1988; 39: 125-189Crossref PubMed Scopus (799) Google Scholar). Alternatively, the ammonium group of Lys could form a hydrogen bond with a carbonyl function of the peptide backbone. The predicted interaction between Lys128and 125I-ZOTA is in good agreement with 3D antagonists/V1a receptor models that we have constructed previously for V1a-selective linear and cyclic peptide antagonists (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar). Moreover in the present study, we have performed the photolabeling of the K128A mutant V1areceptor using a high concentration of 125I-ZOTA (around 2–3 nm), but we were not able to directly measure theK d of the radioligand in saturation studies. This suggests that the affinity of the ligand 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 (29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar), mutation of Lys128 of the human V1areceptor 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 125I-HO-LVA (45Barberis 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. Elands J. Neuroendocrinology. 1995; 62: 135-146Crossref PubMed Scopus (92) Google Scholar), and K i was 43 ± 6.8 nm (n = 3). This value is in agreement with that measured in COS cells (29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar). Altogether, these data magnify the role of Lys128 (Lys116 in OTR) and demonstrate again that this residue contributes to the overlap of agonist- and antagonist-binding sites in the human V1areceptor binding pocket (29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar). The role of Lys128 could be compared with 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 (46Strader C.D. Sigal I.S. Candelore M.R. Rands E. Hill W.S. Dixon R.A.F. J. Biol. Chem. 1988; 263: 10267-10271Abstract Full Text PDF PubMed Google Scholar).As depicted in Fig. 10, most of the interactions between linear or cyclic peptide antagonist ligands and the V1a receptor (25Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 28Chini B. Mouillac B. Ala Y. Balestre M.N. Trumpp-Kallmeyer S. Hoflack J. Elands J. Hibert M. Manning M. Jard S. Barberis C. EMBO J. 1995; 14: 2176-2182Crossref PubMed Scopus (162) Google Scholar, 29Cotte N. Balestre M.N. Aumelas A. Mahe E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 1-12Crossref PubMed Scopus (60) Google Scholar, 30Barberis C. Mouillac B. Durroux T. J. Endocrinol. 1998; 156: 223-229Crossref PubMed Scopus (234) Google Scholar) 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 125I-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 (18Postina R. Kojro E. Fahrenholz F. J. Biol. Chem. 1996; 271: 31593-31601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) are in agreement with such a model. Based on photoaffinity labeling results, 3D docking of two linear peptide antagonists into the V1areceptor was equivalent to that of the natural hormone AVP. When bound to the receptor despite an open chain, the two linear peptide antagonists 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 125I-ZOTA is also able to enter this central binding pocket and interact with the V1areceptor in a similar way while establishing its own network of molecular interactions. Because (i) 125I-ZOTA displays an equivalent affinity for the human OTR and the human V1areceptor, (ii) several residues involved in the binding of all classes of antagonists are conserved throughout the AVP/OT receptor family, and (iii) OTR and V1a share a significant sequence identity in the upper part of TMD II, TMD III, TMD VI, and TMD VII as well as in the first extracellular loop, we propose that binding sites for125I-ZOTA in the OTR and V1a receptor could be equivalent. Howe" @default.
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• W2100492328 title "Direct Identification of Human Oxytocin Receptor-binding Domains Using a Photoactivatable Cyclic Peptide Antagonist" @default.
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