B lymphocyte antigen receptors, membrane immunoglobulins (mIg), function in transduction of information across the plasma membrane leading to altered gene expression and cell activation, and in focussing and internalization of antigen for subsequent processing and presentation to T cells. Recent findings indicate that under certain circumstances ligation of membrane immunoglobulin also leads to inactivation of the ability of the cell's unligated receptors to transduce signals. This """"""""desensitization"""""""" may be the basis of tolerance mediated by clonal anergy in the B cell pool. The performance of all of these functions requires that mIg molecules communicate physically with molecules in the cytoplasm. This protein, or could be indirect, involving secondary membrane proteins which bridge receptor mIg with cytosolic protein, or could be indirect, occurring via interaction of the cytoplasmic tail of mIg with cytosolic protein, or could be indirect, involving secondary membrane proteins which bridge receptor mIg to cytosolic proteins. The lack of significant cytoplasmic structure in mIg molecules virtually excludes the former possibility and, until recently, only fragmentary data existed in support of the latter possibility. We have found that membrane immunoglobulins are noncovalently associated with a complex of proteins composed of two disulfide bonded heterodimers and that components of these complexes differ slightly between mIgM-associated and mIgD-associated forms. mIgM is associated with heterodimers of IgMalpha(32kDa) and Igbeta(37kDa), and IgMalpha and Iggamma (34kDa) proteins. mIgD is associated with heterodimers of IgDalpha (33kDa), and Igbeta, and IgDalpha and Iggamma proteins. Recent studies indicate that IgMalpha and IgDalpha are products of the mb-1 gene(s), and that Igbeta and Igalpha are products of the B29 gene(s). Distinct alpha subunit usage may determine the previously noted differences in biological responses which follow mIgM vs. mIgD ligation. Consistent with a role in signal transduction and receptor desensitization, multiple subunits of the above complex are inducibly phosphorylated on tyrosine residues. In this application, we propose to determine the function of these proteins and specific structural motifs which they exhibit, in mediating receptor function. These studies will involve the cloning of IgMalpha, IgDalpha, Igbeta and Iggamma encoding proteins of the genes. Based on deduced sequence, we will produce antibodies specific for subunits of the complex and use these antibodies to study subunit distribution and function. We will utilize biochemical approaches to identify specific sites phosphorylated during signal transduction, desensitization, and cytoskeletal interaction. We will determine the role of specific phosphorylation events in receptor function by production and analysis of transfected B cell which express mutant receptor subunits in which specific phosphorylation sites are altered or absent. The proposed studies should contribute significantly to our understanding of the molecular basis of B lymphocyte activation, antigen processing and tolerance.
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