title: A single amino acid in the cytoplasmic domain of the beta 2 integrin lymphocyte function-associated antigen-1 regulates avidity-dependent inside-out signaling.
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.
authors.affiliation: Department of Tumor Immunology, University Medical Center Nijmegen, Philips van Leydenlaan 25, Nijmegen 6525 EX, The Netherlands.
authors.forename: D A
authors.initials: DA
authors.lastname: Bleijs
authors.forename: G C
authors.initials: GC
authors.lastname: van Duijnhoven
authors.forename: S J
authors.initials: SJ
authors.lastname: van Vliet
authors.forename: J P
authors.initials: JP
authors.lastname: Thijssen
authors.forename: C G
authors.initials: CG
authors.lastname: Figdor
authors.forename: Y
authors.initials: Y
authors.lastname: van Kooyk
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002154:Calpain / Q000737:chemistry; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D020014:K562 Cells; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009153:Mutagens; D009154:Mutation; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate; D014162:Transfection
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D016169:Lymphocyte Function-Associated Antigen-1; D009153:Mutagens; D018799:Intercellular Adhesion Molecule-1; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate