Abstract
The leukocyte-specific beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)/beta(2)) mediates activation-dependent adhesion to intercellular adhesion molecule (ICAM)-1. In leukocytes, LFA-1 requires activation by intracellular messengers to bind ICAM-1. We observed malfunctioning of LFA-1 activation in leukemic T cells and K562-transfected cells. This defective inside-out integrin activation is only restricted to beta(2) integrins, since beta(1) integrins expressed in K562 readily respond to activation signals, such as phorbol 12-myristate 13-acetate. To unravel these differences in inside-out signaling between beta(1) and beta(2) integrins, we searched for amino acids in the beta(2) cytoplasmic domain that are critical in the activation of LFA-1. We provide evidence that substitution of a single amino acid (L732R) in the beta(2) cytoplasmic DLRE motif, creating the DRRE motif, is sufficient to completely restore PMA responsiveness of LFA-1 expressed in K562. In addition, an intact TTT motif in the C-terminal domain is necessary for the acquired PMA responsiveness. We observed that restoration of the PMA response altered neither LFA-1 affinity nor the phosphorylation status of LFA-1. In contrast, strong differences were observed in the capacity of LFA-1 to form clusters, which indicates that inside-out activation of LFA-1 strongly depends on cytoskeletal induced receptor reorganization that was induced by activation of the Ca(2+)-dependent protease calpain.
Highlights
lymphocyte function-associated antigen-1 (LFA-1) has to be activated via outside-in or inside-out signals to efficiently bind intercellular adhesion molecule (ICAM)-1
Our finding that expression of chimeric LFA-1 containing the 1 cytoplasmic domain (␣L/2/1) can completely restore the PMA responsiveness of LFA-1 in K562 cells prompted us to search for single amino acids that differ between the 1 and 2 cytoplasmic domain
Using a cell transfection system in which inside-out signaling of the 2 integrin LFA-1 could be modified by substitution of the 2 for the 1 cytoplasmic domain, we searched for single amino acids in the 2 cytoplasmic domain that regulate insideout signaling
Summary
Monoclonal Antibodies—The monoclonal antibodies (mAbs) SPV-L7 (IgG1), NKI-L15 (IgG2a), and NKI-L16 (IgG2a) reactive with the ␣-chain of LFA-1 were raised as described previously [20]. Cells (2 ϫ 105) were incubated (30 min, 4 °C) in phosphate-buffered saline, containing 0.5% (w/v) bovine serum albumin (Roche Molecular Biochemicals) and 0.01% sodium azide (10 mM; Merck) with appropriate dilutions of either an anti-integrin mAb or an isotype-matched control antibody, followed by incubation with FITC-labeled goat F(abЈ) anti-mouse IgG mAb (Zymed Laboratories Inc., San Francisco, CA) for 30 min at 4 °C. Different concentrations of purified soluble ICAM-1Fc were added together with medium or the LFA-1activating mAb KIM185 (15 g/ml), and the suspension was incubated for 30 min at 37 °C. Fixed cells were stained with TS2/4 mAb (10 g/ml) for 30 min at 37 °C, followed by incubation with FITC-labeled goat F(abЈ) anti-mouse IgG mAb (Zymed Laboratories Inc., San Francisco, CA) for 30 min at room temperature. The same instrument settings of the CLSM were used throughout the distinct experiments
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