Alanine scanning mutagenesis of the chemokine receptor CCR3 reveals distinct extracellular residues involved in recognition of the eotaxin family of chemokines

Despite considerable differences in primary structure, the chemokines eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 signal via a single receptor, CCR3, but exhibit different potencies and efficacies. To examine receptor/ligand interactions in more detail, we performed alanine scanning mutagenesis of 21 charged residues within the extracellular loops (ECLs) of CCR3. Following transient expression in the L1.2 cell line, CCR3 mutants were assessed for their ability to be expressed at the cell surface, bind CCL11 and induce chemotactic responses to CCL11, CCL24 and CCL26.The majority of constructs were well expressed at the cell surface and bound CCL11 with low nanomolar affinity. Exceptions to this rule included the mutants E175A and E176A (ECL2) which were poorly expressed and responded weakly to all three ligands in chemotaxis assays. In contrast, the mutants K26 (amino-terminus) E179 and E180 (ECL2) responded in chemotaxis assays to CCL11 and CCL24, but not to CCL26. Mutation of residues in ECL3 was informative, with the D272A, K277A and D280A mutants exhibiting reduced chemotactic responses to two or more of the three ligands examined, despite being expressed on the cell surface at levels similar to WT CCR3. This suggests a major role for ECL3 in the recognition of all three eotaxins.In summary, distinct acidic and basic residues within CCR3 determine both receptor expression and activation by the eotaxins. Determining how these chemokines interact with their receptor at the molecular level should increase our understanding of the process of chemokine receptor activation.