Abstract
We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit β-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.
Highlights
The oxytocin receptor couples to multiple G proteins, leading to different physiological responses
We found that the Gi functional selective ligands generated G protein activation without -arrestin recruitment or oxytocin receptor (OTR) internalization, indicating that they have a bias toward -arrestin activity
No differences between the bioluminescent resonance energy transfer (BRET) ratio of OTR-GFP2 and that of the negative control CD4-GFP10, which is plasma membrane-located as the OTR but does not interact with G protein complexes, were found
Summary
The oxytocin receptor couples to multiple G proteins, leading to different physiological responses. Two peptides (DNalOVT and atosiban) activated only Gi1 or Gi3, failed to recruit -arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual Gi/o family members Both analogs inhibited cell proliferation, showing that a single Gi subtype-mediated pathway is sufficient to prompt this physiological response. In human embryonic kidney HEK293 cells stably transfected with human OTRs, receptor coupling to Gi is responsible for inhibiting cell growth, whereas receptor coupling to a pertussis toxin (PTX)-insensitive complex (possibly Gq) stimulates cell growth [5, 7] Because of this heterogeneity in the final outcome of receptor activation, functional selective ligands will be of great help in identifying the roles of the different OTR-elicited pathways in physiological functions; as they may have distinct therapeutic actions, they may lead to new therapeutic approaches. We found that the Gi functional selective ligands generated G protein activation without -arrestin recruitment or OTR internalization, indicating that they have a bias toward -arrestin activity
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