Cellular Regulation of Mutationally Activated G Proteins

About This Grant

Summary Heterotrimeric G proteins (αβγ) canonically link G protein-coupled receptors (GPCRs), activated by a wide variety of extracellular signals, to a variety of intracellular responses. Although in the classical model, GPCR activation of G proteins and subsequent G protein-mediated signaling occurs at the cell’s plasma membrane, it is becoming increasingly clear that GPCR-mediated G protein signaling also happens, with important physiological implications, at the cytoplasmic surface of intracellular organelles, such as endosomes and Golgi. However, much less understood is how G protein α (Gα) subunits function independently of GPCRs when they contain mutations that render them constitutively active. Understanding signaling and regulation of mutationally activated Gα in cells is critical to enhance our knowledge of G protein function in cells and also because somatic mutations have been found in many Gα in human disease contexts, most prominently in cancer. All Gα appear to use almost identical molecular mechanisms for binding GDP or GTP and for hydrolyzing GTP back to GDP; mutations at one of two hotspot residues, a catalytic glutamine (Q) or arginine (R), cause inhibition of the GTP hydrolysis turn-off mechanism resulting in a constitutively active Gα and dysregulated signaling. In one of the most dramatic examples of mutational activation of Gα, the highly similar Gαq and Gα11 subunits have been identified in over 90% of patients with uveal melanoma where the constitutively active Gαq and Gα11 serve as oncogenic drivers. Despite the pathophysiological importance of mutationally activated Gα, the cellular function and regulation of such mutant Gα remain poorly understood. Generally, it has been assumed that mutationally activated Gα are essentially GPCR-activated Gα and indeed mutant Gα are typically used as tools in model systems as surrogates for GPCR-activated Gα. Recently, our work and the work of others is challenging this idea, by uncovering unique properties and mechanisms of constitutively active Gα mutants. This research project will take a broad approach and comprehensive analysis to identify new cellular mechanisms that regulate mutationally activated Gα by investigating multiple Gα families, most prominently Gαq and Gαs subunits, and multiple activating mutations, naturally occurring and not. We will investigate signaling by mutationally activated Gα at intracellular locations, define how distinct mutations direct differences in signaling and regulation, and gain further insight into unique mechanisms of regulation by addressing the paradoxical ability of the natural product YM-254890 to inhibit mutationally activated forms of Gαq. Our work will address these issues using a variety approaches, including biochemical approaches, signaling assays, fluorescence microscopy, live cell imaging, and proteomic approaches to identify novel interactomes and signaling pathways. Ultimately, a better understanding of the cellular mechanisms regulating signaling by mutationally activated Gα may lead to ways to therapeutically target the mutant Gα while leaving the wild type Gα intact.