Phosphorylation of CCR5 in monocytes treated with fMLF.
(A, B) Monocytes preincubated with the designated concentrations of fMLF (1 hour at 37°C) (panel A) or with 10−6 mol/L fMLF (panel B) for designated time intervals were lysed, and lysates (200 μg) were subjected to immunoprecipitation with an antiphosphoserine antibody followed by immunoblotting with anti-CCR5 antibody. Chemokine MIP-1α at 1 μg/mL (37°C, 1 minute) was used as a positive control. Inset in panel B shows the detection by immunoblotting of CCR5 in 20 μg cell lysates prior to immunoprecipitation. Relative fold increase in levels of phosphorylated CCR5 normalized to receptor contents were as follows: for fMLF, 1.2 (0.5 minutes), 5 (1 minute), 7 (15 minutes), and 10 (60 minutes); for MIP-1α, 11 (1 minute). (C) Monocytes preincubated with or without calphostin C (Cal C; 50 ng/mL, 2.5 hour at 37°C) were stimulated with MIP-1β (1 μg/mL, 37°C, 1 minute) or fMLF (10−6 mol/L, 37°C, 60 minutes), and the cell lysates were examined for serine phosphorylation of CCR5. (D) Monocytes were treated with different concentrations of synthetic T20 peptide derived from HIV-1 gp41 and were then measured for CCR5 phosphorylation. MIP-1α treatment at 1 μg/mL (37°C, 1 minute) was used as a positive control.
