A High-Content, Live-Cell, and Real-Time Approach to the Quantitation of Ligand-Induced β-Arrestin2 and Class A/Class B GPCR Mobilization

We report the development of a method to analyze receptor and beta-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged beta 2-adrenergic receptor (beta(2)-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged beta-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, beta-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to beta-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting beta-arrestin interaction found with beta 2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.