Figure 1
Binding and dissociation of the LDV-FITC probe in response to Gα s -coupled receptor agonists. Experiments were conducted as described under "Methods". A, LDV-FITC probe binding and dissociation on U937 cells stably transfected with CXCR4 receptor plotted as mean channel fluorescence (MCF) versus time. The experiment involved sequential additions of Gαs-coupled receptor ligands (isoproterenol (100 nM) or amthamine (500 nM)), then, fluorescent LDV-FITC probe (4 nM, below saturation, added 3 min prior to addition of Gαi-coupled receptor ligands). Control cells were treated with vehicle (DMSO). Next, cells were activated with SDF-1 (25 nM, arrows). Rapid and reversible binding of the probe reflects VLA-4 affinity change [6]. Curves are means out of two independent runs calculated on a point-by-point basis. B, LDV-FITC probe binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant of FPR (ΔST) [33] plotted as mean channel fluorescence (MCF) versus time. The experiment involved sequential additions of fluorescent LDV-FITC probe (4 nM), fMLFF (100 nM), and different concentrations of amthamine, or DMSO (control) (arrows). The MCF value corresponding to cell autofluorescence is indicated by the horizontal arrow. One representative experiment out of three experiments is shown. Experiments shown in the different panels were performed using different instruments, and therefore MCF values are not identical.