Role of G proteins in the binding of gp120s and chemokines to CCR5 and CXCR4 and identification of antigenically distinct conformations of CXCR4 on CD4TL (Related to Fig 4).
A Coupling of CCR5 to G-proteins is required for the binding of CCL3, but not for the binding of R5 gp120s. The panel represents the specific binding of 125I-CCL3 (0.1 nM) or of the R5 35S-gp120s (10 nM in complex with 30 nM sCD4) from patients P#1, #341, #458 and #1031 to membrane preparations from CCR5-expressing HEK 293T cells, in the presence or absence of 100 μM of 5’-guanylylimidodiphosphate (Gpp(NH)p), a nonhydrolysable GTP analog that uncouples permanently G proteins from receptors. Specific binding was deduced by subtracting from total binding the non-specific binding measured in the presence of 10 μM maraviroc. A representative experiment out two is shown. Results (expressed as % binding relative to binding in the absence of Gpp(NH)p) are means ± SEM of technical triplicates. B In contrast to CCL3, the binding of CXCL12 (0.5 nM) or of the X4 35S-gp120s (80 nM in complex with 300 nM sCD4) from patients P #39 and #208 to membrane preparations from PBMCs is poorly sensitive to treatment with 100 μM Gpp(NH)p. Specific binding was calculated as in panel A. Non specific binding of CXCR4-using ligands was determined with 10 μM AMD3100. Means ± SEM of two independent experiments are shown. C Saturation binding of anti-CXCR4 mAbs to CD4TL. PHA/IL-2-treated CD4TL (1 x 105 cells) in PBS supplemented with 1% human serum were incubated for 2 h at room temperature with increasing concentrations of mAbs (in μg/ml and in nM) targeting the second extracellular loop (ECL2) of CXCR4 (mAbs 44 708, 44716 and 44717), its N-terminus (Nt) (4G10) or a conformational epitope encompassing ECL2, ECL3 and the Nt (12G5). Cells were then stained with an AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody for 30 min at 4°C. GMFI values for each mAb were determined by flow cytometry analysis, and subtracted of the GMFI obtained for the secondary antibody alone. Binding levels of mAbs were inferred from GMFI values and are expressed as percent of maximal binding of mAb 44717. Means ± SEM of two independent experiments using cells from distinct donors are shown. Fitting of results to a one-site binding model and determination of equilibrium dissociation constants Kd were done using GraphPad Prism.
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