PROFILING G PROTEINS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER

Abstract

GPCRs are receptors that act in signal transduction pathways via guanosine nucleotide-binding proteins (G proteins). Extracellular ligands act on GPCRs resulting in activation of one or more G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) affecting the concentration of intracellular second messenger molecules ultimately altering cellular function. Cellular responses to external signals are typically studied indirectly by measuring concentration changes in second messengers. However, this approach can be problematic as many GPCRs can activate multiple G protein subtypes, and many second messenger pathways engage in crosstalk. To address this issue, we used Bioluminescence Resonance Energy Transfer (BRET) to directly measure coupling between 5-hydroxytryptamine (5-HT; serotonin) receptors and different G protein family subtypes. We co-transfected cells with plasmid DNA encoding the 5-HT2B or 5-HT4 receptors fused to the bioluminescent protein nanoluciferase (NLuc) as well as plasmid DNA containing G protein subtypes fused to the fluorescent protein Venus. In BRET assays, we found that mGsq couples to 5-HT2B and mGscouples with 5-HT4 in response to 5-HT activation. These results are consistent with the literature. Interestingly, initial studies suggest that activated 5-HT4 shows secondary coupling to mGsi highlighting the potential novel signaling pathways that can be elucidated using this technique.