FIGURE 4.

PSC-RANTES stabilizes spare CCR5 conformations. A, displacement of 1 nm PSC-RANTES-biot binding to CCR5-expressing A3.01 cells by increasing concentrations of the indicated chemokines. Data with unlabeled PSC-RANTES used as a competitor were fitted according to a one-site competitive binding model (mean ± S.E., n = 4). B, PSC-RANTES-biot or [¹²⁵I]CCL3 dissociation from CCR5. CCR5-expressing A3.01 cells were incubated for 90 min with 1 nm PSC-RANTES-biot or for 120 min with 0.5 nm [¹²⁵I]CCL3. Dissociation was initiated by addition of 100 nm 5P12 (a high-affinity CCR5 antagonist). PSC-RANTES-biot or [¹²⁵I]CCL3 remaining bound to CCR5 was measured over time and analyzed as in Figs. 4A and 1B (one representative experiment of two). C and D, dose-dependent CCR5 internalization in CCR5-349-expressing HEK293 cells (C) or in CCR5-expresssing A3.01 cells (D) mediated by 20 min (C) or 5 min (D) of stimulation with PSC-RANTES at 37 °C. C, receptor internalization was measured after incubation, or no incubation, of CCR5-349-expressing HEK293 cells with 100 nm CCL5 at 4 °C for 90 min (data were fitted according to a sigmoidal dose-response model with a variable slope) (mean ± S.E., n = 4). D, receptor internalization was measured after incubation, or no incubation, of A3.01 cells with 300 nm CCL4 at 4 °C for 90 min (data were fitted according to a sigmoidal dose-response model with a variable slope) (mean ± S.E., n = 3). E–G, calcium responses measured in Fura-2-loaded CCR5-expressing HEK293 cells (E and F) or primary T cells (G) stimulated twice (2-min intervals between the first and the second stimulation) with 15 nm chemokines. E, results were obtained by imaging cells coated on a glass coverslip. Each trace is the mean of at least 25 single-cell calcium responses. F and G, results were obtained by spectrofluorimetry on the entire resuspended HEK293 cell population (F) (n = 3 independent experiments) or primary T cell population (G) (one representative experiment of two performed on two different donors). *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant; unpaired, two-tailed Student's t test. C, CCL5; P, PSC-RANTES.