Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-98). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 262-280). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 262-280). Also available online.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

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Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: John J.G. Tesmer. Vita. Includes bibliographical references. Also available from UMI.

Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibition of high voltage-dependent calcium (CaV) channels. Furthermore, they have tissue-specific distribution with Rem and Rad most highly expressed in the heart. Indeed, the importance of Rem and Rad in the cardiovascular system is underscored by a number of studies linking them to disease states including cardiac hypertrophy, cardiac fibrosis, and inflammation. A hallmark feature of RGK proteins is their ability to inhibit current through CaV channels (ICa) and in fact, they are recognized as the most potent endogenous inhibitors of ICa. However, how RGK proteins are regulated and what their physiological role is are unknown. Understanding these points is critical for defining the patho-physiological roles of RGK proteins. My thesis work contributes towards the RGK field on two fronts: First, we demonstrate that RGK proteins are non-canonical G-proteins in the context of their ability to undergo nucleotide regulation and second, we reveal a novel paradigm of RGK-mediated inhibition on CaV channels. In Chapters 2 and 3, we show that Rem and Rad are are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding pocket (GNBP). Canonical Raslike G-proteins contain a conserved G-domain that encompass a GNBP and is important for guanine nucleotide binding and hydrolysis. Since RGK proteins also possess a G-domain and GNBP as well as demonstrate bona fide nucleotide binding, it was initially thought that they were regulated in a manner similar to other Ras proteins. However, subsequent studies suggested that RGK proteins may not obey such a classical model and as a result, the regulatory role of their GNBP in the G-domain was unclear. By using a wide range of functional measurements (CaV1.2 currents, Ca2+ transients, β-subunit binding), we demonstrate that RGK proteins Rem and Rad are non-canonical G-proteins. Utilizing point mutants that abolish GTPbinding and prevent GTPase activity (RemT94N and RadS105N), we show that only some cellular functions are dependent on an operational nucleotide binding pocket while others are unperturbed. Specifically, Rem- and Rad-mediated inhibition of ICa is independent of guanine nucleotide regulation whereas protein interactions with the b-subunit of CaV channels (CaVβ) and protein stability are sensitive to nucleotide regulation. We also discover skeletal and cardiac actin to be novel binding partners of Rem. And lastly, we observe differences between the effects of Rem and Rad on their degree of ICa inhibition in cardiac myocytes. Thus, Rem and Rad are non-canonical G-proteins with respect to the regulatory role of their GNBP. In collaboration with a close colleague, Akil Puckerin, Chapter 4 reveals a novel mechanism behind RGK-mediated inhibition of ICa. Together, we show RGK proteins display different modes of inhibition against specific CaV channels and that we can utilize this property to design calcium channel blockers which inhibit CaV channels in an isoform specific manner. We demonstrate this by designing Rem and Rad mutants which have diminished CaVβ capacity, termed Rem-βNULL and Rad-βNULL, respectively. Characterization of these mutants using wholecell patch clamp experiments revealed that Rem-βNULL inhibits only CaV1.2 whereas Rad-βNULL inhibits only CaV1.2 and CaV2.2. Thus, our results describe the first genetically encoded calcium channel blocker that can selectively distinguish amongst L-type channels. Altogether, this thesis work contributes towards our understanding of RGK protein regulation function and the underlying mechanisms by which they inhibit ICa. These findings advance the field both from a mechanistic and physiological standpoint, and will be of great importance towards investigating the patho-physiological role of RGK proteins.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

The adaptor subunit of phosphatidylinositol 3'-kinases (PI3K), p85, is involved in many different biological processes. Recent studies have shown that one of these functions is to serve as a GTPase activating protein (GAP) towards Rab5, a small monomeric G-protein. Rab5, like other G-proteins, can bind to either GDP or GTP in vivo, assuming its inactive and active form, respectively. The p85 protein has been shown to associate with both the nucleotide-bound and nucleotide-free states of Rab5. It has also been shown that p85 associates with activated, phosphorylated platelet-derived growth factor receptors (PDGFRs) via its two SH2 domains, and that upon binding there is a conformational change in the p85 protein which leads to a derepression of p110 kinase activity. The purpose of this study was to analyze if binding of the activated PDGFR peptides to p85 affects its Rab5GAP activity, as well as to measure the binding affinity of p85 towards Rab5 in each of its nucleotide-bound states. GAP assays were performed to measure the effect that peptide analogs of both the activated and inactivated PDGFR had on p85 Rab5GAP activity, while the binding affinity of p85 towards Rab5 was measured using surface plasmon resonance. The results of this study suggest that PDGFR peptides have no significant effect on p85 Rab5GAP activity. Furthermore, p85 appears to have a higher magnitude of binding to nucleotide-associated Rab5 proteins, than nucleotide-free Rab5 proteins. It also appears that p85 forms more stable complexes with Rab5-GTP than with Rab5-GDP. These results further support previous studies that show p85 to be an important regulator of Rab5-mediated endosomal fusion and show that this activity is not regulated by binding to the activated PDGFR itself.