EP4 receptor conformation sensor suited for ligand screening and imaging of extracellular prostaglandins.

Prostaglandins are important lipid mediators with a wide range of functions in the human body. They act mainly via plasma membrane localized prostaglandin receptors, which belong to the G-protein coupled receptor class. Due to their localized formation and short lifetime, it is important to be able to measure the distribution and abundance of prostaglandins in time and or space. In this study, we present a Foerster resonance energy transfer (FRET)-based conformation sensor of the human EP4 receptor, which was capable of detecting prostaglandin E 2 (PGE 2 )-induced receptor activation in the low nanomolar range with a good signal to noise ratio. The sensor retained the typical selectivity for PGE 2 among arachidonic acid products. Human embryonic kidney (HEK293) cells stably expressing the sensor did not produce detectable amounts of prostaglandins making them suitable for a co-culture approach allowing us, over time, to detect prostaglandin formation in Madin-Darby canine kidney cells and primary mouse macrophages. Furthermore, the EP4 receptor sensor proved to be suited to detect experimentally generated PGE 2 gradients by means of FRET-microscopy, indicating the potential to measure gradients of PGE 2 within tissues. In addition to FRET-based imaging of prostanoid release, the sensor allowed not only for determination of PGE 2 concentrations, but also proved to be capable of measuring ligand binding kinetics. The good signal to noise ratio at a commercial plate reader and the ability to directly determine ligand efficacy shows the significant potential of this sensor interest for screening and characterization of novel ligands of the pharmacologically important human EP4 receptor. Significance Statement We present a biosensor based on the prostaglandin EP4 receptor, which is well suited to measure extracellular prostaglandin E 2 (PGE 2 ) concentration with high temporal and spatial resolution. It can be used for the imaging of PGE 2 levels and gradients by means of Foerster resonance energy transfer (FRET) microscopy, for determining PGE 2 release of primary cells as well as for screening purposes in a plate reader setting.