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Originally published In Press as doi:10.1074/jbc.M404387200 on May 27, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32435-32443, July 30, 2004
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Conformational Regulation of {alpha}4{beta}1-Integrin Affinity by Reducing Agents

"INSIDE-OUT" SIGNALING IS INDEPENDENT OF AND ADDITIVE TO REDUCTION-REGULATED INTEGRIN ACTIVATION*

Alexandre Chigaev{ddagger}, Gordon J. Zwartz{ddagger}, Tione Buranda{ddagger}, Bruce S. Edwards{ddagger}, Eric R. Prossnitz§, and Larry A. Sklar{ddagger}||

From the {ddagger}Department of Pathology and the Cancer Center and the §Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131 and the National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Received for publication, April 21, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}4{beta}1-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670–48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn2+ in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {alpha}4{beta}1-integrin (very late antigen-4 (VLA-4)1, CD49d/CD29) is a heterodimeric protein and a member of the family of adhesion receptors that is broadly expressed in lymphocytes and dendritic cells (1) and stem cells (2). VLA-4 has a flexible molecular structure that allows initial capture, tethering, rolling, and firm attachment of cells using the same counterstructure (3, 4). These properties appear to result from regulation of affinity and conformation by "inside-out" signaling (5, 6). Although the precise molecular mechanism of integrin conformational activation is unknown, significant understanding of this mechanism has been achieved in the last several years.

One model involves the "mechano-conformational" regulation of integrin affinity and conformation. It is based on the idea that the separation of the intracellular {alpha}- and {beta}-subunit tails may initiate "a piston-like or scissors-like motion" of the transmembrane domains (7). This motion results in a large conformational rearrangement of the integrin, accompanied by a "switchblade-like" opening of the molecule (8) and its conformational activation (5, 6, 9, 10). This "tail separation model" is supported by the experiments by Lu et al. (11) and Takagi et al. (12), in which unclasping of the link between C-terminal parts of the integrin subunits results in the conformational activation of the molecule. A direct demonstration of the spatial separation of the lymphocyte function-associated antigen-1 integrin tails by fluorescence resonance energy transfer (FRET) has been recently published (10). The tail separation might be induced by binding of adaptor proteins such as talin (a common adaptor protein that binds to the {beta}-subunits) (13) and paxillin (an {alpha}4-specific adaptor, whose binding is regulated by phosphorylation of the integrin) (14–16).

Another model suggests that integrin conformation is regulated by reduction of the disulfide bonds and possibly involves protein-disulfide isomerase (PDI) (17–19). PDI regulates disulfide exchange and conformationally induced shedding of L-selectin (20). Moreover, dithiothreitol (DTT) and other reducing agents elevate integrin-mediated cell adhesion avidity (21–23), and non-penetrating blockers of the free sulfhydryl groups inhibit integrin-mediated adhesion (22, 24). Bacitracin, an inhibitor of the reductive function of the plasma membrane, and anti-PDI monoclonal antibody (mAb) cause inhibition of ligand binding to the {beta}3-integrin (19).

A number of mutational studies have shown that disruption of disulfides in the integrin {beta}3-subunit results in constitutively active integrins (Cys5, Cys435, Cys560, Cys598, Cys663, and Cys687) (25–28). Truncated {beta}3-subunits (amino acids 1–469) lacking the Cys-rich domain form heterodimers that bind fibrinogen with high affinity (29). In addition, all 56 cysteines in the integrin {beta}-subunits were found to be well conserved throughout evolution (see Fig. 1 in Ref. 30). Thus, there is good evidence that reduction or disruption of the disulfide bonds could be an important mechanism in the regulation of the integrin-dependent cell adhesion.

Recently, we developed a new approach for monitoring the relationship between VLA-4 affinity, cell avidity, and molecular conformation. Using a VLA-4-specific fluorescent probe based on an LDV-containing compound, we were able to monitor changes in integrin affinity and conformation in real time in live cells in response to cell activation by inside-out signaling (31, 32). We have also demonstrated a strong correlation between VLA-4 affinity and cell adhesion avidity (33).

The goal of this study was to investigate the role of reducing agents in regulating integrin conformation and affinity. We found that reducing agents induced multiple affinity states of VLA-4 and that the affinity changes were accompanied by an extension of VLA-4 detected using FRET. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of reducing agents and had no effect on the inside-out signaling generated using G protein-coupled receptors (GPCRs). Based on these results and differences in the kinetics of integrin activation, we conclude that the activation of VLA-4 by inside-out signaling is independent of activation by reducing agents.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The VLA-4-specific ligand 4-((N'-2-methylphenyl)ureido)phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (referred to as LDV-containing small molecule) (31–33) and its FITC-conjugated analog (LDV-FITC-containing small molecule) were synthesized at Commonwealth Biotechnologies, Inc. (Richmond, VA). Octadecyl rhodamine B chloride (R18) was from Molecular Probes, Inc. (Eugene, OR). All restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA). All other reagents were from Sigma.

Cell Lines and Transfectant Construct—The human monoblastoid cell line U937 was purchased from American Type Culture Collection (Manassas, VA). Site-directed mutants of the formyl peptide receptor (FPR) in U937 cells were prepared as described (34). High expressers were selected using the MoFlo flow cytometer (Cytomation, Inc., Fort Collins, CO). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (pH 7.4), and 10% heat-inactivated fetal bovine serum; harvested; resuspended in 1 ml of HEPES buffer (110 mM NaCl, 10 mM KCl, 10 mM glucose, 1 mM MgCl2, and 30 mM HEPES (pH 7.4)) containing 0.1% human serum albumin; and stored on ice. The buffer was depleted of lipopolysaccharide by affinity chromatography over polymyxin B-Sepharose (Detoxigel, Pierce). Cells were counted using a Coulter Multisizer/Z2 analyzer. For experiments, cells were suspended in HEPES buffer at 1 x 106 cells/ml and warmed to 37 °C. The expression of VLA-4 was measured with fluorescent mAbs and quantified by comparison with a standard curve generated with Quantum Simply Cellular microspheres (Bangs Laboratories, Inc., Fishers, IN) stained in parallel with the same mAb. This produces an estimate of the total mAb-binding sites/cell. Typically, we find 40,000–60,000 VLA-4 sites/U937 cell.

Kinetic Analysis of Binding and Dissociation—Kinetic analysis of the binding and dissociation of the LDV-FITC-containing small molecule was described previously (31). Briefly, U937 cells (1 x 106 cells/ml) were preincubated in HEPES buffer containing 0.1% human serum albumin under different conditions for 10–40 min at 37 °C: divalent cations (Mn2+, Ca2+), DTT (up to 3 mM), or 2,3-dimercapto-1-propanesulfonic acid (DMPS; up to 50 mM). Flow cytometric data were acquired for up to 1000sat37 °C while the samples were stirred continuously at 300 rpm with a 5 x 2-mm magnetic stir bar (Bel-Art Products, Pequannock, NJ). Samples were analyzed for 30–120 s to establish a base line. The fluorescent ligand was added, and acquisition was re-established, creating a 5–10-s gap in the time course. For real-time activation experiments, U937 cells were preincubated with 4 nM LDV-FITC-containing small molecule for 15 min at 37 °C. Then, data were acquired for 30–120 s to establish a base line, and DTT (3 mM), N-formyl-L-methionyl-L-leucyl-L-phenylalanyl-L-phenylalanine (fMLFF; 100 nM), or ATP (1 µM) was added. Acquisition was re-established, and data were acquired continuously for up to 1000 s. The concentration of the LDV-FITC-containing small molecule chosen for the experiments (4 nM) is below the dissociation constant (Kd) for binding to resting VLA-4 (low affinity; Kd ~ 12 nM) and above the Kd for the physiologically activated receptor (high affinity; Kd ~ 1–2 nM) (31). Therefore, the transition from the low to high affinity receptor state leads to increased binding of the probe (from ~25 to ~70–80% of receptor occupancy, respectively), which is detected as an increase in the mean channel fluorescence. For dissociation kinetic measurements, cell samples were preincubated with the fluorescent ligand (4–10 nM) and treated with excess unlabeled LDV-containing small molecule (2 µM), and the dissociation of the fluorescent molecule was followed. The resulting data were converted to mean channel fluorescence versus time using FCSQuery software (developed by one of us, B. S. E.).

Cell Pretreatment with Bacitracin—U937 cells were preincubated on ice for 1.5 h with 1 mM bacitracin. Prior to the experiment, 4 nM LDV-FITC-containing small molecule was added, and cells were incubated at 37 °C for an additional 15 min. Data were acquired using a flow cytometer for 30 s to establish a base line. Then, DTT (3 mM), fMLFF (100 nM), or ATP (1 µM) was added. For dissociation experiments, cells were preincubated with bacitracin and the LDV-FITC-containing small molecule as described above and treated with excess unlabeled LDV-containing small molecule (2 µM). The dissociation of the fluorescent molecule was followed.

FRET Detection of Integrin Conformational Activation—The FRET assay was performed with a peptide donor (the LDV-FITC-containing small molecule, which specifically binds to the {alpha}4-integrin head group) and octadecyl rhodamine B acceptors incorporated into the plasma membrane previously described in detail (32). Briefly, U937 cells were preincubated with 50–100 nM LDV-FITC-containing small molecule to saturate low affinity sites of the integrin in HEPES buffer containing 0.1% human serum albumin supplemented with 1 mM Mn2+, 1 mM Ca2+, 1–3 mM DTT, or a combination of the reagents for up to 50 min at 37 °C. Next, samples were incubated with different concentrations of R18 (up to 20 µM) for 1 min. Donor intensities (FL1) were measured using a BD Biosciences FACScan flow cytometer at 37 °C.

The quenching curves generated using the following procedure characterize the distance of closest approach between the integrin head group and the surface lipid membrane as was shown previously (32). For real-time FRET experiments, U937 cells were stably transfected with the wild-type FPR or its non-desensitizing mutant (FPR{Delta}ST) (35, 36). The U937 cells were preincubated with 50–100 nM LDV-FITC-containing small molecule in HEPES buffer containing 1.5 mM CaCl2 and 1 mM MgCl2 at 37 °C. Samples were analyzed for 60–120 s to establish a base line, and then saturating R18 (10 µM final concentration) was added to yield maximal quenching. 1 min after R18 was added, the fMLFF peptide (0.1 µM), ATP (1 µM), or DTT (3 mM) was added. Flow cytometer acquisition was re-established after a 5–10-s gap. The cells were also tested using low concentrations of the LDV-FITC-containing small molecule (3–5 nM) to determine the affinity change as described above.

Statistical Analysis—Curve fits and statistics were determined using GraphPad Prism. Mean values are presented in Table I and the figures. Each experiment was repeated three times. The experimental curves represent the mean of two independent runs. S.E. values were calculated using GraphPad Prism.


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  		TABLE I
Summary of dissociation rate constants for U937 cells treated with different concentrations of DTT and DMPS

The data were fit to the equation MCF = Span1·e–k1t + Span2·e–k2t + Span3·e–k3t + Plateau, where MCF (mean channel fluorescence) represents total binding, kn is the dissociation rate constant, t is time, and Plateau is nonspecific fluorescence. Span = Span1 + Span2 + Span3 is the difference between binding at time 0 and Plateau. Dissociation rate constants were fixed at k1 = 0.06 s–1, k2 = 0.01 s–1, and k3 = 0.002 s–1. The Span value was assigned to equal 1 (Span1 + Span2 + Span3 = 1). Next, a fraction corresponding to Span1, Span2, and Span3 was calculated. These values, corresponding to a fraction of VLA-4 receptors in each affinity state, are shown below. For comparison, a two-component fit is shown for 3 mM DTT (dissociation rate shown in parentheses).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Reducing Agents Generate Multiple Affinity States of the VLA-4 Integrin as Detected Using the Fluorescent Ligand— Multiple affinity states of VLA-4 have been detected using the LDV-FITC-containing molecule in real time in response to activation by divalent cations; activating mAbs; or inside-out signaling in response to stimulation of CXC chemokine receptor-2 and -4, FPR, and IgE and interleukin-5 receptors (31). The affinity of the LDV-FITC probe for the integrin varies in parallel with the affinity of a native ligand, vascular cell adhesion molecule-1 (VCAM-1). Cell adhesion avidity was found to be strongly dependent on the affinity of the integrin (33, 37). Here, we used the same LDV-FITC-containing molecule to probe the affinity of VLA-4 on the surface of U937 cells treated with different concentrations of DTT and DMPS. DMPS is known to be a membrane-impermeable reducing agent due to the presence of a charged acidic group. Fig. 1A shows a typical binding and dissociation experiment in which the LDV-FITC-containing molecule was added to a cell suspension after 30 s of stirring. Excess unlabeled competitor was added 3 min later. By fitting the dissociation kinetics to double exponential curves, we extracted rate constants corresponding to states of different affinity (Table I). In all experiments, with the exception of those performed with untreated cells, a combination of high and low affinity state receptors was detected (Fig. 1, A–C). For untreated cells, only a single exponential fit was needed, and koff ~ 0.06–0.1 s–1 was obtained. This off-rate corresponds to the resting receptor state (31). In addition, a resting state was detected in cells treated with low concentrations of reducing agents (300 µM DTT and 1–20 mM DMPS) (Table I).



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  		FIG. 1.
Binding and dissociation of the LDV-FITC-containing small molecule in U937 cells. Experiments were conducted as described under "Experimental Procedures." A, LDV-FITC-containing small molecule binding and dissociation in U937 cells plotted as mean channel fluorescence (MCF) versus time after sequential additions of fluorescent (4 nM) and non-fluorescent (2 µM) LDV-containing small molecules (arrows). U937 cells were pretreated in HEPES buffer with 1 mM DTT for 10 min at 37 °C (open circles) or in vehicle (Untreated; closed circles). Values of mean channel fluorescence corresponding to the cell autofluorescence and nonspecific binding of the LDV-FITC-containing small molecule are indicated by dashed arrows. B, LDV-FITC-containing small molecule dissociation plotted as mean channel fluorescence versus time. U937 cells were preincubated for 40 min at 37 °C with the indicated concentrations of DTT in the presence of 4 nM LDV-FITC-containing small molecule. Next, 2 µM non-fluorescent LDV-containing small molecule was added to induce probe dissociation (arrows). Curves were fitted to a one-phase exponential curve (Untreated; +), or a two-phase exponential curve (all other symbols). Calculated off-rate constants are presented in Table I. C, the same experiment as shown in B, but with DMPS instead of DTT. D, schematic showing a hypothetical mechanism implying that sequential reduction of the disulfides (-S–S- -> -SH + HS-) results in a change in the affinity/conformation of the integrin.

 
Changes in VLA-4 affinity were strongly dose-dependent, and fits required at least two dissociation rates. Under the strongest reducing conditions (3 mM DTT), the dissociation could be fit with two rates (0.014/s and 0.002/s) or with three rates, resembling the resting, intermediate, and high affinity states. For consistency, all the data were fit with three fixed rates, with higher concentrations of DTT and DMPS or longer incubation times resulting in a larger fraction of high affinity receptors (Table I). The progression of quantitatively similar states led to the idea that a sequential reduction of each of the disulfide bonds generates a distinctive conformation of the molecule (Fig. 1D).

Kinetics of the Affinity Changes Induced by Mn2+ and DTT in Real Time—Next, real-time activation was used to measure the kinetics of the VLA-4 affinity change. U937 cells were preincubated with the LDV-FITC-containing small molecule and treated with DTT alone or in combination with 1 mM Mn2+ (Fig. 2). Mn2+ is used to induce a higher affinity state of VLA-4 with a distinctive extended conformation (31, 32). The effect of Mn2+ was stable and irreversible for >1000 s. The addition of DTT induced a slow and gradual increase in the LDV-FITC-containing small molecule binding. This was completely different from a rapid activation induced by Mn2+ (Fig. 2A). Next, when the two stimuli were added together, biphasic binding kinetics were observed. A rapid binding phase (60–120 s) resembling the Mn2+-alone curve was followed by a slow gradual signal increase similar to the DTT-alone curve (Fig. 2A). Analysis of the dissociation kinetics (Fig. 2B) confirmed that DTT added together with Mn2+ created higher VLA-4 affinity than Mn2+ added alone (a slower dissociation rate corresponds to a state of higher affinity (31)).



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  		FIG. 2.
Response kinetics of the LDV-FITC-containing small molecule binding to U937 cells following stimulation by Mn2+ and DTT. A, U937 cells were preincubated with 4 nM LDV-FITC-containing small molecule in HEPES buffer containing 1 mM CaCl2 and 0.1% human serum albumin for 5–10 min at 37 °C. Next, 1 mM DTT (arrow 2, closed triangles), 1 mM Mn2+ (arrow 2, closed circles), or sequentially 1 mM DTT (arrow 1, open circles) and 1 mM Mn2+ (arrow 2, open circles) were added. B, shown is the dissociation of the LDV-FITC-containing small molecule initiated by the addition of 2 µM non-fluorescent LDV-containing small molecule (arrow) to cells treated as described for A. Dissociation rate constants shown on the graph were obtained by fitting data to single exponential curves. C, the data corresponding to the Mn2+ experiment (A, closed circles) were subtracted from the data for cells treated with DTT and Mn2+ (A, open circles) and are plotted (open circles) on the same panel with the DTT-alone data (A, closed triangles). The base-line value 140 (shown in A by the dashed line) was subtracted from the DTT-alone data (inverted closed triangles). The slope of the curve for DTT and Mn2+ minus Mn2+ remains constant over time. The slope (~0.07) of the control curve for DTT alone in C (inverted closed triangles) is approximately one-third of the slope (~0.2) in Fig. 3C (closed triangles) because of the lower DTT concentration used (3 mM for the experiment shown in Fig. 3 and 1 mM in Fig. 2). D, shown is the LDV-FITC-containing small molecule dissociation from U937 cells treated with 1 mM Mn2+ in HEPES buffer in the absence of other divalent cations (Ca2+ and Mg2+) in the presence or absence of DTT (3 mM for 40 min at 37 °C). Dissociation rate constants shown on the graph were obtained by fitting the data to single exponential curves. Binding is plotted as mean channel fluorescence (MCF) versus time.

 
Exposure of disulfides to solvent is a factor that regulates the rate of disulfide reduction by reducing agents. Therefore, we investigated whether a conformational change in the integrin molecule induced by cations affects the response to DTT. We subtracted the curve corresponding to activation by Mn2+ alone (Fig. 2A, closed circles) from the curve corresponding to DTT and Mn2+ (open circles). The resulting curve is plotted in Fig. 2C (open circles) together with the DTT-alone curve from Fig. 2A (closed triangles). We found that the slope of the curve corresponding to DTT and Mn2+ minus Mn2+ was approximately two times larger than that of the curve corresponding to DTT alone. Thus, the rate of DTT-induced activation of integrin was higher for the extended conformation induced by Mn2+. This suggests that the conformational change in VLA-4 induced by ions facilitates a subsequent change in the affinity induced by DTT. Presumably, this could be achieved by exposing disulfides, which are less accessible to DTT in the resting conformation.

Next, cells were preincubated with Mn2+ in the presence or absence of DTT (Fig. 2D) to test whether the addition of DTT can generate a higher affinity state than that induced by Mn2+ alone. In our previous experiments, the dissociation constant (Kd) for the binding of the LDV-FITC-containing small molecule in 1 mM Mn2+ (in the absence of other divalent ions) was ~0.1–0.3 nM, and koff was ~0.0005–0.0007 s–1 (33). Fig. 2D shows that the koff was at least five times slower for DTT-treated cells (koff ~ 0.0001 s–1). This value corresponds to a KD of ~20–60 pM. Thus, the addition of DTT induced a higher affinity state compared with divalent cations only. This suggests that the mechanism of integrin activation by reducing agents is independent of and additive to the one induced by ions.

Kinetics of the Affinity Changes Induced through Inside-out Signaling Differ from those Induced by Activation by DTT—Next, to determine whether DTT affects integrin activation through inside-out signaling, cells were treated with DTT alone or in combination with activation using two GPCR ligands: fMLFF (ligand for the FPR) (Fig. 3) and ATP (ligand for P2Y receptors) (Supplemental Fig. 1). Whereas U937 cells are transfected with the FPR (34, 36), a family of P2Y receptors (purinergic receptors) is constitutively expressed in U937 cells (38–40). These activation experiments were performed in real time.



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  		FIG. 3.
Response kinetics of the LDV-FITC-containing small molecule binding to U937 cells following stimulation by fMLFF and DTT. A, U937 cells transfected with the wild-type FPR were preincubated with 4 nM LDV-FITC-containing small molecule for 10 min at 37 °C. Next, 3 mM DTT (arrow 2, closed triangles), 0.1 µM fMLFF (arrow 2, closed circles), or sequentially 3 mM DTT (arrow 1, open circles) and 0.1 µM fMLFF (arrow 2, open circles) were added. B, shown is the dissociation of the LDV-FITC-containing small molecule initiated by the addition of 2 µM non-fluorescent LDV-containing small molecule (arrow) to cells treated as described for A for 500 s. Dissociation rate constants shown on the graph were obtained by fitting data to single (fMLFF only; closed circles) or double exponential curves. Numbers in parentheses represent a fraction of VLA-4 receptors in each affinity state calculated as described in the legend for Table I. C, the data corresponding to the fMLFF experiment (A, closed circles) were subtracted from the data corresponding to the cells treated with DTT and fMLFF (A, open circles). The results (closed triangles) are plotted on the same panel with the data from DTT alone (A, closed triangles). The base-line value 220 (shown in A by the dotted line) was subtracted from the DTT-alone data (open circles). The different slopes of the curve for DTT and fMLFF minus fMLFF correspond to different rates of integrin activation by DTT. Binding is plotted as mean channel fluorescence (MCF) versus time. D, shown is a schematic of a hypothetical mechanism that links rapid and reversible inside-out signaling with exposure of the disulfide bonds and reductive activation of the integrin.

 
Treatment of cells with DTT induced a gradual increase in LDV-FITC-containing small molecule binding (Fig. 3A). In these experiments, we used a higher concentration of DTT (3 mM) in comparison with Mn2+ experiments (1 mM). Therefore, the slope of the curve for the DTT-treated cells was approximately three times higher (compare Fig. 2C (slope of ~0.07) with Fig. 3C (slope of ~0.23) and Supplemental Fig. 1C (slope of 0.20)). As shown previously (31), cell activation through GPCRs induced rapid and reversible changes in integrin affinity (FPR (Fig. 2) and P2Y receptor (Supplemental Fig. 1)). Fig. 3B shows the presence of two affinity states in the experiments in which DTT or DTT and fMLFF were added (low affinity, koff ~ 0.04–0.06 s–1, corresponding to the resting receptor state; and high affinity, koff ~ 0.004 s–1). A larger fraction of high affinity receptors was detected when DTT and fMLFF were added together compared with DTT or fMLFF alone (compare values in Fig. 3B next to the dissociation curves). When cells were activated through the FPR (Fig. 3B, closed circles), only one dissociation component (koff ~ 0.04 s–1) was detected. This result is consistent with rapid desensitization of the wild-type FPR (31). Thus, the kinetics of DTT-induced LDV-FITC-containing small molecule binding, which reflect the kinetics of the VLA-4 affinity changes (31), are dramatically different from those of activation by inside-out signaling through GPCRs.

Fig. 3C shows the fMLFF-alone curve (Fig. 3A, closed circles) subtracted from the curve corresponding to DTT and fMLFF (open circles) and plotted together with the DTT-alone curve. This curve (Fig. 3C, open circles) has two slopes: a higher slope starting from 70 to 240 s (~0.39), which was interpreted as a faster rate of disulfide reduction caused by the conformational change induced through inside-out signaling, and a lower slope (~0.23) with exactly the same value as obtained with DTT alone (compare open circles after 240 s and closed triangles). We hypothesize that the inside-out signaling generated by FPR activation results in a conformational rearrangement of the integrin and that this leads to the exposure of integrin disulfides, as in the case of activation by Mn2+ (Fig. 3D). Thus, a conformational change facilitates activation of the integrin by DTT. After desensitization and termination of receptor signaling (after ~240 s), integrins return to their resting affinity (31) and conformational (32) state. As a result, disulfide bonds become less accessible to the reducing agent. After ~240 s, the slope of the line (Fig. 3C, open circles) became ~0.23. This value corresponds to the resting non-extended molecule (Figs. 3, C (closed triangles) D). Essentially the same behavior of integrins with faster kinetics was observed when VLA-4 was activated by ATP through P2Y receptors. P2Y2 and P2Y6 are nucleotide GPCRs constitutively expressed in U937 cells (Supplemental Fig. 1) (38–40).

It is worth noting that the ratio between the two slopes of the curves for DTT and fMLFF minus fMLFF and for DTT alone (0.39/0.2 ~ 2, from 70 to 240 s) (Fig. 3C) was approximately the same as for Mn2+ activation (0.16/0.07 ~ 2) (Fig. 2C). We detected an ~2-fold difference in the rate of reduction between the folded and extended conformations. Thus, the extension of integrins induced by ions and that induced by inside-out signaling result in similarly facilitated reduction, but with different kinetics: long and persistent in the case of ions and short and reversible in the case of GPCR activation.

Bacitracin Diminishes the Effect of DTT on Integrin Activation, but Has No Effect on the Response Induced by Inside-out Signaling—Recently, it was proposed that PDI present on the cell surface participates in the regulation of integrin-dependent adhesion (17–19, 24) and could be part of "outside-in" and/or inside-out signaling pathways (17). To clarify the role of PDI in the inside-out activation of the integrin, we used bacitracin, an inhibitor of the reductive function of the plasma membrane (20, 41). Preincubation of U937 cells with 1 mM bacitracin significantly diminished the rate of DTT-induced activation of VLA-4 (compare slopes in Fig. 4A). In contrast, no statistically significant inhibition of inside-out integrin activation through the FPR or P2Y receptors was detected (Fig. 4, B and C). Bacitracin had no effect on the integrin affinity of resting cells; the dissociation rate was similar for treated and untreated cells (koff ~ 0.04 s–1) (Fig. 4D). Nonspecific binding of fluorescent substances present in the bacitracin solution resulted in different base lines for treated and untreated cells (Fig. 4D). Thus, the reductive capacity of the plasma membrane had no effect on VLA-4 activation by intracellular signaling. This result is more consistent with the mechano-conformational theory of integrin regulation than with involvement of reduction-related mechanisms in inside-out integrin activation.



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  		FIG. 4.
Effect of bacitracin on integrin activation by DTT and inside-out signaling detected using the LDV-FITC-containing small molecule. A–C, U937 cells transfected with the wild-type FPR were preincubated on ice for 1.5 h with or without 1 mM bacitracin (Bac). The cells were then incubated at 37 °C with 4 nM LDV-FITC-containing small molecule. A, cells activated with 3 mM DTT; B, cells activated with 0.1 µM fMLFF; C, cells activated with 1 µM ATP (P2Y nucleotide receptors constitutively expressed in U937 cells (38–40)). D, shown is the dissociation of the LDV-FITC-containing small molecule from cells preincubated with or without bacitracin (as described for A–C). The difference in the base lines for bacitracin-treated and untreated cells was due to nonspecific binding of fluorescent substances present in the bacitracin solution (compare plateaus of the dissociation curves in D). The dissociation rate constant shown on the graph was obtained by fitting data to a single exponential curve. Binding is plotted as mean channel fluorescence (MCF) versus time.

 
FRET-based Detection of Integrin Extension Induced by DTT—A FRET-based method was used to detect molecular extension of integrins (32). The LDV-FITC-containing small molecule was used as a fluorescence donor, and R18 incorporated into the membrane was used as an acceptor. U937 cells were treated with different concentrations of DTT and divalent ions (Fig. 5). As we have shown previously for ions and inside-out signaling, activation of VLA-4 by DTT results in decreased FRET efficiency (32). This was interpreted as an increase in the distance of closest approach between the integrin ligand-binding site and the surface of the membrane. An estimate of the distance between the head group of VLA-4 and the cell membrane for 1 mM Mn2+ and 3 mM DTT was ~60–90 Å (32). This estimate was based on calibration of acceptor surface densities for the resting receptor in 1 mM Ca2+ and was defined to be 0-Å separation distance. Thus, the activation of VLA-4 using DTT results in the extension of the integrin, in which the headpiece moves away from the membrane (Fig. 5C).



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  		FIG. 5.
Energy transfer in U937 cells between the LDV-FITC-containing small molecule donor and the octadecyl rhodamine B chloride (R18) acceptor. Measurements were made as described under "Experimental Procedures" and in Ref. 32. A, fluorescence intensity plotted as a function of R18 concentration under three conditions: 1 mM Ca2+, 1 mM Mn2+, and 1 mM Mn2+ + 3 mM DTT for 40 min at 37 °C. Data are plotted as specific fluorescence of the LDV-FITC-containing small molecule. (The fluorescence signal corresponding to the sample blocked with 2 µM non-fluorescent LDV-containing small molecule was subtracted; the fluorescence signal was normalized to the intensity of the donor in the absence of acceptors.) Inset, data from A replotted as relative quantum yield versus acceptors/R02. Curves represent simulation of energy transfer as a function of donor distance of closest approach expressed in terms of R0 according to the Wolber and Hudson model (46). The surface densities were estimated based on the lateral FRET using fluorescein C18/rhodamine C18 in U937 cells (see Fig. 3 in Ref. 32). Because the Wolber and Hudson model is valid only for acceptor densities <0.5 acceptors/R20, the analysis of the data in A is truncated. The data shown in the inset represent the analysis of the boxed data in A as limited by the FRET model. B, quenching data plotted for two DTT concentrations and untreated cells in buffer containing 1 mM Ca2+ and 1 mM Mg2+ (incubation for 40 min at 37 °C). Inset, data from B replotted as relative quantum yield versus acceptors/R20. C, schematic of FRET methodology. Shown is an integrin heterodimer in the inactive conformation (bent). Upon activation, the integrin assumes an extended (upright) conformation. Changes in FRET efficiency between the LDV-FITC-containing small molecule donor bound to the headpiece of the molecule and the octadecyl rhodamine B chloride acceptor (R18) incorporated into the membrane were used to estimate the distance of closest approach of the donor and acceptor molecules (32).

 
Kinetics of DTT-induced Extension Detected Using FRET Coincide with Those of Affinity Changes—Finally, for a real-time FRET-based assay (32), cells were preincubated with a large excess of the LDV-FITC-containing small molecule. Next, the fluorescence signal was quenched using R18. Cells were then activated through different GPCRs or DTT. The addition of DTT induced slow and gradual unquenching of the fluorescence signal (Fig. 6B, closed triangles). However, the inside-out signaling promoted an instant unquenching, reflecting rapid extension of the integrin molecule, as was shown previously (32). Thus, the kinetics of the conformational extension of VLA-4, detected using FRET, were different between inside-out signaling and activation by DTT as shown for the affinity change.



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  		FIG. 6.
Real-time FRET experiments with integrin activation by inside-out signaling and reducing agents. U937 cells were stably transfected with the non-desensitizing mutant of FPR (FPR{Delta}ST) (36) and preincubated at 37 °C with 100 nM LDV-FITC-containing small molecule to saturate low affinity sites in buffer containing 1 mM Ca2+ and 1 mM Mg2+. Next, the LDV-FITC-containing small molecule fluorescence was quenched after the addition of 10 µM octadecyl rhodamine B chloride (R18) (arrow). Cells were then activated by the addition of 0.1 µM of fMLFF or 3 mM DTT. A, data plotted as mean channel fluorescence (MCF) versus time for two conditions: quenched and then activated by fMLFF (open circles) and quenched only (base line; inverted closed triangles). B, comparison of integrin conformational activation by fMLFF and 3 mM DTT in real time. Data were plotted by subtracting the base-line data from the activated cell data; therefore, the y axis is labeled {Delta}MCF. Because the FPR{Delta}ST mutant does not desensitize, VLA-4 remains in a state of constant affinity (31).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Multiple Affinity States of the VLA-4 Integrin—In circulating lymphocytes, VLA-4 has the potential to exhibit multiple affinity states that mediate tethering, rolling, and arrest on its endothelial ligand VCAM-1 (42–44). We have used the LDV-FITC-containing small molecule as a model ligand showing the affinity state of VLA-4 under different activating conditions (31). The fluorescent probe was based on the structure of BIO1211, a highly specific {alpha}4{beta}1-integrin inhibitor developed by Biogen Inc. (43, 45). Previously, we found that the LDV-FITC-containing small molecule can be used to determine the affinity of the natural VLA-4 ligand VCAM-1 and that changes in the integrin binding affinity for VCAM-1 coincide with changes in cell adhesion avidity (33) and molecular conformation (32). For activation by DTT, most of the variation in the affinity of the probe arose from changes in the dissociation rate rather than the association rate, as shown for activation by divalent ions, activating antibodies, and inside-out signaling (31, 33). The differences in dissociation constant values for BIO1211 are also governed almost exclusively by dissociation rates (43). This situation is probably typical for the type of receptors in which the conformation of the ligand-binding pocket determines the residence time of the ligand. For integrins, the change in ligand affinity and residence time could be sufficient to slow down cell rolling and to result in cell arrest and firm adhesion of the leukocytes. The reported difference between the highest and resting affinity states of VLA-4 is >2 orders of magnitude (31, 33). This concept is additionally supported by the result that stable cell aggregates can be formed between VLA-4- and VCAM-1-expressing cells connected only by one or two bonds at states of different affinity (37).

In contrast, when integrins were activated by reducing agents as shown here, several distinctive affinity states of VLA-4 were detected in the cell population at the same time (Table I). The kinetics of activation by DTT and the changes in VLA-4 affinity were slow and were dependent on time and concentration. Longer incubation times at higher concentrations of reducing agents resulted in a larger fraction of high affinity VLA-4. These data differ from those obtained with integrin activation using divalent ions, where usually only one affinity state was detected. Fits to the dissociation data require one exponential curve (see Fig. 1 (B and C) in Ref. 33 and Ref. 43). The VLA-4-activating mechanism may explain the above difference: for quickly diffusing divalent ions at high concentrations (usually 1–3 mM), equilibrium is reached rapidly, resulting in a similar state for all receptors. For reductive activation involving disulfide exchange reactions and possibly enzymatic reactions catalyzed by PDI (17–19, 24), several discrete states of integrin activation occur, presumably by reducing different numbers of disulfide bonds in different molecules. Fig. 1D shows a hypothetical mechanism that relates the number of reduced disulfides to the conformational state of VLA-4.

Previously, the rapid interconversion between the resting state and the physiologically activated state was demonstrated using the LDV-FITC-containing small molecule (see Fig. 5 in Ref. 31). However, in this case, only two affinity states (koff1 ~ 0.06 s–1 and koff2 ~ 0.01 s–1) were detected. The affinity state generated using the highest concentration of DTT was at least 10 times higher (Table I) than that of the physiologically activated receptor. Thus, the magnitude of the affinity changes after reductive activation of VLA-4 was significantly different from that generated through inside-out signaling.

Kinetics of Affinity Changes and Cell Activation—Because it has been recognized that integrin affinity can be regulated by a mechanism related to disulfide bond reduction (17, 19, 21, 24), our goal was to determine whether affinity regulation by DTT could occur in a proper time frame to be physiologically relevant. We found that the kinetics of integrin activation induced by high concentrations of reducing agents (up to 3 mM DTT and up to 50 mM DMPS) were very slow in comparison with those of activation through GPCRs (Figs. 2, 3, 4 and Supplemental Fig. 1). Moreover, with the knowledge that reducing agents generate populations of different affinity receptors, our data support the idea that two different mechanisms result in the state of higher integrin affinity. For inside-out signaling, it could be separation of C-terminal tails of the {alpha}- and {beta}-subunits, resulting in a large conformational change (7, 10–12). For reducing agents, it could be an enzymatic mechanism that involves PDI-catalyzed disulfide exchange reactions (17–19, 21, 24). To further test this hypothesis, we used bacitracin, a drug that is known to inhibit the reductive function of the membrane (20, 41).

Inside-out Signaling and Reducing Agents—Several ideas connecting inside-out signaling and integrin activation led us to investigate the effect of bacitracin on integrin activation induced by DTT simultaneously with signaling though GPCRs. These include a "DTT-sensitive regulatory element" (21) as well as requirements for PDI enzymatic activity in integrin activation (19), adhesion (24), and aggregation (17). We showed that bacitracin reduced the rate of DTT-induced conformational activation of the integrin (Fig. 4A), but had no inhibitory effect on integrin activation by inside-out signaling (Fig. 4, B and C). In fact, bacitracin caused slightly slowed desensitization of the LDV-FITC-containing small molecule signal in the case of fMLFF stimulation (Fig. 4B, open circles between 100 and 300 s). These data show that regulation of integrins by reducing agents was essentially independent of inside-out signaling, whereas the GPCR- or Mn2+-induced conformational change increased the rate of integrin activation by reducing agents (Figs. 2 and 3 and Supplemental Fig. 1).

Integrins: Three or More Independent Activating Mechanisms—Integrin conformational change and activation can be achieved under different conditions: divalent ions, reducing agents, inside-out signaling, mutations in extracellular domains and C-terminal tails, and disulfide disruption (11, 23, 25, 26, 43). Several opposing mechanisms may contribute to integrin conformational change with divalent ions. The central MIDAS (metal ion-dependent adhesion site) has two geometries and is regulated by two other polar sites: a site adjacent to the MIDAS site and a ligand-induced metal-binding site (47). In this scenario, the Ca2+/Mn2+ competition is critical for the regulation of ion-mediated cell adhesion. However, another report has shown that the Ras-like small GTPase Rap1 is necessary for the activation of integrins by Mn2+ or activating antibodies (48). In this scenario, intracellular signaling could be involved in the regulation of integrin-dependent cell adhesion in response to Mn2+ or mAb TS2/16. Our data show that changes in VLA-4 affinity can be detected after incubating cells on ice with Mn2+ or activating antibodies, suggesting that an ion/antibody-induced conformational change in the molecule rather than intracellular signaling is sufficient for increased affinity (31). Moreover, the VLA-4 affinity state induced by Mn2+ or TS2/16 is several orders higher than the "physiologically activated" state induced by the inside-out signal. Thus, the affinity/conformational state induced by Mn2+ or activating mAbs is "non-physiological," although it may reflect the continuum of states available to the flexible molecule under physiological conditions. It was impossible to achieve the high affinity similar to the Mn2+- or mAb-induced state via only inside-out signaling through GPCRs (31–33).

There is a similar dichotomy for the relevance of conformation and signaling to disulfide reduction. One line of research showed that activation of integrin-dependent cell adhesion by DTT or other reducing agents requires cell signaling, the cytoskeleton, and PDI activation (17–19, 21, 22, 24). Since PDI participates in the regulation of L-selectin shedding (20), it is tempting to propose a PDI-related mechanism as a general regulatory feature of both selectins and integrins, the two main classes of adhesion molecules. Another report suggested that the redox site within the extracellular domain of the integrin molecule functions as an "on/off switch that regulates ligand binding affinity" (49, 50). The reduction of disulfides could "mechanically" lead to global conformational changes and to the opening of ligand-binding sites.

Our data show that the affinity state of VLA-4 generated using membrane-permeable and -impermeable reducing agents was much higher than the state induced by inside-out signaling. The kinetics of DTT-induced VLA-4 activation were slow in comparison with those of GPCR stimulation. Moreover, the presence of large amounts of reducing agent during cell activation had no significant effect on inside-out activation. Bacitracin had no effect on integrin activation via GPCR signaling, but significantly reduced DTT-induced activation. These data suggest that integrin activation by inside-out signaling is not associated with disulfide reduction (5, 9, 12). The exposure and reduction of disulfides within VLA-4 upon activation (49) are more likely to be a result of conformational rearrangement than the cause of it. For reducing agents, the slow reduction of disulfides was facilitated by the conformational change and the associated extension of VLA-4 that was detected using FRET. In our opinion, the states produced by disulfide reduction are therefore not physiological, although the affinities observed may represent a continuum of affinities accessible to the flexible VLA-4 molecule and encompass those induced by Mn2+, activating antibodies, and molecular stretching (see below).

Regulation of VLA-4 Affinity and Conformation Provides a "Catch-bond" Mechanism—Previously, we showed a progressive increase in VLA-4 affinity, a decrease in the ligand dissociation rate, and an increase in the distance of closest approach of the ligand-binding site to the membrane as the integrin was activated by divalent ions or GPCRs (31, 32). In this study, we used a mechanistically different approach to activate integrins: activation by reducing agents. We found that a progressive decrease in the LDV-FITC-containing small molecule dissociation rate was accompanied by extension of the integrin molecule detected using end-point and real-time FRET-based assays (Figs. 1, 5, and 6). A strong correlation between the affinity states of VLA-4 and the degree of the molecular extension supports the idea that the conformational change involving VLA-4 extension also affects ligand binding affinity (Fig. 7). These data provide a novel mechanism accounting for an adhesion catch bond (51): a mechanical stretching of a flexible integrin molecule during cell rolling or under shear that would induce a high affinity conformation of the integrin and result in higher cell adhesion avidity.



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  		FIG. 7.
Correlation between the affinity states of VLA-4 and the degree of molecular extension determined using FRET. Separation distance (rc) is plotted as fraction R0 versus logarithm of the dissociation rate (koff, s –1) of the LDV-FITC-containing small molecule for five different affinity states. For the fluorescein-rhodamine pair, R0 ~ 55 Å. Putative VLA-4 conformations are depicted as schematics. Data from several previous publications (31–33) and this study were used.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants P50 HL56384, IR01EB02022, and IR24 CA88339 (to L. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1. Back

|| To whom correspondence and reprint requests should be addressed: Dept. of Pathology and Cancer Center, University of New Mexico HSC, Albuquerque, NM 87131. Tel.: 505-272-6892; Fax: 505-272-6995; E-mail: lsklar{at}salud.unm.edu.

1 The abbreviations used are: VLA-4, very late antigen-4; FRET, fluorescence resonance energy transfer; PDI, protein-disulfide isomerase; DTT, dithiothreitol; mAb, monoclonal antibody; GPCR, G protein-coupled receptor; FITC, fluorescein isothiocyanate; FPR, formyl peptide receptor; DMPS, 2,3-dimercapto-1-propanesulfonic acid; fMLFF, N-formyl-L-methionyl-L-leucyl-L-phenylalanyl-L-phenylalanine; VCAM-1, vascular cell adhesion molecule-1. Back


   ACKNOWLEDGMENTS
 
We thank Denise C. Dwyer for help with experiments.



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