Figure 5
Energy transfer, on U937 cells stably transfected with the non-desensitizing mutant of FPR, between the LDV-FITC donor probe and octadecylrhodamine (R18) acceptor probe. Experiments were conducted as described under "Methods", and in [18,19]. A, Cells were preincubated at 37°C with 100 nM LDV-FITC probe to saturate high and low affinity sites. Next, LDV-FITC fluorescence was quenched after addition of octadecyl rhodamine (R18, 10 μM). Then, cells were activated by addition of fMLFF (100 nM). Data are plotted as MCF versus time for three conditions: quenched and then activated by Gαi-coupled receptor agonist (fMLFF (solid line)), quenched, then activated by Gαi-coupled receptor agonist (fMLFF) and then activated by Gαs-coupled receptor agonist (amthamine, 500 nM (dashed line)), and quenched only (R18 only, DMSO vehicle, dotted line). B, Data from panel A were re-plotted by subtracting the baseline data (R18 only) from activated cell data. The data are normalized assuming that average MCF value for FPR activated cells is equal to 100%; therefore, the Y-axis is labeled as "Donor fluorescence, % relative to fMLFF". As shown previously, activation of the cells transfected with the non-desensitizing mutant of FPR [18] led to the rapid unquenching of the FITC signal, which was interpreted as a change in the distance of closest approach between the VLA-4 ligand binding site occupied by LDV-FITC and the membrane surface (so called unbending or extension of the integrin molecule, see Fig. 1 in [18], or Fig. 5C in [19]). Curves are means out of five independent runs calculated on a point-by-point basis. C, Cells were preincubated for 5 min at 37°C with 10 μM of forskolin or DMSO (control). Next, cells were stained with 100 nM LDV-FITC probe and fluorescence was quenched after addition of octadecyl rhodamine (R18, 10 μM). Cells were activated by addition of 100 nM fMLFF, or DMSO (control). Notice the absence of the difference between forskolin treated (forskolin/fMLFF) and DMSO treated (DMSO/fMLFF) cells after addition of fMLFF. Mean and standard error of mean are shown (n = 3). D, Data from panel C were re-plotted as described for panel B.