B lymphocyte activation. Transmembrane signal transduction by membrane immunoglobulin in isolated cell membranes.
We report the development of cell-free systems in which ligation of B cell membrane immunoglobulin leads to demonstrable mono- and polyphosphatidylinositol hydrolysis. Membranes were prepared by differential centrifugation of sonicates of normal murine B cells. Incubation of these membranes with 32P-adenosine triphosphate in the presence of Mg2+ effected the radiolabeling of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP). Alternately, membranes were labeled with exogenous 3H(inositol)-PtdInsP2 in sodium cholate. Stimulation of labeled membranes with anti-immunoglobulin, but not anti-Ia or anti-H2 antibodies, resulted in hydrolysis of phosphatidylinositol, PdtInsP, PtdInsP2 and generation of inositol phosphates indicative of activation of a phospholipase C. The response was rapid, being detectable within 30 sec of stimulation, and independent of Ca2+ and guanosine 5'-triphosphate. Optimal responses were dependent on the presence of a cytosolic factor presumed to be phospholipase C. Development of these systems represents an important step towards reconstitution of membrane immunoglobulin-mediated transmembrane signaling in artificial membranes.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000276:immunology* / Q000378:metabolism; D002474:Cell-Free System; D006160:Guanosine Triphosphate / Q000502:physiology; D006868:Hydrolysis; D008213:Lymphocyte Activation; D051379:Mice; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000276:immunology*; D010738:Type C Phospholipases / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011947:Receptors, Antigen, B-Cell; D006160:Guanosine Triphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium