Abstract Heterotrimeric G proteins () are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological processes. However, it has also become increasingly recognized that G proteins can function independently of GPCRs. Highlighting the physiological relevance of GPCR-independent G protein function, constitutively activating somatic mutations in several G subunits have been identified and shown to contribute to human disease, most prominently in cancer. This research project focuses on constitutively active mutants of the highly similar q and 11 subunits, collectively termed q/11. Mutations at one of two hotspot residues, that result in inhibition of the GTP hydrolysis turn-off mechanism, can convert q/11 into an oncogenic driver of uveal melanoma, and such constitutively activating q/11 mutations have also been associated with vascular tumors, capillary malformation, congenital hemangioma and Sturge Weber syndrome. This research project will focus on uncovering basic cellular mechanisms regulating signaling by mutationally activated q/11. G subunits are typically thought as exceptionally difficult proteins for which to develop pharmacological or molecular inhibitors. However, numerous recent studies have demonstrated that the highly similar natural products YM-254890 and FR900359 are effective inhibitors of wild type and mutationally activated q/11. Our work has uncovered an unexpected mechanism of action of these inhibitors leading to the hypothesis that YM- 254890 and FR900359 inhibit q/11, at least in part, by regulating subcellular localization of q/11. Understanding the novel mechanisms of inhibition and cellular regulation of mutationally activated q/11 may suggest novel therapeutic targets or approaches for disrupting dysregulated signaling. The objectives in this research project will be pursued by a variety of experimental approaches, including using cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, constitutive targeting to specific subcellular locations, biosensors, pharmacological inhibitors, mutational analysis, and biochemical assays.

Project Narrative G protein-mediated signaling pathways regulate numerous physiological responses, including neuron development, neurotransmitter responses, cardiovascular function, cell differentiation, cell migration, immune cell function, and smell, taste, and vision. Dysregulation of G protein-mediated signaling pathways contribute to numerous disease states, including neurological diseases, heart disease, hypertension, cancer, metastasis, endocrine disorders, and blindness. Understanding novel cellular mechanisms that regulate disease causing, mutationally activated G proteins will provide new knowledge and opportunities to therapeutically target unwanted G protein responses.