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.