ACKR4 Recruits GRK3 Prior to ß-Arrestins but Can Scavenge Chemokines in the Absence of ß-Arrestins.

Chemokines are essential for guiding cell migration. Atypical chemokine receptors (ACKRs) contribute to the cell migration process by binding, internalizing and degrading local chemokines, which enables the formation of confined gradients. ACKRs are heptahelical membrane spanning molecules structurally related to G-protein coupled receptors (GPCRs), but seem to be unable to signal through G-proteins upon ligand binding. ACKR4 internalizes the chemokines CCL19, CCL21, and CCL25 and is best known for shaping functional CCL21 gradients. Ligand binding to ACKR4 has been shown to recruit ß-arrestins that has led to the assumption that chemokine scavenging relies on ß-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited ß-arrestin1 and ß-arrestin2 to human ACKR4, but found no evidence for ß-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that ß-arrestins interacted with ACKR4 in the steady-state and contributed to the spontaneous trafficking of the receptor in the absence of chemokines. Deleting the C-terminus of ACKR4 not only interfered with the interaction of ß-arrestins, but also with the uptake of fluorescently labeled cognate chemokines. We identify the GPCR kinase GRK3, and to a lesser extent GRK2, but not GRK4, GRK5, and GRK6, to be recruited to chemokine-stimulated ACKR4. We show that GRK3 recruitment proceded the recruitment of ß-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of ß-arrestins to ACKR4. Overexpressing ß-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that ß-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking ß-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that ß-arrestins are dispensable for chemokine scavenging by ACKR4.

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4.

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.