“Investigating the Interaction between G Proteins and the 5-HT1E and 5-HT2C Serotonin Receptors Using BRET”

Abstract

G protein-coupled receptors (GPCRs) are important mediators in cellular signaling and are common targets of drug action. GPCRs are responsible for the transduction of extracellular signals into intracellular signals, mediated by G proteins of four subtypes: Gs, Gi, Gq, and G12/13. A thorough understanding of a signaling pathway involves determining which G protein is coupled to a signal-activated GPCR. In this project, a technique called Bioluminescence Resonance Energy Transfer (BRET) was used to measure the interaction between an activated GPCR from the serotonin (or hydroxytryptamine, 5-HT) receptor family, and G proteins from each subtype. The cDNA for serotonin receptors 5-HT1E and 5-HT2C was fused with the gene for a luminescent protein called Nanoluciferase (Nluc). Then, the receptor-Nluc DNA along with DNA containing a G protein tagged with a fluorescent protein (Venus) was transfected into mammalian cells for expression. Data from BRET assays suggest that the 5-HT1E receptor couples to the Gi/o subclass of G proteins upon serotonin activation, while the 5-HT2C receptor couples to the Gq subclass of G proteins. Profiling serotonin receptors will deepen our understanding of serotonin receptors, associated diseases, and the drugs that target them.