CNR - Institute of Neuroscience CNR
Institute of Neuroscience


Molecular and cellular pharmacology of oxytocin and vasopressin receptors

With more than 600 potential members in the human genome, seven transmembrane receptors (also known as G-protein-coupled receptors, GPCRs) are key regulators of extracellular signal transduction and cell homeostasis. Due to their widespread involvement in biological processes, mutations or functional alterations of GPCRs underlie several human diseases, including neurodegenerative, neuro-endocrine, cardiovascular and neoplastic disorders. Not surprisingly, more than 60% of the currently marketed drugs exert their therapeutic actions thanks to their ability to interact with specific members of the GPCR superfamily. However, despite their physiological and clinical relevance, several fundamental aspects of GPCR signaling and regulation still need to be clarified. In our laboratory, we are investigating the cell biology and molecular pharmacology of the oxytocin (OT) and vasopressin (AVP) receptors, which includes three vasopressin receptors (V1A, V1B and V2) and one oxytocin receptor (OTR), all belonging to the GPCR superfamily.

Molecular pharmacology of OT/AVP receptors

1) Design and characteriztion of functional selective analogs

It is now widely accepted that GPCRs can couple to different coupled G-proteins, a phenomenon known as "promiscuous coupling". However, the conformational changes characterising these different receptor states are still poorly understood, as, at molecular level, receptor coupling to multiple G-proteins is a very complex phenomenon. One current idea is that GPCRs are in equilibrium among a number of resting, active and inactive states characterized by different and dynamic structural conformations. The receptors capable of coupling to more than one Gα subunit can acquire different active states with different affinities for the different G-protein complexes, and agonists may be capable of "selecting" one of these possible active states, a phenomenon termed "agonist-directed trafficking of receptor stimulus". Analogues capable of promoting selective receptor/Gα coupling, such as the very recently described "biased agonists" have enormous pharmacological and clinical importance because they may activate, for each single receptor subtype, only the "appropriate" downstream signaling pathways, representing a new class of highly selective compounds. In line with this view, we have recently found that atosiban, an OT-derived peptide, posses biased agonist properties at the human OTR (Reversi et al J Biol Chem 2005).


To screen for functional selective OTR peptides, we employed an untagged hOTR and a novel biosensor developed in the laboratory of Celine Gales (Toulouse, France) in which the energy transfer occurs within two subunits of the heterotrimeric G protein complex. In particular, the energy donor is a Galpha subunit fused to Rluc and the acceptor is a Gγ2 subunit fused to GFP10 (GFP10- Gγ2). In this case, Rluc was inserted in the loop connecting helices A and B of the G protein, at position 91 of the aminoacid sequence of Gαi1-3, Gα0A and Gα0B, and at position 97 of the Gαq sequence. The position of Rluc insertion was determined on the basis of the crystal structure of active, inactive, and transition state of several Gα subunits. As shown in Figure 1, when the energy donor is located at this position, the BRET biosensor may monitor the activation of the G protein thanks to the opening of the Gα GTPase site and the subsequent movement of the alpha helices domain through the linker 1 region. This structural re-arrangement, which allows the GDP/GTP exchange, increases the distance between Rluc located at position 91 (or 97 Gαq) and the GFP10 positioned in Gγ2; as a consequence, there will be a decrease in the transfer of energy between the donor and the acceptor. The activation of G protein, due to receptor activation, will thus correspond to a decrease in the basal BRET signal measured in the absence of ligand (Fig. 1). Using this sensor, BRET2 changes between GFP10- Gγ2 and Gαq, Gαi1, Gαi2 and Gαi3-RLUC were monitored in response to the activation of the human OTR by different peptides. These data confirmed the promiscuous interaction of the OTR with Gαq and Gαi1-3, and also monitored the agonist nature of the interaction, i.e. the G protein activation induced by receptor activation. Furthermore, they indicate that these biosensors can be used to screen for functional selective analogues.

2)Design and characterization of "bivalent ligands"

Evidence is accumulating that GPCR do not function as monomers but as dimers/oligomers that may be targeted by specific ligands, such as bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. We recently started to investigate the pharmacological properties of the first bivalent ligands for the human OTR and V1AR, synthesized in the laboratory of Prof. Manning (University of Toledo, Ohio, USA). Suberic acid, served as the spacer joining Orn or Lys residues in OTR/V1AR agonists. Our preliminary data indicate that bivalent analogs promotes IP accumulation at very low concentrations (10-12-10-9 M),and possesses a high intrinsic activity (as shown by the high degree of maximal stimulation). These findings suggest that sub8(dLVT)2 may represent a lead of a new class of highly potent compounds that may act by activating a subpopulation of receptors in an high affinity state, whose further pharmacological characterization and development may represent a real improvement in the design of new analogues to treat disorders in which the OT/AVP peptide/receptor systems are involved.

Intracellular trafficking of OT/AVP receptors

As in the case of most G-protein coupled receptors (GPCRs), agonist stimulation of human oxytocin receptors (OTRs) leads to desensitization and internalization; however, little was known about the subsequent intracellular OTR trafficking, which is crucial for re-establishing agonist responsiveness. We examined receptor resensitization by using HEK293T cells stably expressing human OTRs. Upon agonist activation, the receptors were almost completely sequestered inside intracellular compartments that were not labelled by lysosomal markers, thus indicating that the internalized receptors were not sorted to degrading organelles. Instead, we found that almost 85% of the receptors had returned to the cell surface after four hours indicating an almost complete receptor recycling. Subsequent investigations of receptor recycling pathways showed that OTRs localize 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/lysosomal compartments). Taken together, these data indicate that OTRs are capable of very efficient and complete resensitization due to receptor recycling via the "short cycle".

We have now synthesized fluorescent OT analogs to follow in vivo the internalization of the OTR. Furthermore, we have produced GFP tagged Gα-proteins to follow their destiny after OTR activation.


  • Innamorati G, Giannone F, Guzzi F, Rovati GE, Accomazzo MR, Chini B, Bianchi E, Schiaffino MV, Tridente G, Parenti M (2009) Heterotrimeric G proteins demonstrate differential sensitivity to beta-arrestin dependent desensitization. Cell. Signal. 21:1135-42.
  • Conti F, Sertic S, Reversi A, Chini B (2009) Intracellular trafficking of the human oxytocin receptor: evidence of receptor recycling via a Rab4/Rab5 "short cycle". Am. J. Physiol. Endocrinol. Metab. 296:E532-42.
  • Chini B, Manning M, Guillon G (2008) Affinity and efficacy of selective agonists and antagonists for vasopressin and oxytocin receptors: an "easy guide" to receptor pharmacology. Prog. Brain Res. 170:513-7.
  • Manning M, Stoev S, Chini B, Durroux T, Mouillac B, Guillon G (2008) Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents. Prog. Brain Res. 170:473-512.
  • Reversi A, Rimoldi V, Marrocco T, Cassoni P, Bussolati G, Parenti M, Chini B (2005) The oxytocin receptor antagonist atosiban inhibits cell growth via a "biased agonist" mechanism. J. Biol. Chem. 280:16311-8.


AIRC, Milano

Italian Ministry of University and Scientific Research, PRIN

Fondazione CARIPLO, Milano


  • C. Gales, Dipartimento di Scienze Biomediche e Oncologia Umana, Universit di Torino, Torino, Italy.
  • M. Manning, Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, USA.


PI photo

Bice Chini

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