No description available.

No description available.

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.

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.

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

Rab11 is a critical GTPase involved in the regulation of membrane trafficking in the endocytic pathway, and it’s misregulation is involved in a variety of human diseases including Huntington’s disease and Alzheimer’s disease. Additionally, de novo mutations (DNMs) of Rab11 have been identified in patients with developmental disorders, and interestingly several parasites, viruses, and bacteria can subvert membrane trafficking through Rab11 positive vesicles to allow for replication and evasion from the immune system. Although Rab11 is one of the best characterized Rab GTPases, hindering the capability to completely understand Rab11 regulation and its role in human disease is the lack of detail describing how Rab11 proteins are activated by their cognate guanine nucleotide exchange factors (GEFs). This thesis is therefore focused on revealing the molecular mechanisms of the GEFs responsible for the activation of Rab11: SH3BP5 and TRAPPII. To investigate the recently discovered GEF SH3BP5, we solved the 3.1Å structure of Rab11 bound to SH3BP5 and revealed a coiled coil architecture of SH3BP5 that mediates exchange through a unique Rab-GEF interaction. The structure revealed a unique rearrangement of the switch-I region of Rab11 compared to other solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutational analysis of switch-I revealed the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases, and GEF deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. To interrogate the highly controversial GEF TRAPPII, we recombinantly expressed and purified the 9 subunit, 427 kDa complex in Spodoptera frugiperda 9(Sf9) cells. We found that the TRAPPII complex is a GEF for both Rab1 and Rab11, and we discovered novel activity for another Rab GTPase. To interrogate the role of these GEFs in human disease, we used HDX-MS and nucleotide exchange assays to show that some DNMs destabilize Rab11 either through a complete or partial disruption of nucleotide binding. Importantly, we discovered that one of these DNMs, K13N, completely prevented SH3BP5 and TRAPPII mediated nucleotide exchange, revealing a putative mechanism of disease. Overall the work completed in this thesis leads to a greater understanding of the molecular mechanisms underlying the activation of Rab11 by its cognate GEFs. / Graduate / 2020-11-25

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: References located at the end of each chapter.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 124-138). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;