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Activation and function of the Rap GTPases in B lymphocytes McLeod, Sarah Jean

Abstract

Multiple receptors on the surface of B cells are involved in the regulation of B cell development, trafficking and activation. The chemokine SDF-1 and its receptor CXCR4 mediate the retention of B cell progenitors in the bone marrow during development while the trafficking of mature B cells into and within secondary lymphoid organs, where they encounter antigen, is mediated by SDF-1 as well as the chemokines SLC, ELC, and BLC. B cells recognize antigen through their B cell antigen receptor (BCR). Depending on the nature of this antigen and whether co-stimulatory signals are also received, antigen recognition may lead to B cell activation, anergy, or apoptosis. Both chemokine and antigen signals activate integrins, adhesion receptors on the surface of B cells. Integrins are normally in an inactive, non-adherent state, but signalling initiated by antigen or chemokines can rapidly convert them to an activated, adhesion-competent state. Integrin-mediated adhesion is important for B cell development, trafficking and activation. Chemokine receptors and the BCR mediate their effects through the activation of multiple signalling pathways in B cells. Both of these receptors activate low molecular weight GTPases belonging to the Ras superfamily. GTPases act as molecular switches, cycling between an inactive GDP-bound form and an active GTP-bound form. In this thesis, I have investigated the activation and function of the Rap GTPases in B cells. I have shown that Rap is activated downstream of BCR and CXCR4 engagement in B cells, and that this activation occurs through a phospholipase Cand diacylglycerol-dependent pathway. Rap was first identified by its ability to reverse the transformation of cells by activated forms of Ras, and active, GTP-bound Rap can bind Ras effectors, but cannot activate them. Thus, it was originally hypothesized that Rap would act as a negative regulator of Ras-mediated signalling by binding and sequestering Ras effectors, such as Raf-1, an upstream activator of the ERK1 and ERK2 mitogen-activated protein kinases. However, I found that in B cells Rap does not regulate Ras signalling to ERK. Instead, I found that Rap regulates the ability of B cells to migrate towards the chemokine SDF-1. Lymphocyte migration requires cycles of integrin-mediated adhesion at the leading edge of the cell and deadhesion at the rear of the cell. Rap may regulate B cell migration through its ability to promote the activation of integrins on B cells, and subsequent integrinmediated adhesion. I found that Rap activation following BCR engagement or binding of SDF-1 is necessary for integrin activation and cell adhesion. Rap activation also regulates cell spreading and actin polymerization, processes that may be important for cell migration and cell adhesion. In summary, I have identified the Rap GTPases as targets of the BCR and the receptor for SDF-1, CXCR4. Activation of the Rap GTPases in B cells appears to be important for B cells to migrate to chemokines and for integrin-mediated B cell adhesion. Since chemokine-directed migration and integrin-mediated adhesion are important for B cell development, trafficking and activation, Rap may play a role at multiple stages in the life of a B cell.

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