UBC Theses and Dissertations
Membrane localization of RasGRPs by C1 domains Goulding, Rebecca Ellen
Ras and Rap GTPases are membrane-bound activators of signal transduction pathways that regulate several cell processes including proliferation, apoptosis and adhesion. Guanine nucleotide exchange factors (GEF5) positively regulate Ras and Rap GTPases by exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). In order to activate Ras and Rap GTPases, GEFs must be at the same membrane compartments where their target GTPases are located. The Ras guanine nucleotide releasing protein (RasGRP) family of four GEFs regulate both Ras and Rap GTPases, with differential specificities. All RasGRPs contain Cl domains, which have the potential to bind the lipid second messenger diacylglycerol (DAG) that is generated at membranes in response to the ligation of many cell surface receptors. Binding of their Cl domains to DAG could serve to co-localize RasGRPs with membrane bound Ras and Rap GTPases. While some evidence exists for each member of the RasGRP family being potentially regulated by their Cl domains binding to DAG, there is contradictory evidence for RasGRP2. My thesis research focused on Cl domain-mediated mechanisms of RasGRP membrane localization, with special focus on RasGRP2. I found that the Cl domains of RasGRP2 and the β splice variant of RasGRP4 do not bind either DAG or phorbol ester, a DAG analog. However, all RasGRP Cl domains were shown to bind anionic phospholipids. I determined that the Cl domain of RasGRP2 is required for constitutive plasma membrane localization in NIH 3T3 fibroblasts and T-cells, and also for translocation to the plasma membrane in SDF-1α-stimulated T-cells. I also identified a putative PDZ protein binding site which is required for RasGRP2 localization at the Golgi. My experiments showed that while RasGRP2 localization can occur at the plasma membrane and Golgi of NIH 3T3s, RasGRP2 mediated Rapi activation at the plasma membrane via its Cl domain is required for changes in cell morphology that are induced by RasGRP2 expression. My thesis research has demonstrated that all four members of the RasGRP family utilize their Cl domains to localize to membranes, although in the case of RasGRP2 this occurs via a DAG-independent mechanism, which targets RasGRP2 to the plasma membrane.
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