Real-time detection of G protein activation using monomolecular Gγ FRET sensors

Abstract

G protein-coupled receptors (GPCRs) are involved in many diseases, and are the target of a large percentage of modern drugs. While only a few fluorescence resonance energy transfer (FRET) sensors have been made for real-time detection of GPCR activation, the human genome encodes roughly 950 GPCRs and a need for a broad real-time detection system is needed. In this study, we developed and characterized a new type of G protein sensor based on the Gγ subunit containing an intra-molecular FRET pair to allow detection of real-time G protein activation in multiple cell lines using time-resolved fluorescence spectroscopy. Our Gγ sensors were able to detect G protein activation to aluminum fluoride, a G protein activator, in human embryonic kidney 293 cells (HEK293). Moreover, our sensors were able to couple to the bradykinin B2, parathyroid type 1 and adrenergic 2A GPCRs and detect G protein activation in response to the cognate ligands; bradykinin, parathyroid and noradrenaline hormones, respectively. Furthermore, our sensors also detected ligand-independent G protein activation by fluid shear stress. G protein inhibitors, pertussis toxin and guanosine 5′ [β-thio] diphosphate, inhibited the FRET response to G protein stimulants indicating that the sensor response is specific to G protein activation. These findings suggest that the described Gγ sensors can be used to monitor real-time G protein activation by a variety of G protein stimulants or inhibitors.