Rules of engagement: determinants of chemokine receptor activation and selectivity by CCL27 and CCL28.

The distinct functional roles of chemokines CCL27 and CCL28 in epithelial immunity of skin and mucosal tissues, respectively, are coordinated by their shared receptor, CCR10, and the CCL28-specific receptor, CCR3. To identify determinants of receptor activation, internalization and binding specificity, we conducted structure-function studies focused on the N-termini of these two chemokines. Deletion of two N-terminal residues of CCL27 resulted in a CCR10 antagonist, highlighting the critical roles of these residues in driving receptor pharmacology. Extension with a Phe produced a CCR10 superagonist by occupying a unique subpocket in the receptor. Swapping the CCL28 N-terminus onto the CCL27 globular domain (NT28-CCL27) also resulted in a superagonist of CCR10, but the opposite swap (NT27-CCL28) showed equivalent or reduced activity compared to WT CCL28, indicating that the CCL28 N-terminus is a stronger driver of CCR10 signaling. The effects of these mutations were rationalized by AlphaFold models of the CCR10 complexes. Modeling also revealed that the reduced size of the binding pocket, and more basic nature of the N-terminus and extracellular loops of CCR3 compared to CCR10, contribute to its specificity for CCL28 while CCR10 accommodates both ligands. The basic nature of CCL28 also contributes to its high affinity for glycosaminoglycans, and is likely important for its retention in mucosal tissues. These data illustrate how the modular nature of these chemokines enables their overlapping but non-redundant functions, and how this modularity can be exploited to produce engineered chemokines for probing or targeting CCR10 in disease.