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

Published online by Cambridge University Press:  28 January 2013

Anthony P. Leonard ,
Kathryn M. Appleton ,
Louis M. Luttrell  and
Yuri K. Peterson
Anthony P. Leonard
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
Kathryn M. Appleton
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
Louis M. Luttrell
Affiliation:
Medical Universityof South Carolina, Medicine, Charleston, SC 29425, USA
Yuri K. Peterson*
Affiliation:
Medical Universityof South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA
*
*Corresponding author. E-mail: petersy@musc.edu
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Abstract

We report the development of a method to analyze receptor and β-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged β2-adrenergic receptor (β2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged β-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, β-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to β-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting β-arrestin interaction found with β2-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.

Keywords

high contentGPCRadrenergicparathyroidarrestincolocalizationkineticsimage processingmicroscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 1 , February 2013 , pp. 150 - 170
Copyright
Copyright © Microscopy Society of America 2013

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Footnotes

†

These authors contributed equally to this work.

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