B lymphocytes synthesize antibodies both as secreted proteins and as cell surface receptors for antigen. Crosslinkage of the membrane form of immunoglobulin (mIg) results in triggering of hydrolysis of phosphoinositides and the generation of second messengers including diacylglycerol and cytoplasmic free calcium ions. The goals of this project are: 1.) to determine how mIg activates the phospholipase C enzyme that breaks down the phosphoinositides. This involves identifying and characterizing the phospholipase C, the G protein that mediates between mIg and phospholipase C, and putative accessory proteins that associate with crosslinked mIg and permit interaction with the G protein. A better understanding of the molecular mechanisms of mIg signal transduction should give us better insight into what molecular structures make particularly good or poor antigens, which would aid vaccine design. 2.) to determine whether phosphoinositide signaling reactions mediate the biological response of a B lymphoma cell line that is a model for an immature B cell undergoing tolerance induction. Phosphoinositide breakdown will be specifically interrupted by microinjection of antibodies that bind to these phospholipids or by genetic knockout of phospholipase C. In addition, other receptors that can trigger phosphoinositide breakdown will be introduced into the cell line by DNA-mediated gene transfer, with the expectation that they will also induce the growth inhibitory response normally induced by mIg, provided these signaling reactions actually mediate the growth inhibition. 3.) to examine the mechanisms by which phosphoinositide second messengers regulate the growth of B cells. These experiments will involve isolation of genes induced by mIg signal transduction, characterization of which second messengers are responsible for this induction, and use of these genes as molecular markers for characterizing the defects in mutants that have lost the growth inhibitory response to mIg signaling. 4.) to determine whether phosphoinositide breakdown is also triggered by mIg in authentic immature B cells undergoing tolerance induction, and whether these biochemical reactions are mediating the response here as well. These latter studies should give us additional insight into the process of """"""""clonal deletion"""""""" whereby self-reactive lymphocytes are removed at an immature stage of their development. The failure of this process may play a role in the etiology of autoimmune diseases.
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