A single amino acid in the cytoplasmic domain of the beta 2 integrin lymphocyte function-associated antigen-1 regulates avidity-dependent inside-out signaling

D A Bleijs, G C van Duijnhoven, S J van Vliet, J P Thijssen, C G Figdor, Y van Kooyk

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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 languageEnglish
Pages (from-to)10338-46
Number of pages9
JournalJournal of Biological Chemistry
Issue number13
Publication statusPublished - 30 Mar 2001


  • 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

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