Research ArticleGPCR SIGNALING

Biased antagonism of CXCR4 avoids antagonist tolerance

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Science Signaling  16 Oct 2018:
Vol. 11, Issue 552, eaat2214
DOI: 10.1126/scisignal.aat2214
  • Fig. 1 Antagonist tolerance develops after inhibition of endocytosis.

    (A) IC50 values calculated from Transwell chemotaxis assays of Jurkat cell migration toward CXCL12 after pretreatment with AMD3100 or X4-2-6 at the indicated concentrations for the indicated times. Data are means ± SEM of three independent experiments. *P < 0.001 by Student’s t test. (B) Flow cytometry analysis of CXCR4 cell surface expression on Jurkat cells treated with AMD3100 (▲) or X4-2-6 (●) for 72 hours as compared to vehicle-treated cells. Data are means ± SD of three experiments performed in triplicate at each condition. (C) Flow cytometry analysis of CXCR4 endocytosis stimulated by CXCL12 in Jurkat cells treated with AMD3100 (▲) or X4-2-6 (●) as compared to cells stimulated with CXCL12 alone. Data are means ± SD from three experiments performed in triplicate for each condition. (D) Transwell migration assay of Jurkat cell chemotaxis toward CXCL12 after treatment with the indicated concentration of AMD3100 with or without dynasore for 1 hour. Chemotaxis is plotted relative to chemotaxis in the presence of CXCL12 alone. Data are means ± SD from three independent experiments performed in triplicate for each condition. *P < 0.04 by Student’s t test. Flow cytometry analysis of CXCR4 cell surface expression before and after dynasore treatment (upper left inset) is representative of all experiments. IgGκB, immunoglobulin G κB; PE, phycoerythrin.

  • Fig. 2 BA1/2 recruitment and function downstream of CXCR4 is not substantially affected by X4-2-6.

    (A) Confocal microscopy analysis of CXCR4 and BA2 in Jurkat cells pretreated with vehicle, AMD3100, or X4-2-6 and stimulated with CXCL12, as indicated. Arrows indicate CXCR4 (red), BA2 (green), or colocalization (orange). Images are representative of three independent experiments. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride. (B) PRESTO-Tango assay analysis of the recruitment of BA1/2 to CXCR4 after treatment with increasing concentrations of CXCL12 in the presence of vehicle (■), AMD3100 (▲), or X4-2-6 (●). Data are means ± SD of three independent experiments performed on six replicates per condition. (C) Western blotting analysis of CXCR4 Ser324/325 and Ser339 phosphorylation in lysates of Jurkat cells treated as indicated. Blots are representative of at least three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D and E) Western blotting analysis of ERK1/2 Thr202/Tyr204 phosphorylation in the lysates of Jurkat cells treated as indicated. (D) Blots are representative of at least three independent experiments. (E) Quantification of the relative abundance of pERK1/2 normalized to that of total ERK1/2. Data are means ± SD from all experiments. *P < 0.05 and **P < 0.01 by one-way analysis of variance (ANOVA) with post hoc Tukey test.

  • Fig. 3 X4-2-6 specifically inhibits G protein activation downstream of CXCR4.

    (A and B) Western blotting analysis for the GTP loading of Gαi in Jurkat cells pretreated with vehicle, AMD3100, or X4-2-6 and stimulated with CXCL12. (A) Blots are representative of three independent experiments. IP, immunoprecipitation; IB, immunblotting. (B) Quantified data are means ± SD from all experiments. *P < 0.01 by ANOVA. (C) Enzyme-linked immunosorbent assay (ELISA)–based analysis of the intracellular concentrations of cAMP in Jurkat cells treated as indicated. Data are means ± SD of three independent experiments with six replicates per condition. *P < 0.05 and **P < 0.005 by Student’s t test. (D) Transwell migration assay of Jurkat cell chemotaxis toward CXCL12 after treatment with AMD3100 or X4-2-6. Data are means ± SD of three independent experiments each performed in triplicate. (E) Ca2+ flux analysis in THP-1 cells treated with vehicle or X4-2-6 and stimulated with CXCL12 as indicated. Data are means ± SD from 12 biological replicates. *P ≤ 5 × 10−8 by Student’s t test. (F) Ca2+ flux analysis in THP-1 cells treated with vehicle or X4-2-6 and stimulated with CCL2 as indicated. Data are means ± SD from 12 biological replicates. *P ≤ 5 × 10−8 by Student’s t test.

  • Fig. 4 X4-2-6 forms a ternary complex with CXCL12 and CXCR4 to function as a biased antagonist.

    (A to C) NMR spectroscopy analysis of CXCL12 alone (gray) and in the presence of X4-2-6 (blue). Peak superimposition (A) and changes in CXCL12 HSQC signal intensity caused by the addition of X4-2-6 (B) are representative of two independent experiments. ppm, parts per million. (C) Substantially altered residues are mapped onto the NMR structure of CXCL12 (Protein Data Bank ID: 2KEE), and the cartoon models the interaction between the N-loop of CXCL12 and X4-2-6. (D to F) NMR spectroscopy analysis of CXCL12 alone (gray) and with membrane preparations containing CXCR4 (red). Peak superimposition (D) and changes in the signal intensity of CXCL12 residues caused by the addition of CXCR4 (E) are representative of two independent experiments. (F) Substantially altered residues are mapped onto the structure of CXCL12, and the cartoon models the interaction involving insertion of the N terminus of the chemokine into the receptor transmembrane helical bundle. (G to I) NMR spectroscopy analysis of CXCL12 alone (gray) and with CXCR4-containing membranes and X4-2-6 (magenta). Peak superimposition (G) and changes in CXCL12 signal intensity caused by the addition of CXCR4 and X4-2-6 (H) are representative of two independent experiments. (I) The most substantial changes are mapped onto the NMR structure of CXCL12, and the cartoon models X4-2-6 binding to CXCR4 and CXCL12 to partially inhibit the binding of the extreme N terminus of the chemokine to the receptor.

  • Fig. 5 Proposed model for the mechanism of biased antagonism and development of tolerance to unbiased antagonists.

    (A) The current paradigm of CXCL12-mediated CXCR4 signaling suggests that the CXCL12 N terminus and N-loop insert into the CXCR4 transmembrane helical bundle, whereas the receptor N terminus binds to the globular domain of the chemokine. This leads to the activation of CXCR4 and subsequent G protein signaling, BA1/2 recruitment, and receptor endocytosis. (B) Our data suggest that X4-2-6 binds to CXCL12 and CXCR4 to form a ternary complex and displaces the extreme N-terminal portion of CXCL12 away from the transmembrane helical bundle of CXCR4. Thus, X4-2-6 functions as a biased antagonist by inhibiting G protein signaling but not BA1/2 recruitment to CXCR4. (C) In contrast, AMD3100 displaces the entire CXCL12 N terminus to inhibit all CXCR4 signaling. Over time, the inhibition of BA1/2 and the subsequent endocytosis result in the accumulation of CXCR4 on the cell surface, CXCL12 binding to the receptor, and the development of tolerance to AMD3100. GDP, guanosine diphosphate.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/552/eaat2214/DC1

    Fig. S1. AMD3100 increases CXCR4 abundance in Jurkat cells.

    Fig. S2. Neither AMD3100 nor X4-2-6 causes CXCR4 internalization.

    Fig. S3. AMD3100 but not X4-2-6 inhibits CXCL12-stimulated CXCR4 endocytosis.

    Fig. S4. BA2 recruitment downstream of CXCR4 is not substantially affected by X4-2-6.

    Fig. S5. X4-2-6 does not inhibit BA2 recruitment to CXCR4.

    Fig. S6. BA1/2 regulates ERK phosphorylation downstream of CXCR4.

    Fig. S7. CXCR4 and CCR2 share 63% amino acid sequence similarity in the region of the receptor corresponding to X4-2-6.

    Fig. S8. X4-2-6 inhibits the CXCL12-mediated chemotaxis of THP-1 cells.

    Fig. S9. X4-2-6 binds directly to CXCL12.

    Fig. S10. AMD3100 displaces the N terminus of CXCL12 from the binding site of CXCR4.

    Fig. S11. The small molecule SEN071 is a biased antagonist of CXCR4 that avoids antagonist tolerance.

  • This PDF file includes:

    • Fig. S1. AMD3100 increases CXCR4 abundance in Jurkat cells.
    • Fig. S2. Neither AMD3100 nor X4-2-6 causes CXCR4 internalization.
    • Fig. S3. AMD3100 but not X4-2-6 inhibits CXCL12-stimulated CXCR4 endocytosis.
    • Fig. S4. BA2 recruitment downstream of CXCR4 is not substantially affected by X4-2-6.
    • Fig. S5. X4-2-6 does not inhibit BA2 recruitment to CXCR4.
    • Fig. S6. BA1/2 regulates ERK phosphorylation downstream of CXCR4.
    • Fig. S7. CXCR4 and CCR2 share 63% amino acid sequence similarity in the region of the receptor corresponding to X4-2-6.
    • Fig. S8. X4-2-6 inhibits the CXCL12-mediated chemotaxis of THP-1 cells.
    • Fig. S9. X4-2-6 binds directly to CXCL12.
    • Fig. S10. AMD3100 displaces the N terminus of CXCL12 from the binding site of CXCR4.
    • Fig. S11. The small molecule SEN071 is a biased antagonist of CXCR4 that avoids antagonist tolerance.

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