Characterization of Serotonin Receptors in Response to Ligand Binding Using BRET

Abstract

G protein-coupled receptors (GPCRs) are receptors that act in signal transduction pathways via activation of guanosine nucleotide-binding proteins, known as G proteins. An extra-cellular signal (a ligand) activates a receptor from outside the cell and working through a G protein, the external signal is transmitted inside the cell. There are four main classes of G proteins: Gs, Gi, Gqand G12. When activated, eachof these G protein types is responsible for a specific intracellular event, often determined by measuring how the concentration of a second messenger changes as a function of the concentration of the external signal. This indirect approach limits our understanding of the role of each type of G protein in signaling pathways. Our group is currently using Bioluminescence Resonance Energy Transfer (BRET) to directly measure G protein activation by GPCRs in response to external stimuli (includingboth endogenous and synthetic ligands). We have generated recombinant DNA for nanoluciferase fused to GPCRs in the serotonin (5-hydroxytryptamine, 5-HT) receptor family. These genetic fusions, along with fusions of yellow fluorescent protein and various G proteins were co-transfected into HEK293 cells for BRET assays. Initial results show that activation of receptors 5-HT1D and 5-HT1F with serotonin are coupled to Gi. Future studies will include a G protein profile for all twelve receptors in the serotonin family.