Synthesis and application of a radioiodinated photoreactive oxytocin antagonist

Kojro, Elżbieta; Hackenberg, Mario; Klein, Uwe; Fahrenholz, Falk

doi:10.1007/978-94-011-3034-9_279

Cited by 4 publications

(8 citation statements)

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“…Binding Assay-In saturation and displacement assays [ Photoaffinity Labeling-The synthesis of the radioiodinated photoreactive oxytocin antagonist was performed by introducing a photoreactive 4-azidophenylamidino group at Orn 8 of OTA and by radioiodination at Tyr 9 (12). Photoaffinity labeling was performed as described elsewhere (12).…”

Section: Methodsmentioning

confidence: 99%

“…Photoaffinity labeling was performed as described elsewhere (12). Briefly, 100 g of membrane protein from transfected COS.M6 cells containing between 1.3 and 2.0 pmol/mg chimeric receptors were incubated with 0.25 nM 125 I-labeled photoreactive antagonist in 200 l of binding buffer (50 mM Hepes/NaOH, pH 7.4, 10 mM MnCl 2 ) for 30 min at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cloning of the oxytocin receptor from five mammalian species has shown that it belongs to the G protein-coupled receptor family with seven transmembrane helices (7)(8)(9)(10)(11). Photoaffinity labeling studies with a photoreactive OT antagonist identified the oxytocin receptor as a glycoprotein with an apparent molecular weight between 68,000 and 80,000 (12). Functional domains, especially the topography of the ligand-binding site, are presently unknown.…”

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confidence: 99%

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Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Postina

Kojro

Fahrenholz

1996

Journal of Biological Chemistry

119

102

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The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V 2 vasopressin receptor, chimeric "gain in function" V 2 /OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. Oxytocin belongs to the family of neurohypophyseal nonapeptide hormones, which are characterized by a cyclic hexapeptidic part with a disulfide bridge between Cys 1 and Cys 6 and a C-terminal linear tripeptidic extension (Fig. 1). Its major physiological role is to induce contraction of uterine smooth muscle and mammary myoepithelium. As pregnancy nears term, uterine smooth muscle exhibits enhanced sensitivity to oxytocin (1). Oxytocin is widely used to induce labor, and conversely, oxytocin antagonists are being evaluated for the treatment of preterm labor (2, 3). Furthermore, OT 1 has been shown to play an important role in reproduction biology by influencing sexual behavior and response, as well as the formation of social bonds (4). These oxytocinergic effects are mediated by a specific cell surface receptor, the OT receptor which is also found in other organs such as brain (5) and ovary (6). Cloning of the oxytocin receptor from five mammalian species has shown that it belongs to the G protein-coupled receptor family with seven transmembrane helices (7-11). Photoaffinity labeling studies with a photoreactive OT antagonist identified the oxytocin receptor as a glycoprotein with an apparent molecular weight between 68,000 and 80,000 (12). Functional domains, especially the topography of the ligand-binding site, are presently unknown.The aim of our investigations was to determine domains of the OT receptor that are involved in high-affinity binding of both agonists and antagonists. Since all known OT receptors share almost identical primary structure and bind OT with high affinity, it can be assumed that alterations of this naturally optimized binding site will result in a loss of high-affinity oxytocin binding. This effect can be caused either by disturbances of specific receptor-ligand interactions but also by changes in the overall receptor conformation. Therefore, we decided to produce "gain in function" receptors, to identify OT receptor domains that contribute to high-affinity OT and oxytocin antagonist binding.As starting protein for the identification of oxytocin binding domains, we used the related V 2 vasopressin receptor. This receptor mediates the antidiuretic action of [8-arginine]vasopressin in kidney. It shows 40% overall sequence identity to the oxytocin receptor with the highest sequence similarity in the transmembrane regions and the extracellular loops. Although vasopressin and oxytocin differ only at position 3 of the cyclic peptide part and position 8 of the C-terminal tripeptide amide, the V 2 vasopressin receptor dis...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Postina

Kojro

Fahrenholz

1996

Journal of Biological Chemistry

119

102

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“…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 masses of Ϸ48 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 of irradiation), the human V 1a receptor was degraded during incubation with the ligand, leading to two bands of molecular masses of Ϸ85-90 and 46 kDa.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Binding and Antagonistic Properties Of The New Photoactivatablementioning

confidence: 99%

Direct Identification of Human Oxytocin Receptor-binding Domains Using a Photoactivatable Cyclic Peptide Antagonist

Breton

Chellil

Kabbaj-Benmansour

et al. 2001

Journal of Biological Chemistry

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Design, Synthesis and Pharmacological Characterization of a Potent Radioiodinated and Photoactivatable Peptidic Oxytocin Antagonist

et al. 2001

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Using a segment strategy, we have synthesized four iodinated photoactivatable cyclic peptidic ligands of oxytocin, bearing a beta-mercapto-betabeta-cyclopentamethylene propionic group (Pmp) on their N-terminus. All the syntheses were RP-HPLC monitored, and the compounds were HPLC purified. They were characterized by 1H NMR, MALDI-TOF, or FAB mass spectrometries. The affinities of Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (20), Pmp-Tyr-Ile-Thr-Asn-Cys-Gly-Orn-Phe(3I,4N3)-NH2 (21), Pmp-Tyr(Me)-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (22), and Pmp-Tyr-Ile-Thr-Asn-Cys-Pro-Orn-Phe(3I,4N3)-NH2 (23) were evaluated as inhibition constants (K(i), in nM) for the human oxytocin receptor expressed in Chinese hamster ovary cells by displacement of a radioiodinated disulfide-cyclized antagonist (Elands et al. Eur. J. Pharmacol. 1987, 147, 197-207). The most potent of them, compound 22, was synthesized by another method in order to allow its radiolabeling by 125I. Its dissociation constant (K(d)) for the human oxytocin receptor, directly measured in saturation studies, was 0.25 +/- 0.04 nM, and its antagonist properties were determined by inactivation of phospholipase C, thus obtaining an inactivation constant (K(inact)) of 0.18 +/- 0.02 nM, evaluated by inositol phosphate accumulation. This compound is a very good tool for the mapping of peptidic antagonist binding sites in the human oxytocin receptor.

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Synthesis and application of a radioiodinated photoreactive oxytocin antagonist

Cited by 4 publications

References 5 publications

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

Direct Identification of Human Oxytocin Receptor-binding Domains Using a Photoactivatable Cyclic Peptide Antagonist

Design, Synthesis and Pharmacological Characterization of a Potent Radioiodinated and Photoactivatable Peptidic Oxytocin Antagonist

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