Ancestral Origin of the CXCL17–GPR25 System Traced to the Lobe-Finned Fish Latimeria chalumnae

The roles of C-X-C motif chemokine ligand 17 (CXCL17) and its receptor, G protein-coupled receptor 25 (GPR25), in immune regulation are gaining recognition, but their evolutionary origins and phylogenetic distributions have yet to be determined. In this study, we identified a CXCL17 gene in the lobe-finned fish Latimeria chalumnae (coelacanth), a close living relative of tetrapods. This gene produces two alternatively spliced secretory proteins, designated Lc-CXCL17a and Lc-CXCL17b, which display limited sequence similarity to known CXCL17s from mammals or ray-finned fishes. Recombinant forms of Lc-CXCL17a and Lc-CXCL17b were generated via bacterial overexpression, purification, and enzymatic processing. Functional assays, including NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment, ligand–receptor binding, and chemotaxis assays, demonstrated that both proteins tightly bound to and potently activated coelacanth GPR25 in a manner dependent on their C-terminal residues, because removal of these residues markedly diminished activities. These findings provide the first evidence that a functional CXCL17–GPR25 system exists in lobe-finned fishes, supporting the hypothesis that this ligand–receptor pair originated in ancient fishes and was transmitted to vertebrate lineages through lobe-finned fish ancestors. This work broadens understanding of chemokine evolution and highlights the coelacanth as a valuable model for tracing conserved immune signaling pathways.