The development and activation of B lymphocytes is regulated by the antigen receptors, the membrane forms of IgM and IgD. Recently we and others have shown that crosslinkage of mIgM with anti-IgM antibodies (presumably acting as a surrogate for antigen) stimulates the hydrolysis of phosphatidylinositol 4,5- bisphosphate. This reaction generates two second messengers, diacylglycerol, which activates protein kinase C, and inositol 1,4,5-triphosphate (IP3), which causes release of Ca++ into the cytoplasm. First, the molecular mechanism by which mIgM stimulates these reactions will be examined. The part of mIgM involved in signal transduction will be determined by site-specific mutagenesis of the mu gene, and the possible involvement of a new member of the G protein coupling component family will be assessed. Efforts will be made to achieve mIgM activation of the G protein and the phospholipase C in isolated membranes, as a step toward dissecting the system. These experiments will be done in a B cell lymphoma, WEHI-231, which stops growing in response to anti-IgM. The biological roles of the second messengers generated by anti-IgM will be examined by using phorbol diesters to mimic diacylglycerol and calcium ionophores to increase cytoplasmic calcium and by determining their ability to cause growth arrest in an authentic way. If these two small molecules are not completely responsible for growth arrest, then IP3 may play an important role in addition to causing Ca++ release. Recently it has been shown that 1,4,5 IP3 can be converted to 1,3,4 IP3, a more long-lived compound. The kinetics and regulation of production of these two isomers of IP3 will be characterized by using HPLC separations. One advantage of the WEHI-231 system is that mutants defective in the signaling or in the growth regulation have been obtained. Many of the experiments described above will be used to determine the nature of the defect in each of the mutants. This may give us considerable insight into the mechanisms of signal transduction and/or growth regulation. Finally, the nature of the growth regulation of these cells by anti-IgM will be probed by introducing several known oncogenes into WEHI-231 cells and examining the effects they have on growth regulation and anti-IgM-stimulated phosphoinositide breakdown. For example, the ras and src oncogenes have been proposed to affect the phosphoinositide pathway, and this will be examined in transfected WEHI-231 cells.