Ligand-specific conformational transitions and intracellular transport are required for atypical chemokine receptor 3–mediated chemokine scavenging

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.

Characterization of a chimeric chemokine as a specific ligand for ACKR3

“…Moreover, the authors suggest that arrestin binding to ACKR3 is not required for chemokine scavenging. 31 The finding somewhat contrasts other observations where it was shown that CXCL11 and CXCL12 binding to ACKR3 enhances arrestin recruitment. [32][33][34][35][36] A known drawback for the investigations of GPCRs is the paucity of antibodies which recognize endogenous receptors expressed at the cell surface.…”

“…CXCL11 induced a faster ACKR3 internalization, but slower recycling than CXCL12. Moreover, the authors suggest that arrestin binding to ACKR3 is not required for chemokine scavenging . The finding somewhat contrasts other observations where it was shown that CXCL11 and CXCL12 binding to ACKR3 enhances arrestin recruitment …”

“…However, at full effective concentrations AMD3100 also targets ACKR3. 31,51 Binding of CXCL11 to CXCR3 can be similarly abolished with small molecule inhibitors, however, ACKR1 also binds the chemokine potentially obscuring interactions with ACKR3. 1 In order to design a selective ligand for ACKR3, we hypothesized that a chimeric chemokine composed of domains derived from CXCL11 and CXCL12 could possess such characteristic.…”

“…Comparing the sequences of CXCL11 and CXCL12 with the data from the radiolytic footprinting suggests that different domains of ACKR3 must be involved in the recognition of the 2 chemokines . A recent study confirmed different modes of binding of CXCL11 and CXCL12 to ACKR3 . CXCL11 induced a faster ACKR3 internalization, but slower recycling than CXCL12.…”

CXCR4/ACKR3 Phosphorylation and Recruitment of Interacting Proteins: Key Mechanisms Regulating Their Functional Status

“…The receptor is then mainly recycled back to the plasma membrane (Luker et al, 2010) even if a partial degradation of ACKR3 can be observed (Hoffmann et al, 2012). Interestingly, Phosphorylation/Interactome Regulate CXCR4/ACKR3 Functions 803 at ASPET Journals on July 3, 2020 molpharm.aspetjournals.org the rate of receptor internalization is faster and recycling is lower in the presence of CXCL11, compared with CXCL12 (Montpas et al, 2018).…”