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Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21—in contrast to CCL19—is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.

The chemokine receptor CCR7, together with its ligands, is responsible for the migration and positioning of adaptive immune cells, and hence critical for launching adaptive immune responses. CCR7 is also induced on certain cancer cells and contributes to metastasis formation. Thus, CCR7 expression and signalling must be tightly regulated for proper function. CCR7, like many other members of the G-protein coupled receptor superfamily, can form homodimers and oligomers. Notably, danger signals associated with pathogen encounter promote oligomerisation of CCR7 and is considered as one layer of regulating its function. Here, we assessed the dimerisation of human CCR7 and several single point mutations using split-luciferase complementation assays. We demonstrate that dimerisation-defective CCR7 mutants can be transported to the cell surface and elicit normal chemokine-driven G-protein activation. By contrast, we discovered that CCR7 mutants whose expression are shifted towards monomers significantly augment their capacities to bind and internalise fluorescently labelled CCL19. Modeling of the receptor suggests that dimerisation-defective CCR7 mutants render the extracellular loops more flexible and less structured, such that the chemokine recognition site located in the binding pocket might become more accessible to its ligand. Overall, we provide new insights into how the dimerisation state of CCR7 affects CCL19 binding and receptor trafficking.

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 β‐arrestin1 and β‐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 β‐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.

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 β-arrestins that has led to the assumption that chemokine scavenging relies on β-arrestin-mediated ACKR4 trafficking, a common internalization route taken by class A GPCRs. Here, we show that CCL19, CCL21, and CCL25 readily recruited β-arrestin1 and β-arrestin2 to human ACKR4, but found no evidence for β-arrestin-dependent or independent ACKR4-mediated activation of the kinases Erk1/2, Akt, or Src. However, we demonstrate that β-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 β-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 β-arrestins upon ACKR4 engagement and that GRK2/3 inhibition partially interfered with steady-state interaction and chemokine-driven recruitment of β-arrestins to ACKR4. Overexpressing β-arrestin2 accelerated the uptake of fluorescently labeled CCL19, indicating that β-arrestins contribute to the chemokine scavenging activity of ACKR4. By contrast, cells lacking β-arrestins were still capable to take up fluorescently labeled CCL19 demonstrating that β-arrestins are dispensable for chemokine scavenging by ACKR4. published

The chemokine receptor CXCR4 plays a fundamental role in homeostasis and pathology by orchestrating recruitment and positioning of immune cells, under the guidance of a CXCL12 gradient. The ability of chemokines to form heterocomplexes, enhancing their function, represents an additional level of regulation on their cognate receptors. In particular, the multi-faceted activity of the heterocomplex formed between CXCL12 and the alarmin HMGB1 is emerging as an unexpected player able to modulate a variety of cell responses, spanning from tissue regeneration to chronic inflammation. Nowadays, little is known on the selective signaling pathways activated when CXCR4 is triggered by the CXCL12/HMGB1 heterocomplex. In the present work, we demonstrate that this heterocomplex acts as a CXCR4 balanced agonist, activating both G protein and β-arrestins-mediated signaling pathways to sustain chemotaxis. We generated β-arrestins knock out HeLa cells by CRISPR/Cas9 technology and show that the CXCL12/HMGB1 heterocomplex-mediated actin polymerization is primarily β-arrestin1 dependent, while chemotaxis requires both β- arrestin1 and β-arrestin2. Triggering of CXCR4 with the CXCL12/HMGB1 heterocomplex leads to an unexpected receptor retention on the cell surface, which depends on β-arrestin2. In conclusion, the CXCL12/HMGB1 heterocomplex engages the β-arrestin proteins differently from CXCL12, promoting a prompt availability of CXCR4 on the cell surface, and enhancing directional cell migration. These data unveil the signaling induced by the CXCL12/HMGB1 heterocomplex in view of identifying biased CXCR4 antagonists or agonists targeting the variety of functions it exerts.