Identification and Functional Characterization of CXCL17 Orthologs in Amphibians

C-X-C motif chemokine ligand 17 (CXCL17) has recently been identified as an agonist of the poorly characterized G protein-coupled receptor 25 (GPR25). Although GPR25 orthologs are widely distributed across vertebrates, non-mammalian CXCL17 orthologs have only been identified in some fish species in our recent studies. In this study, we systematically searched public databases for amphibian CXCL17 orthologs based on conserved C-terminal motif, gene synteny, and genomic architecture. Using this approach, we identified up to eighteen CXCL17 orthologs from diverse amphibian species. These amphibian CXCL17s exhibit no significant overall sequence similarity to known mammalian or fish CXCL17s, thus they were previously classified as uncharacterized proteins or even unannotated. Compared with known mammalian or fish CXCL17s, most amphibian CXCL17s display distinctive features, including four cysteine residues in their mature peptide and an additional residue following the conserved C-terminal Xaa-Pro-Yaa motif. A representative ortholog from the tropical clawed frog (Xenopus tropicalis) was recombinantly expressed and functionally characterized using cell-based assays, inducing ligand-receptor binding, {beta}-arrestin recruitment, and chemotactic cell migration. The recombinant amphibian CXCL17 directly bound to and efficiently activated its cognate GPR25 receptor and induced chemotactic migration of the transfected human embryonic kidney (HEK) 293T cells, but deletion of four C-terminal residues largely abolished its activity, indicating that all CXCL17 orthologs employ a conserved mechanism for receptor binding and activation. These findings establish the presence of a functional CXCL17-GPR25 signaling system in amphibians and provide new insights into the phylogenetic distribution and sequence diversity of CXCL17 orthologs across vertebrate lineages.