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Regulatory mechanisms of the exchange factor RasGRP1

University of British Columbia

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.

Thesis/Dissertation

application/pdf

2008-12-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/2861

Genetics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/