Fig 1.
Sequences and structural features of CXCR6 around the DRF motif.
A: Sequence of TM3 (residues 3.37–3.56) in human chemokine receptors. The DRY motif (residues 3.49–3.51) is highlighted in green, the DRF motif of CXCR6 in red, other motifs at this position in orange, and residues at position 3.56 in blue. B: 2D structure of the human CXCR6 (obtained from the GPCRdb [67]), with DRF motif highlighted in red. C/D: Snapshot from the molecular dynamics simulation of the 3D model of the human CXCR6. The residues in the DRF motif (D1263.49, R1273.50 and F1283.51) are shown as spheres, and a lipid molecule in contact with F1283.51 is shown as sticks (C). The cluster of hydrophobic residues around F1283.51 is shown in D. The rest of protein side chains, lipids, water molecules, and ions are not displayed for clarity. E: 3D structure of the human CCR5 receptor, showing the hydrogen bond between residues at positions 3.51 and 3.56.
Fig 2.
Rescue of the DRY motif in CXCR6 does not affect ligand binding or receptor recycling in HEK293 cells.
HEK293 cells were transduced with lentivirus encoding the human CXCR6 variants or the EV control. A/C-E: Surface expression of CXCR6 in untreated cells (A, n = 4), after receptor internalization induced by 15 min treatment with soluble CXCL16 (C, n = 3), and receptor recycling (D, dyna = dynasore, n = 4) was determined by FACS analysis using an antibody against human CXCR6. Signals were expressed in relation to control (EV in A, untreated cells in C/D). Receptor recycling was further quantified as area under the curve of data shown in D (AUC, E). B: Ligand binding was analyzed by incubation with CXCL16-Fc fusion protein and FACS analysis (n ≥ 3, a.u. = arbitrary units). Statistical differences were analyzed by one-sample t-test (A and C, hypothetical value 1) or Student’s t-test (B, D and E). Asterisks indicate differences to control (*p<0.05, **p<0.01).
Fig 3.
Rescue of the DRY motif in CXCR6 does not affect ligand binding or receptor recycling in THP-1 cells.
THP-1 cells were transduced with lentivirus encoding human CXCR6 variants or EV control. A: Ligand binding of the CXCL16-Fc fusion protein (n ≥ 6, a.u. arbitrary units). B-D: Surface expression of CXCR6 after receptor internalization (B, 1 nM CXCL16-Fc for 15 min at 37°C), and receptor recycling (C, 1 nM CXCL16-Fc for 15 min at 37°C and subsequent removal) was analyzed by incubation with CXCL16-Fc fusion protein and FACS analysis (n = 3). Recycling was determined after 0, 5, 15, 30, and 60 min. Signals were expressed in relation to untreated cells (1 nM CXCL16-Fc for 15 min at 4°C). Receptor recycling was further quantified as area under the curve of data shown in C (AUC, D). Statistical differences were analyzed by one-sample t-test (B, hypothetical value 1) or Student’s t-test (A, C and D). Asterisks indicate differences to control (*p<0.05, ***p<0.001).
Fig 4.
Rescue of the DRY motif in CXCR6 has no major impact on AKT signaling and adhesion.
THP-1 cells were transduced with lentivirus encoding human CXCR6 variants or EV control. A/B: AKT activation upon stimulation with 10 nM soluble CXCL16 (A, n = 7) or with 3 nM CCL2 (B, 10 min, control of general responsiveness, n = 7) was measured as ratio of phosphorylated AKT (pAKT) to total AKT investigated by Western blot analysis. Data were expressed in relation to buffer control stimulated cells. C: Adhesion to immobilized CXCL16-Fc for 30 min was normalized to cells adhering to anti‑human-Fc (set = 1 for each cell-type, n = 4). Cells were pretreated with 100 nM soluble CXCL16 for 15 min to indicate specificity of CXCL16-CXCR6-mediated adhesion. D: Adhesion to immobilized CXCL16-Fc for 5, 15, and 30 min was normalized to cells adhering to anti‑human-Fc (set = 1 for each cell-type, n = 3). Statistical differences were analyzed by one-sample t-test (C for differences to adhesion to anti-human-Fc, hypothetical value 1) or Student’s t-test (A-D, with Welch’s correction in A). Asterisks indicate differences to control (untreated cells in B/C, EV in A/D), hashes indicate differences between receptor variants in A and D and CXCL16 preincubation in C (*/#p<0.05, **p<0.01, ***p<0.001).
Fig 5.
The DRY motif is optimized for calcium signaling and chemotaxis.
THP-1 cells were transduced with lentivirus encoding human CXCR6 variants or EV control. A/B: Cells were loaded with Fluo4-AM and stimulated with 10 nM soluble CXCL16 (A, n = 4), increasing concentrations of soluble CXCL16 (n = 4), or 3 nM CCL2 (control of general responsiveness, n = 3) (B). The increase of the Ca2+ signal was measured as increase in Fluo4-AM-fluorescence. Data were normalized to signals at 9 sec in A and quantified as AUC. In B, maximal fluorescence intensity was determined, and minimal fluorescence intensity was subtracted for each calcium response and expressed in relation to the response of EV cells for each concentration. C: Chemotaxis against increasing concentrations of soluble CXCL16 or 3 nM CCL2 (control of general migratory potential, n ≥ 4) was analyzed in a Boyden chamber assay (n ≥ 4). Statistical differences were analyzed by Student’s t-test. Asterisks indicate differences to control (EV in A, each buffer control in B, random migration in C), hashes indicate differences between receptor variants (*/#p<0.05, **p<0.01).
Fig 6.
The DRF motif impairs chemotaxis.
THP-1 cells were transduced with lentivirus encoding murine CX3CR1 variants or EV control. A: Ligand binding was analyzed by incubation with human CX3CL1-Fc fusion protein and FACS analysis (n = 3). B/C: Chemotaxis against increasing concentrations of soluble CX3CL1 was analyzed in a Boyden chamber assay. In B, wild type cells were investigated for chemotaxis towards soluble murine or human CX3CL1 (n = 3), and in C cells overexpressing murine receptor variants were assayed for chemotaxis towards murine CX3CL1 (n ≥ 3). Statistical differences were analyzed by one-sample t-test (B, hypothetical value 1) or Student’s t-test (A-C). Asterisks indicate differences to control (EV in A, random migration in B/C), hashes indicate differences between receptor variants (*/#p<0.05, **/##p<0.01, ***p<0.001).