Characterising the pleiotropic activity and spatial dynamics of Prostaglandin EP2 receptor signalling

Abstract

EP2 is a member of the G protein coupled receptor superfamily and is implicated in both physiological and pathophysiological signalling in reproductive tissues. Classically reported as Gαs-mediated in reproductive tissues EP2 can also signal via Gαq/11, although the mechanism underlying this promiscuity is unknown. Tight regulation of internalisation and trafficking to endosomal compartments such as the early, or very early endosomes, is fundamental to GPCR signalling capacity, thus discovery of ligands that take advantage of EP2 pleiotropy by preferentially activating specific EP2 pathways could be valuable therapeutically. Therefore, I first characterised EP2 trafficking and signalling using three highly specific ligands – butaprost, AH13205, and PGN9856i. In HEK 293 cells, EP2 undergoes dynamin-dependent constitutive, but limited ligand-directed, internalisation, which was required for full activation of Gαs-cAMP and Gαq/11-Ca2+ pathways. In pregnant myometrium, EP2 signals via Gαs/Gαq/11, activating contrasting pro- and anti- labour pathways until labour, when EP2 Gαs signalling is downregulated. Prostaglandin signalling is a key part of physiological labour, thus understanding the mechanics of EP2 signalling during labour could lead to novel therapeutics to combat preterm birth, which represents a major challenge to public health. I found that EP2 signalling is altered with the onset of labour, favouring pro-inflammatory pathways in early and late labour. OTR activation ‘switched’ EP2 signalling pathways to Gαi/o when activated with butaprost, enhancing pro-labour pathways and dampening Gαs-cAMP, which may be mediated by formation and rearrangement of EP2-OTR heteromers. Whilst AH13205 exhibited an extreme inflammatory profile in both HEK 293 and myometrial cells, and engaged EP2 in crosstalk with OTR, enhancing pro-labour pathways, PGN9856i did not activate Gαq/11-mediated pro-labour/inflammatory pathways in HEK cells or primary myometrial cells, and may actively antagonise OT-mediated pathways. Together, these findings uncover novel aspects of EP2 pleiotropy and suggest that biased EP2 ligands may be valuable tools for targeting reproductive tissues.