Identifying the domains involved in the regulation of RasGRP1 in response to BCR ligation

Guanine nucleotide exchange factors (GEFs) are proteins that act as activators for the Ras family of small GTPases. In order to become activated, they typically need to localize in membrane structures, where their substrates are located. RasGRPl is a guanine nucleotide exchange factor whose activation is associated with translocation to membranes. Previous studies have indicated that translocation of RasGRPI is driven solely by binding o f its Cl domain to diacylglycerol (DAG) generated in membranes following receptor-coupled phospholipase C's activation. In DT40 B cells, ligation of the B cell receptor induces RasGRPl activation, and translocation of RasGRPl occurs exclusively to the plasma membrane. My thesis focused on identifying the domains involved in RasGRPl translocation and activation. My results show that the Cl domain of RasGRPl is essential for efficient BCR-induced plasma membrane translocation and activation, but on its own the Cl domain is unable to drive translocation to the plasma membrane. I identified a domain of RasGRPl, termed the KWEN loop, which acts in conjunction with an adjacent leucine zipper (LZ) to confer BCR-induced translocation to the plasma membrane, either autonomously or in cooperation with the Cl domain. In addition, a newly identified repressor region was shown to dampen KWEN/LZ-mediated translocation and activation. I propose a revised model for RasGRPl translocation and activation: plasma membrane localization in response to BCR ligation is provided by the KWEN loop and LZ . Interaction between DAG and the Cl domain enhances KWEN - mediated binding to the plasma membrane, overriding the suppressive effect of the repressor region and enabling DAG-dependent activation of RasGRPl.Medicine, Faculty ofMedical Genetics, Department ofGraduat