Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines

Abstract

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells

“…Since CCL19 and CCL21 have different N‐termini, they are expected to interact with and thus activate CCR7 in different ways. Earlier studies have presented data on the interaction of CCL19 and CCL21 with CCR7 . Altogether these studies support different docking modes of the two chemokines, with specific amino acid residues in the receptor being differentially important for activation by one ligand without affecting activation by the other.…”

“…Earlier as well as recent studies have provided evidence that these ligands are also functionally distinct signaling molecules. CCL19 seems to be more potent in inducing β‐arrestin 2 recruitment and CCR7 internalization in various established cell lines, and, importantly, a more potent chemotactic cue in guiding DC chemotaxis compared to CCL21 . In contrast, CCL21 seems to be as strong as or stronger than CCL19 in inducing intracellular calcium release and ERK activation in DCs .…”

“…CCL19 seems to be more potent in inducing β‐arrestin 2 recruitment and CCR7 internalization in various established cell lines, and, importantly, a more potent chemotactic cue in guiding DC chemotaxis compared to CCL21 . In contrast, CCL21 seems to be as strong as or stronger than CCL19 in inducing intracellular calcium release and ERK activation in DCs . Thus CCL19 and CCL21 give rise to biased CCR7 signaling; a phenomenon not only observed in CCR7, but also among other chemokine receptors …”

“…Altogether these studies support different docking modes of the two chemokines, with specific amino acid residues in the receptor being differentially important for activation by one ligand without affecting activation by the other. Thus amino acids in the top of transmembrane segment (TM)3 and TM4, in the area of the major binding pocket (encircled by TM3‐7) are exclusively important for CCL21 induced CCR7 activation . Recent modeling of CCL19 and CCL21 interactions with CCR7 suggests that CCL19 also makes ligand specific contacts with CCR7 …”

Remodeling our concept of chemokine receptor function: From monomers to oligomers

“…For example, differential GRK recruitment to CCR7 triggered by CCL19 or CCL21, explains their differences to activate β‐arrestins, although both ligands activate similarly G proteins . This observation is nonetheless controversial as it has been also reported that CCL21 is less potent than CCL19 activating G protein, triggering β‐arrestin recruitment, and inducing 3D chemotaxis of mature human DC, although it induces prolonged and more efficient ERK1/2 activation . This CCL19 bias is reported to rely on robust phosphorylation on Ser/Thr residues in the C terminus domain of CCR7, mediated by GRK3 and GRK6, resulting in stronger β‐arrestin recruitment and ERK activation .…”