B lymphocyte membrane immunoglobulins (mIg) appear to play multiple roles in the generation of humoral immune responses. They focus antigen for subsequent internalization, processing and expression in the context of Ia. The also transduce transmembrane signals which lead to increased expression of proto-oncogenes and Ia antigens, and in some situations to B cell proliferation. Since the last renewal of this grant, many of the principal elements of the transduction cascade utilized by mIg have been defined. These include phosphoinositide hydrolysis, Ca++ mobilization, and protein kinase C activation. However, the basis by which mIg binding initiates this cascade remains an enigma. In view of the relatively small size of the cytoplasmic portion of mIg and mIgM and mIgD, it seem likely that these molecules utilize secondary transducer molecules analogous to the T cell T3 complex and/or GTP- binding (N) proteins to mediate the activation of phosphoinositide hydrolysis. Our preliminary studies indicate that mIgM and mIgD are associated with accessory molecules in the plasma membrane, and that these molecules are phosphorylated following ligand binding to mIg. Further evidence suggests that phosphorylation of these molecules may lead to receptor desensitization. We propose to define these molecules and explore the possibility that they and/or GTP-binding proteins function as transducer molecules utilize a newly-developed isolated membrane system to study the role of GTP and GTP-binding proteins in mIg coupling to phosphoinositide hydrolysis. Receptor desensitization, as evidenced by the failure of cells to mobilize Ca++ in response to ligand, will be characterized in terms of kinetics, and inducing-ligand specificity requirements. MIg associated phosphoproteins will be characterized following isolation from normal B cells and B lymphomas by copurification with mIg using anti-immunoadsorbents. Finally we will utilize cloned transfectants of mutant IgM genes to determine Ig structural requirements for signal transduction and receptor desensitization, and for association with mIg accessory molecules and N proteins. These studies should allow mapping of mIg domains involved in signal transduction and regulation of receptor sensitivity. They should also provide insight regarding the role of accessory molecules and N proteins in these processes.
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