Fig. 1. Chemokine- and gp120-binding CCR5 represent different receptor populations. Binding of 0.1 nM 125I-CCL3 (A) or 10 nM 35S-gp120Bx08 (in the presence of 30 nM soluble CD4) (B, C and D) was displaced by increasing amounts (A, C and D) or a 100 nM concentration (B) of unlabeled chemokines. Results were normalized for non-specific binding (0%) and specific binding in the absence of competitors (B0, 100%) and fitted according to a one-site (panel A, CCL7, 6P4, 2P3 and 5P12 in panels C and D) or a two-site (16.2 < F value < 93.3 with p < 0.0001 for CCL3 in C and CCL4 and PSC in D) competitive binding model. In panel B, data points are means ± SEM of 5 independent determinations. Panels A, C and D show representative experiments out of 3-5 performed independently.
Fig. 2. Steric inhibition of gp120 binding to CCR5 does not contribute to the anti-HIV-1 activity of PSC-RANTES. HeLa P4C5 cells were incubated with MVC, 5P12-or PSC-RANTES at 37 or 4 °C before being infected by Bx08Ren viruses and treated as indicated in the text. Results represent the luciferase activity in the cell lysates, expressed as relative light units (RLU). A representative experiment out of 5 independent determinations is shown. Uninfected cells (NI) served as negative controls in those experiments.
Fig. 3. CCR5 coupling to NFG-proteins differentially influences native agonist chemokine and gp120 binding. (A) Total binding of 0.2 nM 125I-CCL3 to 5.105 A3.01-R5 cells (left panel) or membranes from CD4+ T-cells (15 g of proteins) (right panel) was measured in the presence or absence (control) of GTPS or Gpp(NH)p or after treatment of cells with PTX. Non specific binding was determined using the antagonist TAK779 or MVC. *, p < 0.05; **, p < 0.01 as compared to controls in unpaired two-tailed Student’s t test. Panels (B) and (C) are saturation experiments of 125I-CCL3 and 125I-CCL5 binding to HEK-R5 cell membranes, respectively. Specific binding was measured in the presence or in the absence of Gpp(NH)p. Total binding of 0.1 nM 125I-CCL3 (D) or 10 nM 35S-gp120 from the HIV-1 strains 25, 34 or Bx08 in complex with sCD4 (E) to membranes from HEK 293T cells expressing WT-CCR5 or R126N-CCR5 (R/N) was measured in the presence or absence (control) of Gpp(NH)p and/or MVC (nonspecific binding). Equal amounts of WT-CCR5 and R126N-CCR5 at the cell surface were confirmed by flow cytometry. Saturation binding experiments of 35S-gp120/sCD4 complexes revealed KD values (in nM) of 7.5, 8.3 and 9.9 for gp12025, gp12034 and gp120Bx08, respectively. (F) Displacement of 35S-gp120Bx08 binding by CCL3 was measured in the absence or presence of Gpp(NH)p. Data were fitted according to a two-site competitive binding model (F = 42 with p < 0.0001 and F = 5.5 with p = 0.0062 for data in the absence and in the presence of Gpp(NH)p, respectively). Panels show representative experiments out of at least 3 performed independently.