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Sittampalam GS, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.

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Measurement of β-Arrestin Recruitment for GPCR Targets

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Introduction

β-Arrestins are ubiquitously expressed in all cell types, and function in the desensitization of G- protein coupled receptors (GPCRs), the control of GPCR intracellular trafficking, and the activation of GPCRs to multiple signaling pathways (1-4). Therefore, β-arrestin-mediated signaling constitutes an important part of GPCR signaling in addition to G protein-mediated signaling. As many GPCRs are found to recruit β-arrestin, the β-arrestin recruitment assay has found important use in drug discovery, especially in the discovery of ligands for orphan GPCRs and in situations where the second messenger signaling is unknown (5,6). Furthermore, the discovery of biased GPCR ligands and the findings that distinct G-proteins versus β-arrestin signaling preferences may offer therapeutic advantages over conventional ligands imply that a screening campaign should be designed to focus on the most disease relevant pathways (7-10). In this aspect, the β-arrestin recruitment assay has added an important piece to the repertoire of assay tools in drug discovery.

There are four major in vitro assay technologies available on the market that are capable of measuring ligand-induced β-arrestin recruitment: PathHunter β-arrestin Assay (DiscoverX) (11), Tango GPCR Assay (Thermo Fisher Scientific) (12), LinkLight GPCR/ β-arrestin Signaling Pathway Assay (BioInvenu) (13), and Transfluor Assay (Molecular Devices) (14). The PathHunter β-arrestin Assay, Tango GPCR Assay System and LinkLight GPCR/ β-arrestin Signaling Pathway Assays are homogenous, high throughput assays while the Transfluor Assay is a fluorescence image-based assay. All four assays involve the expression of the β-arrestin as a fusion protein with another protein or fragment, while the PathHunter, Tango and LinkLight assays require fusion of the GPCR to another peptide or protein moiety as well. Table 1 compares the principles of these technologies as well as their advantages and limitations.

Table 1. . Comparison of various β-arrestin assay technologies as well as their advantages and limitations.

Table 1.

Comparison of various β-arrestin assay technologies as well as their advantages and limitations.

This guideline uses PathHunter β-arrestin from DiscoverX to illustrate the concepts for performing GPCR β-arrestin recruitment assay. The principle behind this guideline can be applied to all the β-arrestin assay technologies.

Flowchart of Assay Development Guidelines

Image beta_arrestin-Image001

Overview of the β-Arrestin Recruitment Assay

The PathHunter GPCR β-arrestin assay is based on proprietary Enzyme Fragment Complementation technology from DiscoverX. PathHunter β-arrestin GPCR cells are engineered to co-express the ProLink (PK) tagged GPCR and the Enzyme Acceptor (EA) tagged β-arrestin. Activation of the GPCR-PK induces β-arrestin-EA recruitment, facilitating complementation of the two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes the Galacton Star substrate to generate a chemiluminescent signal (Figure 1).

Figure 1. . The GPCR β-arrestin recruitment assay principle.

Figure 1.

The GPCR β-arrestin recruitment assay principle.

PathHunter β-arrestin GPCR cells can be purchased from DiscoverX (https://www.discoverx.com) directly as stable expressing cell lines, or generated using PathHunter parental cells engineered to express β-arrestin1 or β-arrestin2-EA fusion protein. Also, retroviral particles are available to generate the parental cells in various cellular backgrounds. Various types of ProLink vectors can be chosen to create the GPCR construct carrying the ProLink (PK) tagged GPCR. Specific detection reagents can be purchased from DiscoverX.

The use of positive and negative controls is important to define the dynamic range of the assays in each plate and monitor the overall assay quality. Inclusion of one or multiple reference compounds for concentration-response analysis is valuable for assessing the incidence of assay drift and robustness. More details can be found in the AGM chapters HTS Assay Validation (15) and Assay Operations for SAR Support (16). As in other assays, performing a counter-screen assay with an unrelated GPCR in the same technology readout is crucial to ensure on-target interaction. The signal may be subject to interference from compounds that inhibit the β-galactosidase enzymatic activity, or those that inhibit the GPCR β-arrestin complex formation.

Guidelines and Sample Protocol for Agonist Assays

Step 1: Grow cells in tissue culture flasks for 1-3 days in the corresponding cell culture medium.

  • Select a DiscoverX PathHunter cell line expressing the tagged GPCR target of interest.
  • A negative control cell line as a counter screen may be either the DiscoverX β-arrestin parental cell line devoid of the target or a DiscoverX β-arrestin cell line expressing an unrelated GPCR.
  • Make sure the cells are healthy and active in the log phase of growth and are maintained the same way in all experiments, since the growth state of the cells may affect the receptor response.
  • Cell passage number should be tightly controlled based on assay performance statistics. Ideally, to avoid variation in cellular response due to changes in the state of the cells or level of receptor expression, expand the cell culture to the quantities required for the entire screening campaign in one large batch of a single passage, “assay-ready” cryopreserved cells in appropriate aliquot sizes (17). DiscoverX has a suggested cryopreservation method and cryopreservation reagent for its cell lines outlined in the instruction manual. Other cryopreservation methods and reagents are available from various manufacturers of cell culture reagents. These can be tested as well for suitability to identify a method that provides high viability and receptor functionality. Frozen cell aliquots can be stored for some time and recovered as needed during a screen.

Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes to pellet the cells.

  • Use of Trypsin or other enzymatic/non-enzymatic cell dissociation agents should be tested prior to screening. Some receptor proteins may tolerate trypsinization, but other enzymatic reagents such as TrypLE (Thermo Fisher) or non-enzymatic reagents such as Cellstripper (Corning) may be used if the system under study does not tolerate treatment with proteases.

Step 3: Aspirate the cell culture medium and re-suspend the cells in cell plating medium. Count and dilute the cells to the proper densities in cell plating medium.

  • Cells should be dissociated well enough so that no cell clumps are observed during cell counting. For troublesome cell lines, optimize the dissociation condition and filter the cells through an appropriate strainer (e.g. BD Cell Strainer).
  • In most cases, basal medium supplemented with 0.5 to 10% FBS can be used. Sensitivity of the cells and compound activity to serum should be tested.
  • Cell densities should be determined during assay development in cell titration experiments. Generally, a range of 5,000–20,000 cells/well will be suitable for 384-well assay.

Step 4: Dispense cells in plating medium into 384-well microplate and incubate the cells overnight at 37°C at 95% O2/5% CO2.

  • Cells and other reagents can be dispensed by various peristaltic pump-based liquid handlers, such as Thermo Combi-drop or the BioTek Washer Dispenser, or any of those widely available tip-based liquid handlers.
  • Tissue culture-treated or poly-D-lysine plates may be used depending on the cell type.
  • The PathHunter β-arrestin assay may be run as a suspension assay. In that case, the cells diluted in assay buffer can be seeded directly into assay plates and used immediately for assays.

Step 5: Remove plating medium and replace with serum-free or low serum assay medium if needed.

  • At this step, cells can be serum-starved for 4-16 hours depending on application. Serum starvation helps minimize basal signaling, which is especially important if agonists for the GPCR target are present in the serum.

Step 6: Dispense test compounds into the plates.

  • Compounds dissolved in DMSO can be pre-dotted into assay plates in sub-microliter volume using an acoustic dispenser such as the Labcyte Echo, or pre-diluted in an assay buffer prior to addition.
  • Tolerance of the cellular response to DMSO should be tested. Maintaining DMSO concentration under 1% is preferred. An example of DMSO tolerance test is shown in Figure 2.
Figure 2. . DMSO tolerance test.

Figure 2.

DMSO tolerance test. (A) The effect of DMSO on agonist-stimulated response is shown in relationship to the agonist concentration-dependent responses. In this example, DMSO concentrations at 5% or higher reduce the maximal responses by over 50% without (more...)

Step 7: Incubate the cell plates at 37°C for 90 minutes (or other optimized condition).

  • GPCR-β-arrestin interaction is known to exhibit two distinguishable characteristics, known as Class A and Class B interactions. Class A interaction refers to the formation of transient GPCR-β-arrestin complexes which dissociate during receptor trafficking. Class B interaction is more sustained, and the complex co-localizes to subcellular compartments during receptor translocation (18).
  • Incubation time of the assay should be optimized for each GPCR. For some GPCRs, the assay can also be performed at room temperature.
  • With the PathHunter β-arrestin assay, the PK tag on the engineered GPCR may have some degree of intrinsic affinity for the EA fragment. This may influence the GPCR-β-arrestin interaction. A time-course experiment may be performed to assess how this may affect Class A and Class B profiles.

See figure 3 for determination of compound incubation time.

Figure 3. . Determination of compound incubation time in an agonist assay.

Figure 3.

Determination of compound incubation time in an agonist assay. A known agonist is used to assess the time it takes to achieve steady-state cellular response by incubating the agonist for increasing periods of time. In this example, the maximal response (more...)

Step 8: Prepare a solution of PathHunter Detection Reagent (1 part Galacton Star, 5 parts Emerald II Solution and 19 parts PathHunter Cell Assay Buffer according to manufacturer’s instruction).

  • If cells are incubated with compounds at 20 μl of volume, then 10 μl of detection reagent is needed for each well.
  • The detection reagent can be added directly to the compound-treated cells using any of the peristaltic pump-based liquid handlers (e.g., Combi-drop from Thermo Fisher or BioTek Washer Dispenser).
  • If compound interference is suspected, a wash step using DPBS with Ca2+/Mg2+ can be carried out prior to the addition of detection reagent.

Step 9: Incubate for 60 minutes at room temperature and measure chemiluminescent signal using plate readers such as the PerkinElmer EnVision. The output signal is relative light units (RLU). The absolute RLU scale may vary depending on the detector.

The final cell density, temperature and the selection of optimal time point is based on the Z’ and the expected potency (15).

Guidelines and Sample Protocol for Antagonist Assays

Prior to setting up an antagonist-characterization assay, the agonist to be used for stimulating the cells during the antagonist mode should be tested to determine the potency. The EC50-80 of the agonist (determined from a concentration response curve) can be selected for use in the antagonist assay. The assay procedure is similar to the agonist mode with an additional step for agonist stimulation (EC50-80 value) after incubation of cells with test compounds. All test compounds should also be assessed in the agonist mode to examine any agonist activities.

Step 1: Grow cells in tissue culture flasks for 1-3 days in the corresponding cell culture medium.

  • Select a DiscoverX PathHunter cell line expressing the tagged GPCR target of interest.
  • A negative control cell line as a counter screen may be either the DiscoverX β-arrestin parental cell line devoid of the target or a DiscoverX β-arrestin cell line expressing an unrelated GPCR.
  • Make sure the cells are healthy and active in the log phase of growth and are maintained in the same way in all experiments, as the growth state of the cells may affect the receptor response.
  • Cell passage number should be tightly controlled based on assay performance statistics. Ideally, to avoid variation in cellular response due to changes in the state of the cells or level of receptor expression, expand the cell culture to the quantities required for the entire screening campaign in one large batch of a single passage, “assay-ready” cryopreserved cells in appropriate aliquot sizes (17). DiscoverX has a suggested cryopreservation method and cryopreservation reagent for its cell line that is available in the instruction manual. Other cryopreservation methods and reagents are available from various manufacturers of cell culture reagents. These can be tested as well for suitability to identify a method that provides high viability and receptor functionality. Frozen cell aliquots can be stored for some time and recovered as needed during a screen.

Step 2: Dissociate the cells from the flask and centrifuge at 1000 rpm for 5 minutes to pellet the cells.

  • Use of Trypsin or other enzymatic/non-enzymatic cell dissociation agents should be tested prior to screening.
  • Some receptor proteins may tolerate trypsinization, but other enzymatic reagents such as TrypLE (Thermo Fisher) or non-enzymatic reagent such as Cellstripper (Corning) may be used if needed.

Step 3: Aspirate the cell culture medium and re-suspend the cells in cell plating medium. Count and dilute the cells to the proper densities in cell plating medium.

  • Cells should be dissociated well enough so that no cell clumps are observed during cell counting. For troublesome cell lines, optimize the dissociation and filter the cells through an appropriate strainer (e.g. BD Falcon Cell Strainer).
  • In most cases, basal medium supplemented with 0.5 to 10% FBS can be used. Sensitivity of the cells and compound activity to serum should be tested.
  • Cell densities should be determined during assay development in cell titration experiments. Generally, a range of 5,000–20,000 cells/well will be suitable for 384-well assay.

Step 4: Dispense cells in plating medium into 384-well microplate and incubate the cells overnight at 37°C at 95% O2/5% CO2.

  • Tissue culture-treated or poly-D-lysine plates may be used depending on the cell type.
  • The PathHunter β-arrestin assay may be run as a suspension assay. In that case, the cells diluted in assay buffer can be seeded directly into assay plates and used immediately for assays.

Step 5: Remove plating medium and replace with serum-free or low serum assay medium if needed.

  • At this step, cells can be serum-starved for 4-16 hours depending on application. Serum starvation helps minimize basal signaling, which is especially important if agonists for the GPCR target are present in the serum.

Step 6: Dispense test compounds into the plates.

  • Tolerance of the cellular response to DMSO should be tested. Maintaining DMSO concentration under 1% is preferred.

Step 7: Incubate the cell plates at 37°C for 15-30 minutes (or other optimized condition), then dispense the proper amount of the EC50-80 concentration of agonist.

Step 8: Incubate the cell plate at 37°C (or other optimized condition) for the desired length of time according to the time-course experiment.

  • GPCR-β-arrestin interaction is known to exhibit two distinguishable characteristics. Class A interaction is transient, and the GPCR- β-arrestin complex may dissociate during receptor trafficking. Class B interaction is more sustained, and the complex co-localizes during receptor translocation (18).
  • Appropriate incubation time of the assay can be optimized for each GPCR. For some GPCRs, the assay can also be performed at room temperature.
  • With the PathHunter β-arrestin assay, the PK tag on the engineered GPCR may have some degree of intrinsic affinity for the EA fragment. This may influence the GPCR-β-arrestin interaction. Tests may be performed to assess how this may affect the Class A and Class B profiles.

Step 9: Prepare a solution of PathHunter Detection Reagent (1 part Galacton Star, 5 parts Emerald II Solution and 19 parts PathHunter Cell Assay Buffer according to manufacturer’s specification).

  • The detection reagent can be added directly to the compound-treated cells using any of the peristaltic pump-based liquid handlers (e.g., Combidrop from Thermo Fisher or BioTek Washer Dispenser).
  • If compound interference is suspected, a wash step using DPBS with Ca2+/Mg2+ can be carried out prior to addition of detection reagent.

Step 10: Incubate for 60 minutes at RT and measure chemiluminescent signal using plate readers such as EnVision.

The final cell density, temperature and the selection of optimal time point is based on the Z’ and the expected potency (15).

See figure 4 for examples of agonist and antagonist concentration-response curves in GPCR-β-arrestin recruitment assays.

Figure 4. . Examples of agonist and antagonist concentration-response curves in GPCR-β-arrestin recruitment assays in agonist mode (A, C), antagonist mode (B, D) and Schild dose-shift assay (E).

Figure 4.

Examples of agonist and antagonist concentration-response curves in GPCR-β-arrestin recruitment assays in agonist mode (A, C), antagonist mode (B, D) and Schild dose-shift assay (E). Raw data from chemiluminescence readout are illustrated in Panels (more...)

An overall guideline of the assay workflow is shown in the following table.

Assay StepTypical RangeInstrumentation
Grow cellsCO2 incubator
Harvest cellsCentrifuge
Plate cells5,000 – 20,000 cells per well (384w)Combi-drop (Thermo Fisher), BioTek Washer Dispenser, or tip-based liquid handler
Dispense compounds0.1 nM – 10 μMEcho, ATS Gen5, or tip-based liquid handler
Incubate cellsRoom temperature or 37°CCO2 incubator
Dispense agonist (for antagonist assay)EC50 –EC80Combi-drop (Thermo Fisher), BioTek Washer Dispenser, or tip-based liquid handler
Wash cells with PBS (if compound interference is suspected)BioTek Washer, BlueWasher (BlueCatBio), or manually inverted the plate to remove the liquid
Stop reaction by addition of lysis and detection reagentsCombi-drop (Thermo Fisher), BioTek Washer Dispenser, or tip-based liquid handler
Incubate detection reagentsRoom temperature
Measure chemiluminescenceEnvision, or other luminometer

Troubleshooting

Key assay optimization parameters and troubleshooting guidelines are available in Table 2.

Table 2.

Table 2.

Key assay optimization parameters and troubleshooting guidelines

Acknowledgements

The authors would like to thank Nathan Cheadle, Jie Pan and Melissa Yarde for the review of this guidance.

References

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All Assay Guidance Manual content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported license (CC BY-NC-SA 3.0), which permits copying, distribution, transmission, and adaptation of the work, provided the original work is properly cited and not used for commercial purposes. Any altered, transformed, or adapted form of the work may only be distributed under the same or similar license to this one.

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