DESCRIPTION (provided by applicant): Ca2+ signals in B cells are crucial in mediating the coupling between the B cell antigen receptor (BCR) and activation of B cell responses. The size and duration of Ca2+ entry signals is a primary determinant of the developmental response of B cells to antigen-induced crosslinking of the BCR. The project has two fundamental objectives: (a) to study how B cell Ca2+ entry signals are controlled, and (b) to use B cells as a model system to understand the molecular components of Ca2+ entry. The work entails a combination of molecular, genetic, and physiological approaches using the chicken DT40 B cell line which, through targeted gene deletion, provides a system to dissect the BCR-induced Ca -signaling pathway. Using this system, the studies seek to answer the following specific questions: 1. How do components of the B cell receptor complex control Ca 2+ entry signals? The BCR complex is an organized array of adaptor/effector proteins serving to transduce the BCR-binding signal. The hypothesis to be tested is that Ca 2+ entry signals are controlled by the downstream effector and adaptor proteins of the B cell receptor complex. Studies focus on three classes of BCR-coupled molecules: (a) PLC-gamma2, (b) the BCR-coupled adaptor proteins BLNK and Bam32, and (c) BCR-coupled tyrosine kinases. The work uses a series of clonal DT40 B cell lines in which genes for each BCR-coupled effector/adaptor protein have been deleted, and seeks to examine the role of these proteins by measuring a spectrum of Ca2+ signaling parameters defining the endogenous Ca 2+ entry pathways. 2. Can members of the TRP family of proteins function as Ca2+ entry mediators in B cells? TRP channels are widely expressed in cells, including B cells, but their physiological activation is unresolved. The hypothesis to be tested is that TRP channels can couple to and be controlled by the downstream effector and adaptor proteins of the B cell receptor complex. Using the DT40 model cell system, the studies comprise a combination of controlled expression and repressed expression systems with which the coupling processes that activate TRP channels can be monitored. The studies examine details of how the TRPC3 and TRPC4 channels are activated by and molecularly interact with two key signaling proteins - the InsP3 receptor and the phospholipase C-gamma enzyme. The studies provide information on a novel BCR-induced coupling mechanism in the plasma membrane to control B cell Ca2+ signals, a potentially important pharmacological target. BCR-induced Ca 2+ signals are the primary determinants of the developmental fate of B cells, that is, whether they proliferate, whether they remain anergic, or whether as a result of self-recognition they undergo elimination by apoptosis. Definition of this Ca 2+ entry control process provides a key target through which B cell function and development can be modified providing the potential to control major immunological diseases including primary B cell deficiencies and lymphoproliferative disorders.
|Effective start/end date||1/1/04 → 12/31/08|
- National Institutes of Health: $371,250.00