Noncanonical roles of chemokine regions in CCR9 activation revealed by structural modeling and mutational mapping.

The G protein-coupled chemokine receptor CCR9 plays a major role in inflammatory bowel disease and is implicated in cancer. Despite its therapeutic relevance, the mechanism by which CCR9 is activated by its endogenous chemokine CCL25 remains poorly understood. Here, we combine structural modeling with multimodal pharmacological analysis of CCR9 mutants to map the CCR9-CCL25 interface and delineate key determinants of binding, G protein versus arrestin signaling, and constitutive activity. We show that unlike other chemokines which drive receptor activation through their N-termini, CCL25 activates CCR9 via a distinct region, its 30s loop. Supporting this non-canonical mechanism, CCR9 signaling tolerates alanine mutations in the CCL25 N-terminus but is strongly affected by 30s loop modifications. Engineered N-terminally modified CCL25 analogs remain full agonists, consistent with signaling determinants lying outside the N-terminus. This non-canonical activation signature provides insights for CCR9 drug discovery and may inform structure-based design for other chemokine receptors.