Heterologous desensitization of CCR5 and the effect of staurosporine.

Hos/CD4/CCR5/FPRL1 cells were preincubated in the absence (A) or presence (B) of staurosporine (1.4 ng/mL) at 37°C for 1 hour, followed by treatment with W peptide (1 μM) at 37°C for an additional 60 minutes. Ca⁺⁺ flux in response to RANTES (50 ng/mL) was measured. Hos/CD4/CCR5/FPRL1 cells were preincubated with (C) or without (D) staurosporine (1.4 ng/mL) at 37°C for 1 hour, and RANTES-induced Ca⁺⁺ flux was then measured. (E) Hos/CD4/CCR5/FPRL1 cells were preincubated at 37°C with medium, W peptide (1 μM, 1 hour; W pep), staurosporine (1.4 ng/mL, 1 hour) followed by W peptide (1 μM, 1 hour; Stauro+Wpep), or staurosporine alone (1.4 ng/mL, 1 hour; Stauro alone). After washing, cell migration in response to RANTES (50 ng/mL) was measured. The asterisk indicates a significantly reduced cell migration after W peptide treatment, compared with cells treated with medium only (medium). ▪ indicates medium; ■, W pep; ░, stauro + W pep; ▨, stauro alone. (F) Hos cells expressing CD4/CCR5/FPRL1 were treated at 37°C with RANTES (1 μg/mL, 1 minute) or W peptide (1 μM, 60 minutes). The cell lysates were detected for CCR5 phophorylation by using an antiphosphoserine antibody (anti-PS) for immunoprecipitation (IP) followed by immunoblot with anti-CCR5 antibody.