High fidelity detection of endogenous GPCR-G protein activity with a versatile mouse biosensor platform

Activation of heterotrimeric G-proteins (Gaß?) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of transmembrane signal transduction in eukaryotes with major biomedical implications in therapeutic targeting. We developed a suite of publicly-accessible transgenic mouse lines that, collectively, allow for the conditional expression of biosensors, named BRET sensors with ER/K linker and YFP 3 (BERKY3), for any class of GPCR-activated G-protein (Gs, Gi/o, Gq/11, or G12/13). These permit detection of endogenous G-protein activation by endogenous GPCRs in diverse contexts, like in different brain cell types or in lung cells under native-like tissue conditions. To address whether the expression of BERKY biosensors in cells isolated from the transgenic mice affected the signaling mechanism under investigation, unbiased transcriptomic analyses were performed, revealing the high fidelity of the biosensor readout. In summary, the experimental platform of genetically-modified mice introduced here provides a versatile resource to reproducibly interrogate biological processes or mechanisms of drug action that operate via GPCR/G-protein signaling under native cellular conditions and across physiologically-relevant systems. Overall design: RNA-sequencing was performed for cells (astrocytes for Gas*-BERKY3 and lung fibroblasts for Gai*-BERKY3, Gaq*-BERKY3, and Ga13*-BERKY3) isolated from BERKY3 mice. RNA was isolated from agonist (isoproterenol for Gas*-BERKY3 and TRAP-6 for Gai*-BERKY3, Gaq*-BERKY3, and Ga13*-BERKY3) stimulated or unstimulated cells pre-treated with adeno-associated virus (AAV2/1-CMV-Cre; University of Iowa Viral vector Core, cat# VVC-U of Iowa-237) for Cre-recombinase-mediated biosensor expression or a control empty adeno-associated virus (AAV2/1-CMV-empty; University of Iowa Viral vector Core, cat# VVC-U of Iowa-4223). Three replicate samples were taken for each condition.