Functional analysis of CCR5 R60S protein in 293T cells.
The ability of transiently transfected wild-type (A) and R60S CCR5 (B) cells to stimulate increases in intracellular calcium concentration in response to MIP-1β was measured using calcium-sensitive dye Fluo-3,am, which is converted to Fluo-3 by intracellular esterases. Representative traces of fluorescence (λex 505 nm, λem 525 nm) (y-axis) from fluo-loaded cells in response to injection of 1 mM carbachol (control) and 33 nM MIP-1β as a function of time (x-axis) are depicted. To control for efficient loading of the dye to the cells and thereby standardize the response, the carbachol-dependent calcium flux associated with ligand binding to endogenous muscarinic receptors was also measured. The activity of the wild-type CCR5 receptor was measured as the ratio of the height of the peak for MIP-1 β (0.42 arbitrary fluorescence units) to that of the peak for carbachol (0.12 units) and was calculated as 3.5 in this experiment and defined as 100%. The height of the MIP-1 β peak (0.16 units) divided by the carbachol peak (0.08 units) was calculated to be 2, which translated as 57% of the wild-type CCR5 receptor activity in this experiment. The labeled arrows indicate the addition of 1 mM carbachol and 33 nM MIP-1β.
