Figure 3
Effect of guanylyl cyclase activator on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gα i -coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of the Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of BAY 41-2272 (guanylyl cyclase activator). B, The experiment involved sequential addition of the BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL12/SDF-1 (12 nM). C, The experiment involved sequential addition of BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL8/IL-8 (20 nM). The means are significantly different (p<0.05) at the peak of activation (marked on panels B and C as “*”), and at the steady state (marked in panels B and C as “**”). D, Kinetic analysis of binding and dissociation of the LDV-FITC probe. Cells were sequentially treated with the LDV-FITC probe (25 nM, near saturation), the Gαi-coupled receptor ligand (fMLFF, 100 nM), BAY 41-2272 (guanylyl cyclase activator, 50 μM). At time points indicated by arrows, cells were treated with excess unlabeled LDV containing small molecule (2 μM), and the dissociation of the fluorescent molecule was followed. Dissociation rate constants (koff) were obtained by fitting dissociation curves to a single exponential decay equation. E, Dissociation rate values, obtained in experiments analogous to panel D, summarized as a bar graph showing mean and SEM (n=4). Colors of the dissociation curves in panel D and bars on panel E are matching. The difference between koffs for “resting” and “fMLFF activated”, and between “fMLFF activated” and “fMLFF activated and treated with BAY 41-2272” is statistically significant (P < 0.05) as calculated by one-way ANOVA.