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.
Original language | English |
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Pages (from-to) | 10338-46 |
Number of pages | 9 |
Journal | Journal of Biological Chemistry |
Volume | 276 |
Issue number | 13 |
DOIs | |
Publication status | Published - 30 Mar 2001 |
Keywords
- Amino Acid Motifs
- Amino Acid Sequence
- Amino Acids/chemistry
- Antibodies, Monoclonal/metabolism
- Calpain/chemistry
- Cell Adhesion
- Cell Line, Transformed
- Cells, Cultured
- Cytoplasm/chemistry
- Cytoskeleton/chemistry
- Enzyme Activation
- Flow Cytometry
- Humans
- Intercellular Adhesion Molecule-1/metabolism
- K562 Cells
- Lymphocyte Function-Associated Antigen-1/chemistry
- Microscopy, Confocal
- Microscopy, Fluorescence
- Models, Biological
- Molecular Sequence Data
- Mutagens
- Mutation
- Phosphorylation
- Point Mutation
- Protein Binding
- Protein Structure, Tertiary
- Sequence Homology, Amino Acid
- Signal Transduction
- T-Lymphocytes/metabolism
- Tetradecanoylphorbol Acetate
- Transfection