title: Mutational Analysis of Atypical Chemokine Receptor 3 (ACKR3/CXCR7) Interaction with Its Chemokine Ligands CXCL11 and CXCL12.
abstract: Atypical chemokine receptors do not mediate chemotaxis or G protein signaling, but they recruit arrestin. They also efficiently scavenge their chemokine ligands, thereby contributing to gradient maintenance and termination. ACKR3, also known as CXCR7, binds and degrades the constitutive chemokine CXCL12, which also binds the canonical receptor CXCR4, and CXCL11, which also binds CXCR3. Here we report comprehensive mutational analysis of the ACKR3 interaction with its chemokine ligands, using 30 substitution mutants. Readouts are radioligand binding competition, arrestin recruitment, and chemokine scavenging. Our results suggest different binding modes for both chemokines. CXCL11 depends on the ACKR3 N terminus and some extracellular loop (ECL) positions for primary binding, ECL residues mediate secondary binding and arrestin recruitment potency. CXCL12 binding required key residues Asp-1794.60and Asp-2756.58(residue numbering follows the Ballesteros-Weinstein scheme), with no evident involvement of N-terminal residues, suggesting an uncommon mode of receptor engagement. Mutation of residues corresponding to CRS2 in CXCR4 (positions Ser-1032.63and Gln-3017.39) increased CXCL11 binding, but reduced CXCL12 affinity. Mutant Q301E7.39did not recruit arrestin. Mutant K118A3.26in ECL1 showed moderate baseline arrestin recruitment with ablation of ligand-induced responses. Substitutions that affected CXCL11 binding also diminished scavenging. However, detection of reduced CXCL12 scavenging by mutants with impaired CXCL12 affinity required drastically reduced receptor expression levels, suggesting that scavenging pathways can be saturated and that CXCL12 binding exceeds scavenging at higher receptor expression levels. Arrestin recruitment did not correlate with scavenging; although Q301E7.39degraded chemokines in the absence of arrestin, S103D2.63had reduced CXCL11 scavenging despite intact arrestin responses.
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, University of Montreal, Montréal H3T 1J4, Canada and.|Research Centre, Saint-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada.
authors.forename: Besma
authors.initials: B
authors.lastname: Benredjem
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, University of Montreal, Montréal H3T 1J4, Canada and.|Research Centre, Saint-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Girard
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, University of Montreal, Montréal H3T 1J4, Canada and.|Research Centre, Saint-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Rhainds
authors.affiliation: Research Centre, Saint-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: St-Onge
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, University of Montreal, Montréal H3T 1J4, Canada and nikolaus.heveker@recherche-ste-justine.qc.ca.|Research Centre, Saint-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Heveker
mesh_terms: D000595:Amino Acid Sequence; D054371:Chemokine CXCL11 / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D054387:Receptors, CXCR / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C501896:ACKR3 protein, human; D054371:Chemokine CXCL11; D054377:Chemokine CXCL12; D008024:Ligands; D050505:Mutant Proteins; D054387:Receptors, CXCR
keywords: ACKR3; CXCR7; arrestin; atypical chemokine receptor; bioluminescence resonance energy transfer (BRET); chemokine; chemokine scavenging; receptor; structure-function