Multiple Activation States of Integrin alpha 4beta 1 Detected through Their Different Affinities for a Small Molecule Ligand*

Ling Ling Chen, Adrian Whitty, Roy R. Lobb, Steven P. Adams, and R. Blake PepinskyDagger

From Biogen, Inc., Cambridge, Massachusetts 02142

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have used the highly specific alpha 4beta 1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha 4beta 1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha 4beta 1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 µg/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 × 10-4 s-1 to >140 × 10-4 s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 × 106 M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha 4beta 1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha 4beta 1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins comprise a large family of cell-surface receptors that mediate cell-cell and cell-matrix interactions in diverse biological settings (see Refs. 1 and 2 for reviews). Each integrin is a two-chain heterodimer containing an alpha -chain and a beta -chain. The leukocyte integrin alpha 4beta 1 regulates cell migration into tissues during inflammatory responses and normal lymphocyte trafficking (3-5) and provides a key co-stimulatory signal supporting cell activation (6-10). In vivo studies using blocking monoclonal antibodies (4) and inhibitor peptides (11-13) have demonstrated a critical role for alpha 4 integrins in leukocyte-mediated inflammation. alpha 4beta 1 mediates cell adhesion by binding to either of two protein ligands, vascular cell adhesion molecule-1 (VCAM-1)1 or the alternatively spliced CS1-containing fibronectin variant (14-17). Whereas expression of alpha 4beta 1 is constitutive, its interaction with ligands is strongly enhanced in an activated state that can be induced by various stimuli including antigen, anti-T cell receptor mAbs, phorbol esters, the divalent cation Mn2+, and certain beta 1-specific antibodies (18-20). These changes in affinity and/or avidity ultimately determine whether the interaction is productive and stabilizes the ligand-integrin complex or is nonproductive.

Although all integrins require divalent cations to bind ligand, the regulation of function by metal binding is complex and is not fully understood (21, 22). Integrin alpha -subunits contain multiple EF-hand-like Ca2+ binding loops (1, 23), which are in close proximity to ligand-binding sites (24, 25). This region of the alpha -chain is made up of seven sequence repeats of about 60 amino acids each, which are presumed to be organized in a beta -propeller fold motif found in various enzymes (24, 25). The beta -chain contains a second type of metal binding/ligand binding motif that shares homology with the A-domain of von Willebrand's factor (26, 27). A homologous structure is present in some alpha -subunits as well. This 200-residue protein module has proven to be surprisingly tractable for biochemical evaluation, and the corresponding regions from alpha M, alpha L, and alpha 2, have been successfully crystallized (28-30). The crystal structures revealed a Mg2+ or Mn2+ bound at the apex of a dinucleotide binding motif (28-30). The observed structure defined by the coordination of the cation with the peptide backbone has been postulated to mimic the ligand-occupied structure in what is commonly referred to as the metal ion-dependent adhesion site or MIDAS (see Refs. 21 and 27 for references). Whereas the role of cation-binding sites in regulating integrin function is well established, how the sites are coordinated is unclear. Regulation of ligand binding by cations is further complicated by the fact that submillimolar concentrations of Ca2+ can non-competitively inhibit ligand binding, indicating that certain of the metal-binding sites can play an inhibitory role (22, 31).

Recently, we developed a series of highly selective alpha 4beta 1 inhibitors using the tetrapeptide ILDV ligand binding sequence from the CS1 region of fibronectin as the starting point for inhibitor design (32). This sequence is homologous to the tetrapeptide QIDS, which comprises the alpha 4beta 1-binding site in VCAM-1 (33). Whereas both ILDV and QIDS peptides weakly inhibit ligand binding and cell adhesion (33, 34), a compound that was 106 times more potent, BIO1211, was generated by substituting isoleucine with a 4-((N'-2-methylphenyl)ureido)-phenylacetyl N-terminal cap (35). Here we used a tritiated version of BIO1211 as a probe to assess alpha 4beta 1 function under various states of activation. The data provide new information on the effect that different activation conditions have on the affinity of alpha 4beta 1 for its ligands and establish that these differences in affinity are regulated by changes in the dissociation rate of the ligand-integrin complex.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthesis of [3H]BIO1211-- [3H]BIO1211 (50 Ci/mmol) was synthesized by NEN Life Science Products using [4,5-3H]leucine as a precursor. The radiochemical purity of the compound was >95% as measured by reverse phase HPLC on a C18 column, and the compound yielded the predicted spectrum by 3H NMR. [3H]BIO1211 was dissolved in Me2SO, diluted with 20 volumes of water, and sodium phosphate, pH 8.8, was added to 20 mM (3.66 mCi/ml). The solution was then aliquoted and stored at -70 °C. The binding affinity of the labeled compound was indistinguishable from unlabeled BIO1211 in the alpha 4beta 1-direct binding assay with VCAM-Ig-alkaline phosphatase as a reporter (36).

Binding of [3H]BIO1211 to alpha 4beta 1 Expressing Cells-- Jurkat cells that had been enriched for alpha 4beta 1 expression by FACS sorting were maintained in RPMI 1640 medium plus 10% fetal bovine serum at 37 °C in a tissue culture incubator. K562 cells that had been transfected with either the human alpha 4, human alpha 2, or human alpha 1 gene and selected for high levels of alpha 4beta 1, alpha 2beta 1, and alpha 1beta 1, respectively, by FACS were grown in the same medium supplemented with 1 mg/ml G418, 10 µg/ml gentamicin sulfate, and 50 µg/ml streptomycin. alpha 2 and alpha 4 K562 cells were a gift of Dr. Martin Hemler. For binding studies, the cells were pelleted by centrifugation, washed two times with TBS (50 mM Tris HCl, 150 mM NaCl, 0.1% bovine serum albumin, 2 mM glucose, 10 mM HEPES, pH 7.4), suspended at approximately 2 × 106 cells/ml in TBS, and counted using a Neubauer hemocytometer. The cells were further diluted with TBS to the concentration indicated and treated with [3H]BIO1211 at room temperature. The cells were then pelleted by centrifugation, resuspended in 100 µl of TBS plus Mn2+, and transferred to a scintillation vial containing 2.9 ml of ScintiVerse II (Fisher). Cell-associated radioactivity was quantified by scintillation counting. All studies were performed in siliconized 1.5-ml Eppendorf tubes with a standard 1-ml sample volume. Each condition was tested in at least two independent studies. In the studies indicated, a 100-1000-fold excess of unlabeled BIO1211 was added to samples after the incubation with [3H]BIO1211 to prevent further binding. Binding studies testing the effects of cell number, incubation time, and [3H]BIO1211 concentration were performed in TBS plus 2 mM MnCl2 as described. Nonspecific binding of [3H]BIO1211 to cells was assessed at each cell density and [3H]BIO1211 concentration in TBS but in the absence of added metal ion. Specific counts bound were calculated by subtracting nonspecific counts from total counts bound. Other studies testing the effects of activation on binding were performed as indicated. In the 1 mM Ca2+, 1 mM Mg2+ state where KD = 20-40 nM for binding of BIO1211 to alpha 4beta 1, a high background at the higher BIO1211 concentrations (3,000 cpm at 10 nM) in the standard assay format limited the concentration of [3H]BIO1211 that could be tested to ~10 nM; however, by diluting the specific activity of the label from 50 to 5 Ci/mmol, binding could be evaluated at BIO1211 concentrations up to 100 nM, albeit with reduced precision.

For kinetic on rate measurements, Jurkat cells were treated with 2 nM [3H]BIO1211 at room temperature for the times indicated and then treated with a 500-fold excess of unlabeled BIO1211 to quench further binding by the [3H]BIO1211. Cells were collected by centrifugation and subjected to scintillation counting. For kinetic off rate measurements, Jurkat cells were treated with 5 nM [3H]BIO1211 at room temperature for 1 h. A 500-fold excess of unlabeled BIO1211 was added, and the cells were further incubated for the times indicated. Cells were pelleted at each time point, and cell-associated [3H]BIO1211 was measured by scintillation counting. Binding and dissociation data are represented as a percent of the maximum specific counts bound as a function of time. The data were fitted to an exponential curve by nonlinear regression. For koff, the exponential rate constant is the off rate. For kon, the observed rate constant is the true rate constant multiplied by the [3H]BIO1211 concentration. The effect of added unlabeled BIO1211 on dissociation rates was tested over a wide range of concentrations from 100 nM (20-fold excess) to 50 µM (10,000-fold excess). The dissociation curves were superimposable over this range of concentrations, indicating that the excess unlabeled ligand was exerting no allosteric effect on the rate of dissociation (data not shown).

Assessing alpha 4beta 1-Ligand Interactions by Competition-- [3H]BIO1211 was also used to study alpha 4beta 1 function by competition, using the radioactivity as a reporter for alpha 4beta 1 occupancy. In this format, Jurkat cells (1 × 106/ml) in the buffers indicated were treated with serial dilutions of test compound for 1 h, and then 5 nM [3H]BIO1211, an amount sufficient to bind all unoccupied receptors, was added for 10 min before measuring the bound counts. The cells were then pelleted by centrifugation and subjected to scintillation counting. Counts bound under these conditions measure integrin that is not occupied by the test compound and is therefore free to bind the [3H]BIO1211. The competition format was also used for kinetic binding studies. Binding and dissociation constants were calculated from alpha 4beta 1 that after treatment with test compound was free to bind the [3H]BIO1211.

Analysis of [3H]BIO1211 by Reverse Phase HPLC-- Samples were analyzed by reverse phase HPLC on a C18 column (Vydac, catalog number 218TP54, 0.46 × 25 cm) at 23 °C. The column was developed at 1 ml/min with the following gradient of acetonitrile in 50 mM sodium acetate, pH 4.5: 0-32 min 17.5-21.5%, 32-45 min 21.5-34.5%, 45-45.1 min 34.5-50%, 45.1-48 min 50%, 48-48.1 min 50-17.5%, and 48.1-53 min 17.5%, conditions that maximize the resolution of BIO1211 from potential proteolytic and hydrolytic degradation products (data not shown). The column effluent was monitored at 254 nm, and 0.5-ml fractions were collected. The fractions were mixed with scintillation mixture and analyzed by scintillation counting. Immediately prior to injection on the HPLC, test samples were spiked with 2.5 µg each of cold BIO1211 and cold BIO-1588, an analog of BIO1211 with the C-terminal Val-Pro deleted, and a likely proteolysis product. The elution profiles of the cold inhibitors were used to monitor column performance.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Development of a BIO1211-alpha 4beta 1 Binding Assay Using [3H]BIO1211-- The ability of [3H]BIO1211 to bind Jurkat (alpha 4beta 1 positive) cells and alpha 1-transfected K562 (alpha 4beta 1 negative) cells was examined under diverse conditions known to alter the activation state of alpha 4beta 1 (Fig. 1). [3H]BIO1211 binding to Jurkat cells was greatest in the presence of 2 mM Mn2+ alone, 10 mM Mg2+ alone, and 2 mM Mn2+ + 10 µg/ml mAb TS2/16, treatments that activate alpha 4beta 1. Binding was greatly reduced under non-activating conditions exemplified by treatment with Ca2+, Mg2+ (1 mM each) (see Fig. 1, A and B). No binding was observed to the alpha 1-transfected K562 (alpha 4beta 1-negative) control cell line. The ~2000 cpm background seen in Fig. 1A is residual free [3H]BIO1211, which was removed if samples were washed prior to analysis. In the studies presented below, specific binding was calculated by subtracting background counts from total counts bound. Background binding was determined for each test sample using a control sample that was subjected to the same treatment but in the absence of any divalent cation.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   Binding of [3H]BIO1211 to Jurkat and alpha 1-K562 cells under various states of alpha 4beta 1 activation. Jurkat cells were incubated with [3H]BIO1211 in the indicated buffers and conditions. The cells were then pelleted by centrifugation, resuspended in 100 µl of TBS plus Mn2+, transferred to a scintillation vial containing 2.9 ml of ScintiVerse II, and cell-associated radioactivity was quantified by scintillation counting. A, a series of studies were performed in which 1-ml samples (2 × 106 cells) were incubated with 5 nM [3H]BIO1211 in the indicated buffers for 2 h at room temperature. B, Jurkat cells at 0.75 × 106/ml were incubated at room temperature for 40 min with the indicated concentrations of [3H]BIO1211 in the presence of 1 mM Ca2+, 1 mM Mg2+ (black-square); Ca2+, Mg2+, Mn2+ (1 mM each) (black-triangle); 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 (); 2 mM Mn2+ (black-diamond ); 2 mM Mn2+ plus 10 µg/ml TS2/16 (+). Background counts were determined at each concentration of [3H]BIO1211 tested by incubating identical samples in TBS buffer in the absence of divalent cations. Specific counts bound were calculated by subtracting background counts from total counts bound. C, Jurkat cells were treated with mAb B5G10 under the conditions indicated, then with phycoerythrin-labeled anti-mouse Ig antibody, and analyzed by FACS.

Specific counts bound at saturation provided a direct measure of alpha 4beta 1 expression levels, and based on this number, we estimate that the Jurkat cells used in these studies have approximately 80,000 copies of alpha 4beta 1 per cell. To rule out the possibility that alpha 4beta 1 levels changed under the different treatments, alpha 4beta 1 levels were compared under the 1 mM Ca2+, 1 mM Mg2+ (non-activating), and 2 mM Mn2+ (activating) conditions by FACS, using the non-neutralizing anti-alpha 4 mAb B5G10 as a reporter (Fig. 1C). The alpha 4beta 1 levels in the activated and non-activated samples were indistinguishable.

A series of studies were performed to assess selected variables that might affect the performance of the assay. First, no impact of temperature on BIO1211 binding was observed under any of the activating conditions tested when binding was compared at room temperature and at 37 °C (data not shown). Second, the presence of human serum or plasma at concentrations up to congruent 100% of the total assay volume had no effect on the binding assay (data not shown). Third, bound [3H]BIO1211 could be quantitatively released from Jurkat cells with EDTA, indicating that the BIO1211·alpha 4beta 1 complex remained on the cell surface. The released BIO1211 was further characterized by reverse phase HPLC for the presence of potential degradation products that might impact our interpretation of binding. As shown in Fig. 2, over 95% of the bound [3H]BIO1211 was released by the EDTA treatment unchanged, indicating that only minimal degradation had occurred. Previously, we showed that the kinetically determined KD values for binding of [3H]BIO1211 to Mn2+-activated cells of various types (peripheral blood lymphocytes, ~10,000 copies of alpha 4beta 1/cell; Jurkat cells, ~80,000 copies/cell; alpha 4-K562 cells ~250,000 copies/cell) were similar, showing that BIO1211 binding was not affected by differences in surface expression of alpha 4beta 1 (35). Finally, because of the presence of two carboxylic acid groups in the BIO1211 sequence, we tested whether it could function as a chelator. Using fluorescent metal ion indicators that are sensitive to the concentrations of free Mg2+, Mn2+, and Ca2+ as probes for binding, we were unable to detect an association between BIO1211 and these metal ions (data not shown).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Analysis of [3H]BIO1211 stability by reverse phase HPLC. Jurkat cells (7.5 × 106 cells/ml) in TBS containing 2 mM Mn2+ were incubated at room temperature for 30 min with 1 or 5 nM [3H]BIO1211 as indicated. The cells were pelleted, and the supernatant was collected (supernatant 1). The cells were washed with TBS containing 2 mM Mn2+ and treated with TBS + 2 mM Mn2+ plus 10 mM EDTA for 60 min, with each step followed by a centrifugation step. Supernatants from the initial incubation medium, wash, and EDTA extraction were analyzed by reverse phase HPLC. 0.5-ml fractions were collected, mixed with scintillation mixture, and analyzed by scintillation counting. A shows HPLC elution profile for [3H]BIO1211 without treatment. B shows the corresponding data from supernatant 1 of the 1 nM BIO1211 treatment, and C shows the data from the EDTA extract of the 5 nM treatment.

Assessing the KD of BIO1211 for alpha 4beta 1 by Equilibrium Binding-- The low level of occupancy seen in Fig. 1B, for binding of [3H]BIO1211 to Jurkat cells under non-activating conditions, suggested that the affinity of BIO1211 for alpha 4beta 1 was lower for non-activated than for the activated integrin. In order to confirm this possibility and to obtain a more accurate measure of KD, the analysis was repeated using higher BIO1211 concentrations. The resulting curves showed dose-dependent binding and, at 100 nM [3H]BIO1211, specific counts bound were comparable for the 1 mM Ca2+, 1 mM Mg2+ state and the 2 mM Mn2+ state (data not shown). Based on these binding data, a KD of 20-40 nM was calculated for the 1 mM Ca2+, 1 mM Mg2+ (non-activated) state. Attempts to estimate KD values for activated states using equilibrium binding methods were unsuccessful because the KD values were lower than the concentration of alpha 4beta 1 in the assay. Thus, while we observed dose-dependent and saturable binding (see Fig. 1B), the binding curves were in fact simply measuring a titration of the receptor to full occupancy and could not be used to accurately calculate affinity. A more accurate assessment of the affinity of BIO1211 for activated alpha 4beta 1 was obtained using kinetic measurements (see below). For unknown reasons, total counts bound were 20% lower in the 1 mM Ca2+, 1 mM Mg2+ + TS2/16 state under conditions that should have been saturating for [3H]BIO1211 binding compared with other activated states (Fig. 1B). It was not possible to perform FACS analysis in the presence of TS2/16 to test if TS2/16 treatment had altered surface levels of integrin, since the analysis would have required co-treatment with two murine anti-alpha 4beta 1 mAbs.

Kinetics for Binding of BIO1211 to alpha 4beta 1 in Different Activation States-- Kinetic data for the binding and dissociation of [3H]BIO1211 to alpha 4beta 1 were measured under the following conditions, as shown in Fig. 3: 1 mM Ca2+, 1 mM Mg2+; Ca2+, Mg2+, Mn2+ (1 mM each); 1 mM Ca2+, 1 mM Mg2+ + 10 µg/ml TS2/16; 2 mM Mn2+; and 2 mM Mn2+ + 10 µg/ml TS2/16. Binding reached a plateau level within 10 min of treatment for all test conditions (Fig. 3A). Association rate constants (kon) calculated from these time courses all fell in the narrow range from 0.9 to 2.7 × 106 M-1 s-1 (Table I). Differences in the percent alpha 4beta 1 occupied after 10 min were identical to those seen in the equilibrium experiments shown in Fig. 1B. Time courses for the rates of dissociation of BIO1211 from Jurkat cells are shown in Fig. 3B. Unlike the association rates, which were similar across the different assay conditions, the dissociation rates varied over a wide range and were highly dependent on the activation state of the integrin. Values for koff ranged from 0.17 × 10-4 s-1 for the 2 mM Mn2+ + TS2/16 state to >140 × 10-4 s-1 for the 1 mM Ca2+, 1 mM Mg2+ state (Table I). The kinetic rate constants from these and other binding studies were used to calculate KD values, which are summarized in Table I. The close correspondence between the variations in KD values and in dissociation rates indicates that the affinity of BIO1211 for alpha 4beta 1 is governed almost exclusively by off rates. KD values observed under conditions commonly considered to be activating ranged from 470 pM in the Ca2+, Mg2+, Mn2+ (1 mM each) state to 18 pM in the 2 mM Mn2+ + TS2/16 state. These differences in affinity that resulted from activation were not apparent from the equilibrium binding measurements because the KD values were lower than the concentration of alpha 4beta 1 in the assays and therefore were masked by the format of the assay. Similar problems were encountered when affinities were estimated by measuring the ability of BIO1211 to block cell adhesion to CS1 or VCAM or to block direct binding of VCAM-Ig to Jurkat cells with VCAM-Ig as the reporter (data not shown).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Assessing [3H]BIO1211/alpha 4beta 1 binding on Jurkat cells under various states of activation using kinetic measurements. Jurkat cells were incubated with [3H]BIO1211 in the presence of 1 mM Ca2+, 1 mM Mg2+ (black-square); Ca2+, Mg2+, Mn2+ (1 mM each) (black-triangle); 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 (); 2 mM Mn2+ (black-diamond ); 2 mM Mn2+ plus 10 µg/ml TS2/16 (+). For kon measurements, 1-ml samples (1.35 × 106 cells/ml) were treated with 2 nM [3H]BIO1211 for the times indicated. Cells were collected by centrifugation and subjected to scintillation counting. For koff measurements, 1-ml samples (2 × 106 cells/ml) were treated with 5 nM [3H]BIO1211 for 120 min at room temperature. 5 µM unlabeled BIO1211 was added, and the cells were further incubated for the times indicated. Cells were pelleted at each time point, and cell-associated [3H]BIO1211 was measured by scintillation counting. The data in A (association) and B (dissociation) were fitted to exponential curves by nonlinear regression, and kon and koff values were calculated from the curve fits (Table I). For kon, 100% refers to maximum [3H]BIO1211 bound in the Mn2+-activated state, whereas for koff, 100% bound reflects maximum binding under each test condition (i.e. specific binding at t = 0).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Binding of [3H]BIO1211 to alpha 4beta 1 on Jurkat cells under various states of activation
On rates, off rates, and KD values for the binding of [3H]BIO1211 to alpha 4beta 1 were determined from kinetic measurements as described in the legend to Fig. 3. mAbs TS2/16 and HP1/2 were used at 10 µg/ml. On rates for the Mg2+ states were determined at 1, 10, and 100 mM Mg2+ and were indistinguishable. The value measured at those concentrations has been assigned to the other Mg2+ alone states shown.

To understand better the role metal ions have on activation, we performed the study shown in Fig. 4, where binding was tested as a function of changing Mg2+ concentrations. The dissociation rates were highly dependent on the Mg2+ concentration and changed from 8.3 × 10-4 s-1 to 5.3 × 10-4 s-1 to 4.3 × 10-4 s-1 to 3.2 × 10-4 s-1 to 2.1 × 10-4 s-1 when dissociation rates were measured in the presence of 2, 10, 50, 100, and 300 mM Mg2+. When KD was plotted as a function of Mg2+ concentration, the data fit a hyperbolic curve, suggesting that the measurements were part of a continuum rather than discrete points (Fig. 4B). At high concentrations of added Mg2+, binding appears to be saturating out at an affinity that approximates the value seen at 2 mM Mn2+ (Fig. 4B). Suboptimal concentrations of Mn2+ produced a similar titration of the dissociation rate to that seen with Mg2+, although at a much lower concentration of the metal ion (data not shown). Variations in the concentration of TS2/16 from 0.1 to 10 µg/ml had no effect on KD (data not shown), indicating that 10 µg/ml was a saturating concentration.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Dissociation curves for [3H]BIO1211 from the Mg2+-activated BIO1211·alpha 4beta 1 complexes. One-ml aliquots of Jurkat cells (1 × 106 cells) under the conditions indicated were treated with 5 nM [3H]BIO1211 for 2 h at room temperature. 5 µM of unlabeled BIO1211 was added, and the cells were further incubated for 0, 5, 15, 30, 60, 90, and 120 min. The cells were pelleted, and cell-associated [3H]BIO1211 was measured by scintillation counting. The data were fitted to exponential curves by nonlinear regression. 100% occupied was defined as specific counts bound at t = 0. Over the range of Mg2+ concentrations tested there was no change in this number. 100% binding = 11,000 cpm in the Mg2+ alone samples and 12,000 cpm in the Mg2+ + TS2/16 samples. Background = 1500 cpm for both sets of data. A, 2 mM Mg2+ (black-square); 10 mM Mg2+ (star ); 100 mM Mg2+ (black-triangle); 2 mM Mg2+ plus 10 µg/ml TS2/16 (); 10 mM Mg2+ plus 10 µg/ml TS2/16 (star ); 100 mM Mg2+ plus 10 µg/ml TS2/16 (triangle ). B, hyperbolic dependence of KD on [Mg2+] (solid line), saturating at high [Mg2+] to the value of KD = 100 pM seen at 2 mM Mn2+ (dashed line).

While the maximal affinity for binding of BIO1211 to alpha 4beta 1 in the presence of divalent cations was achieved with 2 mM Mn2+, a further increase in affinity was observed if samples were co-activated with 2 mM Mn2+ + TS2/16, suggesting that the mechanisms by which TS2/16 and divalent cations brought about activation were distinct. Results from these analyses are summarized in Table I. In the presence of 2 mM Mn2+ alone and 2 mM Mn2+ + TS2/16, KD values of 100 and 18 pM, respectively, were observed. The KD value resulting from activation by TS2/16 alone (i.e. 1 mM Mg2+, 1 mM Ca2+ + TS2/16) was 220 pM. A similar increase in affinity was seen after treatment of the 10 mM Mg2+ alone state with TS2/16 (KD = 440, 220, and 40 pM for the 10 mM Mg2+ alone, TS2/16 alone, and 10 mM Mg2+ + TS2/16 states, respectively). Interestingly, the final KD observed after co-activation by divalent cations plus TS2/16 was strongly influenced by the concentration and type of divalent cation. KD values varied with metal ion concentrations in a manner analogous to the data observed in the divalent cation alone state, whereas the TS2/16 treatment appeared to produce a quantum step. This distinction in effect is particularly apparent from the dissociation data shown in Fig. 4A. In contrast to the additive effect seen after co-activation by TS2/16 plus divalent cations, the dissociation rate observed in the presence of 2 mM Mn2+ plus 10 mM Mg2+ was not significantly slower than that seen with 2 mM Mn2+ alone, indicating that activation caused by Mn2+ and Mg2+ was not additive (data not shown).

In the non-activated 1 mM Ca2+, 1 mM Mg2+ state, the kinetically determined affinity of BIO1211 for alpha 4beta 1 was significantly lower (>5 nM) than the affinities determined for the activated states. Whereas the on-rate for the Ca2+, Mg2+ state was similar to that observed under activating conditions (see Table I), dissociation was too rapid to allow an accurate determination of koff, and so the kinetic data gave only a lower limit to KD (see Fig. 3B). The limit of KD >5 nM is consistent with the more accurate estimate of KD = 20-40 nM that was obtained from equilibrium measurements described above. Together the data in Table I demonstrate that the affinity of BIO1211 for alpha 4beta 1 is highly sensitive to the activation state of alpha 4beta 1 and can vary by over 1000-fold depending on the assay conditions.

Since alpha 4beta 1 affinity states are dependent on metal ion binding, we investigated the effects of EDTA treatment on dissociation of the BIO1211·alpha 4beta 1 complex under various activating conditions. Results from this analysis are shown in Fig. 5 and Table II. EDTA treatment of the 2 mM Mn2+ alone state resulted (Fig. 5A), as expected, in a more rapid release of [3H]BIO1211 from the complex, although even after EDTA treatment the rate of release was not as rapid as from the non-activated state. EDTA treatment also resulted in a more rapid release of BIO1211 from the 2 mM Mn2+ + TS2/16 state; however, the rate of release was clearly slower in the presence of TS2/16 than when either the 10 mM Mg2+ alone or 2 mM Mn2+ alone activated states were treated with EDTA.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Dissociation curves for [3H]BIO1211 from the 2 mM Mn2+ + TS2/16-activated BIO1211·alpha 4beta 1 complex after treatment with EDTA and Ca2+. One-ml aliquots of Jurkat cells (1 × 106 cells) in TBS plus 2 mM Mn2+ and 10 µg/ml TS2/16 or with 2 mM Mn2+ alone were treated with 5 nM [3H]BIO1211 for 2 h at room temperature. 5 µM unlabeled BIO1211 was added in the same buffer either alone or with the addition of 10 mM EDTA or 1 mM Ca2+, and the cells were further incubated for 0, 5, 15, 30, 60, 90, and 120 min. The cells were pelleted, and cell-associated [3H]BIO1211 was measured by scintillation counting. The data were fitted to exponential curves by nonlinear regression. A, 2 mM Mn2+ alone (black-diamond ); 2 mM Mn2+ plus EDTA (diamond ); 2 mM Mn2+ plus 10 µg/ml TS2/16 (+); 2 mM Mn2+ plus 10 µg/ml TS2/16 plus EDTA (); 2 mM Mn2+ plus 10 µg/ml TS2/16 plus 1 mM Ca2+ (×). B, 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 (); 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 plus EDTA (open circle ). Data sets for the 2 mM Mn2+ plus 10 µg/ml TS2/16 plus EDTA and 2 mM Mn2+ plus 10 µg/ml TS2/16 plus 1 mM Ca2+ conditions shown in A and for both sets of conditions shown in B were fit to double exponential curves.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Rate constants for the dissociation of BIO1211-integrin complexes under various conditions
One-ml aliquots of Jurkat cells (1 × 106 cells) in TBS with the indicated additives were treated with 5 nM [3H]BIO1211 for 2 h at room temperature. 5 µM unlabeled BIO1211 was added in the same buffer with or without 10 mM EDTA as indicated, and the cells were further incubated for 0, 5, 15, 30, 60, 90, and 120 min. The cells were pelleted, and cell-associated [3H]BIO1211 was measured by scintillation counting. BIO1211 release data were fitted to single or double exponential curves (see text) by nonlinear regression as shown in Fig. 5. koff values were calculated from the curve fits. For dissociation curves fit to a double exponential equation, k1 and k2 are rate constants for the fast and slow phases of dissociation, respectively.

An unexpected feature of the dissociation curve for the release of BIO1211 from the divalent cation plus TS2/16-activated state was that it required a double exponential fit to account for the data. A similar biphasic curve for the release of BIO1211 was observed for alpha 4beta 1·BIO1211 complexes formed in the presence of 1 mM Ca2+, 1 mM Mg2+ + TS2/16 and treated with EDTA (see Fig. 5B). Unlike the large effect of EDTA treatment on the release of BIO1211 from the divalent cation alone and divalent cation plus TS2/16-activated states, the effect of EDTA on the 1 mM Ca2+, 1 mM Mg2+ + TS2/16 state was modest, causing only a 2-3-fold increase in the rate of the fast phase of release. Although we originally had assumed that the dissociation of BIO1211 from alpha 4beta 1·BIO1211 complexes would follow a single exponential based on the release data that had been generated for the Mn2+-activated integrin, a reevaluation of the data summarized in Table I indicated that this was not necessarily true for all methods of activation. Additional experiments with more data points added along the relevant regions of the dissociation curves supported our original finding that BIO1211 release from divalent cation alone activated alpha 4beta 1 follows a single exponential and reconfirmed the observation that the dissociation of BIO1211 from the 1 mM Ca2+, 1 mM Mg2+ + TS2/16 state was biphasic. By extrapolating the line defined by the slowly dissociating component back to the y intercept, we can estimate that about 50% of the integrin-BIO1211 complex for the 1 mM Ca2+, 1 mM Mg2+ + TS2/16 state was in this form (Fig. 5B). For the tight binding states that were promoted by divalent cation plus TS2/16 treatment, koff without EDTA treatment was so slow that it was impossible to distinguish between a single and double exponential due to the limited dissociation that occurred during the 120-min duration of the experiment, so the potential biphasic nature of these states remains unclear.

Independent evidence for the biphasic nature of BIO1211 release after TS2/16 treatment was obtained by repeating the analysis on purified alpha 4beta 1 that had been immobilized on plastic and assayed in an enzyme-linked immunosorbent assay-type format. Dissociation rates for the release of BIO1211 from BIO1211·alpha 4beta 1 complexes that were formed in the presence of 2 mM Mn2+ or 2 mM Mn2+ + TS2/16 were similar to values observed on live cells, and when the same complexes were treated with EDTA, TS2/16 treatment dramatically decreased the rate of release of BIO1211 from the complex (data not shown). The effect of TS2/16 in the plate format was even more pronounced than on cells. Whereas about 50% of the BIO1211 was rapidly dissociable from cells after EDTA treatment, in the plate format only about 10% was in the rapidly dissociable form, and 90% of the alpha 4beta 1·BIO1211 complexes were in the EDTA-resistant form.

The effects of Ca2+ on alpha 4beta 1 activation are complex. For alpha 4beta 1·BIO1211 complexes, formed in the presence of 2 mM Mn2+ + TS2/16, Ca2+ treatment was as effective as EDTA at promoting release of BIO1211 and, like EDTA treatment, required a double exponential fit to account for the data (Fig. 5A). Ca2+ had less of an effect on the divalent cation alone or TS2/16 states (see Table I). In particular for activation by Mn2+, 1 mM Ca2+ treatment resulted in only a modest 3-fold effect on koff (koff = 5.3 and 1.4 × 10-4 s-1 for the Ca2+, Mg2+, Mn2+ (1 mM each) and 2 mM Mn2+ states, respectively). In the studies shown in Fig. 1A, we observed that increasing the Ca2+ concentration from 1 to 10 mM slightly stimulated BIO1211 binding. Although the ability of Ca2+ to induce the release of bound BIO1211 from the 2 mM Mn2+ + TS2/16 state is consistent with the published model based on studies for RGD-binding integrins in which high concentrations of Ca2+ can function as a non-competitive inhibitor of ligand binding (22, 31), the results we observed for Ca2+ on other states show that Ca2+ can have a wide range of effects on activation. Recent findings (37) have further highlighted differences between alpha 4beta 1 and other beta 1 integrins in their responsiveness to calcium. A more thorough study is needed to understand better the interplay between Ca2+ and other metal ion binding and how these effects are connected to ligand binding.

CS1 and VCAM-Ig Detect the Mn2+ + TS2/16 High Affinity State-- As a result of its high affinity and slow rate of dissociation, the [3H]BIO1211 can also be used as a probe for ligand-integrin interactions through competition measurements in which [3H]BIO1211 binding is used as a reporter for receptor occupancy. In this format, Jurkat cells are first incubated with test compound and then subjected to a brief treatment with [3H]BIO1211 and counted. Counts bound under these conditions measure integrin that is not occupied by the test compound and is therefore free to bind the [3H]BIO1211. Typical results from this type of analysis are shown in Fig. 6. Competition for [3H]BIO1211 binding was seen with BIO1211, VCAM-Ig, and CS1 at concentrations that are consistent with their known binding constants for alpha 4beta 1 (35, 36). An interesting aspect of the binding data was that the VCAM-Ig titration required a non-hyperbolic curve fit to account for the data, as had been previously seen in FACS binding experiments using VCAM-Ig (38). The binding equation used to fit the data for VCAM-Ig describes the data expected for a bivalent ligand (39) and differs from the simple hyperbolic competition curve seen for the monovalent CS1. VCAM-Ig binding was also evaluated by competition using a kinetic readout. Surprisingly, the t1/2 for dissociation of the VCAM-Ig·alpha 4beta 1 complex was only 8 min for the Mn2+ state, which identified a limitation in using VCAM-Ig as a reporter of integrin function (t1/2 = 70 min for BIO1211 under these conditions). [3H]BIO1211 as a reporter type readout should have broad applications as a probe for alpha 4beta 1 structure-function.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 6.   Analysis of CS1 and VCAM-Ig binding through competition. The apparent affinities of CS-1 and VCAM-Ig for alpha 4beta 1 were assessed on Jurkat cells through competition with [3H]BIO1211. Jurkat cells (2 × 106 cells/ml) in TBS plus 2 mM Mn2+ buffer were incubated for 1 h at room temperature with serial dilutions of CS1, VCAM-Ig, or unlabeled BIO1211 at the concentrations indicated. At the end of the incubation, 5 nM [3H]BIO1211 was added, and the cells were further incubated for 10 min. The cells were then pelleted, and the bound [3H]BIO1211 was quantified by scintillation counting. The titration data were plotted as a percent of maximum counts bound with no added competitor. black-square, BIO1211; , VCAM-Ig; black-triangle, CS1.

To understand better whether the difference in affinities between the 2 mM Mn2+ alone and 2 mM Mn2+ + TS2/16 states is specific to BIO1211 or whether it reflects a property of alpha 4beta 1 common to its interaction with other ligands, we tested VCAM-Ig and CS1 binding to Jurkat cells under the same conditions described for BIO1211. As shown in Table III, VCAM-Ig and CS1 were also sensitive to differences between the 2 mM Mn2+ alone and 2 mM Mn2+ + TS2/16 states. These observations support the notion that BIO1211 mimics many of the properties of alpha 4beta 1 ligands and that the different activation states of alpha 4beta 1 it distinguishes are indeed relevant to the binding of more physiological ligands.

                              
View this table:
[in this window]
[in a new window]
 
Table III
The Mn2+ + TS2/16 high affinity state is a property of alpha 4beta 1 that affects binding of VCAM-Ig and CS-1
1-ml aliquots of Jurkat cells (1 × 106 cells) in the buffer indicated were incubated for 1 h at room temperature with serial 2-fold dilutions of VCAM-Ig (0.1-300 nM) or CS1 (0.1-40 µM). 5 nM [3H]BIO1211 was added, and the samples were further incubated 10 min. The cells were collected by centrifugation and [3H]BIO1211 binding quantified by scintillation counting. Apparent KD values for VCAM-Ig and CS1 were assigned from the IC50 values for inhibition of BIO1211 binding. Data for BIO1211 were calculated for the 2 mM Mn2+ and 2 mM Mn2+ + TS2/16 states from on and off rates (Table I) and for the 1 mM Ca2+, 1 mM Mg2+ state from equilibrium binding measurements as described in the text. ND, not determined.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have used [3H]BIO1211 as a model LDV-containing ligand to study alpha 4beta 1 integrin function. When binding was tested under various conditions that affect the activation state of alpha 4beta 1, KD values were observed that ranged from a value of 20-40 nM in the 1 mM Mg2+, 1 mM Ca2+ (non-activated) state to 100 pM in the 2 mM Mn2+ (activated) state to 18 pM in a newly identified state detected when binding was measured in the presence of 2 mM Mn2+ + 10 µg/ml mAb TS2/16. The differences in affinity were regulated almost exclusively by changes in the dissociation rate of the complex. Although we had expected to see affinity differences between non-activated and activated alpha 4beta 1, the large variation in affinity among the various activated states was surprising. Over a 25-fold difference in affinity for BIO1211 was detected between the Ca2+, Mg2+, Mn2+ (1 mM each) state and the 2 mM Mn2+ + TS2/16 state. To verify that the variations in the integrin affinity state are relevant to ligands other than BIO1211, we tested VCAM-1 and CS-1 binding under selected conditions and showed that similar trends were also seen for these more physiologically relevant ligands.

A striking feature of the activated alpha 4beta 1 was that activation was not defined by a single high affinity state but rather by a continuum of affinities that was easily manipulated by changes in the assay conditions. This result is particularly apparent from the data shown in Fig. 4B where the affinity changed from 700 to 440 to 360 to 220 to 170 pM by simply changing Mg2+ concentrations. At high concentrations of added Mg2+, BIO1211 affinity and dissociation rate converge to the values seen at 2 mM Mn2+ (Fig. 4B). This observation suggests that the activating effects seen with Mg2+ and Mn2+ at saturation involve formation of the same affinity state of the integrin. This conclusion was supported by the observation that no further increase in affinity was observed when samples that had been treated with 2 mM Mn2+ were also treated with 10 mM Mg2+. Suboptimal concentrations of Mn2+ produced a similar titration of the dissociation rate to that seen with Mg2+, with the most dramatic effects seen at concentrations of less than 50 µM Mn2+ (data not shown). Presumably, this continuum represents the effects of metal ion-dependent interconversion of a finite number of discrete states of the integrin. If the interconversion between the states is fast as few as two states could account for the data shown in Fig. 4B. Further studies are needed to define the exact number of affinity states involved.

In the presence of 10 mM Mg2+ + TS2/16 or 2 mM Mn2+ + TS2/16, a further increase in affinity was seen. The additive effect of activation by divalent cations and by TS2/16 on the KD for [3H]BIO1211 binding indicates that the mechanisms of activation by divalent metals and by antibody are distinct. The kinetic data are less informative about the transition between the "2 mM Mn2+ state" and the higher affinity state observed with Mg2+ or Mn2+ plus TS2/16. However, the observed differences in the affinities and dissociation rates between 2 mM Mn2+ + TS2/16 and 10 mM Mg2+ + TS2/16 suggested that the higher affinity state can likewise lead to the observation of a continuum of affinities. This notion was further supported by studies in which dissociation rates were measured in the presence of 2 mM Mg2+ + TS2/16 (koff = 0.76 × 10-4 s-1), 10 mM Mg2+ + TS2/16 (koff = 0.43 × 10-4 s-1), and 100 mM Mg2+ + TS2/16 (koff = 0.17 × 10-4 s-1). The relative changes in the dissociation rate of the BIO1211·alpha 4beta 1·TS2/16 complex that were observed as Mg2+ was increased from 2 to 10 mM and from 10 to 100 mM are strikingly similar to the relative changes observed at the same Mg2+ concentrations in the absence of TS2/16 (Table I). In fact, when koff values for the Mg2+ + TS2/16 states were plotted against koff values for the corresponding Mg2+ alone states, we observed a straight line with a slope of 0.125. Key features of the binding data are summarized in the schematic drawing shown in Fig. 7. In particular, Fig. 7 highlights the distinct and additive mechanisms by which divalent cations and TS2/16 promote alpha 4beta 1 activation.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Schematic summary of binding data. Dissociation constants for BIO1211 binding are summarized using actual koff measurements to define integrin activation states as described in Table I. I = non-activated alpha 4beta 1 observed in the presence of 1 mM Ca2+, 1 mM Mg2+. IT = activated alpha 4beta 1 observed in the presence of 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16. IM2+ = activated alpha 4beta 1 observed in the presence of divalent cations. IM2+ = activated alpha 4beta 1 observed after co-stimulation with divalent cation plus TS2/16. Hatch marks indicate the different affinity states observed as a result of changes in the type and concentrations of divalent cations. The single IT state was observed over a range of TS2/16 concentrations from 0.1 to 10 µg/ml.

When high affinity alpha 4beta 1·BIO1211 complexes were treated with EDTA, BIO1211 was released; however, the rate of release differed depending on the degree of activation. Dissociation data for cation-activated alpha 4beta 1 could be fitted to a single exponential, indicating that once the integrin is treated with EDTA, BIO1211 is released at a single characteristic rate. In contrast, the data for TS2/16-activated alpha 4beta 1 did not fit a single exponential and required a double exponential fit indicating that more than one population of sites contributes to the release process. The rate constant for the rapidly dissociating population was similar to that for release from the divalent ion alone state after treatment with EDTA, whereas the rate constant for the slowly dissociating population was similar to that for the divalent cation plus TS2/16 state in the absence of EDTA. Similar biphasic release data were obtained regardless of whether samples were activated by TS2/16 alone or by TS2/16 plus divalent cation, indicating that the complex dissociation curves were not unique to the high affinity state. Thus, it appears either that the divalent cations are not accessible to EDTA to the same degree in the different activated states of alpha 4beta 1 or that a different step governs the release rate. Further studies are needed to distinguish between these alternatives. It is not clear whether activation with TS2/16 induces heterogeneity in the integrin or allows preexisting heterogeneity to become visible. Nevertheless, the fact that dissociation data after EDTA treatment can distinguish between divalent cation and TS2/16-activated states provides additional support for the idea that the two stimuli bring about activation of alpha 4beta 1 in distinct ways.

Although the precise mechanism by which the affinity states of integrins are regulated is unknown, a large body of data implicates the cation-binding sites as a key component (21, 22, 31). Since ligand-binding sites map to the same regions on the alpha - and beta -chains as the cation-binding sites, various models have been proposed in which the cations coordinate with ligands or compete with ligand for binding (28-30, 32, 40), or in which cation binding regulates the opening of the integrin dimer to expose the ligand-binding site (21). Our data argue against the latter model since only the affinities and not the association rate constants for ligand binding are altered by changes in the activation state, indicating that the ligand-binding site is equally accessible for binding under all states of activation. In one published study where a peptide spanning the MIDAS site from the beta 3 subunit of alpha IIbbeta 3 was used as a model for the ligand-binding site, the authors suggested ligand binding could directly compete for Mn2+ binding (40). We see no evidence for this competition using [3H]BIO1211 as a reporter for alpha 4beta 1 function.

Although many models have been proposed for how integrin activation is regulated, the simplest explanation for the independent and additive effects of divalent cations and TS2/16 on alpha 4beta 1 activation is that the metal-binding sites on the alpha 4-chain and the MIDAS site on beta 1-chain can be independently regulated. Thus, the dependence of alpha 4beta 1 affinity on divalent cations could reflect regulation on the alpha -chain elements, whereas the dependence of the affinity on TS2/16 binding could reflect regulation on the beta -chain. Because of the multiple putative metal-binding sites on the alpha 4-chain, variations in the type and concentration of metal ions could affect occupancy of these sites and thereby change the affinity of alpha 4beta 1 for ligand. In contrast, since TS2/16 would either be bound or not bound, one would expect a quantum effect of the antibody on affinity through a conformational change induced by antibody binding that can either directly induce ligand binding or, alternatively, that affects metal binding at the MIDAS which would indirectly affect ligand binding. Although the current studies do not allow us to evaluate BIO1211 binding at a molecular level, the availability of alpha -chain mutants (41) that are targeted at these key regulatory sites should allow us to define more precisely the effects of these distinct elements on ligand binding.

Ligand binding and integrin activation induce a cascade of conformational changes within the integrin that ultimately lead to the activation of intracellular signaling pathways. These changes have been studied in detail using mAbs whose epitopes are either exposed (termed LIBS) or lost following ligand binding (21, 42). BIO1211 is a LIBS inducer and therefore might be expected to exhibit the same profile of effects that natural ligands exhibit (35). The small size of BIO1211 makes it particularly well suited for this type of analysis, since it minimizes the chance of steric inhibition of antibody binding. This notion is particularly apparent from the data for HP1/2 shown in Table I. HP1/2 is a B1 class anti-alpha 4-antibody, which is defined as a potent inhibitor of alpha 4beta 1-ligand interactions that does not induce leukocyte homotypic aggregation (42). Although HP1/2 is a potent inhibitor of VCAM-Ig binding (36), our data clearly demonstrate that HP1/2 has no effect on BIO1211 binding and therefore that it blocks ligand binding through steric effects rather than through direct binding at the ligand-binding pocket. The exquisite sensitivity of the [3H]BIO1211 to differences in alpha 4beta 1 activation suggests that the labeled probe will prove to be an extremely valuable readout for this type of analysis.

Although many assays have been used to study alpha 4beta 1 function (36, 43, 44), the data we generated with [3H]BIO1211 revealed various features about ligand binding that were not evident from these conventional assay methods. Most significant were the observations that activation is not defined by a single state but rather by several distinct states that give rise to a range of affinities and that the affinity differences are tightly coupled to dissociation rates of the integrin-ligand complex. As soluble, monovalent probes for alpha 4beta 1 function, BIO1211, and related inhibitors represent novel tools that should aid in further unraveling the complexities associated with integrin activation.

    ACKNOWLEDGEMENTS

We thank Francisco Sanchez-Madrid for the TS2/16 antibody; Martin Hemler for providing the alpha 2- and alpha 4-K562 transfectants and the B5G10 hybridoma; and Phil Gotwels for providing the alpha 1-transfected K562 cell line. We also thank Diane Leone, Andrew Sprague, and William Delahunt for providing data on the biochemical properties of BIO1211 in diverse assays in which IC50 values were calculated using adhesion and direct binding formats with VCAM-Ig as reporters.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142. Tel.: 617-679-3310; Fax: 617-679-2616.

    ABBREVIATIONS

The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; BIO1211, 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline; mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; HPLC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
  2. Ruoshlahti, E. (1991) J. Clin. Invest. 87, 1-5[Medline] [Order article via Infotrieve]
  3. Hemler, M. E., Elices, M. J., Parker, C., and Takada, Y. (1990) Immunol. Rev. 114, 45-65[Medline] [Order article via Infotrieve]
  4. Lobb, R. R., and Hemler, M. E. (1994) J. Clin. Invest. 94, 1722-1728[Medline] [Order article via Infotrieve]
  5. Springer, T. A. (1994) Cell 76, 301-314[Medline] [Order article via Infotrieve]
  6. Damle, N. K., and Aruffo, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6403-6407[Abstract]
  7. Anwar, A. R., Walsh, G. M., Cromwell, O., Kay, A. B., and Wardlaw, A. J. (1994) Immunology 82, 222-228[Medline] [Order article via Infotrieve]
  8. Clark, E. A., and Brugge, J. S. (1995) Science 268, 233-239[Medline] [Order article via Infotrieve]
  9. Udagawa, T., Woodside, D. G., and McIntyre, B. W. (1996) J. Immunol. 157, 1965-1972[Abstract]
  10. Yoshikawa, H., Sakihama, T., Nakajima, Y., and Tasaka, K. (1996) J. Immunol. 156, 1832-1840[Abstract]
  11. Ferguson, T. A., Mizutani, H., and Kupper, T. S. (1991) Proc. Natl. Acad. Sci. U. S. A 88, 8072-8076[Abstract]
  12. Wahl, S. M., Allen, J. B., Hines, K. L., Imamichi, T., Wahl, A. M., Furcht, L. T., and McCarthy, J. B. (1994) J. Clin. Invest. 94, 655-662[Medline] [Order article via Infotrieve]
  13. Molossi, S., Elices, M., Arrhenius, T., Diaz, R., Coulber, C., and Rabinovitch, M. (1995) J. Clin. Invest. 95, 2601-2610[Medline] [Order article via Infotrieve]
  14. Osborn, L., Hession, C., Tizard, R., Vassalio, C., Luhowskyj, S., Chi-Rosso, G., and Lobb, R. (1989) Cell 59, 1203-1211[Medline] [Order article via Infotrieve]
  15. Elices, M. J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M. E., and Lobb, R. R. (1990) Cell 60, 577-584[Medline] [Order article via Infotrieve]
  16. Wayner, E. A., Garcia-Pardo, A., Humphries, M. J., McDonald, J. A., and Carter, W. G. (1989) J. Cell Biol. 109, 1321-1330[Abstract]
  17. Guan, J. L., and Hynes, R. O. (1990) Cell 60, 53-61[Medline] [Order article via Infotrieve]
  18. Chan, B. M. C., Wong, J. G. P., Rao, A., and Hemler, M. E. (1991) J. Immunol. 147, 398-404[Abstract/Free Full Text]
  19. Shimizu, Y., VanSeventer, G. A., Horgan, K. J., and Shaw, S. (1990) Nature 345, 250-253[CrossRef][Medline] [Order article via Infotrieve]
  20. Masumoto, A., and Hemler, M. E. (1993) J. Biol. Chem. 268, 228-234[Abstract/Free Full Text]
  21. Humphries, M. J. (1996) Curr. Opin. Cell Biol. 8, 632-640[CrossRef][Medline] [Order article via Infotrieve]
  22. Hu, D. D., Barbas, C. F., III, and Smith, J. W. (1996) J. Biol. Chem. 271, 21745-21751[Abstract/Free Full Text]
  23. Gulino, D., Boudignon, C., Zhang, L., Concord, E., Rabiet, M.-J., and Margue, G. (1992) J. Biol. Chem. 267, 1001-1007[Abstract/Free Full Text]
  24. Springer, T. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 65-72[Abstract/Free Full Text]
  25. Irie, A., Kamata, T., and Takada, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7198-7203[Abstract/Free Full Text]
  26. Corbi, A. L., Miller, L. J., O'Conner, K., Larson, R. S., and Springer, T. A. (1987) EMBO J. 6, 4023-4028[Abstract]
  27. Tuckwell, D. S., and Humphries, M. J. (1997) FEBS Lett. 400, 297-303[CrossRef][Medline] [Order article via Infotrieve]
  28. Lee, J.-O., Rieu, P., Arnaout, A., and Liddington, R. (1995) Cell 80, 631-638[Medline] [Order article via Infotrieve]
  29. Qu, A., and Leahy, D. J. (1996) Structure 4, 931-942[Medline] [Order article via Infotrieve]
  30. Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517[Abstract/Free Full Text]
  31. Mould, A. P., Akiyama, S. K., and Humphries, M. J. (1995) J. Biol. Chem. 270, 26270-26277[Abstract/Free Full Text]
  32. Chen, L. L., Lobb, R. R., Cuervo, J. H., Adams, S. P., and Pepinsky, R. B. (1998) Biochemistry 37, 8743-8753[CrossRef][Medline] [Order article via Infotrieve]
  33. Wang, J.-H., Pepinsky, B., Stehle, T., Liu, J.-H., Karpusas, M., Browning, B., and Osborn, L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5714-5718[Abstract]
  34. Wayer, E. A., and Kovach, N. L. (1992) J. Cell Biol. 116, 489-497[Abstract]
  35. Lin, K.-C., Ateeq, H. S., Lee, W.-C., Zimmerman, C. N., Castro, A., Hammond, C., Kalkunte, S., Chen, L. L., Pepinsky, R. B., Leone, D. R., Sprague, A. G., Abraham, W. M., Gill, A., Lobb, R. R., and Adams, S. P. (1998) J. Med. Chem. 42, 920-934[CrossRef]
  36. Lobb, R. R., Antognetti, G., Pepinsky, R. B., Burkly, L., Leone, D., and Whitty, A (1995) Cell Adhes. Commun. 3, 385-397[Medline] [Order article via Infotrieve]
  37. Bazzoni, G., Ma, L., Blue, M.-L., and Hemler, M. E. (1998) J. Biol. Chem 273, 6670-6678[Abstract/Free Full Text]
  38. Jakubowski, A., Rosa, M. D., Bixler, S., Lobb, R., and Burkly, L. C. (1995) Cell Adh. Commun 3, 131-142[Medline] [Order article via Infotrieve]
  39. Perelson, A. S., and DeLisi, C. (1980) Math. Biosci. 48, 71-110[CrossRef]
  40. D'Souza, S. E., Haas, T. A., Piotrowics, R. S., Byers-Ward, V., McGrath, D. E., Soule, H. R., Cierniewski, C., Plow, E. F., and Smith, J. W. (1994) Cell 79, 659-667[Medline] [Order article via Infotrieve]
  41. Irie, A., Kamata, T., Puzon-McLaughlin, W., and Takada, Y. (1995) EMBO J. 14, 5550-5556[Abstract]
  42. Newham, P., Craig, S. E., Clark, K., Mould, A. P., and Humphries, M. J. (1998) J. Immunol 160, 4508-4517[Abstract/Free Full Text]
  43. Lobb, R. R., Chi-Rosso, G., Leone, D., Rosa, M., Newman, B., Luhowshyj, S., Osborn, L., Schiffer, S., Benjamin, C., Dougas, I., Hession, C., and Chow, E. P. (1991) Biochem. Biophys. Res. Commun. 178, 1498-1504[Medline] [Order article via Infotrieve]
  44. Masumoto, A., and Hemler, M. E. (1993) J. Biol. Chem. 268, 228-234[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.