Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells.
Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.
Dreskin
S C
SC
University of Colorado Health Sciences Center, Division of Clinical Immunology, Denver 80262.
Metzger
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism* / Q000473:pathology; D008274:Magnesium / Q000378:metabolism; D008854:Microscopy, Electron; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D011961:Receptors, Fc / Q000502:physiology*; D017455:Receptors, IgE; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000944:Antigens, Differentiation, B-Lymphocyte; D010716:Phosphatidylinositols; D011961:Receptors, Fc; D017455:Receptors, IgE; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008274:Magnesium; D002118:Calcium