GPR17 is expressed in hepatocytes and the cortical renal nephron, signaling through a Gipathway

G protein-coupled receptors (GPCRs) are the largest class of proteins in the human body and are considered to be excellent targets for drug discovery. However, the vast majority of GPCRs are understudied. Using a large-scale TaqMan array screen on both male and female C57BL6 murine livers, we identified GPR17 as a highly expressed orphan receptor within the liver (ΔCt = 20.23, normalized to GAPDH). Hepatic expression of Gpr17 was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR) and was also identified in murine whole kidneys from both males and females. While GPR17 has known roles in oligodendrocyte maturation and myelination in the central nervous system, its functions within the liver and kidney remain unknown. To investigate its physiological functions in respective tissues, we started by uncovering GPR17’s localization pattern, ligand profile, and signaling mechanism. Using custom RNAScope in-situ hybridization probes, we found Gpr17 mRNA expression within hepatocytes and cortical nephron segments of the kidney. RNAScope colocalization studies using lotus tetragonolobus lectin (LTL) as a proximal tubule marker revealed robust cortical Gpr17 mRNA expression within both LTL+ and LTL- nephron segments. Additionally, ongoing subcellular localization experiments using Alpha mouse liver 12 (AML12) and Madine-Darby canine kidney (MDCK) cell lines will shed light on GPR17 tissue-specific expression within subcellular departments. To screen for potential ligands, we cloned Gpr17 into flag-tagged PME18s and Presto-Tango vectors. Live-cell surface labeling of HEK293T expressing the GPR17 constructs confirmed surface expression facilitating ligand screening. GPR17 downstream signaling has yet to be studied. Thus, we used the PRESTO-TANGO assay, an exogenous chemical ligand screening assay that relies on a luminescence readout to indicate -arrestin recruitment and receptor activation. A large, non-biased chemical screen of over 500 physiological compounds was tested for activation of GPR17. To date, we identified seven exogenous activators of GPR17. Additionally, literature suggests that synthetic MDL29,951 (MDL) and endogenous oxysterols function as GPR17 ligands. To determine GPR17 signaling mechanisms, we employed Dual Luciferase cAMP-based reporter assays. On their own, GPR17 ligands did not induce a rise in intracellular cAMP. However, when cAMP levels were elevated via forskolin treatment alongside ligand administration, GPR17 activation resulted in a significant reduction of intracellular cAMP, suggesting that GPR17 signaling is coupled to the G i pathway. Results confirmed MDL and select oxysterol ligand behavior and indicated the utilization of a G i signaling mechanism by hepatic and renal GPR17. Ongoing bioluminescence resonance energy transfer studies using compact ONE vector G-protein Optical biosensors are aimed at confirming GPR17’s coupling to the G i pathway. NIH This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.