Comparison of G-Protein Coupled Receptor Desensitization-Related  β- Arrestin Redistribution Using Confocal and Non-Confocal Imaging

Dorothea      Haasen; Michael      Wolff; Martin   J.   Valler; Ralf      Heilker

Abstract

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we compared a confocal and a non-confocal cellular HCS system, the IN Cell Analyzers1 3000™ and 1000™, respectively. As a cellular model system we used the Transfluor™ 2 technology in the 384-well microtiter plate (MTP) format. The Transfluor HCS assay for G-protein coupled receptor (GPCR) activation is based on the recruitment of a green fluorescent protein-labelled arrestin (ArrGFP) from the cytosol to the plasma membrane. We investigated two GPCRs, the wild-type (wt) β2 adrenergic receptor ( β2AR) and the β2AR-enhanced (E), a C-terminally mutated receptor with a higher affinity to arrestin. Upon agonist stimulation, the β2AR-wt induced the redistribution of ArrGFP to coated pits, the β2AR-E maintained the interaction with ArrGFP down to the formation of endocytic vesicles. Our findings reveal that the assay is feasible on both instruments, with sufficiently robust Z statistics. Improved Z statistics, though, are achieved with the confocal system, particularly in case of weak signals. Moreover, throughput is dramatically higher for the IN Cell Analyzer 3000. We conclude that, depending on the needs for throughput and assay biology, either instrument may fulfil a successful role in the drug discovery process. Confocal optics, however, provide a better basis for the detection of smaller subcellular structures with lower fluorescence intensity.

Keywords: High content screening, confocal imaging, β-arrestin redistribution, subcellular translocation

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