Dataset Information

A high-content, live-cell, and real-time approach to the quantitation of ligand-induced ?-Arrestin2 and Class A/Class B GPCR mobilization.

ABSTRACT: We report the development of a method to analyze receptor and ?-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged ?2-adrenergic receptor (??-AR) or parathyroid hormone receptor type 1 (PTH?R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged ?-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, ?-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to ?-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting ?-arrestin interaction found with ??-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH?R. 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.

SUBMITTER: Leonard AP

PROVIDER: S-EPMC4169994 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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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.

Leonard Anthony P AP Appleton Kathryn M KM Luttrell Louis M LM Peterson Yuri K YK

Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada 20130128 1

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 ...[more]

PMID: 23351552

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Project description:Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

Dataset Information

A high-content, live-cell, and real-time approach to the quantitation of ligand-induced ?-Arrestin2 and Class A/Class B GPCR mobilization.

Publications

A high-content, live-cell, and real-time approach to the quantitation of ligand-induced β-Arrestin2 and Class A/Class B GPCR mobilization.

Similar Datasets

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