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Profiling Gαq Cancer Mutations Using a Novel BRET‐based Biosensor
Author(s) -
Maziarz Marcin,
Leyme Anthony,
Patel Prachi P.,
GarciaMarcos Mikel
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.668.8
Subject(s) - heterotrimeric g protein , g protein , g protein coupled receptor , gtpase activating protein , gtpase , mutant , regulator of g protein signaling , signal transduction , cancer research , biology , microbiology and biotechnology , gene , computational biology , genetics
Heterotrimeric G proteins are critical signal transducers and dysregulation of their activity causes many diseases, including cancer. For instance, activating mutations in the Gα subunit Gαq are frequently found in uveal and meningeal melanomas. A current challenge in the field of cancer in general and oncogenic G protein signaling in particular is that the rate of discovery of cancer‐associated mutations clearly outpaces our ability to analyze their functional consequences. This limits our ability to discern driver mutations from passenger mutations, and to elucidate possible mechanisms by which they favor cancer progression. Here, we set out to develop and implement a novel approach to rapidly profile cancer‐associated mutations that impact Gq‐dependent signaling. We envisioned the design of a Gq activity biosensor to detect GTP‐bound Gαq in live cells based on the principle of Bioluminescence Resonance Energy Transfer (BRET). First, we validated that our sensor specifically detects Gαq activity, and that it can faithfully report its modulation by G protein‐coupled receptors (GPCRs) and by Regulators of G protein signaling (RGS) in real time with sub‐second resolution. After benchmarking our sensor against well‐characterized Gαq oncogenic mutants, we implemented it in a battery of cancer‐associated Gαq mutants that had not been characterized before. Our findings reveal that a hotspot mutation present in >10% of hepatocellular carcinomas does not give rise to a hyperactive Gαq, whereas other infrequent mutations enhance Gαq activity. More specifically, the latter mutations are insensitive to deactivation by GTPase activating proteins (GAPs) of the RGS family, which sensitizes Gαq activation and delays inactivation upon GPCR‐mediated modulation. In conclusion, we have developed a G protein activity biosensor that directly monitors the formation of Gαq‐GTP, and showed that it is useful to rapidly profile disease‐associated G protein mutants and reveal novel mechanisms of aberrant activation. Support or Funding Information Funding: R01GM130120, R21MH118745 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .