Research ArticleBiochemistry

Kinetics of CXCL12 binding to atypical chemokine receptor 3 reveal a role for the receptor N terminus in chemokine binding

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Science Signaling  10 Sep 2019:
Vol. 12, Issue 598, eaaw3657
DOI: 10.1126/scisignal.aaw3657
  • Fig. 1 Arrestin recruitment of N-terminally truncated ACKR3 variants.

    (A) Extracellular portion of the ACKR3:CXCL12 complex from experimentally driven molecular model (8) and sequences of ACKR3 truncation mutants. Truncated regions are highlighted according to the color scheme in the model. (B) Representative examples of recruitment of GFP10–β-arrestin–2 to ACKR3-Rluc3 variants stimulated with different concentrations of CXCL12WT, CXCL11, and CCX662 measured by BRET. Each point corresponds to the average and SE of two measurements. (C and D) Potency (C) and efficacy (D) of ligand-induced β-arrestin–2 recruitment determined relative to ACKR3WT from fitting of dose-response curves. pEC50 and Emax values for truncation mutants were determined as ΔpEC50 = pEC50,mutant − pEC50,WT and %Emax = Emax,mutant/Emax,WT × 100. Data are mean and SE of three or more experiments. Significantly lowered potency or efficacy relative to ACKR3WT with the same ligand is noted: *P < 0.05, **P < 0.01, and ***P < 0.001 from one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Pound sign (#) in (C) indicates that CXCL11-induced arrestin recruitment by ACKR3d29 could not be accurately fit, and ΔpEC50 was estimated from the raw data to be >−2.2.

  • Fig. 2 Kinetics of CXCL12 binding to ACKR3.

    (A) Dissociation of CXCL12WT was measured by flow cytometry by monitoring the decrease in geometric mean of FITC fluorescence after addition of the small-molecule ligand CCX777 to live Sf9 cells coexpressing ACKR3WT and HA-tagged CXCL12WT bound to FITC-conjugated antibody. a.u., arbitrary units. (B) Association of CXCL12WT to ACKR3WT after adding HA-tagged CXCL12WT complexed with FITC-conjugated antibody against HA to cells expressing ACKR3WT. (C) kobs values for the slow phase of CXCL12WT association to ACKR3WT at different ligand concentrations determined from fitting the data to a two-component exponential equation. (D) Binding of ACKR3 in nanodiscs to immobilized CXCL12 measured by SPR. RU, response units. (E and F) Association (E) and dissociation rate constants (F) were determined for binding of ACKR3 in nanodiscs to immobilized CXCL12 detected by SPR and for CXCL12 binding to ACKR3 in live Sf9 cells detected by flow cytometry. The asterisk indicates that the dissociation rate is slower in Sf9 cells as determined from unpaired t test with P < 0.05. (G) Kinetics of β-arrestin–2 recruitment to ACKR3WT in HEK293T cells followed by BRET. Curves in (A), (B), (D), and (G) are representative examples of six (A) or three (B, D, and G) independent results, and each point or bar in (C), (E), and (F) is means and SEs of three or more experiments.

  • Fig. 3 Ligand binding kinetics of truncated ACKR3 variants.

    (A) Representative example of SDS-PAGE of purified bril-ACKR3WT, bril-ACKR3d17, and bril-ACKR3d29. Addition of PNGase F deglycosylates the receptor. (B) Representative example of thermal unfolding of ACKR3 variants in complex with CCX662 and CXCL12LRHQ measured using CPM fluorescence (32). (C) Representative example of CXCL12WT association to ACKR3 variants at 5 nM chemokine detected by flow cytometry. (D) Percent of the association curves corresponding to the slower phase of chemokine association determined from fitting association curves at 10 nM CXCL12WT to a two-phase exponential equation. Bars represent the average and SEs of three or more experiments. ACKR3d29 has a larger slow component than ACKR3WT (*P < 0.05 as determined from one-way ANOVA with Dunnett’s multiple comparison test). (E) Mean and SEs from three or more measurements of kobs values for the slow phase in (D). (F) Representative example of arrestin recruitment to ACKR3 variants determined from BRET experiments after addition of 10 nM CXCL12WT. (G) Average and SEs of kobs values from fitting three time-resolved BRET experiments to single exponential equations. (H) Representative example of CXCL12WT dissociation from ACKR3:CXCL12 complexes in Sf9 cells. (I) Mean and SEs of CXCL12WT dissociative half-life determined from fitting three or more dissociation curves to a single-phase exponential equation. The dissociative half-life of bril-ACKR3d29 is significantly shorter than ACKR3WT (***P < 0.001 as determined from one-way ANOVA with Dunnett’s multiple comparison test). (J) Schematic representation of CXCL12 binding equilibria for ACKR3WT and ACKR3d29 highlighting the faster dissociation rate but unchanged association rate of CXCL12 binding to the truncated receptor.

  • Fig. 4 ACKR3 binding kinetics and arrestin recruitment of CXCL12 mutants.

    (A) Extracellular portion of ACKR3:CXCL12 model and N-terminal sequences of CXCL12 variants. Residues in CXCL12WT that differ between the three mutants are highlighted in pink in the model. (B) Representative curves for CXCL12 dissociation from bril-ACKR3WT detected by flow cytometry. (C) Means and SEs from three or more experiments of dissociative half-lives determined from fitting dissociation curves to a single exponential. The dissociative half-life of CXCL12LRHQ was too slow to quantify but was estimated to be longer than 150 min (highlighted by # in the figure). Binding of CXCL12P2G to ACKR3d29 was too low to quantify a dissociation rate [highlighted by the dollar sign ($) in the figure]. (D) Representative CXCL12 dissociation curves from bril-ACKR3d29 detected by flow cytometry. (E) Means and SEs of the percent-specific chemokine binding remaining 20 min after the start of dissociation for three or more experiments. CXCL12LRHQ has a higher fraction of chemokine bound than CXCL12WT for both bril-ACKR3WT and bril-ACKR3d29, and CXCL12P2G has a lower fraction bound for bril-ACKR3WT. Binding of CXCL12P2G to ACKR3d29 was too low to quantify (highlighted by $ in figure). (F) Representative dose-response curves for β-arrestin–2 recruitment to ACKR3WT and ACKR3d29. (G) Emax normalized to ACKR3WT with CXCL12WT (%Emax = Emax,mutant/Emax,WT × 100). Each bar represents the average and SEs of three or more experiments. (H) Mean and SEs of pEC50 relative to ACKR3WT with CXCL12WT (ΔpEC50 = pEC50,mutant − pEC50,WT). CXCL12P2G-mediated recruitment of arrestin to ACKR3d29 was barely detectable, and the ΔpEC50 was estimated to be less than −2 [highlighted by $ in (G) and (H)]. (I) Representative dose-response curves for β-arrestin–2 recruitment to ACKR3WT and ACKR3d29. Significant differences for CXCL12P2G and CXCL12LRHQ compared to CXCL12WT are noted: *P < 0.05, **P < 0.01, and ***P < 0.001 from one-way ANOVA with Dunnett’s multiple comparison test.

  • Fig. 5 Model of the mechanism of chemokine interaction with the receptor.

    Experiments with chemokine mutations (CXCL12P2G and CXCL12LRHQ) and receptor truncations (ACKR3d29) suggested that the chemokine initially engages with the CRS1.5 and CRS2 of the receptor followed by formation of additional interactions with the CRS0.5 and CRS1 epitopes. Initial docking of the receptor N terminus with the core of the chemokine (bottom left corner) is not a key step in the formation of the fully engaged receptor:chemokine complex.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/598/eaaw3657/DC1

    Fig. S1. β-Arrestin recruitment to ACKR3WT induced by CCX662 and CXCL11 in BRET experiments.

    Fig. S2. Expression of ACKR3 variants in HEK cells.

    Fig. S3. Kinetics of association of CXCL12 with ACKR3 on live Sf9 cells.

    Fig. S4. SPR in nanodiscs and detergent micelles.

    Fig. S5. N-terminal cytochrome b562-RIL fusion protein did not affect CXCL12 dissociation rate or folding of ACKR3.

    Fig. S6. BRET signal increase after addition of 2 μM chemokine for ACKR3WT and ACKR3d29.

    Table S1. IC50 values (nM) for equilibrium binding studies reported in literature.

    References (4751)

  • This PDF file includes:

    • Fig. S1. β-Arrestin recruitment to ACKR3WT induced by CCX662 and CXCL11 in BRET experiments.
    • Fig. S2. Expression of ACKR3 variants in HEK cells.
    • Fig. S3. Kinetics of association of CXCL12 with ACKR3 on live Sf9 cells.
    • Fig. S4. SPR in nanodiscs and detergent micelles.
    • Fig. S5. N-terminal cytochrome b562-RIL fusion protein did not affect CXCL12 dissociation rate or folding of ACKR3.
    • Fig. S6. BRET signal increase after addition of 2 μM chemokine for ACKR3WT and ACKR3d29.
    • Table S1. IC50 values (nM) for equilibrium binding studies reported in literature.
    • References (4751)

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