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

Elzbieta Kojro,Falk Fahrenholz,Rolf Postina

doi:10.1074/jbc.271.49.31593

Elzbieta Kojro, Falk Fahrenholz + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.49.31593

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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 V2 vasopressin receptor, chimeric "gain in function" V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.

Highlights

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
The results of this study demonstrate that different receptor domains determine the selectivity and affinity of the oxytocin receptor for peptide agonists and antagonists (Fig. 7)
The synergistic increase in affinity for oxytocin by exchange of the first three V2 receptor domains for those of the OT receptor demonstrates a concerted binding of flexible extracellular domains to a ligand which itself can exist in several conformations [28]

Summary

Peptide Binding Sites of the Oxytocin Receptor

Changed, such chimeric receptors would in principle couple to G␣s and respond to oxytocin with high efficiency activation of the adenylyl cyclase system. This approach could allow the localization of the binding site for antagonists with high specificity for the oxytocin receptor. Whereas for the transfer of a high-affinity oxytocin agonist binding site into the V2 receptor the first three extracellular oxytocin receptor domains were essential; just seven amino acids of the upper part of transmembrane helix seven of the oxytocin receptor were sufficient to introduce a high-affinity binding site for an oxytocin peptide antagonist into the V2 receptor. Our results demonstrate the synergistic contribution of extracellular domains on agonist binding and ligand specificity in a peptide receptor and the different localization of agonist and antagonist binding sites. The necessity of considering the outer membrane regions for further three-dimensional modeling of G protein-coupled receptors became clear

EXPERIMENTAL PROCEDURES

RESULTS

OTA Ki

The Second Extracellular Domain Also Binds to the Acyclic

The Third Extracellular Domain Interacts with the Cyclic

DISCUSSION