Evaluating functional ligand-GPCR interactions in cell-based assays

Abstract

G protein-coupled receptors (GPCRs) are a family of transmembrane proteins that act as major mediators of cellular signaling, and are the primary targets for a large portion of clinical therapeutics. Despite their critical role in biology and medicine, a large number of GPCRs are poorly understood, lacking validated ligands or potent synthetic modulators. Ligand-induced GPCR activation can be measured in cell-based assays to test hypotheses about ligand-receptor interactions or to evaluate efficacy of synthetic agonists or antagonists. However, the techniques necessary to develop and implement a cell-based assay to study a given receptor of interest are not commonplace in all laboratories. This chapter outlines methods to develop a cell-based assay to evaluate agonist-induced activation for a GPCR of interest, which can be useful to evaluate the effectiveness of predicted ligands. Examples of sample preparation protocols and data analysis are provided to help researchers from interdisciplinary fields, especially those in fields with relatively little molecular biology or cell culture experience.

Keywords: Cell-based activity assay; Cellular signaling; G protein-coupled receptor (GPCR); IP1 assay; Ligand-induced activation.