Abstract
The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein–independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein–independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.
Highlights
The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses
The failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine
Substitution mutants were designed of residues that line the activation-relevant polar network lining the central channel/cavity of the receptor and are involved in canonical 7TMR activation (G protein and arrestin recruitment) [30]
Summary
Substitution mutants were designed of residues that line the activation-relevant polar network lining the central channel/cavity of the receptor and are involved in canonical 7TMR activation (G protein and arrestin recruitment) [30]. Overall and cell surface expression of C-terminally YFP-fused ACKR3 and mutants in transfected HEK293 cells were tested. Specific 125ICXCL12 radiolabel tracer binding (50 pM) was compared between wildtype and mutant receptors, as well as the IC50 of competing unlabeled CXCL12 or CXCL11, as proxies for ligand affinity. Quantification of the YFP tags suggested similar overall expression levels and stability of all mutants. Mutants N127S, D141N, and Y143A were less detected at the cell surface, suggesting altered steady-state surface expression levels (Fig. 1B). All transfectants showed substantial specific 125ICXCL12 tracer binding (Fig. 1C) that was sufficient to perform binding competition experiments (Fig. 1 (D and E) and Table 1). All mutants were expressed at the cell surface to some degree, 894 J. Y315A 7.53 a p Ͻ 0.001. b p Ͻ 0.01. c p Ͻ 0.05
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