Identification of the orphan GPR25 as a receptor of the chemokine CXCL17

C-X-C motif chemokine ligand 17 (CXCL17) is a small secretory protein primarily expressed in mucosal tissues and likely functions as a chemoattractant, but its receptor is still controversial. In the present study, we identified the rarely studied orphan G protein-coupled receptor 25 (GPR25) as a receptor of CXCL17 via prediction by the newly developed AlphaFold 3 algorithm and validation by the NanoBiT-based β-arrestin recruitment assay. In the β-arrestin recruitment assay, recombinant human CXCL17 could activate human GPR25 in transfected human embryonic kidney (HEK) 293T cells with an EC₅₀value around 100 nM, but it had no activation effect on 17 other tested G protein-coupled receptors (GPCRs). Deletion of three conserved C-terminal residues of human CXCL17 almost abolished its activity, and alanine replacement of W95 or R178 of human GPR25, two conserved residues in the predicted orthosteric ligand binding pocket, almost abolished its response towards CXCL17. These results are 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 the receptor GPR25. According to the expression pattern of CXCL17 and GPR25 shown at the Human Protein Atlas, CXCL17 might be an endogenous agonist of GPR25 in human and other mammals, but this hypothesis needs to be tested in future studies via more assays. The present deorphanization paves the way for further functional characterization of the orphan receptor GPR25 and the orphan ligand CXCL17.