Mohammed Akli Ayoub, Julien Trebaux, Julie Vallaghe, Fabienne Charrier-Savournin, Khaled Al-Hosaini, Arturo Gonzalez Moya, Jean-Philippe Pin, Kevin D. G. Pfleger, Eric Trinquet
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.
Pharmacology, Toxicology and Pharmaceutical Science
9
18%
Chemistry
7
14%
Medicine and Dentistry
2
4%
Other
5
10%
Unknown
4
8%
Attention Score in Context
Attention Score in Context
This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 23 June 2014.
All research outputs
#17,690,023
of 25,932,719 outputs
Outputs from Frontiers in endocrinology
#5,432
of 13,317 outputs
Outputs of similar age
#148,672
of 244,638 outputs
Outputs of similar age from Frontiers in endocrinology
#36
of 53 outputs
Altmetric has tracked 25,932,719 research outputs across all sources so far. This one is in the 21st percentile – i.e., 21% of other outputs scored the same or lower than it.
So far Altmetric has tracked 13,317 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 5.1. This one has gotten more attention than average, scoring higher than 55% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 244,638 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 30th percentile – i.e., 30% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 53 others from the same source and published within six weeks on either side of this one. This one is in the 28th percentile – i.e., 28% of its contemporaries scored the same or lower than it.
