CCL19 and CCL21 Display Reciprocal Dependence on R209 in ECL2 of CCR7 for Receptor Activation, Relating to Differences in the Chemokine N‐Terminal Region

ABSTRACT CCL19 and CCL21 interact differentially with their shared receptor CCR7. A key amino acid important for CCL19‐mediated receptor activation is arginine 209 (R209) in extracellular loop 2 (ECL2) of CCR7. CCL21 activation of CCR7, in contrast, does not depend on R209. Using chimeric versions of CCL19 and CCL21, we investigate the importance of the N‐terminal region, core domain and C‐terminal region of CCL19 and CCL21 in contributing to this distinction. Overall, signalling via G αi and β‐arrestin2 recruitment induced by CCL19 and all variants containing the N‐terminal region of CCL19 is negatively impacted by substituting arginine 209 with an alanine (R209A), whereas signalling induced by ligands with the N‐terminal region of CCL21 is either unaffected or positively affected by this substitution. Of interest, the R209A substitution greatly improves the ability of CCL21 and all chemokine variants carrying the N‐terminal region of CCL21 to induce β‐arrestin2 recruitment by CCR7, with a general two‐fold increase in efficacy at 100‐nM chemokine concentrations. Our data fit with the proposed model for CCL19 and CCL21 docking at CCR7, where the more negatively charged N‐terminus of CCL19 contacts R209 during insertion into the CCR7 binding pocket, while CCL21 contacts the binding pocket in a different manner.