A rapid fluorogenic GPCR–β-arrestin interaction assay

Abstract

Detection of protein–protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein-coupled receptor (GPCR)–β-arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal-to-noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease-activated receptor-1 activation developed in minutes and required specific serine–threonine residues in the receptor carboxyl tail, consistent with a classical G protein-coupled receptor kinase dependent β-arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.