Engineering potent avian GPR25 agonists using an ostrich CXCL17 gene relic
C-X-C motif chemokine ligand 17 (CXCL17) activates G protein-coupled receptor 25 (GPR25) in most vertebrate lineages. Although birds appear to have lost functional CXCL17, CXCL17 gene relics and intact GPR25 genes persist in certain taxa. This study investigated whether a fragment encoded by an avian CXCL17 gene relic could guide the engineering of potent agonists for avian GPR25s. A 22-residue C-terminal fragment encoded by African ostrich CXCL17 gene relic was grafted onto the corresponding position of loggerhead turtle or human CXCL17, and the resulting hybrids were prepared via bacterial overexpression and functionally characterized via cell-based assays. The turtle-ostrich and human-ostrich CXCL17 hybrids showed no detectable activity at turtle or human GPR25 but exhibited substantially greater potency than their parental proteins at two avian GPR25s in {beta}-arrestin recruitment assays, with half-maximal effective concentrations of approximately 25 and 100 nM at the ostrich receptor and 5 and 13 nM at the mallard receptor, respectively. Both hybrids bound directly to the ostrich or mallard GPR25 in homogenous ligand-receptor binding assays and induced migration of transfected cells expressing the ostrich or mallard receptor in chemotaxis assays. These findings demonstrate that a fragment encoded by the ostrich CXCL17 gene relic confers potent avian GPR25 agonism and establishes the C-terminus as the primary driver of species selectivity. These hybrids provide functional surrogate tools for probing avian GPR25 biology.