A single amino acid in the cytoplasmic domain of the β2 integrin lymphocyte function-associated antigen-1 regulates avidity-dependent inside-out signaling

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 KS62 readily respond to activation signals, such as phorbol la-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 p, 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 Ca2+-dependent protease calpain.