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:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Anthony P Leonard; Kathryn M Appleton; Louis M Luttrell; Yuri K Peterson
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-01-28
Journal Detail:
Title:  Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada     Volume:  19     ISSN:  1435-8115     ISO Abbreviation:  Microsc. Microanal.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-05     Completed Date:  2013-07-22     Revised Date:  2014-06-12    
Medline Journal Info:
Nlm Unique ID:  9712707     Medline TA:  Microsc Microanal     Country:  United States    
Other Details:
Languages:  eng     Pagination:  150-70     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Arrestins / metabolism*
Automation / methods
Cell Line
Humans
Kinetics
Microscopy, Fluorescence / methods*
Protein Binding
Receptor, Parathyroid Hormone, Type 1 / metabolism*
Receptors, Adrenergic, beta-1 / metabolism*
Time-Lapse Imaging / methods*
Grant Support
ID/Acronym/Agency:
C06 RR015455/RR/NCRR NIH HHS; C06 RR015455/RR/NCRR NIH HHS; R01 DK055524/DK/NIDDK NIH HHS; R01 DK55524/DK/NIDDK NIH HHS; T32 GM008716/GM/NIGMS NIH HHS; T32 GM08716/GM/NIGMS NIH HHS; TL1 RR029881/RR/NCRR NIH HHS; TL1 RR029881/RR/NCRR NIH HHS; TL1 TR000061/TR/NCATS NIH HHS; TL1 TR000061/TR/NCATS NIH HHS
Chemical
Reg. No./Substance:
0/Arrestins; 0/PTH1R protein, human; 0/Receptor, Parathyroid Hormone, Type 1; 0/Receptors, Adrenergic, beta-1; 0/beta-arrestin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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