Phoenixine-20-amide does not bind heterologously expressed GPR173

GPR173 (SREB3) is an orphan GPCR of the ‘Superconserved Receptors Expressed in the Brain’ family with high mRNA levels expressed in the hypothalamus, cortex and cerebellum. Phoenixin-20-amide (P20A), an endogenous hypothalamic ligand regulating ovarian cyclicity (Ullah et al., 2017; Stein et al., 2016), has been suggested to activate GPR173. In vitro studies on gonadotrophs showed that P20A stimulates GPR173, thereby increasing cAMP production, resulting in an elevated hypothalamic ‘Gonadotrophin Releasing Hormone Receptor’ (GnRHR) and kisspeptine expression, eventually increasing luteinizing hormone (LH) secretion (Treen et al., 2016; Stein et al., 2016). However, using the Promega GloSensor™ Technology, we failed to observe GPR173 mediated changes of cyclic AMP concentrations in HEK293 cellular models upon stimulation with P20A. Furthermore, it has been suggested that GPR173 could be stimulated by GnRH1_5 to trigger the recruitment of arrestin-3 in neurons co-expressing GnRHR, thereby inhibiting developmental neuronal migration (Larco et al., 2013). In our hands, with an arrestin-3-based firefly luciferase complementation assay in heterologously tranfected HEK293 cells, GPR173 was not able to recruit arrestin-3 upon stimulation with neither P20A nor GnRH1_5. Finally, GPR173 did neither bind any promiscuous g-proteins upon stimulation with P20A nor show any constitutive activity. Whether P20A influences the secretion of LH has not been studied, but P20A did not directly activate GPR173 in none of our assays. Nevertheless, transfected cellular models should be distinguished from native systems. Further studies are therefore necessary to determine if an endogenous partner is missing in our models and might be responsible for the previously reported results