Trimeric G proteins as Novel Targets in Cancer Progression

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ABSTRACT: Heterotrimeric G proteins are molecular switches that control signal transduction. Dysregulation of the G protein pathway can lead to aberrant signal transduction and herald many diseases and oncogenesis. Although G proteins are traditionally known to transduce signals initiated by G protein coupled receptors (GPCRs), a growing body of work by my mentor's group and others have established that they also transduce signaling downstream of yet another large group of receptors, the growth factor receptor tyrosine kinases (RTKs), via mechanisms that are poorly understood. Recent studies have demonstrated that Gα-Interacting Vesicle associated protein (GIV, a.k.a Girdin) is an unusual signal transducer that can bind both RTKs and G proteins. As a direct consequence of an unusual modular makeup of GIV, signals initiated by multiple RTKs converge on the GIV-platform to trigger non-canonical transactivation of trimeric G protein, Gαi. Working downstream of a variety of growth factors and ligands, it has been demonstrated that the consequences of such signaling are far reaching, and that the impact on a diverse set of biological processes, in both health and disease, is enormous. Despite the insights gained, the mechanism of G protein activation in close proximity of RTKs remains unclear, how may this pathway affect signal transduction or cellular phenotype, and what might be the structural basis for this unusual RTK-GIV-Gi pathway and their pathophysiologic consequences remain unexplored. Preliminary results indicate that the proximity between the receptor and the G protein is essential for phosphorylation of Gαi by multiple RTKs at three unique tyrosines, that such phosphorylation requires GIV to recruit G proteins to the RTKs, and that one of the major. These findings will be studied in-depth through the experiments in the following 3 aims – 1) assess the consequence(s) of phosphorylation of Gαi by multiple growth factor RTKs using in vitro and in vivo phosphorylation assays, protein-protein interaction assays with various modulators of G proteins, measures of Gi activation, and phenotypic assays to evaluate migration, invasion, mitosis, and survival in cells expressing WT or Y mutants of Gi; 2) investigate how transactivation of G proteins by RTKs is deregulated in cancers by studying the profile of RTK-triggered tyrosine phosphorylation of Gαi in tumor cells during metastasis and by characterization of a novel somatic mutation in Gαi where the tyr (Y) that is targeted by RTKs is mutated to his (H) using similar biochemical and cell biological assays as outlined in Aim 1; and 3) elucidate the structural basis for phosphotyrosine-dependent transactivation of G proteins using a combination of protein chemistry, functional binding assays with rationally designed mutant proteins, and x-ray crystallography. The overall goal of this proposal is to dissect the mechanisms by which multiple growth factor RTKs transactivate trimeric G proteins via the novel linker/platform, GIV from an atomic level to tumor cell phenotype.