Section9:Phospho-ERK Assays

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Phospho-ERK Assays

Introduction

ERK 1/2 (p42/44) is a protein kinase in the MAP kinase family. Measurement of the phosphorylation of ERK 1/2 is widely used because it is a measurable endpoint from many multiple cellular cascades. Activation of ERK1/2 is used to measure the functional outcomes for many signaling pathways, kinase targets, and receptor systems. Our focus will be on p-ERK as a measurement of functional responses for G-protein coupled receptors (GPCRs).

For GPCR targets, a utility of measuring phospho-ERK is its use across multiple receptor classes (Gq, Gi/o, and some Gs). The α-subunit of G proteins can be categorized into different subclasses: Gαi/o, Gαq, and Gαs. These subclasses each trigger a different signaling cascade. Gαs-coupled receptors increase cAMP and Gαi receptors decrease cAMP levels through the stimulation or inhibition of the adenylate cyclase pathway. Gαq coupled receptors are known to work through the activation of the Phospholipase C (PLC) pathway causing increases in intracellular calcium. The advantage of measuring phosphorylated ERK (p-ERK) is that it is a common endpoint despite initialization from different α-subunits and therefore different cascades. Now due to newer technology, there are options for developing cell-based screening assays with high throughput capability.

Measuring p-ERK can provide an alternative read-out for receptors that do not optimally couple through calcium or cAMP pathways and other challenging G-coupled GPCR targets. Detection of p-ERK can also potentially provide some advantages that supplement calcium and cAMP assay results when assessing GPCR drug candidates. Calcium assays require dye loading or cell transfection and time-consuming measurements. They also cannot measure orphan or Gαq-coupled receptors without forcing use of a different pathway via promiscuous or chimeric G proteins.

Overview of Technology

There are several methods currently used to measure p-ERK:

• Western Blot Analysis – This method requires an antibody and can be labor intensive.
• ELISA - Enzyme linked immunosorbent assays require wash steps and often require overnight incubations. However, measurement with an Acumen reader can provide images of cells if this is desirable.
• Meso-Scale Discovery Assays – Electrochemiluminesence-based method providing medium- to high-throughput screening.
• LICOR - Infrared fluorescence-based method providing medium-to high- throughput screening.
• AlphaScreen® SureFire™ ERK Assay – Newer high throughput capability using bead proximity-based AlphaScreen® technology. This method is becoming a popular choice.

AlphaScreen® SureFire™ ERK Assay

General Background

See the following links for more information, please visit http://www.PerkinElmer.com and http://www.TGR-Biosciences.com.

Advantages of the AlphaScreen® SureFire™ ERK Assay

Measurement of phospho-ERK can be done in many ways, but the kit combining Perkin Elmer’s AlphaScreen® technology and TGR BioSciences’ SureFire™ cellular ERK assay is a popular method of measurement. Published references (see Osmond et al.) indicated that ERK 1/2 activation is pharmacologically similar to previously established responses in other assay formats. Here are some advantages to this assay format:

• Primary or cultured cells (adherent or non-adherent)
• Cloned or endogenous receptors (transient and stable)
• Non-radioactive
• Detects agonists, antagonists, or orphans
• Homogeneous assay
• No wash steps
• One day assay
• Automatable for high throughput (384 or 1536)
• AlphaScreen® technology
• Highly sensitive
• Low background
• Broad affinities
• Easy data handling

Sample Protocol

See Perkin Elmer Protocol for AlphaScreen® SureFire™ p-ERK Assay Kits or TGR Biosciences SureFire™ Cellular ERK Assay Protocol for more detailed information. Additional information is also available for one vs. two plate protocols and non-adherent cells.

Suggested Assay plate: PerkinElmer, Proxiplate-384 well plate (half volume), #6006280

Schematic Diagram Outline of the SureFire Cellular ERK Assay

• Plate cells into 96 or 384-well proxiplate
  • 24 hrs at 37oC
• Starve cells with low serum or serum-free medium (optional)
  • time varies based on cell type
• Add inhibitors
  • time may vary
• Add agonist
  • 5-15 min
• Lyse cells
  • 10 min at RT
• Transfer 6ul lysate to assay plate
• Add 10ul reaction mix containing beads
  • 2 hrs at RT
• Read plate

Assay Formats

Agonist Mode:

• Cells are stimulated with agonist and optimal time and temperature for stimulation is determined.
• Max response is maximum p-ERK produced by full agonist stimulation.
• Min response is p-ERK produced in the presence of stimulation buffer without agonist.
• Relative EC50 is obtained from the concentration response curve.

Example: Agonist concentration response curve

1. ERK1/2 activation with cells expressing endogenous GPCRs

   
   

2. Detection of GPCR-induced ERK1/2 activation in transfected cell lines

   
   Antagonist Mode:

   • Cells are pretreated with diluted test antagonist at 37oC for 1-2 hrs.
   • Agonist is added at EC80 concentration and incubated at RT for 15 min.
   • Inhibition of the agonist response is quantified.
   • Max response is maximum p-ERK produced by full agonist stimulation.
   • Min response is p-ERK produced in the presence of stimulation buffer without agonist.
   • Relative IC50 is determined from concentration response curve.

   Example: Antagonist concentration response curve

   
   

   

   
   

   Assay Optimization

   The following conditions should be optimized for best assay performance:

   • Cell titration to minimize the baseline ERK1/2 phosphorylation level and to maximize signal window.
   • Serum starvation parameters (if required). This may be necessary to reduce background.
   • DMSO tolerance study.
   • Time course for compound incubation.
   • Time course for agonist stimulation.
   • Optimization of instrument set-up.

   
   

   Helpful Hints for Performing SureFire AlphaScreen® ERK Assay

   • Cells are grown to confluence in microplate wells prior to assaying for ERK. This is important because contact inhibition significantly lowers the level of background ERK 1/2 phosphorylation, synchronizes the responsiveness of the cells, and maximizes the signal window.
   • Cells should be harvested from flasks for seeding into microplates at approximately 70-90% confluence. Cells should be detached from the flasks using conditions as mild as possible and allowed to adhere to plates for at least 24 hrs prior to the assay.
   • Monitor the cell passage number; determine empirically whether the cells will lose responsiveness at passages beyond a maximum limit.
   • Assay incubation temperature should be at least 22oC.
   • To eliminate “edge effect,” increase reaction volume from 11ul (4ul cell lysate and 7ul reaction mix) to 16ul (6ul cell lysate and 10ul reaction mix).
   • Avoid bubbles in assay wells.
   • Because AlphaScreen® beads are light sensitive, add beads and incubate assay plate in light-proof area.
   • Read the plates using the Envision Alpha Turbo module to avoid an “edge effect” in data consistency.

   
   

   References

   1. 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.
   2. Luttrell, DK and Luttrell, LM. (2003) Signaling in time and space: G protein-coupled receptors and mitogen-activated protein kinases. Assay Drug Dev Tech 1: 327-338.
   3. Luttrell, LM. (2003) G protein-coupled receptor signaling in neuroendocrine systems. ‘Location, location, location’: activation and targeting of MAP kinases by G protein-coupled receptors. J. Molec. Endocrinology 30: 117-126.
   4. Schulte, G and Fredholm, BB. (2003) Signalling from adenosine receptors to mitogen-activated protein kinases. Cellular Signalling 15:813-827.

   
   Web Sites

   1. www.TGR-Biosciences.com
   2. www.PerkinElmer.com