RasGRP1 is an intracellular signaling protein expressed in lymphocytes that is responsible
for activating Ras GTPases. Positive regulation of RasGRP 1 requires translocation to
cellular membranes where lipid-anchored Ras can be accessed. Plasma membrane
localization of RasGRP 1 in response to antigen receptors requires both the Cl domain and
the plasma-membrane targeting (PT) domain. The Cl domain binds to diacylglycerol
(DAG) at membranes. The PT domain binds its putative ligand at the plasma membrane and
is negatively regulated by an adjacent suppressor of PT (SuPT) domain. RasGRP1 also
contains a pair of EF-hands, with Ca²⁺-binding capability, but with no known regulatory
role. In DT4O cells, RasGRP1 translocates to the plasma membrane and activates the Ras
ERK pathway in response to B cell receptor (BCR) signaling. By introducing point
mutations in the Ca²⁺-binding loops of each of the EF-hands, I found that a potential Ca²⁺-
interaction loop in the first EF-hand is required for RasGRP1 translocation and the
consequential activation of the Ras-ERK pathway in response to BCR signaling. However,
RasGRP1 translocation is not regulated by BCR-generated Ca²⁺ flux. EF-hands were not
required for Cl domain-mediated membrane localization, but were needed for PT-mediated
plasma membrane targeting. EF-hands enhanced PT-domain mediated plasma membrane
localization by repressing the SuPT domain. The REM and GEF domains, which co
ordinately bind to and catalyze guanine nucleotide exchange on Ras GTPases, needed to be
present and Ras-bound for this EF-hand mechanism to be effective. When not bound to Ras,
the REM-GEF domain complex suppressed both plasma membrane and endomembrane
targeting of RasGRP 1 by an EF-hand independent mechanism. Finally, membrane
localization and activation of a naturally occurring splice variant of RasGRP 1, found overexpressed
in systemic lupus erythematosus (SEE) patients, was examined. This splice
variant lacks exon 11, which encodes the segment of RasGRP1 between the GEF domain
and the first EF-hand. Removal of exon 11 resulted in a defect in plasma membrane
localization that was partially overridden by deletion of SuPT, while membrane localization
control via the REM-GEF complex was not affected. Therefore, exon 11 deletion via
alternative splicing appears to functionally disable the first EF-hand of RasGRP1. / Medicine, Faculty of / Medical Genetics, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/2861 |
| Date | 11 1900 |
| Creators | Tazmini, Ghazaleh |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Format | 3100343 bytes, application/pdf |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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