Institute of Reproductive and Developmental Biology, Department Metabolism, Digestion and Reproduction, Imperial College London, London, UK.
Department of Bioengineering, Imperial College London, London, UK.
J Mol Endocrinol. 2024 May 15;73(1). doi: 10.1530/JME-23-0153. Print 2024 Jul 1.
The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, β-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.
前列腺素 G 蛋白偶联受体（GPCR）EP2 广泛表达，并与子宫内膜异位症、骨质疏松症、肥胖症、早产和癌症有关。内化和细胞内转运对于塑造 GPCR 活性至关重要，但对于 EP2 信号的空间编程知之甚少，也不知道是否可以通过药理学方法来利用这一点。使用三种选择性激活不同 EP2 途径的 EP2 配体，我们表明 EP2 经历了有限的激动剂驱动的内化，但通过依赖于胞质分裂蛋白的、β-arrestin 独立的途径持续内化。EP2 持续被转运到早期和非常早期内体（VEE），配体激活不会改变这一点。APPL1 是 VEE 的关键衔接蛋白和调节蛋白，它不会影响 EP2 激动剂介导的 cAMP。内化对于急性 butaprost 和 AH13205 介导的 cAMP 信号的约 70%是必需的，但 PGN9856i，一种 Gαs 偏向激动剂，对于其 cAMP 信号的依赖程度较低，特别是在天然表达 EP2 的人足月妊娠子宫细胞中。抑制 EP2 内化部分减少了 butaprost 或 AH13205 激活的钙信号，对 PGE2 分泌没有影响。这表明 Gαs 和 Gαq/11 信号需要激动剂依赖性的不同空间要求，以及质膜起始的 Gαq/11-Ca2+-介导的 PGE2 分泌的作用。这些发现揭示了 EP2 持续内化在其信号中的关键作用及其介导下游功能的潜在空间偏向。反过来，这可能会突出未来针对 EP2 信号通路的选择性靶向的重要考虑因素。
