GPR39 Activation Induces AQP2 Internalization and Alterations in Cytoskeleton Dynamics

G protein-coupled receptors (GPCRs) represent the largest class of membrane proteins in mammals, with the ghrelin family receptor GPR39 standing out due to its unique non-responsiveness to traditional peptide hormones and neuropeptides. Despite being well-expressed in the kidney, a renal function for GPR39 has not yet been reported. To pursue this, we first utilized RNAScope ( in situ hybridization) to localize Gpr39 transcripts in mice. Double staining with an AQP2 antibody shows colocalization between Gpr39 and AQP2, with the most abundant Gpr39 expression found in the inner medullary collecting duct. Next, we overexpressed a GFP-tagged GPR39 construct in polarized MDCK cells grown on filters and demonstrated that GPR39 traffics to the basolateral membrane using immunofluorescence (IF). Based on these localization data, we hypothesized a role for GPR39 in AQP2 membrane trafficking. To test this hypothesis, we used murine principal kidney cortical collecting duct cells (mpkCCD), which only express AQP2 at the apical membrane in response to vasopressin (dDAVP). Treatment with the GPR39-specific agonist cpd1324 at 1µM + 10µM Zn 2+ led to notable physiological changes. Within 4 hours, cpd1324 impaired transepithelial resistance (WT/veh=12.5±0.7, WT/cpd = 0.92±0.35, KO/veh = 7.9±0.2, KO/cpd = 8.9±0.3, n=3 per condition (kΩxcm 2 ), destabilized tight junctions (ZO-1 IF), altered apical membrane morphology (wheat germ agglutinin IF), inhibited dDAVP-mediated AQP2 trafficking (AQP2 and p256-AQP2 IF), and reduced cortical F-actin expression (phallodin). Long term treatments with cpd1324 for 24 hours resulted in AQP2 degradation (by Western Blot (WB)) and a recovery of F-actin and ZO-1 localization and TER. These effects were not seen in GPR39 KO mpkCCD cells. Additionally, Gpr39 KO cells express higher levels of AQP2 in response to vasopressin as compared to WT cells by WB. In cAMP assays, 15-minute dDAVP treatment induced cAMP accumulation from 206.5±0.3 pmol/mL to 213.11±1.3 pmol/mL; however, 4h and 24h pretreatment with cpd1324 only reduced dDAVP-mediated cAMP pools to 211.3±0.6 and 210.0±1.5 pmol/mL, respectively. These data suggest that the observed effect on AQP2 is only partially due to inhibition of V2 receptor signaling. Whether this signaling inhibition is the result of direct action on the V2 receptor or a secondary effect of the changes in cellular morphology is currently under investigation. This evidence supports GPR39's involvement in modulating water reabsorption and suggests it as a potential target for managing renal water balance disorders. NHLBIT32HL007534 and 5F31DK137460-02 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.