As in the case of most G-protein coupled receptors (GPCRs), agonist stimulation of human oxytocin receptors (OTRs) leads to desensitization and internalization. We examined OTR internalization in HEK293T cells where, upon agonist activation, the receptors were almost completely sequestered inside intracellular compartments. Binding and fluorescence assays showed that almost 85% of the internalized receptors returned to the cell surface after four hours, by which time cell responsiveness to the agonist was completely restored. Finally, investigations of receptor recycling pathways showed that OTRs were located in vesicles containing the Rab5 and Rab4 small GTPases (markers of the "short cycle"), whereas there was no co-localization with Rab11 (a marker of the "long cycle") or Rab7 (a marker of vesicles directed to endosomal/lysosomalcompartments). Taken together, these data indicate that OTRs are capable of very efficient and complete resensitization due to receptor recycling via the "short cycle".

The oxytocin receptor (OTR) is a promiscuous G-protein coupled receptor that couples to both Gaq and Gai and whose stimulation leads to the activation of different intracellular signaling pathways. OT –derived peptides that activate selectively either the Gai or Gaq pathways were characterized in our laboratory (Reversi et al 2005 J Biol Chem). In order to assay the capability of these analogs to promote receptor internalization we developed two fluorescent agonists: dLVT-Alexa568 and atosiban-Alexa568). After stimulation of the cells with dLVT-Alexa568, that promotes both OTR/Gaq and OTR/Gai coupling, we observed internalization of both the receptor and the fluorescent peptide. The two molecules were initially colocalized in vesicles, but this colocalization was lost after two hours when the receptor and the ligand were found in different vesicles, suggesting that they followed different trafficking pathways. On the contrary, atosiban-Alexa568, a selective OTR/Gai, did not lead to any change in receptor localization at the plasma membrane even after one hour of stimulation. Since OTRs are expressed in the CNS in neuronal and glial cells, we finally used a neuronal cell line (the mouse neuroblastoma Neuro2A cells) transiently tranfected with human OTR to further investigate OTR trafficking. Both the OTR-EGPF and OTR tagged at its N-terminus with an HA epitope were localized at the plasma membrane as expected, but, again, receptor internalization was observed only after stimulation with dLVT-Alexa568. All together, these data suggest that Gaq activation plays an important role in OTR internalization.