AlphaScreen SureFire Phospho-ERK 1/2 assay is proven effective in G Protein-Coupled Receptor screening.

Generic readout for GPCR activation
Can be used with endogenous receptors, including primary cells
Homogeneous, no wash steps

G protein-coupled receptors (GPCRs) are the largest family of cell membrane bound receptors and are implicated in numerous disease states. Their sheer diversity as signaling proteins has made them the target of the largest group of therapeutic compounds affecting all areas of modern medicine. Subsequently, more than 30% of drugs on the market today are targeted to GPCRs. Current methods employed in GPCR screening measure various cellular signaling events including the measurement of cyclic adenosine monophosphate (cAMP), inositol phosphates (IP₁ and IP₃), and intracellular Ca²⁺ mobilization. As a general rule, cAMP measurement provides useful data when screening receptors are known to couple to G_αi/o or G_αs, whereas Ca²⁺ mobilization or IP₃ formation assays are most useful for measuring activation of G_αq/11-coupled receptors.

In recent years, a key method has been developed to measure GPCR activation through phosphorylation of p42/44 extracellular signal-regulated kinase (ERK 1/2). Evidence has demonstrated that the signaling pathways activated by the stimulation of a wide variety of GPCRs converge at ERK 1/2 despite coupling to different G-protein subclasses. In a screening context, this convergence at ERK 1/2 offers a broad continuum of GPCR functional screens. (Fig. 1)

Fig 1: The major signaling pathways used by GPCRs, demonstrating the potential for ERK 1/2 phosphorylation with each major class.

AlphaScreen SureFire Cellular Assay

PerkinElmer exclusively offers the AlphaScreen SureFire cellular ERK 1/2 assay. This assay is capable of detecting phosphorylation of ERK 1/2 in a cell-based format and allows the detection of not only G_αq/11-coupled receptor activation but other more difficult to detect G_αi/o-coupled receptors and even some G_αs-coupled receptors.

The naturally high intracellular concentration of ERK 1/2 means that no transfections, dye loading, or pre-stimulations are required to perform the assay. This, in turn, allows the generation of cell-based assay data that has a high degree of physiological relevance and allows measurement of ERK 1/2 activation by endogenous GPCRs and/or Receptor Tyrosine Kinases (RTKs) in various cell types, even including primary cells.

The AlphaScreen SureFire ERK 1/2 assay is homogeneous (no wash steps) and is ideal for automation. Not only can it be utilized in lab-scale experiments, it is ideal for high-throughput screens.

The AlphaScreen SureFire cellular ERK 1/2 assay can be performed on cells grown in either 96-, 384-, or 1536-well microplates. Generally, cultured cells are stimulated with the appropriate agonist for an optimal time, usually 5 to 10 minutes, and lysed with the SureFire Lysis buffer. For inhibition experiments, a similar protocol is followed, but the cells are pre-incubated with the inhibitor prior to stimulation. To determine the extent of ERK 1/2 phosphorylation, the lysed samples are activated with the SureFire Activation buffer and incubated with the SureFire Reaction buffer, containing AlphaScreen beads, for two hours at room temperature. The signal in the wells can then be measured using a plate reader capable of AlphaScreen measurements such as EnVision Alpha. There are no wash steps required and a minimum of liquid handling, affording easy automation and reduced handling errors.

Assay Applications

Screening GPCRs via ERK 1/2 has several advantages over time-proven methods such as measurement of second messengers. Most obviously, phosphorylation of ERK 1/2 provides a generic and direct means of measuring physiologically relevant agonist activation of G_αs-, G_αi- and G_αq/11-coupled GPCRs.

The most common method to assay G_αi/o-coupled GPCRs is by measuring the reduction of cAMP levels resulting from the inhibition of forskolin-induced stimulation of adenylate cyclase. This approach is very indirect and often suffers from a very narrow signal window, making the assay unsuitable for HTS. In contrast, many G_αi/o-coupled receptors will directly activate ERK 1/2 through the Ras/Raf pathway, or via transactivation of the EGF receptor. Therefore, the SureFire cellular ERK 1/2 assay offers a simpler and more direct way to measure G_αi/o-coupled receptor activation. (Fig. 2)

Fig. 2: HeLa cells endogenously expressing CXCR4 chemokine G_αi-coupled receptor were stimulated with SDF-1 agonist for 5–10 mins, and phospho-ERK accumulation measured using the AlphaScreen SureFire Phospho-ERK 1/2 assay.

In addition, activation of G_αq/11-coupled receptors will also cause a consistent increase in the phosphorylation of ERK 1/2. Therefore, most or all receptors that can currently be screened using existing technologies, such as Ca²⁺ mobilization or phosphoinositides formation (e.g., IP₁, IP₃), may also be screened using the SureFire cellular ERK 1/2 assay. (Figs. 3 + 4)

Fig. 3: HEK 293 cells stably transfected with neurotensin NT1 G_αq-coupled receptor were stimulated with neurotensin agonist for 5–10 mins, and phospho-ERK accumulation measured using the AlphaScreen SureFire Phospho-ERK 1/2 assay.

Fig.4: COS-7 cells transiently transfected with cholecystokinin B (CCKB) G_αq-coupled receptor were stimulated with sulpho-CCK-8 agonist for 5–10 mins in the absence and presence of 10 μM CR2945 inhibitor, and phospho-ERK accumulation measured using the AlphaScreen SureFire Phospho-ERK 1/2 assay.

G_αs-coupled receptors stimulate adenylate cyclase activity and are readily detected by assays that detect the formation of cAMP. However, certain G_αs-coupled receptors can also give rise to increases in the level of phosphorylated ERK 1/2, although the mechanism of the increase is not always clear.

Taken together, the SureFire cellular ERK 1/2 assay provides a powerful tool for the measurement of activation of many GPCR classes.

Orphan GPCR Screening

An additional benefit of the assay is that it can be used for screening orphan receptors when performed with a cell line that offers low levels of endogenous GPCR agonist-induced ERK 1/2 phosphorylation, such as HEK 293 cells. (Fig. 5)

Effectively, HEK 293 cells show low levels of endogenous ERK 1/2 phosphorylation in response to common stimuli from GPCR agonists and serum, namely. However, when GPCRs are transfected into HEK 293 cells, a strong agonist-mediated signal is observed. This offers a unique system for GPCR orphan receptor screening, because so few endogenous ligands activate ERK 1/2, and a broad array of transfected receptors can activate the ERK 1/2. Thus, no a priori knowledge about the GPCR coupling mechanism is required per se.

Fig. 5: Orphan receptor screening in HEK 293 cells using ERK 1/2 as an analyte.

Assay Troubleshooting

While the measurement of ERK 1/2 phosphorylation offers a generic screening platform for detection of GPCR activation, not all receptors may behave as expected. For instance, all G_αq/11-coupled receptors tested so far were shown to stimulate ERK 1/2 phosphorylation while the success rate with G_αi/o-coupled receptors is about 70%. This percentage varies as a function of the cell line used for transfection. The mechanism of G_αs-coupled receptor activation of ERK 1/2 remains unclear, as is how widespread the phenomenon is for this family of GPCRs.

The SureFire cellular ERK 1/2 assay detects ERK 1/2 phosphorylation in cells with a sensitivity equivalent to a Western blot. Compared to their G_αi/o or G_αq/11 counterparts, G_αs-coupled receptors may have a lower propensity to couple to ERK 1/2, which may limit the use of SureFire ERK as a screening tool for these particular receptors.

Optimizing Phosphorylated ERK 1/2 Signal

Several factors can affect the level of ERK 1/2 phosphorylation within cells. In most cases, problems caused by low signals, high backgrounds, or signal variability can be remedied by optimizing cell culture procedures.

Low-background levels of phosphorylated ERK 1/2 are crucial to achieving a good, uniform response in cells and high signal to background windows. There are several ways to achieve low backgrounds. Confluent monolayers of cells can promote contact inhibition with consequent reduction in background levels of ERK 1/2 phosphorylation. Removal of serum prior to assaying by cell starvation can lower background levels. However, serum removal is not mandatory if the cells are grown for a few days and are confluent. Furthermore, the formation of a confluent monolayer of cells also has the effect of reducing signal variability.

Other factors that can affect assay performance include stimulation time, which can vary according to the receptor system but is usually maximal for ERK 1/2 phosphorylation between 5 to 10 minutes. The cell line used can also influence the efficiency of G protein-coupling to ERK 1/2; for example, a receptor system that gives a strong signal in CHO cells may not in HEK 293 cells.

Summary

The SureFire cellular ERK 1/2 assay offers a new and generic approach to successfully screen many different receptor types and classes. Importantly, in addition to G_αq/11-coupled receptors and certain G_αs-coupled receptors, it provides an easy and physiologically relevant means of screening G_αi/o-coupled receptors and adds to the tools available for GPCR research performed with a wide variety of cell lines including primary cells.

For optimal assay performance, the EnVision Multilabel Plate Reader with AlphaScreen option enables high throughput across a broad range of applications. EnVision with AlphaScreen can be customized for your throughput needs:

Standard Option for medium-throughput screening of up to 37,000 samples per day
HTS “Turbo” Option for high-throughput screening of almost 100,000 samples per day

Click here to learn more about AlphaScreen SureFire Assays.

References:
Leroy D, Missotten M, Waltzinger C, Matrin T, Scheer A. (2007) G protein-coupled receptor-mediated ERK 1/2 phosphorylation: towards a generic sensor of GPCR activation. J. Recept. Signal Transduct. Res. 27(1):83-97.
Crouch MF, Dyer AR, Sheehan AJ, Osmond RIW. (2006) A new HTS platform for screening GPCRs measuring ERK ½ phosphorylation: the SureFire Cellular ERK assay utilizing PerkinElmers' AlphaScreen technology. Fifth Annual ScreenTech Conference.
Osmond RIW, Dyer AR, Sexton PM, Christopoulos A, Crouch MF. (2006) Effective GPCR Screening using ERK 1/2: Applying SureFire technology to GPCR screening. Genetic Eng. News 26 (1), 29-30.
Crouch MF, Osmond RIW, Dyer AR. (2005) High throughput homogeneous cell-based assays of receptor signaling pathways: a discovery platform for kinase drug discovery. Society for Biomolecular Screening 11^th Annual Conference and Exhibition.
Crouch MF, Osmond RIW, Sheehan AJ, Dyer AR. (2005) High throughput homogeneous cell-based assays of ERK activation: A GPCR discovery platform to replace Ca²⁺. Society for Biomolecular Screening 11^th Annual Conference and Exhibition.
Leroy D, Waltzinger C, Humbert Y, Missotten M, Martin T, Francon B, Nichols A, Lang P, Scheer A. (2005) ERK phosphorylation: A multi-card signal to study the pharmacology of small molecules modulating GPCR activated pathways. Society for Biomolecular Screening 11^th Annual Conference and Exhibition.
Osmond RIW, Sheehan A, Borowicz R, Barnett E, Harvey G, Turner C, Brown A, Crouch MF, Dyer AR. (2005) GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors. J. Biomol. Screen. 10(7):730-737.
Chong MP, Barritt GJ, Crouch MF. (2004) Insulin potentiates EGFR activation and signaling in fibroblasts. Biochem. Biophys. Res. Comm. 322;535-541.

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