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Front Endocrinol (Lausanne). 2014 Jun 23;5:94. doi: 10.3389/fendo.2014.00094. eCollection 2014.

Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.

Author information

1
Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia , Nedlands, WA , Australia.
2
Cisbio Bioassays , Codolet , France.
3
Department of Molecular Pharmacology, CNRS UMR5203, INSERM U661, Institute of Functional Genomics, Universities Montpellier 1 & 2 , Montpellier , France.

Abstract

The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose-response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z'-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling.

KEYWORDS:

EGFR; ERK1/2; GPCR; HTRF®; RTK

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