Rapid kinetic BRET measurements to monitor G protein activation by GPCR and non-GPCR proteins

摘要

Heterotrimeric G proteins are central hubs of signal transduction whose activity is controlled by G-protein coupled receptors (GPCRs) as well as by a complex network of regulatory proteins. Recently, Bioluminescence Resonance Energy Transfer (BRET)-based assays have been used to monitor real-time activation of heterotrimeric G proteins in cells. Here we describe the use of a previously established BRET assay to monitor G protein activation upon GPCR stimulation and its adaptation to measure G protein activation by non-GPCR proteins, such as by cytoplasmic guanine nucleotide exchange factors (GEFs) like GIV/Girdin. The BRET assay monitors the release of free Gβγ from Gα-Gβγ heterotrimers as a readout of G protein activation, which is readily observable upon agonist stimulation of GPCRs. To control the signal input for non-GPCR activators, we describe the use of a chemically-induced dimerization (CID) strategy to promote rapid membrane translocation of proteins containing the Gα Binding and Activating (GBA) motif found in some non-receptor GEFs. The assay described here allows the kinetic measurement of G protein activation with sub-second temporal resolution and to compare the levels of activation induced by GPCR agonists versus those induced by the membrane recruitment of non-receptor G protein signaling activators.