BIOLOGICAL FUNCTION OF B CELL RECEPTOR DESTABILIZATION

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DESCRIPTION (adapted from the applicant's abstract): Signals transduced by the B cell antigen receptor (BCR) contribute to an immune response that normally leads to proliferation or differentiation. Deregulation of the immune response due to oncoprotein transformation or failure to discriminate self from foreign proteins can lead to lymphoproliferative disorders and autoimmune disease. The candidate's interests are to define molecular mechanisms that "silence" self- specific B lymphocytes. Dr. Vilen s immediate career goals are to further characterize her recent observation that ligation of the B cell antigen receptor leads to destabilization of the BCR complex, as evidenced by the diminished ability to co-immunoprecipitate mIg and Ig-alpha/Ig-beta. The first Specific Aim is to define changes in the integrity of the BCR complex following antigen-induced desensitization. This will allow assessment as to whether BCR destabilization represents a physical dissociation of mIg from Ig- alpha and Ig-beta, or a biochemical destabilization. One method that will be employed uses chemical crosslinkers to estimate the distance between mIg and Ig-alpha/Ig-beta in receptor destabilized cells. A second approach will employ fluorescence microscopy to address if the individual components of destabilized receptors segregate independently upon aggregation. In the second Specific Aim, the applicant will test the hypothesis that BCR destabilization occurs on the cell surface and is temporally correlated with B cell unresponsiveness and receptor desensitization, suggesting that BCR destabilization may be responsible for the desensitized phenotype. This will be assessed by creating a panel of chimeric receptors that are not susceptible to destabilization. These receptors will contain either the Ig-alpha or Ig- beta cytoplasmic tail fused to the mIg extracellular domain and an MHC class I transmembrane domain. The chimeric receptors will be stably transfected into a B cell line, and the chimeric receptors will be assayed for their ability to be desensitized. The applicant's long range goals are to continue to define the molecular basis of B cell receptor desensitization as a model for anergy by defining the molecular mechanism(s) responsible for BCR destabilization. The timing of BCR destabilization and the requirement for kinase activation suggest that a phosphorylation event on either mIg or Ig-alpha/Ig-beta may be responsible for this event. Therefore, her future plans, beyond the duration of the work described in this application, involve establishing if a novel phosphorylation event occurs coincident with receptor destabilization, identifying the targeted site, and confirming its role in receptor destabilization by creating a mutant receptor. Additional future studies include assessing the role of BCR destabilization in an immune response by generating a "knock-in" mouse harboring a mutation that prevents BCR destabilization. Finally, the applicant is interested in defining the endocytic pathways that mIg and Ig- alpha/Ig-beta follow when removed from the cell surface.