A novel BRET biosensor for Gαq‐GTP reveals unique properties of cancer‐associated GNAQ mutants

Abstract

Heterotrimeric G proteins are critical signal transducers and dysregulation of their activity is emerging a critical driver of cancer. In fact, the mutational landscape of heterotrimeric G protein signaling networks in cancer has expanded dramatically in the recent years through large scale sequencing campaigns. However, the rate at which novel mutations are identified clearly outpaces the ability to analyze their functional consequences in a systematic and reliable manner, making evident the need for developing novel tools for their investigation. Optical reporters that directly monitor G protein activity in intact cells are ideally suited for this purpose as they allow the rapid characterization of signaling mechanisms with high sensitivity and temporal resolution. Despite that nucleotide exchange in the α-subunit is the event that defines G protein activation, all reporters described to date rely on measuring the dissociation and/or rearrangement of Gα-Gβγ complexes as an indirect readout of G protein activation. Here we describe a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor that specifically detects GTP-bound Gαq in cells with sub-second resolution and its implementation to characterize cancer-associated Gαq mutants. Using this novel biosensor, we show that Gαq activation by GPCR stimulation leads to a robust increase in BRET that is rapidly reserved by receptor antagonists. This sensor can also detect modulation of the amplitude and kinetics of Gαq responses by GTPase Activating Proteins (GAPs). By combining this Gαq-GTP BRET sensor with other approaches, we dissected the mechanisms of action for several cancer-associated Gαq mutants to make novel and, in some cases, unexpected observations. For example, we have identified mutants that display normal basal activity and GPCR-mediated activation, but deactivate slowly due to GAP-insensitivity, thereby suggesting a novel cancer-promoting mechanism. Strikingly, we found that Gαq Q209L and Q209P, the two most frequent mutations in uveal melanoma, lead to signaling hyperactivation via different molecular mechanisms. Our data suggests that this difference arises from unique structural features of the Q209P mutant, which might represent a novel vulnerability of the 20–25% uveal melanomas that bear this mutation. In summary, we have validated a first-in-class biosensor for the direct measurement of the bona fide active species of Gα and demonstrated its implementation to gain further insights into the properties of G protein mutants in cancer. Support or Funding Information NIH grants R01GM112631 and R01GM108733, American Cancer Society grants RSG-13-362-01-TBE and IRG-72-001-36, and the Karin Grunebaum Cancer Research Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.