Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design

• Published in: Cell 2020-08, Vol.182 (3), p.770-785.e16
• Main Authors: Maziarz, Marcin, Park, Jong-Chan, Leyme, Anthony, Marivin, Arthur, Garcia-Lopez, Alberto, Patel, Prachi P., Garcia-Marcos, Mikel
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Animals; Bioluminescence Resonance Energy Transfer Techniques - instrumentation; Bioluminescence Resonance Energy Transfer Techniques - methods; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; biosensor; BRET; cancer; Cells, Cultured; G protein; GPCR; GTP-Binding Protein alpha Subunits, Gq-G11 - chemistry; GTP-Binding Protein alpha Subunits, Gq-G11 - genetics; GTPase; Guanine Nucleotide Exchange Factors - antagonists & inhibitors; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; HEK293 Cells; HeLa Cells; Heterotrimeric GTP-Binding Proteins - metabolism; Humans; Mice; Mice, Inbred C57BL; Mutation; Neoplasms - genetics; Neoplasms - metabolism; neurobiology; Neurons - chemistry; Neurons - metabolism; Neurons - physiology; neurotransmitter; oncogene; Receptors, G-Protein-Coupled - metabolism; Signal Transduction; Urinary Bladder Neoplasms - genetics; Urinary Bladder Neoplasms - metabolism
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2020.06.020

Heterotrimeric G-proteins (Gαβγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gβγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience. [Display omitted] •Optical biosensors directly detect active G-proteins in real time in live cells•Unimolecular sensors report activity of natively expressed GPCRs/G-proteins•Versatile modular design allows customization for multiple G-protein specificities•Proof-of-principle implementation for cancer and neurobiology applications A suite of modular optical biosensors enables detection of G-protein activity in live cells in contexts ranging from tumor cells to primary neurons.