Mutational analysis of the extracellular disulphide bridges of the atypical chemokine receptor ACKR3/CXCR7 uncovers multiple binding and activation modes for its chemokine and endogenous non-chemokine agonists

Abstract

The atypical chemokine receptor ACKR3/CXCR7 plays crucial roles in numerous physiological processes but also in viral infection and cancer. ACKR3 shows strong propensity for activation and, unlike classical chemokine receptors, can respond to chemokines from both the CXC and CC families as well as to the endogenous peptides BAM22 and adrenomedullin. Moreover, despite belonging to the G protein coupled receptor family, its function appears to be mainly dependent on β-arrestin. ACKR3 has also been shown to continuously cycle between the plasma membrane and the endosomal compartments, suggesting a possible role as a scavenging receptor. So far, the molecular basis accounting for these atypical binding and signalling properties remains elusive. Noteworthy, ACKR3 extracellular domains bear three disulphide bridges. Two of them lie on top of the two main binding subpockets and are conserved among chemokine receptors, and one, specific to ACKR3, forms an intra-N terminus four-residue-loop of so far unknown function. Here, by mutational and functional studies, we examined the impact of the different disulphide bridges for ACKR3 folding, ligand binding and activation. We showed that, in contrast to most classical chemokine receptors, none of the extracellular disulphide bridges was essential for ACKR3 function. However, the disruption of the unique ACKR3 N-terminal loop drastically reduced the binding of CC chemokines whereas it only had a mild impact on CXC chemokine binding. Mutagenesis also uncovered that chemokine and endogenous non-chemokine ligands interact and activate ACKR3 according to distinct binding modes characterized by different transmembrane domain subpocket occupancy and N-terminal loop contribution, with BAM22 mimicking the binding mode of CC chemokine N terminus.

Predominance of the heterozygous CCR5 delta‐24 deletion in African individuals resistant to HIV infection might be related to a defect in CCR5 addressing at the cell surface

“…These results suggest that the hCCR5Δ24 deletion, located at the top of TM2 close to the disulphide bridge linking ECL1 to ECL2, induced a conformational change in ECL2 . This conformational change could lead to protein misfolding and may interfere with its surface export, resulting in its intracellular accumulation . These observations were confirmed by imaging cytometry (Figure E and Figure B).…”

CXCL14 Preferentially Synergizes With Homeostatic Chemokine Receptor Systems

“…In its role as a positive allosteric modulator, CXCL14 synergizes with CXCL12 by direct binding to CXCR4, without inducing CXCR4-mediated signaling in the absence of CXCL12 ( 29 ). To expand our CXCL14 synergy studies to other chemokine/chemokine receptor systems, we examined all known human chemokine and atypical chemokine receptors in a global screen involving β-arrestin recruitment as a functional read-out ( 36 , 37 ). β-arrestin recruitment is an early cellular response to chemokines, requires prior phosphorylation of chemokine receptors by G-protein coupled receptor (GPCR) kinases and results in receptor internalization.…”

“…β-arrestin recruitment to chemokine receptors in response to CCL19, CXCL14 or positive control chemokines was monitored by NanoLuc complementation assay [NanoBiT, Promega, Madison, WI, United States ( 35 )] as previously described ( 36 ). In brief, 6.5 × 10 5 HEK293T cells were plated per well in a 12-well dish (6 × 10 6 per 10 cm dish for concentration response curves) and 24 h later co-transfected with pNBe vectors encoding a chemokine receptor C-terminally tagged to the luciferase fragment SmBiT and human β-arrestin-2 N-terminally fused to LgBiT.…”

A Novel ACKR2-Dependent Role of Fibroblast-Derived CXCL14 in Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer

“…Chemokine-induced b-arrestin-1 recruitment to ACKR2 was monitored by Nanoluciferase complementation assay (NanoBiT, Promega) as described previously (26,27). A total of 5 Â 10 6 HEK cells were plated in 10-cm culture dishes and 24 hours later transfected with plasmids containing human b-arrestin-1 N-terminally fused to LgBiT and ACKR2 C-terminally fused to SmBiT.…”