Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

Center for Physiology and Pharmacology Medical University of Vienna Schwarzspanierstrasse 17 1090 Vienna Austria

Center for Physiology and Pharmacology Medical University of Vienna Schwarzspanierstrasse 17 1090 Vienna Austria School of Biomedical Sciences The University of Queensland QLD 4072 Brisbane Australia

CUBE Division of Computational Systems Biology Department of Microbiology and Ecosystem Science University of Vienna Althanstrasse 14 1090 Vienna Austria

Department of Drug Design and Pharmacology University of Copenhagen Jagtvej 162 2100 Copenhagen Denmark

Harris Wellbeing Preterm Birth Research Centre Department of Cellular and Molecular Physiology Institute of Translational Medicine University of Liverpool L69 3BX United Kingdom

Institute for Molecular Bioscience The University of Queensland QLD 4072 Brisbane Australia

IST Austria Am Campus 1 3400 Klosterneuburg Austria CUBE Division of Computational Systems Biology Department of Microbiology and Ecosystem Science University of Vienna Althanstrasse 14 1090 Vienna Austria

Laboratory of Radioisotopes Institute of Organic Chemistry and Biochemistry CAS Flemingovo nám 2 CZ 16610 Prague 6 Czech Republic

School of Biomedical Sciences The University of Queensland QLD 4072 Brisbane Australia

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