Heterotrimeric G proteins are molecular switches that control signal transduction and their dysregulation can herald oncogenesis. Another pathway with undisputed roles and also widely implicated in driving cancer growth and invasion comprises of growth factor receptor tyrosine kinases (RTKs) and the myriad of signals/pathways they initiate. Furthermore, cross-talk between these two seemingly distinct pathways can transactivate each other, and unrestricted signaling due to recruitment of multiple receptors via transactivation is critical in mediating cancer progression. Although indirect transactivation of RTKs by G protein intermediates is well established, insights into how G proteins are transactivated after direct stimulation of RTKs is lacking. The proposed research aims to expose the mechanisms by which growth factor receptors regulate G protein signaling. The preliminary work showcased in this grant reports the discovery of a number of unique signaling complexes at the cross-roads of G protein- and growth factor signaling pathways and unravels the implications of these complexes during cell migration, mitosis and autophagy;processes that are vital for tumor progression. Our studies with GIV, a non-receptor guanidine exchange factor (GEF), trimeric G protein, G?i, and their regulation by the epidermal growth factor receptor, the prototype member of the growth factor RTK family have allowed us to define a novel stratum for signal amplification. The overall goal in this proposal is to gain mechanistic insights into the workings of this stratum and its relevance in cancer progression.
Our specific aims are: 1) to assess the consequence(s) of phosphorylation of trimeric G protein, G?i by RTKs during cell migration using in vitro and in vivo phosphorylation assays, linear ion-trap technology for phospho-Mass Spectrometry, biophysical analysis of phosphoprotein, protein-protein interaction assays with GEFs, GTPase activating proteins (GAPs), guanidine dissociation inhibitors (GDIs) and GPCRs, computational modeling, and scratch-wound and mitosis assays;2) to identify and characterize the phosphomodifications on GIV triggered by growth factors during tumor cell migration and proliferation using a combination of aforementioned approaches, immunofluorescence, G protein enzymology, and protein-protein interaction assays;and 3) to study how, when and where the RTK-GIV- G?i ternary complexes are assembled in cells, and their consequences on PI3K signaling, cell migration/mitosis by a combination of above approaches, FRET assays by live cell imaging, and NMR spectroscopy. These studies are expected to yield novel information and mechanistic insights into the cross-talk between G proteins and growth factors in regulating signaling transduction. The proposed study is expected to unravel unique interfaces for signal transduction assembled at the cross-roads of the two pathways which may not only serve as attractive and effective therapeutic targets, but insights gained will also help decipher, access, and manipulate the cancer cell's complex signaling code.

Public Health Relevance

Aberrant signal transduction is a critical trigger in the pathophysiology of almost all cancers tha afflict many Americans each year. Identification of key signaling molecules that can be effectively exploited to alter the course of human cancers (growth or spread) remains the cornerstone of developing targeted molecular therapy against cancer. The overall goal of the studies planned in this application is to expose how cancer cells utilize the G protein pathways to intercept signals triggered by multitude of growth factor receptors and amplify those signals in an unrestricted manner. This study seeks to discover unique signaling complexes assembled at the intersection of G protein and growth factor pathways which may not only help decipher, access, and manipulate cancer cell's complex signaling code but also serve as attractive and effective therapeutic targets, in the treatment of cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Woodhouse, Elizabeth
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University of California San Diego
Internal Medicine/Medicine
Schools of Medicine
La Jolla
United States
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Wang, Honghui; Misaki, Taro; Taupin, Vanessa et al. (2015) GIV/girdin links vascular endothelial growth factor signaling to Akt survival signaling in podocytes independent of nephrin. J Am Soc Nephrol 26:314-27
Lopez-Sanchez, Inmaculada; Dunkel, Ying; Roh, Yoon-Seok et al. (2014) GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis. Nat Commun 5:4451
Alakus, Hakan; Babicky, Michele L; Ghosh, Pradipta et al. (2014) Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin. Genome Med 6:43
Lin, Changsheng; Ear, Jason; Midde, Krishna et al. (2014) Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin. Mol Biol Cell 25:3654-71