Document Detail

A High-Content, Live-Cell, and Real-Time Approach to the Quantitation of Ligand-Induced β-Arrestin2 and Class A/Class B GPCR Mobilization.
MedLine Citation:
PMID:  23351552     Owner:  NLM     Status:  Publisher    
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.
Anthony P Leonard; Kathryn M Appleton; Louis M Luttrell; Yuri K Peterson
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-28
Journal Detail:
Title:  Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada     Volume:  -     ISSN:  1435-8115     ISO Abbreviation:  Microsc. Microanal.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9712707     Medline TA:  Microsc Microanal     Country:  -    
Other Details:
Languages:  ENG     Pagination:  1-21     Citation Subset:  -    
Medical University of South Carolina, Pharmaceutical and Biomedical Sciences, Charleston, SC 29425, USA.
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