Bivalent ligands targeting oxytocin receptor homodimers selectively activate distinct G protein and β-arrestin pathways

G protein-coupled receptors can assemble into both homodimers and heterodimers. While heterodimerization has been extensively characterized, the functional consequences of homodimerization on G protein coupling, signaling, and trafficking remain poorly understood. Oxytocin receptors (OTRs) form homodimers, and we previously generated a series of homobivalent compounds composed of two modified oxytocin analogs (dOTK) linked by spacers of varying length. We already demonstrated that the bivalent ligands dOTK 2 -C8 and dOTK 2 -C10 possess an optimal spacer length that allows simultaneous binding of the dOTK moieties to each protomer of OTR homodimers, with an interface involving transmembrane domains 1 and 2 as well as helix 8, and act as superagonists at OTR/Gq-protein signaling. Moreover, dOTK 2 -C8 exhibited approximately 100- and 40-fold increase in potency compared to OT in promoting social behavior in mice and zebrafish, respectively. Here we used BRET biosensors to further explore the OTR signaling pathways activated by all bivalent ligands. We found that, in addition to Gq proteins, they activate Gi2 and Gi3, but not Gi1, GoA, or GoB, and that dOTK 2 -C8 and dOTK 2 -C10 were the only compounds capable of promoting the recruitment of β-arrestin 1 in addition to β-arrestin 2. Altogether, these results indicate that dOTK 2 -C8 and dOTK 2 -C10, stabilizing a distinct OTR dimer conformation, can selectively promote G protein and β-arrestin coupling and provide a framework for the rational development of therapeutic agents with unprecedented selectivity and potency, highly relevant in the context of neurodevelopmental and psychiatric disorders characterized by social deficits, such as autism spectrum disorder and schizophrenia.