Section9:β-Arrestin Recruitment

From Assay Guidance Wiki

β-Arrestin Recruitment

Introduction

β-arrestins are multifunctional intracellular proteins that interact with a structurally diverse group of cell surface receptors including G protein-coupled receptors, to regulate cellular functions. Activated GPCR’s are phosphorylated by G protein-coupled receptor kinases (GRK’s), resulting in recruitment of β-arrestin to the cell surface where it binds to the receptor, leading to desensitization and targeting of the receptor for internalization. In addition to its role in receptor desensitization, termination of G protein-coupled signaling, and internalization, β-arrestin also acts as a scaffolding protein to link activated GPCR’s to additional intracellular signaling pathways such as activation of c-Src, ERK 1/2 and Akt in a G protein-independent manner. The binding of β-arrestin to the GPCR is proximal to receptor activation and occurs with both G protein-dependent (regardless of the type of G protein subunit coupling), and G protein-independent signaling. Detection of the interaction between activated receptor and β-arrestin therefore offers the opportunity for a universal GPCR signaling assay format.

Advantages of measuring β-arrestin as output for receptor activation:

• Universal assay format
• Proximal event to receptor activation (like GTP binding)
• Gain of signal format for Gi coupled receptors
• May reveal agonists, partial agonists and inverse agonists of G protein independent signaling that would be missed using assays that measure effectors of G-protein coupled signaling (cAMP, Ca++, IP3, etc).

Overview of Technology

There are several methods to measure β-arrestin recruitment, including:

• TransFluor® Assay (Molecular Devices). In this technology, β-arrestin is coupled to GFP and upon receptor activation, diffuse cytoplasmic distribution of GFP changes to formation of GFP containing pits or vesicles that are visualized with high content imaging systems.
• Tango™ GPCR Assay System (Invitrogen). A transcription factor is attached at the C-terminal tail of the receptor with a protease cleavage site. Cells are transfected with tagged receptor, protease-tagged β-arrestin and a beta-lactamase reporter construct under the control of the transcription factor. After activation of the GPCR, the protease tagged β-arrestin is recruited to the receptor where it cleaves the transcription factor from the GPCR, activating the beta-lactamase and producing a signal from the substrate.
• Bioluminescence Resonance Energy Transfer (BRET)
• PathHunter™ Enzyme Fragment Complementation (DiscoveRx, see below).

Pathhunter™ β-Arrestin Assay (DiscoveRx)

General Background

The principle of this technology is the conversion of β-arrestin translocation into an easily quantifiable β-galactosidase enzyme fragment complementation assay. A GPCR is tagged at the C-terminus with a small (4 kDa) fragment of the β-galactosidase enzyme. The receptor is expressed in a proprietary cell line that stably expresses β-arrestin tagged with the larger fragment of β-galactosidase. Upon activation of the GPCR, labeled β-arrestin is recruited to the plasma membrane to bind to the activated GPCR, and the two parts of β-galactosidase are brought into proximity. This results in cleavage of the substrate by fully complemented β-galactosidase to generate a chemiluminescent signal.

Advantages of the DiscoveRx PathHunter™ β-Arrestin Assay

• Homogeneous format
• Enzyme amplified signal with very robust signal
• Specificity for GPCR of interest because of tag
• ProLink- β-arrestin complex is reversible, so inverse agonist activity is measurable. (Overnight compound incubation may be required.)
• Can be multiplexed with no-wash calcium assay in the same well.
• Chemiluminescent signal results in low background and lack of interference by fluorescent compounds.
• Plates are read on standard luminescence plate readers.

Sample Protocol

See DiscoveRx PathHunter™ β-Arrestin Assay Development Guide for more detailed information.

1. Subclone GPCR into ProLink Cloning vector or purchase prepared GPCR expression vector from DiscoveRx.
2. Transfect PathHunter EA-Arrestin Parental cells with the GPCR-ProLink construct and generate pools followed by clone selection (or purchase stable pools or clones).
   Reprinted from DiscoveRx with permission
3. Test cells for functional activity
   1. Plate cells in culture media with serum or serum free media (Opti-MEM). During assay optimization, cells should be tested in the presence or absence of serum to determine which condition provides the best results for your receptor. Suggested plates (white, clear-bottom):
      • 96-well plates: Costar #3610
      • 384-well plates: Costar #3707
      • Note: for HEK cells, Poly- D-Lysine white, clear-bottom may be used: BD # 356651 (96-well) or #356660 (384-well).
   2. Suggested cell densities:
      • 96-well: 20,000-30,000 cells/well (100 μl/well)
      • 384-well: 5,000-10,000 cells/well (20 µl/well)
   3. Incubate plate overnight at 37°C, 5% CO2.
   4. Replace media with fresh media before assay (optional). Allow for volume of compound/agonist addition so that stimulation volume is 20 µl (384-well) or 100 µl (96-well).
   5. Add compounds at 10X. Do not exceed 1% DMSO final concentration.
   6. Add 10X agonist to wells.
   7. Incubate 90 minutes at 37°C.
   8. Prepare working solution of PathHunter Detection Reagent: combine 1 part Galacton Star Substrate with 5 parts Emerald II Solution and 19 parts PathHunter Cell Assay Buffer.
   9. Add 12 µl/well (384-well) or 50 µl/well (96-well) of prepared Detection Reagent to wells and incubate 60 min at room temperature.
   10. Read chemiluminescence for 1 sec/well for PMT based instrument (MicroBeta, Envision,) or 5-20 sec/well on imaging instrument (ViewLux, LeadSeeker).

Assay Formats

Agonist Mode:

• Cells are stimulated with agonist for optimum time and increase in chemiluminescence is measured.
• Max response is maximum RLU produced by full agonist stimulation.
• Min response is RLU in the presence of assay buffer and the absence of agonist.
• Relative EC50 and Relative Efficacy (% maximum activity of a test compound relative to the reference agonist.) may be determined from concentration response curve.

Example: Agonist concentration response curves

Antagonist Mode:

• Cells are pre-incubated with test antagonist compound for approximately 15 minutes. Note - DMSO should be < 1% final.
• Agonist is then added at approximately EC80 concentration and incubated for optimum time.
• Inhibition of the agonist response is quantified.
• Max response is RLU produced by EC80 concentration of agonist in the absence of compound.
• Min response is RLU produced in the presence of assay buffer and the absence of agonist or test compound.
• Relative IC50 may be determined from concentration response curve and used to calculate antagonist Kb.

Example: Antagonist concentration response curve

Assay Optimization

The following parameters should be optimized to maximize the signal window and reduce variability:

• Cell number
• Assay in the presence or absence of serum
• Time course for agonist stimulation
• Flash vs glow detection reagents
• Plate types including poly-d-lysine and non-binding surface
• Solubility of compounds

Helpful Hints for Performing the PathHunter™ β-Arrestin Assay

• Allowing cell plates to rest for at least 15 minutes at room temperature before placing in the incubator for overnight incubation allows cells to settle evenly to the bottom of the well and improves variability.
• Evaporation control: Media evaporation during overnight incubation can lead to “edge effects” due to effects on cell growth. Use of MicroClime™ Environmental Lids (Labcyte Inc) or BREATHseal™ (Greiner Bio-One) is recommended on cell plates to reduce evaporation.

References

1. Englen RM (2007). Assessing GPCR activation using protein complementation: a novel technique for HTS. Biochem Soc Trans 35:746-748.
2. Lefkowitz RJ and Shenoy SK (2005). Transduction of receptor signals by beta-arrestins. Science 308: 512 - 517.
3. Olson KR and Eglen RM (2007). β galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay and Drug Development Technologies 5:137-143.
4. Yan YX et al. (2002) Cell-based high throughput screening assay system for monitoring G protein-coupled receptor activation using β-galactosidase enzyme complementation technology. Journal of Biomolecular Screening 7:451-459
5. PathHunter™ β-Arrestin GPCR Assay Development Guide. DiscoveRx Corporation.