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Allosteric mechanisms and consequences of Gi activation via the Guanine-nucleotide Exchange Modulator, GIV


Heterotrimeric G proteins act as molecular switches that gate the flow of information from extracellular cues to intracellular effectors that control cell behavior. Canonically, G Protein-Coupled Receptors (GPCRs) activate G proteins by stimulating GDP to GTP exchange on the G subunit. It has also been extensively documented that G proteins can be non-canonically activated downstream of non-GPCRs, including Receptor Tyrosine Kinases (RTKs). RTKs are traditionally thought to transduce completely distinct signals via phosphorylation of downstream signaling adaptors, but increasing evidence suggests that these two signaling hubs cross-talk to form an integrated signaling network. One recently discovered cross-talk mechanism is mediated via the novel guanine-nucleotide exchange modulator (GEM), GIV. GIV’s C-terminus possesses a unique molecular make-up, containing an SH2-like domain and a GEM motif. The combination of these protein-binding modules allows the formation of RTK-GIV-Gi complexes where GIV can activate Gi in response to growth factor stimulation. Unlike canonical GPCR-mediated G protein signaling however, the structural basis for non-canonical GIV-mediated G protein activation, particularly downstream of growth factor stimulation, remained largely unknown. My dissertation work sought to fill this gap in knowledge by unravelling what binding of GIV may structurally do to Gi to stimulate GDP release, as well as investigating alternative RTK-dependent and GIV-dependent mechanisms of G protein activation. Using structural, computational, and biochemical approaches, I revealed the structural and dynamical basis for GPCR-independent Gi activation by GEMs and found key similarities and differences between GPCR-dependent and -independent G protein activation, specifically identifying the hydrophobic core of Gi as a common allosteric route toward GDP release utilized by both GPCRs and GEMs. Furthermore, I investigated an alternative but parallel GIV-dependent mechanism of RTK-mediated G protein activation via direct RTK phosphorylation of tyrosine residues within the interdomain cleft of Gi. These RTK phosphorylated tyrosines are essential for Gi activation and signaling in cells, and cancer mutation of these tyrosines results in hyperactive G protein. Taken together, my dissertation has formed a holistic understanding, at the atomic level, of the diverse allosteric mechanisms and consequences of non-canonical GIV-mediated G protein activation.

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