Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein–dependent and β-arrestin–dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein–dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance–based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical “two-site model” for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

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.

Oversulfated chondroitin sulfate (OSCS) contaminated heparin has been associated with severe anaphylactoid reaction (AR), mainly in dialysed patients. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. In this study, we investigated the kinin forming capacity of OSCS and OSCS contaminated heparin when incubated in vitro with a pool of human plasma. At 100μg/mL, OSCS liberates BK in a profile similar but not identical to dextran sulfate, a well known activator of the plasma contact system. The results have highlighted that the quantity of BK accumulated during contact system activation depends not only on the concentration of OSCS but also on the plasma dilution and the presence of an angiotensin converting enzyme inhibitor. We demonstrate a highly significant correlation between the concentration of OSCS present in the contaminated heparin and BK released concentration. In conclusion, for the first time, we show that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated heparin.

The CXC chemokine receptor 7 (CXCR7)/ACKR3 is a chemokine receptor that recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and interferon-inducible T-cell α chemoattractant (I-TAC)/CXCL11. Here, we report the development of novel CXCR7-selective ligands with a cyclic pentapeptide scaffold through an SAR study of CXC chemokine receptor 4 (CXCR4) selective antagonist FC131 [cyclo(-d-Tyr-l-Arg-l-Arg-l-Nal-Gly-), Nal = 3-(2-naphthyl)alanine]. Substitution of Gly with l-Pro switched the receptor preference of the peptides from CXCR4 to CXCR7. The SAR study led to the identification of a potent CXCR7 ligand, FC313 [cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal-l-Pro-)], which recruits β-arrestin to CXCR7. Investigations via receptor mutagenesis and molecular modeling experiments suggest a possible binding mode of the cyclic pentapeptide CXCR7 agonist.