Bromophenacyl bromide binding to the actin-bundling protein l-plastin inhibits inositol trisphosphate-independent increase in Ca2+ in human neutrophils.
Ligation of IgG Fc receptors on polymorphonuclear leukocytes causes an increase in the concentration of free intracytoplasmic Ca2+ ([Ca2+]i) which arises from release of intracellular stores but is independent of inositol 1,4,5-trisphosphate. We found that bromophenacyl bromide (BPB), an alkylating agent which inhibits leukocyte degranulation, adherence, and phagocytosis, inhibited IgG-stimulated increases in [Ca2+]i with an IC50 of 0.2 microM. In contrast, BPB had no effect on inositol 1,4,5-trisphosphate-dependent [Ca2+]i increases induced by fMet-Leu-Phe, complement fragment C5a, ATP, or platelet-activating factor. Using a monoclonal antibody specific for BPB, we identified in polymorphonuclear leukocytes a single cytosolic protein of 66 kDa and isoelectric point pH 5.6 which bound BPB when intact cells were treated with the alkylating agent. This BPB-binding protein was identified as l-plastin, a Ca(2+)-regulated actin-bundling protein. l-Plastin was found associated with the Triton X-100-insoluble cytoskeleton in polymorphonuclear leukocytes adherent to immune complexes, suggesting that BPB affects Fc receptor-mediated signal transduction by altering the actin cytoskeleton. Consistent with this hypothesis, both cytochalasin B and cytochalasin D inhibited the IgG-dependent increase in [Ca2+]i, without any effect on fMet-Leu-Phe-induced Ca2+ release. These data suggest that the actin cytoskeleton is essential for signal transduction from plasma membrane Fc receptors and that l-plastin has a critical role in activation of this pathway.
Rosales
C
C
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Jones
S L
SL
McCourt
D
D
Brown
E J
EJ
D000098:Acetophenones / Q000737:chemistry*; D000199:Actins / Q000502:physiology*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism*; D011961:Receptors, Fc / Q000502:physiology*; D015398:Signal Transduction
D000098:Acetophenones; D000199:Actins; D003572:Cytochalasins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011961:Receptors, Fc; C026742:plastin; D015544:Inositol 1,4,5-Trisphosphate; C006821:4-bromophenacyl bromide; D002118:Calcium