The role of connexin 43 in B cell receptor signaling and the high throughput screen for novel B cell receptor signaling inhibitors

B cells migrate throughout the body, following chemokine gradients, to search for foreign antigens. When a B cell encounters an antigen-presenting cell (APC) bearing antigens that bind its B cell receptors (BCRs), it spreads across the surface of the APC to scan for additional antigens. This spreading is followed by contraction in which BCR microclusters recruit signaling enzymes and form an immune synapse. The resulting BCR signaling initiates an activation process in which B cells differentiate into antibody-producing plasma cells, which are directed to the bone marrow by chemokines, where they survive and produce antibodies for long periods of time. Both chemokine-induced B cell migration and BCR-induced cell spreading involve changes in the cytoskeleton. The gap junction protein connexin 43 (Cx43) regulates the migration of neuronal cells during brain development and interacts with many cytoskeletal-regulating partners. Previous work in our lab showed that Cx43 is important for BCR-induced spreading and chemokine-induced B cell migration, and for sustained activation of Rap1 GTPases, master regulators of cytoskeletal organization. However, the mechanism by which Cx43 enhances BCR- and chemokine-induced Rap1 activation was not known. The goal of my project was to determine whether Cx43 modulates all BCR signaling reactions, perhaps by acting on BCR-proximal signaling components, or whether it selectively regulates the activation of Rap1. I found that Cx43 influences the activation of certain BCR signaling pathways, specifically the PLCγ pathway, while having less effect on other targets of BCR signaling. This is the first evidence that Cx43 modulates BCR signaling and provides new insights into how Cx43 contributes to BCR-induced cytoskeletal regulation. BCR signaling is also important for B lymphoma cell survival and for the negative selection processes that eliminate autoreactive B cells. Although B cell depleting drugs have been used to treat B cell malignancies and B cell-mediated autoimmune diseases, there is variable efficacy. Small molecule inhibitors might be useful alternatives or additions to B cell depletion. Although several inhibitors of B cell signaling enzymes are being clinically tested, I have developed and optimized assay conditions to enable a large-scale screen for additional inhibitors of key components of BCR signaling pathways.Medicine, Faculty ofGraduat