Binding Characteristics of Two Oxytocin Variants and Vasopressin at Oxytocin Receptors from Four Primate Species with Different Social Behavior Patterns

Abstract

A clade of New World monkeys (NWMs) exhibits considerable diversity in both oxytocin (OT) ligand and oxytocin receptor (OTR) structure. Most notable is the variant Pro⁸-OT, with proline instead of leucine at the eighth position, resulting in a rigid bend in the peptide backbone. A higher proportion of species that express Pro⁸-OT also engage in biparental care and social monogamy. When marmosets (genus Callithrix), a biparental and monogamous Pro⁸-OT NWM species, are administered the ancestral Leu⁸-OT, there is no change in social behavior compared with saline treatment. However, when Pro⁸-OT is administered, marmosets' sociosexual and prosocial behaviors are altered. The studies here tested the hypothesis that OTR binding affinities and OT-induced intracellular Ca²⁺ potencies would favor the native OT ligand in OTRs from four primate species, each representing a unique combination of ancestral lineage, breeding system, and native OT ligand: humans (Leu⁸-OT, monogamous, apes), macaques (Leu⁸-OT, nonmonogamous, Old World monkey), marmosets (Pro⁸-OT, monogamous, NWM), and titi monkeys (Leu⁸-OT, monogamous, NWM). OTRs were expressed in immortalized Chinese hamster ovary cells and tested for intact-cell binding affinities for Pro⁸-OT, Leu⁸-OT, and arginine vasopressin (AVP), as well as intracellular Ca²⁺ signaling after stimulation with Pro⁸-OT, Leu⁸-OT, and AVP. Contrary to our hypothesis, Pro⁸-OT bound at modestly higher affinities and stimulated calcium signaling at modestly higher potencies compared with Leu⁸-OT in all four primate OTRs. Thus, differences downstream from a ligand-receptor binding event are more likely to explain the different behavioral responses to these two ligands.

Fig. 1.

Representative saturation assays for ¹²⁵I-OVTA binding to OTRs from each of the four species. Cells on 96-well plates were incubated on ice in 50 µl binding medium with the indicated concentrations of ¹²⁵I-OVTA for 3 hours, and specific binding was then quantified. Data are from a single experiment with all four receptors tested side by side in triplicate. Values for this experiment are in good agreement with the average values in the Results and Table 1: humans, B_max = 9.7 fmol/well and K_d = 0.12 nM; macaques, B_max = 6.9 fmol/well and K_d = 0.144 nM; marmosets (R10), B_max = 34 fmol/well and K_d = 0.30 nM; and titi monkeys, B_max = 30 fmol/well and K_d = 0.24 nM.

Fig. 2.

Competition curves for Pro⁸-OT and Leu⁸-OT for each primate species OTR. Increasing concentrations of competitor ligand (Pro⁸-OT, Leu⁸-OT, or AVP) were added to a constant concentration of ¹²⁵I-OVTA in intact CHO cells expressing one of four primate OTRs. All values are expressed as the percentage of the maximal binding in the absence of OT or AVP.

Fig. 3.

Intracellular Ca²⁺ increases for each primate species OTR. Increasing concentrations of Pro⁸,-OT, Leu⁸-OT, or AVP were used to stimulate intracellular Ca²⁺ mobilization in CHO cells expressing one of four primate OTRs. All values are expressed as the percentage of the maximal response to 10⁻⁶ M Leu⁸-OT for each species.