Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format

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

Differential Effects of Camel Milk on Insulin Receptor Signaling – Toward Understanding the Insulin-Like Properties of Camel Milk

“…HEK293 cells transiently co-expressing hIR–Rluc8 and Grb2–Venus were used for ERK1/2 and Akt activation using the classical SDS-PAGE and western blotting technique as well as the homogenous time-resolved fluorescence (HTRF ® )-based assay (CisBio Bioassays, Codolet, France) as previously described ( 31 , 32 ), respectively. For SDS-PAGE and western blot, cells were first cultured in 6-well plate and starved overnight in serum-free DMEM.…”

“…The western blot signals were quantified with GeneTools software (release 4.01.02) and expressed in arbitrary units after normalization. In HTRF ® -based assay, the two-plate protocol was used as recently described ( 32 ) after pre-treatment or not with defatted camel milk and stimulation or not with 100 nM insulin at the indicated times. The plate were then incubated for 2 h at room temperature before reading the fluorescence emission at 620 and 665 nm using the appropriate HTRF programs on Mithras LB 943 plate reader.…”

Angiotensin II regulates phosphorylation of actin‐associated proteins in human podocytes

“…Temporal dynamics of phosphorylation events upon GPCR activation are highly complex (31)(32)(33); therefore, we performed time course experiments and demonstrated the phosphorylation of ERK within the first 15 min after AngII stimulation (14). Thus, we chose this time point for our proteomic study.…”

“…C ) Analysis of the phosphorylation site sequence motif, depicted for all peptides with a .1.5 AngII-to-vehicle ratio was performed with PhosphoLogo. D) Venn diagram of all proteins identified in this study (AngII-PhosProt) compared with a non-phospho-enriched proteome characterization of primary murine podocytes (cPodoProt) from Rinschen et al(33). E ) Venn diagram comparing the phosphoproteins that were increased in this study (AngII-PhosProt) compared with phosphoproteins that were increased in a study of AT1R signaling in HEK293 cells (HEK-AT1R) from Christensen et al(34).…”

HTRF® Total and Phospho-YAP (Ser127) Cellular Assays

“…Thus, HTRF® is used to generate signaling assays suitable for HTS (https://www.cisbio.com/drugdiscovery/htrf-technology) [14,15]. Four main advantages can be pointed out: first HTRF® displays a good signal to noise ratio due to its properties explained above; second, HTRF® assays are easy-torun assays, avoiding purification or washing steps, allowing miniaturizing, and thus are ideal for HTS; third, for most of the assays, great specificity is given by a coincidence detector principle; and fourth, such assays are ideally suited to record endogenous signaling events in native systems.…”