Identification of orphan GPR25 as a receptor for the chemokine CXCL17

C-X-C motif chemokine ligand 17 (CXCL17) is a small secretory protein primarily expressed in mucosal tissues, which likely functions as a chemoattractant; however, its receptor is controversial. Herein, we identified the rarely studied orphan G protein-coupled receptor 25 (GPR25) as a receptor of CXCL17 via prediction using the newly developed AlphaFold 3 algorithm and experimental validation. In the NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay, recombinant human CXCL17 could activate human GPR25 in transfected human embryonic kidney (HEK) 293T cells with an EC₅₀ value around 100 nm, but it had no activation effect on the other 17 tested G protein-coupled receptors. Deletion of three conserved C-terminal residues from human CXCL17 almost abolished its activation effect. Alanine replacement of W95 or R178 of human GPR25, two conserved residues in the predicted orthosteric ligand binding pocket, almost abolished its response to CXCL17. Only the pairing of wild-type CXCL17 with wild-type GPR25 could cause shedding of transforming growth factor α and induce chemotactic movement of transfected HEK293T cells. These results were consistent with the AlphaFold 3-predicted binding model, in which the highly conserved C-terminal fragment of CXCL17 inserts into the orthosteric ligand binding pocket of GPR25. According to their expression pattern shown in the Human Protein Atlas, CXCL17 may be an endogenous agonist of GPR25 in humans and other mammals; however, this hypothesis needs to be tested experimentally in future studies. The present deorphanization paves the way for further functional characterization of the orphan receptor GPR25 and the orphan ligand CXCL17.