Lyb-2 is a B lymphocyte lineage-specific cell surface glycoprotein expressed on early B cell precursors through the stage of mature B cells. Expression is lost upon terminal differentiation into plasma cells. Lyb-2 appears to play an important role in B cell proliferation and activation, and may be a receptor for a lymphokine or growth factor. The major goal of this project will be to define the molecular mechanisms involved in the regulation of expression of Lyb-2 and to further explore the function of the protein. The structure of the Lyb-2 protein will be better characterized by immunoprecipitation studies of B cells and transfectants. A panel of monoclonal antibodies specific for Lyb-2 will be generated and their functional effects upon B cells will be examined. The structure and sequence of he gene will be determined. The level at which expression of the gene is regulated in different cell types and at different stages of B cell development will be examined, and the molecular mechanisms involved int he lineage and stage specificity of expression Lyb-2 will be determined. These studies will begin in tissue culture cells, where the sequence from the Lyb-2 gene that are necessary for proper expression (i.e., the same pattern of expression as Lyb-2 protein and mRNA) of a reporter gene in transfected cells will be determined. Potential regulatory regions will also be identified by analyzing the gene for differences in DNAse hypersensitive sites in cells that express Lyb-2 as compared to cells that do not. The importance of identified sequences will be confirmed by site-specific mutagenesis. Lyb-2 regulation will then be examined in transgenic mice.
The aim of these studies is to determine both the sequences necessary for proper expression of Lyb-2 using its own control sequences, and the consequences of inappropriate expression of the protein resulting from use of control regions of other genes, such as immunoglobulins, T cell receptor and beta-actin. For example, will continued expression of Lyb-2 in B lineage cells, using an immunoglobulin promoter and enhancer, lead to lack or proper B cell differentiation in to antibody-secreting cells? Will such continued expression lead to B cell tumors? Similarly, if Lyb-2 is a growth factor or lymphokine receptor, will expression on T cells or other inappropriate cells lead to tumor development because of continued growth stimulation? These studies should enhance our understanding of the normal mechanisms of control of B cell proliferation and differentiation as well as the abnormalities in these pathways that are observed in B cell neoplastic and autoimmune diseases.
