Regulation of Cell Adhesion by Affinity and Conformational Unbending of α₄β₁ Integrin

Binding and dissociation of the LDV-FITC probe on U937 cells; energy transfer on U937 cells between the LDV-FITC donor probe and octadecylrhodamine (R18) acceptor probe; and intracellular Ca²⁺ kinetics. Experiments were conducted as described under Materials and Methods. A, LDV-FITC probe binding and dissociation on U937 cells stably transfected with the nondesensitizing mutant of FPR (ΔST) (23 ) plotted as MCF vs time. The experiment involves sequential additions of fluorescent LDV-FITC probe (4 nM), fMLFF (100 nM, solid line), or PMA (100 nM, dashed), and nonfluorescent (2 μM) LDV-containing small molecule (arrows). The MCF value corresponding to cell autofluorescence is indicated by arrow. Dissociation rate constants (k_off) obtained by fitting dissociation curves to a single exponential decay equation are shown in parentheses. B, Response kinetics of LDV-FITC probe binding to U937 cells following stimulation by different concentrations of PMA (0.5 nM, −1 μM) plotted as MCF vs time. Experiments were performed as described in A. C, Real-time FRET unbending analysis on VLA-4 in response to inside-out signaling and PMA. U937 cells stably transfected with the nondesensitizing mutant of FPR (ΔST) (23 ) were preincubated at 37°C with 100 nM LDV-FITC probe to saturate low-affinity sites in HEPES buffer containing 1 mM Ca²⁺ and 1 mM Mg²⁺. Next, LDV-FITC fluorescence was quenched after addition of 10 μM octadecyl rhodamine (R18, arrow). Then, cells were activated by addition of 100 nM fMLFF or 1 μM PMA. Data are plotted as MCF vs time for three conditions: quenched and then activated by fMLFF (solid line), quenched and then activated by PMA (gray solid line), and quenched only (R18 only, DMSO vehicle, dashed line). D, Normalized data from C processed as described in the text. SE is shown for every 20-s time point (n = 2). LDV-FITC probe binding and dissociation experiments, together with FRET experiments, were performed on the same day using the same cells and the same set of activating reagents. E, Kinetics of intracellular Ca²⁺ response detected using fluo-4 AM, after sequential additions of PMA (1 μM), followed by fMLFF (100 nM) (solid line), and fMLFF (100 nM), followed by PMA (1 μM) (dashed line), in U937 cells transfected with FPR (ΔST). Notice the absence of the signal increase (rather some decrease) after addition of PMA. One representative experiment of three experiments is shown.

Response kinetics of LDV-FITC probe binding to U937 cells following stimulation by fMLFF and Ca²⁺ ionophore (A23187); energy transfer on U937 cells between LDV-FITC donor probe and octadecylrhodamine (R18) acceptor probe. A, Response kinetics of LDV-FITC binding to U937 cells transfected with the nondesensitizing mutant of FPR (ΔST) following stimulation by fMLFF and A23187 are plotted as MCF vs time. The experiment involved sequential additions of LDV-FITC (4 nM), fMLFF (100 nM), or A23187 (10–40 μg/ml). The MCF value corresponding to the cell autofluorescence is indicated by arrow. B, Real-time FRET experiments are as described in Fig. 2C, except that A23187 (40 μg/ml, solid line) was used to activate VLA-4. C, Real-time FRET experiments are as described in Fig. 2D, except cells treated with PMA (1 μM) were treated additionally with A23187 (40 μg/ml) (PMA, A23187, solid line). Data were processed as described for Fig. 2, C and D, and therefore, the y-axis labeled as “Donor fluorescence, % relative to fMLFF.” The positive control (fMLFF, 100 nM) is also shown. Curves are means of two independent runs calculated on a point-by-point basis. SE is shown for every 20-s time point (n = 2).

Response kinetics of LDV-FITC probe binding to U937 cells following treatment with fMLFF and PLC inhibitor U-73122. A, U937 cells transfected with FPR (ΔST) were preincubated for 30 min with 2 μM U-73122 (dashed line) or 2 μl of DMSO (solid line) at 37°C. Next, LDV-FITC probe (4 nM) and fMLFF (100 nM) were sequentially added. B, U937 cells transfected with FPR (ΔST) were sequentially treated with LDV-FITC probe (4 nM), fMLFF (100 nM), and different concentrations of U-73122. Data are plotted as MCF vs time. The MCF value corresponding to the cell autofluorescence is indicated by arrow.

Kinetics of intracellular Ca²⁺ response, detected using fluo-4, AM; LDV-FITC probe binding and dissociation following cell treatment by fMLFF, PLC inhibitor U-73122, and Ca²⁺ ionophore A23187. A, Kinetics of fluo-4 signal changes in response to the fMLFF activation of U937 cells transfected with FPR (ΔST). B, Kinetics of fluo-4 signal changes after sequential addition of fMLFF (100 nM), U-73122 (1 μM), and A23187 (40 μg/ml). C, Response kinetics of LDV-FITC probe binding to U937 cells following stimulation by fMLFF, or D, after sequential addition of fMLFF (100 nM), U-73122 (1 μM), and A23187 (40 μg/ml). Before the stimulation cells in (C and D) were preincubated with 4 nM LDV-FITC probe for 5 min at 37°C. Data are plotted as MCF vs time.

Effect of U-73122 on the kinetics of energy transfer, LDV-FITC probe binding, and kinetics of intracellular Ca²⁺ response following stimulation with fMLFF in U937 cells transfected with FPR (ΔST). A, Kinetics of the FRET signal change after sequential addition of fMLFF (100 nM), U-73122 (1 μM), and in the presence or absence of A23187 (40 μg/ml). The positive control (100 nM fMLFF) is also shown. Data were processed as described for Fig. 2, C and D, and therefore, the y-axis labeled as “Donor fluorescence, % relative to fMLFF.” B–D, Cell were preincubated with 1 μM U-73122 for 10 min at 37°C. Control cell were preincubated with DMSO. B, Response kinetics of LDV-FITC probe binding to U937 cells following treatment with fMLFF. Cells were treated sequentially with LDV-FITC probe (4 nM) and fMLFF (100 nM). C, Real-time FRET experiments are as described in Fig. 2D, except cells treated with fMLFF (100 nM). Data were processed as described for Fig. 2, C and D, and therefore, the y-axis labeled as “Donor fluorescence, % relative to fMLFF.” D, Kinetics of intracellular Ca²⁺ response, detected using fluo-4, AM; after addition of fMLFF (100 nM). A and B, SE is shown for every 20-s time point (n = 2). C and D, SE is shown for every point (n = 3). Experiments shown on B–D were performed on the same day using the same cells and the same set of reagents.

Real-time kinetics of VLA-4 affinity change, energy transfer, and intracellular Ca²⁺ response after activation of purinergic receptors constitutively expressed on U937 cells (25 ) transfected with FPR (ΔST). A, Response kinetics of LDV-FITC probe binding to U937 cells following treatment with ATP or fMLFF (positive control). Before the experiment, cells were preincubated with 4 nM LDV-FITC probe for 5 min at 37°C. Data are plotted as MCF vs time. B, Kinetics of the FRET signal change after addition of ATP (10 μM). The positive control (100 nM fMLFF) is also shown. Data were processed as described for Fig. 2, C and D, and therefore, the y-axis labeled as “Donor fluorescence, % relative to fMLFF.” C, Kinetics of intracellular Ca²⁺ response, detected using fluo-4, AM, as described in Materials and Methods. The baseline value (dashed line) represents the mean value before addition of the stimuli. Data are plotted as MCF vs time. Representative experiments of three independent experiments are shown.

Changes in cell adhesion between U937 FPR (ΔST) and VCAM-1-transfected B78H1 cells at resting state and in response to receptor stimulation. A, Data are plotted as percentage of aggregates vs time. Four different experimental conditions are shown (resting state (no stimulus), stimulation with 100 nM fMLFF, stimulation with 1 μM ATP, and preincubation with blocking LDV small molecule). The nondesensitizing FPR mutant is used to maintain VLA-4 in a state of constant affinity. B, Data are plotted as aggregates and U937 FPR (ΔST) singlets vs time. Singlet depletion curve exhibited a single exponential kinetics. C, The effect of PLC inhibitor U-73122 upon cell adhesion. Data are plotted as aggregates and U937 FPR (ΔST) singlets vs time. Small arrows indicate the moment of stimulus addition. Representative experiments of three independent experiments are shown.

Estimation of the initial rate of cell aggregation between U937 FPR (ΔST) and VCAM-1-transfected B78H1 cells. A, Singlet cell depletion data as shown on a Fig. 8B were normalized assuming that average singlets count for blocked sample is equal to 0%, and 0 singlet count is equal to 100% (no singlets left in solution, therefore all cells are in the aggregates); therefore, the y-axis is labeled as “ΔST FPR U937 singlets in the aggregate, %.” Four different experimental conditions are shown (resting state (no stimulus), stimulation with 100 nM fMLFF added at the time of the cell mixing, stimulation with 1 μM PMA for 10 min at 37°C before cell mixing (this time is sufficient to generate high-affinity state of VLA-4 (Fig. 2B)) and preincubation with blocking LDV small molecule). Curves show a single exponential fit to the data. B, Estimation of the initial rate of cell aggregation. Using the curve fits from A, the absolute rates of the cell aggregation (percentage of cell singlet incorporated into the aggregate per second) were calculated for each time interval and plotted vs time. The initial rate of the cell aggregation extrapolated to the time 0 point is shown next to each curve. Representative experiment of four independent experiments is shown.