Oxytocin receptor signalling

doi:10.1016/S0079-6123(08)00415-9

Progress in Brain Research

Volume 170, 2008, Pages 167-176

https://doi.org/10.1016/S0079-6123(08)00415-9 Get rights and content

Abstract

The great diversity of the expression sites and proposed function of the oxytocin (OXT) receptor (OXTR) is paralleled by a diversity of its signalling pathways, many of which have still remained unexplored. We have used different approaches to discover novel pathways. By means of a phosphoproteomics approach, we have detected several distinct OXT-induced changes in tyrosine as well as threonine phosphorylation states of intracellular protein in myometrial cells. The most prominent change involved dephosphorylation of a 95-kDa phosphothreonine moiety. By N-terminal amino acid microsequence analysis, this moiety was shown to correspond to eukaryotic translation factor eEF2. This protein is a key regulator of protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. These findings define a novel mechanism by which OXT assumes a so far unrecognized trophic function. We next elucidated the intracellular pathway(s) involved. We found that this effect is not mediated by any of the known pathways known to induce eEF2 dephosphorylation (mTOR, ERK1/2 or p38) but by protein kinase C. Consistent with this idea, we also found that direct stimulation of protein kinase C with a phorbol ester induced eEF2 dephosphorylation in myometrial cells. Using phosphoERK antibodies, we discovered by Western blotting that OXT induced phosphorylation of a higher molecular weight ERK-related protein. We were able to show that this band corresponded to “big MAP kinase1” or ERK5. ERK5 is part of a distinct MAPK cascade and promotes expression of the myosin light chain gene and plays an obligatory role in muscle cell development and differentiation. The role of ERK5 in myometrium has remained unexplored, but it is likely to represent an important novel pathway mediating OXT's effects on smooth muscle function. Further elucidation of these novel signalling pathways will have significant relevance for the development of novel pathway-specific OXTR agonists and antagonists.

Section snippets

General thoughts on oxytocin receptor signalling

The oxytocin (OXT) receptor (OXTR) is a member of the family of G protein-coupled receptors (GPCRs) and, together with the three vasopressin receptor subtypes V1a, V1b and V2, the OXTR forms a subfamily of structurally related GPCRs. The OXTR mediates a very wide spectrum of physiological actions: OXTRs are expressed in different specific brain regions where they mediate behavioural functions, ranging from maternal behaviour to specific sexual and social behaviours. In the periphery, OXTRs

Novel OXTR signalling target: eEF2

In an attempt to characterize more completely the spectrum of signalling pathways set into action following activation of the OXTR, we have determined, on a global level, to what extent OXTR signalling involves phosphorylation or dephosphorylation of specific signalling components. We analyzed OXT-induced changes in the protein phosphorylation pattern in lysates of CHO cells stably transfected with the rat OXTR (CHO-OTR cells), using one- and two-dimensional polyacrylamide gel electrophoresis

Novel OXTR-linked signalling pathway: ERK5

Using phosphoMAPK antibodies, we discovered by Western blotting that OXT induced phosphorylation of a higher molecular weight ERK-related protein. We were able to show that this band corresponded to “big MAP kinase1” or ERK5. ERK5 is part of a distinct MAPK cascade and promotes expression of the myosin light chain gene and plays an obligatory role in muscle cell development and differentiation. The role of ERK5 in myometrium has remained unexplored, but it is likely to represent an important

In vitro contraction assay

To assess the involvement of ERK in mediating OXT-induced contractions, we have now developed successfully a sensitive in vitro myometrial cell contraction assay (Devost and Zingg, 2007). In brief, myometrial hTERT-C3 cells were layered on top of collagen matrices in 24-well plates. Two hours after plating, the matrix was detached and allowed to float. OXT was then added at different concentrations for 1–18 h. Following fixation, collagen matrices were digitally scanned and the surface area was

Conclusions

Further elucidation of novel signalling pathways linked to the OXTR will have significant relevance for the development for novel pathway-specific OXTR agonists and antagonists. The effect of new drug candidates will have to be assessed with respect to each of these additional pathways. This is particularly relevant in the light of the recent realization that receptor ligands can act in a pathway-specific fashion and exert differential agonistic or antagonistic effects on distinct pathways

Abbreviations

β₂AR

beta-2 adrenergic receptor

eEF2

eukaryotic elongation factor 2

ERK 1/2

extracellular signal-regulated kinases 1 and 2

ERK 5

extracellular signal-regulated kinase 5

GPCR

G protein-coupled receptor

MAPK

mitogen-activated protein kinase

MEF2-C

myogenic enhancer factor 2-C

mTOR

mammalian target of rapamycin

OXT

oxytocin

OXTR

oxytocin receptor

PKC

protein kinase C

PLC

phospholipase C

s.e.m.

standard error of mean

Acknowledgement

Supported by a grant from the Canadian Institutes for Health Research (CIHR).

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Oxytocin receptor signalling

Abstract

Section snippets

General thoughts on oxytocin receptor signalling

Novel OXTR signalling target: eEF2

Novel OXTR-linked signalling pathway: ERK5

In vitro contraction assay

Conclusions

Abbreviations

Acknowledgement

J. Biol. Chem.

Am. J. Obstet. Gynecol.

Horm. Behav.

FEBS Lett.

J. Biol. Chem.

Trends Endocrinol. Metab.

Regulation of peptide-chain elongation in mammalian cells

Eur. J. Biochem.

Oxytocin and oxytocin receptors in cancer cells and proliferation

J. Neuroendocrinol.

Oxytocin-induced activation of eukaryotic elongation factor eEF2 in myometrial cells is mediated by protein kinase C

Endocrinology

Oxytocin induces dephosphorylation of eukaryotic elongation factor 2 in human myometrial cells