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
Highlights
The activation of mitogen-activated protein kinases (MAPKs) constitutes one of the major intracellular signaling pathways that couples signals from cell surface receptors, such as receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), to gene expression regulation and other intracellular events [1,2,3,4,5,6]
The assay has been validated by studying multiple aspects including kinetics, dose-dependent effects of agonists and inhibitors, phosphorylated versus total ERK1/2, and the implication of heterotrimeric G proteins
PRINCIPLE OF homogenous time-resolved fluorescence (HTRF)®-BASED PHOSPHO-ERK1/2 ASSAY (PHOSPHO-the extracellular signal-regulated kinase (ERK) ASSAY) Phospho-ERK is an in vitro assay developed to assess the phosphorylation of ERK1/2, a fingerprint of the MAP kinase signaling pathway promoted by a variety of cell surface receptor families such as GPCRs and RTKs
Summary
The activation of mitogen-activated protein kinases (MAPKs) constitutes one of the major intracellular signaling pathways that couples signals from cell surface receptors, such as receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), to gene expression regulation and other intracellular events [1,2,3,4,5,6]. The Phospho-ERK assay has been validated using the two major cell surface receptor families, RTKs and GPCRs, where both agonist-induced ERK1/2 phosphorylation and inhibition by receptor-selective inhibitors/antagonists as well as the comparison with the classical western blot technique were investigated in cell lines endogenously, transiently, or stably expressing the different receptors.
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