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

Abstract

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...

The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to Gq, but Not Gi

“…Whereas truncation of some GPCRs has been shown to modify any one or more of its functions, shortening of the COOH terminus of other family members has no apparent effects (8 -10). Thus, no uniform hypothesis regarding the importance of the COOHterminal region can be applied a priori to the OTR, which has not been previously examined in detail (46,47). In view of the high degree of homology of the COOH-terminal domain of OTRs from several species, we reasoned that conservation would be associated with some important function(s).…”

A Distributed Network for Social Cognition Enriched for Oxytocin Receptors

“…Specifically, we identified epitopes that were maximally different from corresponding regions of the vasopressin V1a/V1b receptors. The affinity of the oxytocin receptor for oxytocin is only 10-fold greater than that for vasopressin (Postina et al, 1996), indicating that the putative ligand-binding domain of the oxytocin receptor is not a good candidate for generating specific antibodies. Correspondingly, the areas of highest homology between oxytocin and vasopressin receptors are located in the extracellular loops and the transmembrane helices, whereas the N terminus, C terminus, and the intracellular loops are less conserved (Gimpl and Fahrenholz, 2001).…”

Docking of Linear Peptide Antagonists into the Human V1a Vasopressin Receptor

“…As shown in Fig. 3A 11,574, and Ͼ2000 nM for human OT, V 1b , and V 2 receptor subtypes, respectively. These affinities were 60 -10,000-fold lower than that measured for the human V 1a AVP receptor (0.18 nM), establishing the [Lys(3N 3 Phpa) 8 ]HO-LVA as a selective ligand.…”