Thermodynamics of the interaction between oxytocin and its myometrial receptor in sheep: A stepwise binding mechanism Vladimir Pliska a,b, *, Gerd Folkers a,b , Vojte ˇch Spiwok c a Collegium Helveticum, Swiss Federal Institute of Technology (ETH-Zu ¨rich) and University of Zurich, Schmelzbergstrasse 25, CH-8092 Zu ¨rich, Switzerland b Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH-Zu ¨rich), Campus Ho ¨nggerberg, CH-8093 Zu ¨rich, Switzerland c Department of Biochemistry and Microbiology, Institute of Chemical Technology (VS ˇ CHT-Prague), Technicka ´ 5, CZ-166 28 Praha 6, Czech Republic 1. Introduction The contractile response of uterus to oxytocin is initiated on receptor sites located in the membrane of myometrial smooth muscle cells. In human uterus, this membrane receptor (OXTR) has a heptahelical structure [1,2] and is associated, via G q or G i protein, with the inositol phosphate signaling system. High and low affinity oxytocin binding sites occur in rat, sheep, and calf uterus [3–5] differing in their dissociation constants (K d ) and binding capacities (B). Studies on cultured myometrial cells and their membrane fractions indicate high affinity binding sites with a K d of 8  10 10 to 3  10 9 mol l 1 , and low ones with K d in the ranging from 5  10 8 to 5  10 7 mol l 1 . A clear-cut effect of GTP on both K d ’s and B’s in experiments on rat myometrial membranes could not so far be demonstrated [5]. Receptor systems displaying several sites with different binding affinities are well known from the literature, and models attempting to describe some of their specific classes were thoroughly reviewed [6–10]. Interdependent receptor forms in equilibrium were suggested in several models, most recently in a ternary model involving G-protein-activated receptor forms [11– 13]. However, outcomes of binding experiments based on measurements of the total bound ligand (not of individual complexes) cannot be matched by models in which a rapid transition between individual forms of a single receptor is Biochemical Pharmacology 91 (2014) 119–127 A R T I C L E I N F O Article history: Received 12 April 2014 Accepted 30 June 2014 Available online 7 July 2014 Chemical compounds studied in this article: Oxytocin Arginine vasopressin Tritium-labeled oxytocin: [2-tyrosine- 2,6- 3 H]oxytocin Tritium-labeled arginine vasopressin: [8-arginine, 3-phenylalanine- 3,4,5- 3 H]vasopressin Atosiban: [1-mercaptopropionic acid, 2-D- (O-ethyl)-tyrosine,4-threonine, 8-ornithine]oxytocin GTP-gS: guanosine-5 0 -O-(3- thiotriphosphate) Keywords: Oxytocin receptor Oxytocin inhibitors Myometrium cell membranes G protein-coupled receptors Ligand-receptor thermodynamics Receptor models A B S T R A C T Entropy (DS), enthalpy (DH) and heat capacity (DC p ) changes attending the oxytocin interaction with its two binding sites on myometrial cell membranes in sheep were derived from the temperature dependence of K d values. The high affinity oxytocin site (K d on the order of 10 9 mol l 1 , 25 8C), ascribed to the oxytocin receptor (OXTR), is entropy-driven in the temperature range 0–37 8C. Enthalpy component prevails as a driving force in the binding to the low affinity site (K d  10 7 ) within the higher temperature range. DC p values in both cases do not differ significantly from zero but become highly relevant in the presence of a GTP analog (10 4 M GTP-gS). Under these conditions, DC p in the low site interaction becomes negative and DS is shifted toward negative values (enthalpy drift); DC p of the high affinity site rises to a high positive value and the interaction is even more strongly entropy driven. Atosiban, a competitive antagonist of oxytocin at OXTR displays a single significant binding site on myometrial cells (K d about 10 7 mol l 1 ). Thermodynamic profiles of atosiban and the low affinity oxytocin site show conspicuous similarities, indicating that the inhibitor is bound to the low affinity site, and not, with a lower affinity, to the putative receptor protein. It is suggested that the interaction of oxytocin with its responding system on myometrial membranes follows in two distinct steps that are likely to be associated with several independent binding domains in the GPCR receptor. ß 2014 Elsevier Inc. All rights reserved. * Corresponding author at: Collegium Helveticum, ETZH–STW, Schmelzberg- strasse 25, Zu ¨ rich, Switzerland. Tel.: +41 44 830 15 40. E-mail addresses: pliska@collegium.ethz.ch, v.pliska@bluewin.ch (V. Pliska). Contents lists available at ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m http://dx.doi.org/10.1016/j.bcp.2014.06.025 0006-2952/ß 2014 Elsevier Inc. All rights reserved.