Oxytocin receptors: ligand binding, signalling and cholesterol dependence

doi:10.1016/S0079-6123(08)00417-2

Progress in Brain Research

Volume 170, 2008, Pages 193-204

https://doi.org/10.1016/S0079-6123(08)00417-2 Get rights and content

Abstract

The G protein coupled oxytocin receptor (OTR) reveals some specific molecular and physiological characteristics. Ligand–receptor interaction has been analysed by photoaffinity labelling, site-directed mutagenesis, the construction of receptor chimeras and molecular modelling. Major results of these studies will be summarized. The N-terminus of the OTR is mainly involved in agonist binding. Notably, antagonists that are derived from the ground structure of oxytocin, bind the receptor at distinct sites partly non-overlapping with the agonist binding site. OTRs are able to couple to different G proteins, with a subsequent stimulation of phospholipase C-β isoforms. In dependence on G protein coupling, OTRs can transduce growth-inhibitory or proliferatory signals. Some evidence is provided that OTRs are also present in form of dimeric or oligomeric complexes at the cell surface. The affinity of the receptor for ligands is strongly dependent on the presence of divalent cations (Mg²⁺) and cholesterol that both act like positive allosteric modulators. While the high-affinity state of the receptor for agonists requires divalent cations and cholesterol, the high-affinity state for antagonists is only dependent on a sufficient amount of cholesterol. Cholesterol affects ligand-binding affinity, receptor signalling and stability. Since the purification of the OTR has never been achieved, alternative methods to study the receptor in its native environment are necessary. Promising strategies for the site-specific labelling of the OTR will be presented. The employment of diverse reporter molecules introduced at different positions within the OTR might allow us in the near future to measure conformational changes of the receptor in its native lipid environment.

Section snippets

Oxytocin

Oxytocin (OT) and oxytocin-like hormones are primarily produced in magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei and are secreted from the posterior pituitary into the systemic circulation in response to a variety of stimuli such as suckling, parturition or certain kinds of stresses. Maternal OT exerts a powerful action on foetal neurons by inducing a transient excitatory-to-inhibitory switch in their GABA signalling shortly before delivery. Reduced neuronal

Oxytocin receptors

The OTR belongs to the class A (rhodopsin-like) superfamily of G protein coupled receptors (GPCRs). Within this class the vasopressin/OTR forms a small family that comprises three types of vasopressin receptors, designated as V1A, V1B and V2, and the OTR. The vasopressin V2 receptor shows only 40% overall sequence identity with the OTR, but strongly discriminates between the ligands [Arg8]vasopressin and OT (structures in Fig. 1). [Arg8]vasopressin is bound with nearly 400-fold higher affinity

Oxytocin receptor research: quo vadis?

The major goal of receptor biochemistry is to obtain structural informations about the receptor, e.g. their conformational changes induced by ligand binding or G protein association. Despite much efforts purification has been achieved only for a handful of receptors. In case of the OTR, all attempts to purify the functional receptor have been unsuccessful to date. However, even when receptor purification would be successful, uncertainty remains whether structural informations obtained with

Abbreviations

BBS

bungarotoxin-binding site

FRET

fluorescence resonance energy transfer

GFP

green fluorescent protein

oxytocin

OTR

oxytocin receptor

Ppant

phosphopantetheinyl

Sfp

surfactin production genetic locus from B. subtilis

Acknowledgements

We thank Christa Wolpert (Mainz) for technical assistance, Dr. Torsten Reinheimer (Ferring Pharmaceuticals, Copenhagen, Denmark) for cooperative support, Dr. Christopher T. Walsh (Harvard Medical School, Boston, USA) and Dr. Jun Yin (University of Chicago, USA) for the supply of the Sfp posphopantetheinyl transferase cDNA.

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G protein coupled receptors (GPCRs) are a class of membrane proteins that sense extracellular signals ranging from light to odorants and small molecules and activate intracellular signaling pathways that control important physiological responses. Being composed of 7 transmembrane helices linked by extracellular and intracellular loops, the great majority of the sequence of these receptors is embedded in the lipid membrane. Therefore, it is expected GPCR structure and function to be impacted by the surrounding lipid environment and the lipid membrane physico-chemical and mechanical properties. A large number of examples from the literature is provided to highlight the role of the lipid nature (lipid headgroup, membrane polyunsaturation and cholesterol) and membrane physical and mechanical properties (curvature elastic stress, membrane thickness and hydrophobic mismatch, fluidity) in the activity of different GPCRs. In addition, lipids are important co-factors being identified in very specific locations in several GPCR structures. GPCRs and G proteins can also be lipid post-translationally modified and such events can significantly impact membrane binding, trafficking and signaling. These aspects are all treated in this review. Understanding how the lipid can modulate GPCR activity is important not only from a fundamental point of view but also due to the fact that certain pathologies, where GPCRs are central targets, have been associated with important lipid imbalance. Establishing a link between the lipid pathological imbalance and the receptor functioning in such environment is thus essential as it can open avenues to potentially innovative therapeutic strategies.
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The complexity of oxytocin-mediated functions is strongly associated with its modulatory effects on other neurotransmission systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Signalling between oxytocin (OT) and 5-HT has been demonstrated during neurodevelopment and in the regulation of specific emotion-based behaviours. It is suggested that crosstalk between neurotransmitters is driven by interaction between their specific receptors, particularly the oxytocin receptor (OTR) and the 5-hydroxytryptamine 2C receptor (5-HTR_2C), but evidence for this and the downstream signalling consequences that follow are lacking. Considering the overlapping central expression profiles and shared involvement of OTR and 5-HTR_2C in certain endocrine functions and behaviours, including eating behaviour, social interaction and locomotor activity, we investigated the existence of functionally active OTR/5-HTR_2C heterocomplexes. Here, we demonstrate evidence for a potential physical interaction between OTR and 5-HTR_2C in vitro in a cellular expression system using flow cytometry-based FRET (fcFRET). We could recapitulate this finding under endogenous expression levels of both receptors via in silico analysis of single cell transcriptomic data and ex vivo proximity ligation assay (PLA). Next, we show that co-expression of the OTR/5-HTR_2C pair resulted in a significant depletion of OTR-mediated Gαq-signalling and significant changes in receptor trafficking. Of note, attenuation of OTR-mediated downstream signalling was restored following pharmacological blockade of the 5-HTR_2C. Finally, we demonstrated a functional relevance of this novel heterocomplex, in vivo, as 5-HTR_2C antagonism increased OT-mediated hypoactivity in mice. Overall, we provide compelling evidence for the formation of functionally active OTR/5-HTR_2C heterocomplexes, adding another level of complexity to OTR and 5-HTR_2C signalling functionality.
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Binding of agonist WAY-267,464 and antagonist WAY-methylated to oxytocin receptor probed by all-atom molecular dynamics simulations
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Citation Excerpt :
OTR peptide antagonists are currently used to delay labor—specifically preventing preterm labor. However, these peptide antagonists are typically not selective for the OTR only, resulting in many undesirable side effects: they cannot be administered orally as they get degraded too quickly within the gastrointestinal tract, and are hard to get past the blood-brain barrier [1,12,13,17,20,25–27]. Non-peptide ligands have promising potential to become orally bioavailable drugs with improved selectivity, and longer lasting effects.
Non-peptide ligands of oxytocin receptor (OTR) have promising potentialities as therapeutic agents with improved pharmacological properties. WAY-267,464 is a non-peptide agonist which loses its agonist activity when its resorcinol moiety is methylated, yielding a partial antagonist (denoted here, WAY-Methylated). This study attempts to rationalize these opposing activities by comparative analyses of structural dynamicsof OTR in complex with these ligands.
Glide extra precision (XP) docking with and without positional constraints was employed to probe alternative binding poses of both WAY-267,464 and WAY-Methylated. The more preferred configuration of each system was subjected to an extended 2 μs MD simulation and the physics-based Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) binding energy was used to rank the complexes with improved accuracy, in addition to empirical-based Glide docking score. Network analysis was performed, and the identified critical residues were cross-referenced with the experimental mutagenesis data.
The added methyl groups in the antagonist WAY-Methylated enhanced hydrophobicity, resulting in a flipped binding pose deeper in the binding pocket. Interestingly, OTR responded to the methylation by stabilizing the initial inactive conformation, decreasing fluctuations and increasing the overall secondary structural composition. Conversely, the agonist WAY-267,464 produced larger fluctuations to allow the receptor to change from the default inactive state to a state of partial activation. These transitions were further supported by the identified critical residues overlapping with experimental mutagenesis data.
These findings provide insights into the activation mechanism of OTR by WAY-267.464 and its antagonism by WAY-Methylated.
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The neuropeptide oxytocin (OXT) plays a key role in adaptive processes associated with reward, tolerance, memory and stress responses. Through interactions with brain reward and stress systems, OXT is known to play a role in several neuropsychiatric disorders, particularly those that involve altered social integration, such as alcohol and drug addiction (Heilig et al., 2016). As such, there is growing interest in the oxytocin system as a potential therapeutic target for the treatment of alcohol and substance use disorders. Accumulating preclinical evidence suggests that administration of OXT influences the development of tolerance, sensitization and withdrawal symptoms, and modulates numerous alcohol/drug-seeking and alcohol/drug-taking behaviors. Further, there is some evidence to suggest that OXT may help to reverse neuroadaptations that occur as a result of chronic alcohol or drug exposure. To date, there have been only a handful of clinical studies conducted in alcohol and drug dependent populations. This review summarizes the preclinical and clinical literature on the effects of OXT administration on alcohol- and drug-related behaviors. In addition, we discuss OXT interactions with the hypothalamic-pituitaryadrenal axis and multiple neurotransmitter systems within addiction circuitry.
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Oxytocin receptors: ligand binding, signalling and cholesterol dependence

Abstract

Section snippets

Oxytocin

Oxytocin receptors

Oxytocin receptor research: quo vadis?

Abbreviations

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Curr. Opin. Struct. Biol.

Biochim. Biophys. Acta

Eur. J. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

BJOG

J. Biol. Chem.

Biochim. Biophys. Acta.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Eur. J. Pharmacol.

Trends Endocrinol. Metab.

Receptor binding of oxytocin and vasopressin antagonists and inhibitory effects on isolated myometrium from preterm and term pregnant women

Br. J. Obstet Gynaecol.

Regulation of oxytocin receptors and oxytocin receptor signaling

Semin. Reprod. Med.

G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there?

J. Mol. Endocrinol.

Identification of dimeric and oligomeric complexes of the human oxytocin receptor by co-immunoprecipitation and bioluminescence resonance energy transfer

J. Mol. Endocrinol.

Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts

Proc. Natl. Acad. Sci. U.S.A.

Activation mechanism of human oxytocin receptor: a combined study of experimental and computer-simulated mutagenesis

Mol. Pharmacol.

Atomic-force microscopy: rhodopsin dimers in native disc membranes

Nature

Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor

Proc. Natl. Acad. Sci. U.S.A.

Cholesterol as modulator of receptor function

Biochemistry

Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane

Eur. J. Biochem.

The oxytocin receptor system: structure, function, and regulation

Physiol. Rev.

Expression of the human oxytocin receptor in baculovirus-infected insect cells: high-affinity binding is induced by a cholesterol–cyclodextrin complex

Biochemistry

Molecular cloning and functional characterization of V2 [8-lysine] vasopressin and oxytocin receptors from a pig kidney cell line

Eur. J. Biochem.