Development of Transgenic Mouse Models for the Expression of Optical Biosensors of Endogenous G Protein Activity

Abstract

G protein‐coupled receptors (GPCRs) are prime pharmacological targets, which is illustrated by the fact that they are the target for ~35% of FDA‐approved drugs and for countless drugs under investigation. These receptors initiate signaling pathways primarily by activating heterotrimeric G‐proteins (Gαβγ) inside the cell. Biosensors that directly measure G protein activation (i.e., formation of Gα‐GTP or Gβγ dissociation) have proven of great value as their readouts are not distorted by signal amplification or pathway cross‐talk, which is in stark contrast to what occurs by indirect measurements of downstream readouts like second messengers. However, these biosensors often require expression of exogenous G proteins which can distort readouts, and are also difficult to implement in many physiologically‐relevant systems, like primary cell cultures. To start addressing these issues, our laboratory has recently developed biosensors based on bioluminescence resonance energy transfer (BRET), called BRET biosensor with ER/K linker and YFP (or BERKY), that can detect endogenous levels of Gα‐GTP or free Gβγ by expressing a single polypeptide chain construct in cells. Here, we set out to further improve the applicability and reproducibility of this technology by generating transgenic mice for the conditional expression of BERKY sensors in virtually any cell type. Since BERKY sensors consist of the same core sequence and only differ in their detector modules, we envisioned a strategy to express two different biosensors from a single transgenic insert by leveraging a Flp recombination of FRT5 sites engineered within the constructs. We confirmed that the introduction of the FRT5 sequence did not interfere with the performance of any of the four biosensors (Gβγ, Gαq‐GTP, Gα13‐GTP, and Gαi‐GTP). Two mouse lines were initially engineered: in one, the Gβγ sensor is expressed before Flp recombination, and the Gαq‐GTP sensor is expressed after, whereas in the second Gα13‐GTP and Gαi‐GTP are expressed before and after Flp‐mediated recombination, respectively. To allow for precise control of the expression of the sensors, the ubiquitous CAG promoter was followed by a loxP‐STOP‐loxP cassette to prevent expression until Cre mediated recombination. After a series of breeding crosses following a specific genotyping strategy, we have successfully established four mouse lines, one for each of the biosensors described above. In conclusion, we have generated mouse lines for the expression of optical biosensors for the detection of endogenous G protein activation. We propose that this experimental platform should prove useful for investigating complex regulatory mechanisms with high fidelity and in any primary cell type fit for cell culture.