Evaluating functional ligand-GPCR interactions in cell-based assays

Abstract

G protein-coupled receptors (GPCRs) are a family of transmembrane proteins that act as major mediators of cellular signaling, and are the primary targets for a large portion of clinical therapeutics. Despite their critical role in biology and medicine, a large number of GPCRs are poorly understood, lacking validated ligands or potent synthetic modulators. Ligand-induced GPCR activation can be measured in cell-based assays to test hypotheses about ligand-receptor interactions or to evaluate efficacy of synthetic agonists or antagonists. However, the techniques necessary to develop and implement a cell-based assay to study a given receptor of interest are not commonplace in all laboratories. This chapter outlines methods to develop a cell-based assay to evaluate agonist-induced activation for a GPCR of interest, which can be useful to evaluate the effectiveness of predicted ligands. Examples of sample preparation protocols and data analysis are provided to help researchers from interdisciplinary fields, especially those in fields with relatively little molecular biology or cell culture experience.

1. Introduction

G protein-coupled receptors (GPCRs) are membrane-bound signaling proteins that play critical roles in a wide array of biological processes. At present more than 30% of approved drugs in the United States utilize GPCRs as their primary targets, and many additional receptors represent promising targets for future drug discovery efforts (Hauser et al., 2017). Over 90 Class A GPCRs have been designated “orphan” receptors, a term used to define receptors without known or rigorously validated ligands (Alexander et al., 2020; Laschet et al., 2018). In addition, there are a large number of biologically active compounds (e.g., newly discovered natural products, cell-cell signaling peptides, etc.) without known receptors (Fricker et al., 2018; Muratspahić et al., 2019). Identifying and validating functional ligand-receptor pairings remain important directions both for basic science and for medicine (Bauknecht et al., 2015; Foster et al., 2019; Gomes et al., 2013; Gomes et al., 2016; Quiroga Artigas et al., 2020; Shiraishi et al., 2019). Even for well-established receptors, developing new synthetic modulators and evaluating the effectiveness of these compounds remain critical research efforts.

Once putative ligand-receptor interactions are proposed or new modulators for a target GPCR are developed, a powerful step toward evaluating this prediction is to monitor ligand-induced receptor activation in a recombinant system. The purpose of this chapter is to guide the researcher from an initial hypothesis regarding a functional ligand-GPCR interaction to directly testing the proposed GPCR for ligand-induced activation in cell-based assays. We outline methods to isolate the transcript of interest from tissue, clone this gene into a plasmid for expression in mammalian cells, and evaluate receptor activation in living cells in response to exogenous ligands. With the increasingly interdisciplinary nature of research, many laboratories without strong molecular biology or cell culture backgrounds may be interested in developing and testing ligand analogues for GPCR activation. Inspired by our own such experiences, our goal is to provide guidance to help researchers from a variety of fields to develop the techniques required for running these experiments in their own labs.

2. Overview of the protocol

The goal of this protocol is to develop a cell-based assay to evaluate ligand-induced activation of a GPCR of interest. GPCR activation can be monitored by measuring the intracellular accumulation of second messengers in response to stimulus. Upon activation, most GPCRs signal through a heterotrimeric G protein, which leads to a variety of downstream effects depending on the coupled Gα subunit. This protocol utilizes transient co-expression of the GPCR with a promiscuous Gα_q-family protein that couples to most GPCRs to activate phospholipase C (PLC) (Conklin et al., 1993; Heydorn et al., 2004; Kostenis et al., 2005a; Kostenis et al., 2005b; Offermanns et al., 1995; Wedegaertner et al., 1993). Activation of PLC leads to intracellular accumulation of d-myo-inositol 1-monophosphate (IP1) in the presence of lithium chloride (LiCl). Intracellular IP1 concentration (which is proportional to receptor activation) is measured via commercially available homogenous time-resolved fluorescence (HTRF) assay (Figure 1) (Trinquet et al., 2006). Dose-response experiments are then used to evaluate and compare ligand potencies.

Figure 1.

Graphical overview of the method. (A) The central question answered by the method: for a proposed ligand-GPCR interaction, does the ligand functionally activate the receptor? (B) The present method describes transient co-transfection of the GPCR of interest with a promiscuous Gα_q-family protein in mammalian cells. Upon ligand stimulation, coupling to the Gα_q pathway activates phospholipase C (PLC), which leads to an accumulation of IP1. IP1 is then measured using an HTRF assay.

There are several limitations to the described protocol. The most prominent limitation is that receptor expression is evaluated and optimized via functional activation. For understudied GPCRs, appropriate positive control ligands to verify expression in this manner may not be available. Thus, while positive results can be used to confirm a ligand-receptor interaction, negative results in receptor stimulation experiments could be due to several factors, such as non-optimal recombinant expression in cell lines. As a result, false negatives are possible without additional experiments to verify receptor expression (which are beyond the scope of this protocol). This limitation, and several others, are addressed in more detail in Section 4.

3. Step-by-step methods

3.1. Preparing cDNA library to isolate gene of interest directly from cell or tissue

If the researcher is already confident in the desired receptor sequence they wish to express, this step may be replaced with purchasing the gene of interest as a synthetic oligonucleotide. However, for some systems (especially non-model organisms without well validated genomes or transcriptomes), it may be beneficial to amplify the gene of interest directly from the tissue itself. Amplifying the transcript directly from tissue may reveal mutations or errors in the database-deposited sequence and inform the researcher of the true sequence of the receptor. This section describes a method to isolate mRNA transcripts from cells/tissue and convert this mRNA to a library of cDNA that may be used for subsequent amplification of a desired receptor sequence.

3.1.1. Materials and reagents

All glassware and plasticware used in this section should either be autoclaved or purchased sterile. Note that all aqueous solutions that come in contact with RNA are prepared using commercially available nuclease-free water or treated with diethylpyrocarbonate (DEPC). To inactivate nucleases with DEPC, dilute DEPC to a final concentration of 0.1% in the solution of interest, stir vigorously at room temperature overnight, and autoclave.

• Nuclease-free water or DEPC-treated water.

• TRIzol RNA isolation reagent (ThermoFisher, 15596026).

• Chloroform.

• Aqueous solution containing 0.8 M sodium citrate, 1.2 M NaCl.

• Isopropanol.

• 5 mg/mL glycogen solution (ThermoFisher, AM9510).

• 70% ethanol/water solution.

• RQ1 DNase, RNAse-free (Promega, M6101) or equivalent.

• Zymo RNA clean and concentrator kit (Zymo Research, R1013) or equivalent.

• iScript cDNA synthesis kit (Bio-Rad, 1708890).

• Plastic tissue homogenization pestle (SP Bel-Art, F19923-0000) or equivalent.

• Microcentrifuge tubes.

• Micropipettes and tips capable of accurately dispensing 1–1000 μL.

• Temperature-controlled microcentrifuge capable of 15,000 × g, 4 °C.

• Microvolume UV-Vis Spectrophotometer capable of measuring absorbance from ≤ 3 μL. of sample (e.g., ThermoFisher NanoDrop instrument or Biotek plate reader with Take3 plate).

3.1.2. Before you begin

Regardless of if a researcher purchases a gene or decides to amplify from cDNA, a few steps can be taken before beginning the cloning procedure to maximize chances of success.

1. Ensure gene sequence of interest contains a full open reading frame (ORF), beginning with an ATG start codon and ending with a stop codon.

2. This protocol is designed to test exclusively for GPCR activation. Thus, the protein of interest must be a GPCR to allow monitoring of receptor activation. GPCRs are characterized by seven transmembrane helices, and these regions can often be predicted based on the receptor’s primary amino acid sequence using computational tools. Freely available online tools such as GPCRHMM (https://gpcrhmm.sbc.su.se/) (Wistrand et al., 2006) and the NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.gov/cdd/)(Marchler-Bauer et al., 2017; Marchler-Bauer et al., 2015; Marchler-Bauer et al., 2011) can be used to aid in protein family prediction based on amino acid sequence.

3.1.3. Protocol

• 1.Suspend cell pellet or tissue in 500 μL TRIzol. Samples can be homogenized immediately or stored at −80 °C until homogenization.
• 2.Homogenize tissues with plastic pestle.
• 3.Add an additional 500 μL TRIzol and incubate at room temperature for 5 min.
• 4.Centrifuge suspension at 12,000 × G, 4 °C, 8 min. Transfer the supernatant to clean microcentrifuge tubes.
• 5.To the supernatant from the previous step, add 250 μL chloroform and shake vigorously by hand for several seconds to mix.
• 6.Centrifuge at 12,000 × G, 4 °C, 15 min.
• 7.Remove the tube from the centrifuge, ensuring not to disrupt the resulting phase separation. Carefully transfer the top layer to clean microcentrifuge tubes for use in the next steps. Discard the remaining solution.
• 8.To the top layer from the previous step, add 0.5 volume equivalents of the 0.8 M sodium citrate + 1.2 M NaCl solution (e.g., for 500 μL of sample, add 250 μL of the 0.8 M sodium citrate + 1.2 M NaCl solution), followed by the same volume of isopropanol (e.g., 250 μL in previous example).
• 9.Add 4 μL of 5 mg/mL glycogen solution and incubate at room temperature for 10 min.
• 10.Centrifuge at 15,000 × g, 4 °C, 12 min to pellet RNA.
• 11.Remove supernatant, being careful not to disrupt RNA pellet at the bottom of the tube.
• 12.Resuspend pellet in 1 mL of 70% ethanol/water.
• 13.Centrifuge at 15,000 × g, 4 °C, 12 min.
• 14.Remove supernatant, being careful not to disrupt RNA pellet at the bottom of the tube.
• 15.Allow RNA pellet to dry open to the air for 5-30 min.
• 16.Dissolve pellet in nuclease-free water and treat with RQ1 DNase, according to manufacturer’s instructions.
• 17.Purify RNA using RNA clean and concentrator kit, according to manufacturer’s instructions. Elute RNA with 16 μL of nuclease-free water.
• 18.Determine concentration and quality of eluted RNA using Nanodrop instrument or equivalent.

Note: A₂₆₀/A₂₈₀ ratio should be ≥1.8. If A₂₆₀/A₂₈₀ ratio is <1.8, repurify RNA with RNA clean and concentrator kit.

• 19.Synthesize cDNA using iScript cDNA Synthesis kit, according to manufacturer’s instructions. We have had good success using 2 μg of RNA in a 40 μL reaction. After reverse transcriptase reaction, cDNA is diluted to a total of 100 μL using nuclease-free water and stored at −20 °C until use.

3.2. Cloning receptor sequence into expression plasmid

This section describes one of several possible methods to clone the receptor gene of interest into pcDNA3.1(+) vector for mammalian expression. This method starts with either a synthetic oligonucleotide corresponding to the ORF or a cDNA library containing the gene of interest (as generated in the previous section). All glassware and plasticware used in this section should either be autoclaved or purchased sterile.

3.2.1. Materials and reagents

• Forward and reverse primers designed to amplify gene open reading frame (ORF) and to install restriction enzyme sites on the resulting PCR product (see primer design step below).

• Phusion high-fidelity DNA polymerase (New England Biolabs, E0553S), or equivalent.

• Restriction enzymes corresponding to restriction sites in designed primers. In the example given below, we used BamHI-HF (New England Biolabs, R3136) and NotI-HF (New England Biolabs, R3189S). Other restriction enzymes can be used, depending on their compatibly with the receptor sequence.

• pcDNA3.1(+) mammalian expression vector (ThermoFisher, V79020).

• Shrimp alkaline phosphatase (SAP) (ThermoFisher, 783901000UN).

• Zymo DNA clean and concentrator kit (Zymo Research, D4013).

• Agarose dissolving buffer (Zymo Research, D4001-1-100).

• T4 DNA ligase (New England Biolabs, M0202L).

• Plasmid mini-prep kit (e.g., GeneJET plasmid miniprep kit, ThermoFisher, K0502).

• DH5α competent E. coli cells (ThermoFisher, 18265017).

• LB Broth base (ThermoFisher, 12780052).

• LB agar, powder (ThermoFisher, 22700025).

• Ampicillin sodium salt (Fisher Scientific, BP1760-5).

• Razor blades, or alternative disposable utensil appropriate for excising bands from agarose gel.

• Supplies and equipment to run a 1% agarose gel, and appropriate imager to visualize the results.

• Microcentrifuge tubes.

• Micropipettes and tips capable of accurately dispensing 1–1000 μL.

• Microcentrifuge capable of ≥12,000 × g (or speeds required for chosen DNA clean and concentrator and plasmid mini-prep kits).

• Thermal cycler for PCR amplification.

• Microvolume UV-Vis Spectrophotometer capable of measuring absorbance from ≤ 3 μL of sample (e.g., ThermoFisher NanoDrop instrument or Biotek plate reader with Take3 plate).

• Variable temperature water bath or heat block.

• Incubator/shaker set to 37 °C.

• Optional: S.O.C. medium (ThermoFisher, 15544034).

• Optional: PCR cloning kit (New England Biolabs, E1202S).

• Optional: Plasmid DNA Maxi-prep kit (e.g., E.Z.N.A. Plasmid Maxi kit, Omega Biotek, D6922-02).

3.2.2. Before you begin

1. Prepare 100 mg/mL ampicillin solution (1000×) by dissolving 1 g ampicillin in 10 mL ultrapure water. Sterile filter with 0.22 μm syringe filters. Aliquot and store at −20 °C until use.

2. Prepare LB-agar plates with 100 μg/mL ampicillin.

3. Design and order DNA primers for PCR amplification of receptor ORF and installation of appropriate restriction enzyme sites.

   1. Examine the DNA sequence for gene of interest and identify regions outside of ORF (5’ of the ATG start codon and 3’ of the stop codon) for primer annealing.
   2. Choose a stretch of ~20 nucleotides in each of your chosen regions as targets for designed primers. There are many tools available to help with appropriate primer design (e.g., Primer3), and many different methods to design effective primers. We aim for a T_m of ~60 °C, ~50% GC content, and 1-2 G or C nucleotides on the 3’ end (a “CG clamp”) of each primer.
   3. Choose appropriate restriction enzymes for insertion of gene into pcDNA3.1(+), paying attention to orientation of restriction enzymes relative to CMV promotor (refer to vector map in the pcDNA3.1(+) user manual to determine relative order of restriction enzyme sites). After ligation, the 5’ end of the ORF should be oriented closer to the CMV promotor than the 3’ end. For example, we may choose BamHI to incorporate at the 5’ end via the forward primer and NotI to incorporate at the 3’ end via the reverse primer.
   4. Ensure chosen restriction enzyme sites are not naturally present in ORF, as these will lead to undesired cleavage events during restriction enzyme digestion. If restriction enzyme sites are found within ORF, choose alternative restriction enzymes.
   5. To design final primers, synthesize 5’-CTC-[X]-[Y]-3’ where [X] is the chosen restriction enzyme sequence and [Y] is the chosen sequence from gene of interest. See Figure 2 for an example.

Figure 2.

Example primer design for amplification of receptor open reading frame. (A) Excerpt of the deposited mRNA sequence encoding for the Aplsyia californica allatotropin-related peptide receptor (ATRPR, XM_005106157.3)(Checco et al., 2018a). The predicted ORF is bold and underlined, while the chosen sites for forward and reverse primers are shown italicized and in color. (B) Selected forward and reverse primers for cloning the ATRPR ORF into pcDNA3.1(+) vector. Both primers incorporate a restriction enzyme site (BamHI for the forward primer, and NotI for the reverse primer), depicted in bold and underlined.

3.2.3. Protocol

• 1.PCR amplify the receptor ORF using Phusion high-fidelity DNA polymerase and the designed primers, following instructions provided for the polymerase. This step will amplify your ORF while simultaneously incorporating appropriate restriction enzyme sites into the amplicon.

Note: If starting from synthetic oligonucleotide as template, start with 2 ng template in 50 μL reaction. Analyze product by 1% agarose gel to ensure amplification. PCR conditions may need to be optimized.

Note: If starting from cDNA library as template, amount of cDNA mixture used in reaction will vary based on gene expression level and polymerase. It may be useful to screen multiple conditions initially to identify those that amplify gene of interest. Analyze product by 1% agarose gel to evaluate amplification. PCR conditions may need to be optimized.

• 2.Clean-up amplified PCR product with DNA clean and concentrator kit according to manufacturer’s instructions, eluting with 10 μL of nuclease-free water.
• 3.Determine concentration and quality of eluted PCR product using Nanodrop instrument or equivalent.

Note: A₂₆₀/A₂₈₀ ratio should be ≥1.8. If A₂₆₀/A₂₈₀ ratio is <1.8, repurify with DNA clean and concentrator kit.

Pause point: Purified PCR products can be stored at −20 °C until further use.

• 4.Perform a digest of PCR product using both selected restriction enzymes simultaneously, following manufacturer’s protocol for selected enzymes. We have had success digesting ~300-400 ng PCR product in 60 μL reaction with two restriction enzymes.
• 5.Perform a digest of pcDNA3.1(+) using both selected restriction enzymes simultaneously, following manufacturer’s protocol for selected enzymes. We have had success digesting ~3 μg plasmid in 60 μL reaction with two restriction enzymes.

Note: Steps 4 and 5 may be performed simultaneously if desired.

• 6.Treat double-digested pcDNA3.1(+) vector with shrimp alkaline phosphatase (SAP), according to manufacturer’s instructions.
• 7.
  Purify double-digested PCR product (from Step 4) and double-digested, SAP-treated vector (from Step 6) using 1% agarose gel in the following manner:

  1. Pour 1% agarose gel in wells large enough to hold entire digestion reaction.
  2. Add loading dye to each digest and load into separate wells on gel. If space permits, it is helpful to leave empty lanes between different samples to avoid the possibility of cross-contamination when excising bands.
  3. Run the gel long enough to ensure adequate separation of bands (e.g., 120 V for 50 min for a 7×10 cm gel).
  4. Visualize products under UV light and excise desired bands with razor or similar tool for the vector backbone and the digested PCR product.

Note: When excising bands from gel, cut as close to desired band as feasible. Use a clean blade for excising each band to minimize chances of cross contamination.

• 8.Dissolve excised bands in 3-5 volumes of agarose dissolving buffer, incubating at 37-55 °C until fully dissolved.
• 9.Clean-up amplified PCR product with DNA clean and concentrator kit according to manufacturer’s instructions, eluting with ≥6 μL of nuclease-free water.
• 10.Determine concentration and quality of eluted PCR product using Nanodrop instrument or equivalent.

Note: A₂₆₀/A₂₈₀ ratio should be ≥1.8. If A₂₆₀/A₂₈₀ ratio is <1.8, repurify with DNA clean and concentrator kit.

Pause point: Purified PCR products can be stored at −20 °C until further use.

• 11.Ligate digested insert and vector using T4 DNA ligase, following manufacturer’s instructions. We have had success using a 1:5 ratio of vector to insert (e.g., 0.02 pmol vector and 0.1 pmol insert), incubating for 10 min – 2 h at room temperature, for a 20 μL-scale reaction.

Note: In addition to “Vector+Insert” reaction, it is often helpful to include a “Vector only” control during this and subsequent steps.

• 12.Thaw competent DH5α cells on ice. Proceed with next step immediately after cells are thawed.
• 13.Add 1-5 μL of above ligation reaction mixture to 50 μL aliquot of DH5α cells. Gently tap sides of tube to mix.
• 14.Incubate cell/ligation reaction mixture on ice for 15 min.
• 15.Heat shock DH5α cells by rapidly transferring tube from ice to 42 °C for 30-90 sec (e.g., using water bath or heat block), followed by ice for 2 min.
• 16.Add 200 μL LB (without antibiotics) or S.O.C. medium.
• 17.Incubate at 37 °C for 30-60 min with shaking.
• 18.Plate onto pre-warmed LB-agar plates supplemented with 100 μg/mL ampicillin.

Note: It is often helpful to plate several plates for each ligation reaction (e.g., one plate using 200 μL of the reaction mixture and another plate using 20 μL of the reaction mixture). This maximizes chances of obtaining a plate with appropriate density of colonies to facilitate isolation of a single colony.

• 19.Incubate plates at 37 °C overnight.

Note 01: As noted above, it is helpful to perform this step with a “Vector only” control. This control is expected to show no (or very few) colonies. In contrast, successful ligation reactions should show a number of colonies after incubation. Colony growth in the “Vector only” control could indicate insufficient restriction enzyme digestion or unintended religation. If a large amount of colony growth is observed on “Vector only” control, optimization of restriction enzyme digestion or SAP treatment step is likely needed. In the event of no growth on “Vector+Insert” reactions, optimization of PCR amplification or ligation step is likely necessary.

Note 02: If amplifying reaction from cDNA, a low amount of PCR product may make it difficult to successfully ligate into the pcDNA3.1(+) vector using this method. If repeated attempts of restriction enzyme cloning into pcDNA3.1(+) vector are unsuccessful, an alternative approach is to directly clone PCR amplicons via blunt- or TA-end cloning into pMiniT 2.0 vector using a PCR cloning kit (New England Biolabs, E1202S), following manufacturer’s instructions. Direct cloning of PCR product into this vector will allow for the amplification and purification of large amounts of plasmid containing the gene of interest. The desired ORF can then be subcloned into pcDNA3.1(+).

Pause point: LB-agar plates with DH5α colonies can be stored at 4 °C for a least one week.

• 20.Using a sterile pipette tip, transfer an individual colony from LB-agar plate to 5 mL LB supplemented with 100 μg/mL ampicillin. Repeat for three total individual colonies.
• 21.Incubate 5 mL cultures at 37 °C with shaking overnight.
• 22.After overnight incubation, isolate plasmid using plasmid Miniprep kit, following manufacturer’s instructions.
• 23.After plasmid isolation, determine concentration and quality of eluted plasmid product using Nanodrop instrument or equivalent.

Note: A₂₆₀/A₂₈₀ ratio should be ≥1.8. If A₂₆₀/A₂₈₀ ratio is <1.8, repurify with DNA clean and concentrator kit.

• 24.Submit isolated plasmids for DNA sequencing. For pcDNA3.1(+), sequencing should be done in both forward and reverse directions using T7-promotor and BGH-reverse primers, respectively. Confirm that full insert is positioned at correct location in the vector and that no mutations have been introduced during the cloning process. Ensure complete coverage of the ORF by sequencing reactions. If portions of the middle of the gene are not confidently sequenced, it may be necessary to design a custom primer to sequence this region.

Note: Mini-prep kits can provide up to ~20 μg of plasmid DNA, which is likely enough for DNA sequencing and for initial screens. However, once successful cloning has been confirmed by DNA sequencing, it is recommended to re-transform DH5α cells with the validated plasmid and purify from a large-scale culture (e.g., 200 mL) using a DNA Maxi kit. Performing such a large-scale purification will provide ample material for the experiments described below.

3.3. Testing receptor activation and determining optimal expression conditions

This section will describe how to perform initial tests for ligand-induced receptor activation using cells transiently transfected with both the receptor of interest and a promiscuous Gα_q subunit. Because optimal transfection conditions may differ for each receptor chosen, this protocol aids in optimizing transfection conditions to generate the maximum IP1 response. Optimal transfection conditions determined here will then be used to generate dose-response curves for a given ligand in Section 3.4. Steps 1-21 of this protocol are performed in a biosafety hood and sterile conditions are maintained throughout the experiment. Steps 21-29 of the protocol can be performed on benchtop and under non-sterile conditions. Figure 3 gives a broad timeline of transient transfection, ligand stimulation, and IP1 accumulation measurement steps.

Figure 3.

Timeline for IP1 accumulation assays.

3.3.1. Materials and reagents

• CHO-K1 cell line (ATCC, CCL-61), or similar easily maintained and transfectable cell line.

• Plasmid containing the receptor of interest in pcDNA3.1(+) vector (generated in Section 3.2)

• pcDNA3.1(+) vector with gene encoding for a promiscuous Gα_q-family protein. (Refer to discussion in Section 3.3.2, as well as additional discussion in Section 4.)

• F-12K nutrient medium (ATCC, 30-2004).

• TurboFect Transfection reagent (ThermoFisher, R0531).

• Fetal bovine serum (FBS) (VWR, 97068-085).

• HyClone Penicillin Streptomycin 100× Solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin) (Fisher Scientific, SV30010).

• Opti-MEM reduced serum medium (ThermoFisher, 31985070).

• 0.05% Trypsin solution, with 0.53 mM EDTA in HBSS without calcium, magnesium, or sodium bicarbonate (Fisher Scientific, MT25052CI).

• IP-One Gq kit (Cisbio, 62IPAPEC).

• 75 cm² tissue culture flasks (T75) (Fisher Scientific, FB012937).

• 35 mm diameter tissue culture-treated dishes (Fisher Scientific, FB012920).

• 96-well, cell culture-treated, flat-bottom, half-area, white microplates (Fisher Scientific, 07-200-309).

• Sterile, single-use 0.2 μm vacuum filter units with media bottles (Fisher Scientific, 09-741-02).

• Disposable sterile serological pipettes (for cell culture).

• Micropipettes and tips capable of accurately dispensing 1–1000 μL.

• Hemacytometer (Fisher Scientific, 02-671-6), or alternative method for cell counting.

• HTRF-compatible microplate reader (e.g., Biotek Synergy Neo2). For more information on HTRF-compatible plate readers, see assay guidance for Cisbio’s IP-One Gq kit.

• Optional: Plasmid encoding for expression of green fluorescent protein (GFP) or similar protein in mammalian cells, as a positive control for transfections.

3.3.2. Before you begin

1. Prepare CHO-K1 cell culture medium with antibiotics: F-12K nutrient medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (F-12K + 10% FBS + P/S). Combine solutions under sterile conditions and filter through 0.2 μm vacuum filtration units into media bottles. Store this medium at 4 °C when not in use. Medium should be used within one month of preparation.

2. Prepare CHO-K1 cell culture medium without antibiotics: F-12K nutrient medium supplemented with 10% FBS (F-12K + 10% FBS). Combine solutions under sterile conditions and filter through 0.2 μm vacuum filtration units into media bottles. Store this medium at 4 °C when not in use. Medium should be used within one month of preparation.

3. Reconstitute and aliquot components from the IP-One Gq kit according to manufacturer’s instructions: IPl-d2, anti-IP1-cryptate, and IP1 standard solution. Store these solutions at −20 °C.

4. Choose and purchase/clone a pcDNA3.1(+) vector for expression of a promiscuous Gα_q-family subunit. These Gα proteins associate with a wide variety of GPCRs to facilitate signaling through the PLC pathway. This means that even GPCRs who natively signal through other downstream signaling pathways can be studied by monitoring PLC activation (e.g., through IP1 accumulation, as performed in this protocol). There are several Gα proteins reported that may be useful, including Gα-15/16, Gα_q, and chimeric Gα_q-family proteins with mutations designed to enhance promiscuity (Kostenis et al., 2005b; Offermanns et al., 1995). Our lab uses a Gα_q-derived chimeric protein bearing a G66D mutation (Heydorn et al., 2004; Kostenis et al., 2005a), N- and C-termini from Gα_i proteins (Conklin et al., 1993; Kostenis et al., 2005a), and an internal HA tag (Wedegaertner et al., 1993) (Figure 4).

5. Maintain cultures of CHO-K1 cells in F-12K + 10% FBS + P/S in T75 flasks, incubating at 37 °C, 5% CO₂. Passage cells regularly (every 2-3 days) to avoid reaching overconfluency.

6. Note that the volumes and concentrations of reagents used in the protocol are provided as a starting guide for the researcher. The reader can use different dilutions based upon the need of their experiment and the availability of materials and reagents.

Figure 4.

Primary amino acid sequence of one of several promiscuous Gα_q-family proteins that can be used to couple GPCRs to PLC pathway. Substitutions from natural Gα_q sequence are shown in red, most of which are designed to increase promiscuity (Conklin et al., 1993; Heydorn et al., 2004; Kostenis et al., 2005a; Kostenis et al., 2005b; Offermanns et al., 1995; Wedegaertner et al., 1993).

3.3.3. Protocol

• 1.On the day prior to transfection, passage CHO-K1 cells into 35 mm diameter tissue culture-treated dishes with 2 mL F-12K + 10% FBS + P/S. Incubate at 37 °C in 5% CO₂ overnight to allow cells to attach and recover.

Note 01: Optimal confluence for transfection with TurboFect is 70-90%. Thus, it may be helpful to practice seeding several dishes at different densities to optimize cell density at the time of transfection.

Note 02: The number of dishes prepared will depend on how many different transfection conditions need to be tested (one dish for each transfection condition). For the example shown in Figures 5–7, we prepared six dishes.

Figure 5.

Example 96-well plate map for testing transfection conditions. (A) After transfecting six different dishes of cells with varying conditions, each condition is seeded into a separate column. (B) During ligand stimulation, three wells for each transfection condition are stimulated with high ligand concentration (e.g., 50 μM) in stimulation buffer, while three different wells are treated with stimulation buffer alone (“No ligand”). Empty wells are left on one side of the plate to place the IP1 standards to generate the standard curve.

• 2.The next day, when cells are 70-90% confluent, exchange the medium for 2 mL of F-12K + 10% FBS (no antibiotics).
• 3.Prepare mixtures of plasmid DNA and Turbofect in 200 μL Opti-MEM. We recommend testing different amounts of total plasmid DNA, as well as different DNA/TurboFect ratios.

Note: We generally vary total DNA plasmid from 2-4 μg of total DNA (with 1:1 ratio of receptor plasmid to promiscuous Gα protein) and 4-8 αL of TurboFect. For the example shown in this section (Figures 5–7), six different DNA/TurboFect mixtures were prepared covering these ranges. It is also a good idea to include a “No transfection” control to account for endogenous signaling by ligand on CHO-K1 cells.

• 4.Carefully mix each DNA + Turbofect mixture by slowly pipetting up and down several times.
• 5.Allow the mixtures to sit undisturbed at room temperature for 15 minutes.
• 6.Evenly distribute each DNA + Turbofect mixture in a dropwise manner to one dish of CHO-K1 cells prepared above.
• 7.Incubate the dishes at 37 °C in 5% CO₂ overnight.
• 8.The next day, rinse cells with 2 mL of sterile PBS and detach the cells from the dish using 1 mL of 0.05% trypsin solution.
• 9.Quench the trypsin reaction by adding 2 mL of F-12K + 10% FBS.
• 10.Centrifuge cells at 800 × g, 3 min.
• 11.Aspirate media and resuspend cells in 1 mL of F-12K + 10% FBS.
• 12.Count cell density using hemocytometer.
• 13.Dilute cells to a concentration of 200,000 cells/mL in F-12K + 10% FBS. Ensure thorough mixing of cells by gently pipetting up and down, or with gentle inversion.
• 14.Distribute 100 μL (20,000 cells) into individual wells of an opaque white 96-well half-area tissue culture-treated plates.

Note: An example of a 96-well plate layout is shown in Figure 5.

• 15.Incubate the 96-well plate at 37 °C in 5% CO₂ overnight.
• 16.The next day, prepare a 1× solution of stimulation buffer from the 5× stimulation buffer provided in the IP-One Gq kit.

Note: The amount of 1× stimulation buffer prepared will vary depending upon the number of transfection conditions to be tested.

• 17.Prepare a 2× stock solution of the ligand in 1× stimulation buffer.

For example: In our assay depicted in Figures 5–7, 50 μM was chosen as the concentration of ligand tested, therefore, a 100 μM (2×) stock was prepared in 1× stimulation buffer.

• 18.Remove the 96-well plate seeded with the transfected cells from the incubator and carefully remove the media from each well.

Note: During this step, it is important not to dislodge the cells from the bottom of the well. Using a single-channel pipette, we carefully touch the bottom edge of the well, and slowly remove the media. Use of aspirators can be helpful to speed up the process and reduce labor, but care should be taken to minimize any cell loss during this step.

• 19.Add 14 μL of 1× stimulation buffer to each well.

Note: Ensure that the stimulation buffer is added quickly to each well once the media is removed. Allowing the cells to dry reduces their viability and may affect the final IP1 response observed.

• 20.Add 14 μL of 2× ligand (for stimulated wells) or 1× stimulation buffer (for control wells) to each well. Run each condition at least in duplicate.
• 21.Incubate the 96-well plate at 37 °C in 5% CO₂ for 30–60 minutes.
• 22.During this time, prepare a 1× solution of IP1-d2 and a 1× solution of anti-IP1-cryptate in lysis buffer provided in the kit.

Note 01: It is advised to prepare fresh stocks of IP1-d2 and anti-IP1-cryptate before each experiment.

Note 02: We have found that using the manufacturer’s recommended amount of IP1-d2 and anti-IP1-cryptate (1× each) provides high sensitivity. However, we have also noted comparable overall results using 0.5× IP1-d2 and 0.5× anti-IP1-cryptate (Checco et al., 2018a; Checco et al., 2018b; Do et al., 2018).

• 23.Prepare serial dilutions of IP1 standard (provided in IP-One Gq kit) in 1× stimulation buffer. We recommend using a highest concentration of 11,000 nM with five–six 4-fold dilutions.
• 24.After cells have been stimulated for 30–60 minutes, remove the plate from incubator and cool to room temperature.
• 25.Add 28 μL of each prepared IP1 standard solution into empty wells on the plate.
• 26.Add 6 μL of IP1-d2 in lysis buffer solution to each well (both experimental and those containing IP1 standards). IP1-d2 should be added to each well before anti-IP1-cryptate.
• 27.Add 6 μL anti-IP1-cryptate in lysis buffer solution to each well (both experimental and those containing IP1 standards).
• 28.Cover the prepared 96-well plate with aluminum foil and incubate at room temperature for at least 60 minutes.
• 29.Read the homogeneous time-resolved fluorescence (HTRF) signal of each well on HTRF-compatible plate reader, using an excitation wavelength of 330 nm and measuring fluorescence emission at both 665 nm and 620 nm.

Note: The settings of the plate reader will vary with the instrument. Follow guidelines provided with IP-One Gq kit for chosen plate reader.

3.3.4. Data analysis and interpretation

1. Export HTRF data into appropriate format for data processing and conversion (e.g., Microsoft Excel).

2. Calculate ratio of fluorescence emission at 665 nm/620 nm for each well. This is the “HTRF ratio”.

3. Using a program suitable for non-linear regression analysis (e.g., GraphPad Prism), plot the IP1 standard curve: log[IP1] versus HTRF ratio (Figure 6).

4. Fit the IP1 standard curve to a four-parameter logistical regression model (Eq. 1, Figure 6) (in GraphPad Prism, this is “log(inhibitor) vs. response – variable slope (four parameters)”) to estimate Bottom, Top, Hill coefficient, and IC₅₀ values for the IP1 standard curve.

5. Using the Eq. 2 (Figure 6) and the measured HTRF ratios, calculate the IP1 concentration in each experimental well. These data can be plotted for analysis (Figure 7).

6. A reproducible increase in IP1 concentration indicates that the ligand activates the chosen receptor. In this case, examination of the different transfection conditions tested can guide the researcher to choosing the optimal conditions for future experiments (e.g., conditions that give the greatest dynamic range).

7. A lack of reproducible IP1 accumulation in transfected cells indicates that the ligand may not be a natural agonist of the chosen receptor. However, a lack of response here may also indicate inefficient transfection, a requirement for co-receptors, or that the chosen promiscuous Gα protein does not associate with this receptor. Refer to Section 4 for more discussion.

Figure 6.

An IP1 standard curve is used to relate IP1 concentration to HTRF ratio (665 nm/620 nm fluorescence intensities). (A) An example of an IP1 standard curve, along with associated values after fitting to a 4-parameter logistical model. (B) The equation for the 4-parameter logistical model (Eq. 1). (C) Using an IP1 standard curve together with Eq. 2, the concentration of IP1 in a given experimental well can be calculated (Burford et al., 2017).

Figure 7.

Example data showing the results of a transfection screen. Amounts of receptor plasmid, promiscuous Gα_q-family protein plasmid, and transfection reagent Turbofect are varied. Activation of the GPCR is then evaluated in the presence or absence of a high concentration of predicted ligand (peptide GdFFD-OH), monitoring IP1 accumulation via HTRF assay. Using an IP1 standard curve (as shown in Figure 6), HTRF ratio values can be converted to IP1 concentrations to evaluate the optimal transfection conditions. These data screen conditions for expression of the Aplysia GPCR apALNR (Bauknecht et al., 2015; Checco et al., 2018b) in CHO-G5A cells (Bauknecht et al., 2015). Bars represent the mean ± standard deviation of three replicate wells on the plate.

Note: If the lab is not confident in performing transient transfections, we recommend practicing with a plasmid encoding for GFP or similar protein. One can practice transfection steps using this plasmid and evaluate relative expression rapidly using fluorescence microscopy or a similar method.

3.4. Determining ligand potency through dose-response curves

Once a ligand-receptor pairing has been validated in a single-point assay and optimal transfection conditions have been established (refer to Section 3.3), ligand potency can be assessed via dose-response experiments. Potency measurements (i.e., EC₅₀ values) can then be compared among different ligands. Steps 1-7 in this section are performed in a biosafety hood under sterile conditions. Steps 8-16 can be performed on benchtop and under non-sterile conditions.

3.4.1. Materials and reagents

• CHO-K1 cell line (ATCC, CCL-61), or similar easily maintained and transfectable cell line.

• Plasmid containing receptor of interest in pcDNA3.1(+) vector (generated in Section 3.2).

• pcDNA3.1(+) vector with gene encoding for a promiscuous Gα_q-family protein (Refer to discussion in Section 3.3.2, as well as additional discussion in Section 4).

• F-12K nutrient medium (ATCC, 30-2004).

• TurboFect Transfection reagent (ThermoFisher, R0531).

• Fetal bovine serum (FBS) (VWR, 97068-085).

• HyClone Penicillin Streptomycin 100× Solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin) (Fisher Scientific, SV30010).

• Opti-MEM reduced serum medium (ThermoFisher, 31985070).

• 0.05% Trypsin solution, with 0.53 mM EDTA in HBSS without calcium, magnesium, or sodium bicarbonate (Fisher Scientific, MT25052CI).

• IP-One Gq kit (Cisbio, 62IPAPEC).

• 75 cm² tissue culture flasks (T75) (Fisher Scientific, FB012937).

• 35 mm diameter tissue culture-treated dishes (Fisher Scientific, FB012920).

• 96-well, cell culture-treated, flat-bottom, half-area, white microplates (Fisher Scientific, 07-200-309).

• Sterile, single-use 0.2 μm vacuum filter units with media bottles (Fisher Scientific, 09-741-02).

• Disposable sterile serological pipettes (for cell culture).

• Micropipettes and tips capable of accurately dispensing 1–1000 μL.

• Hemacytometer (Fisher Scientific, 02-671-6), or alternative method for cell counting.

• HTRF-compatible microplate reader (e.g., Biotek Synergy Neo2). For more information on HTRF-compatible plate readers, see assay guidance for Cisbio’s IP-One Gq kit.

3.4.2. Before you begin

1. Prepare media and culture CHO-K1 cells, as described in Section 3.3.2. Determine optimal transfection conditions, as described in Section 3.3.

2. Note that the concentrations and volumes of reagents used in our protocol are provided as a starting guide for the researcher. The reader can use different dilutions based upon the need of their experiment and the availability of materials and reagents.

3.4.3. Protocol

• 1.Follow steps 1–15 of Section 3.3.3 to transfect and plate CHO-K1 cells in advance of the experiment. Transfect cells with optimized DNA + Turbofect ratio identified in Section 3.3. Plate transfected cells (20,000 cells/well) in 96-well half-area plates, as described above, and incubate overnight at 37 °C, 5% CO₂.

Note: If the receptor of interest naturally couples to the PLC pathway through endogenous Gα_q proteins, the promiscuous Gα protein can be omitted during transfections. This can be tested experimentally by comparing transfection conditions with and without co-expression of promiscuous Gα protein.

• 2.The next day, prepare a 1× solution of stimulation buffer from the 5× stimulation buffer provided in the IP-One Gq kit.

Note: The amount of 1× stimulation buffer prepared will vary depending upon the number of conditions to be tested.

• 3.Prepare serial dilutions of ligands at 2× the intended final concentration (for example, if the desired maximum concentration of ligand is 1 μM, prepare a working stock of 2 μM, and prepare serial dilutions from this working stock) in 1× stimulation buffer.

Note 01: To obtain accurate EC₅₀ values, it is important to use a range of ligand concentrations which elicit the entire response range (allowing for accurate fit of “Top” and “Bottom” of the curve). We generally prepare a six-point dose-response curve using 10-fold serial dilutions. However, a researcher may modify this set-up to increase the number of points in the curve or cover an alternative range. It is not recommended to generate dose-response curves with less than 6 points.

Note 02: While preparing serial dilutions, it is important to change pipette tips between each dilution to avoid cross contamination.

• 4.Remove the 96-well plate seeded with the transfected cells from the incubator and carefully remove the media from each well.

Note: During this step, it is important not to dislodge the cells from the bottom of the well. Using a single-channel pipette, we carefully touch the bottom edge of the well, and slowly remove the media. Use of aspirators can be helpful to speed up the process and reduce labor, but care should be taken to minimize any cell loss during this step.

• 5.Add 14 μL of 1× stimulation buffer to each well.

Note: Ensure that the stimulation buffer is added quickly to each well once the media is removed. Allowing the cells to dry reduces their viability and may affect the final IP1 response observed.

• 6.Add 14 μL of each ligand dilution to respective wells in the plate, running each condition at least in duplicate.

Note: A simple example of a plate set-up for testing four different ligands is given in Figure 8. Plate layouts should be modified to test the desired number of ligands.

Figure 8.

Example 96-well plate map for evaluating potency of predicted ligands at GPCR of interest. During ligand stimulation, two replicate wells for each ligand concentration are evaluated at 6-8 different ligand concentrations. Empty wells are left on one side of the plate to place the IP1 standards to generate the standard curve.

• 7.Incubate the 96-well plate at 37 °C in 5% CO₂ for 30–60 minutes.
• 8.During this time, prepare working solutions of IP1-d2 and anti-IP1-cryptate in lysis buffer provided in the kit.

Note 01: It is advised to prepare fresh stocks of IP1-d2 and anti-IP1-cryptate before each experiment.

Note 02: We have found that using the manufacturer’s recommended amount of IP1-d2 and anti-IP1-cryptate (1× each) provides high sensitivity. However, we have also noted comparable overall results using 0.5× IP1-d2 and 0.5× anti-IP1-cryptate (Checco et al., 2018a; Checco et al., 2018b; Do et al., 2018).

• 9.After 30–60 minutes of stimulation, remove the cell plate from incubator.
• 10.Prepare serial dilutions of IP1 standard (provided in IP-One Gq kit) in 1× stimulation buffer. We recommend using a highest concentration of 11,000 nM with five-six 4-fold dilutions.
• 11.After cells have been stimulated for 30–60 minutes, remove the plate from incubator and cool to room temperature.
• 12.Add 28 μL of each prepared IP1 standard solution into empty wells on the plate.

Note: It is critical that an IP1 standard curve is included with every experiment (Burford et al., 2017). 665/620 ratios can differ between experiments. As a result, an IP1 standard curve from an older experiment cannot be used to accurately determine IP1 concentrations for future experiments.

• 13.Add 6 μL of IP1-d2 in lysis buffer solution to each well (both experimental and those containing IP1 standards). IP1-d2 should be added to each well before anti-IP1-cryptate.
• 14.Add 6 μL anti-IP1-cryptate in lysis buffer solution to each well (both experimental and those containing IP1 standards).
• 15.Cover the prepared 96-well plate with aluminum foil and incubate at room temperature for at least 60 minutes.
• 16.Read the homogeneous time-resolved fluorescence (HTRF) signal of each well on HTRF-compatible plate reader, using an excitation wavelength of 330 nm and measuring fluorescence emission at both 665 nm and 620 nm.

Note: The settings of the plate reader will vary with the instrument. Follow guidelines provided with IP-One Gq kit for chosen plate reader.

3.4.4. Data analysis and interpretation

1. Export HTRF data into appropriate format for data processing and conversion (e.g., Microsoft Excel).

2. Calculate ratio of fluorescence emission at 665 nm/620 nm for each well. This is the “HTRF ratio”.

3. Using a program suitable for non-linear regression analysis (e.g., GraphPad Prism), plot the IP1 standard curve: log[IP1] versus HTRF ratio (Figure 9A).

4. Fit the IP1 standard curve to a four-parameter logistical regression model (Eq. 1, Figure 6) (in GraphPad Prism, this is “log(inhibitor) vs. response – variable slope (four parameters)”) to estimate Bottom, Top, Hill coefficient, and IC₅₀ values for the IP1 standard curve.

5. Using Eq. 2 (Figure 6) and the measured HTRF ratios, calculate the IP1 concentration in each experimental well.

6. Plot the log[ligand] versus calculated [IP1] for each well in GraphPad Prism. Fit the resulting data to either a 3-parameter or 4-parameter dose-response model (Figure 9B).

Figure 9.

Example data and analysis for activation of Aplysia ATRPR by one of its endogenous ligands, peptide L-ATRP (Checco et al., 2018a). (A) An IP1 standard curve is generated from the experimental plate and fit to a 4-parameter logistical equation, as in Figure 6. (B) Six 10-fold serial dilutions of ligand are prepared and used to stimulate CHO-K1 cells expressing ATRPR. Raw 665/620 ratio (HTRF ratio) is converted to IP1 concentration using the standard curve and Eq. 2. The resulting dose-response curve for L-ATRP is then plotted and fit to a 3-parameter logistical equation. Data previously reported in Checco et al., 2018a, and replotted here.

Note 01: To calculate an accurate EC₅₀ value, the dose-response curve must have both a clearly defined top and bottom. Thus, ligands that do not reach maximum response should not be modeled to the dose-response model. In these cases, the EC₅₀ values should be reported as > the maximum concentration tested. Ligands that fail to activate the receptor (i.e., show no increase in IP1) should likewise not be fit to the dose-response model.

Note 02: Due to the inherent variability in cell-based assays, it is a good practice to report EC₅₀ values obtained from multiple independent experiments (independent transfections, plating, and ligand stimulation performed on different days). Most often, we report the mean and standard deviation for logEC₅₀ values from three or more independent experiments. Note that one should not calculate the mean or standard deviation of a non-log transformed EC₅₀ value, since EC₅₀ values are not normally distributed. We often then convert the mean logEC₅₀ back to a non-log value for ease of interpretation.

4. Limitations

As mentioned above, the primary limitation for this method is that gene expression is indirectly confirmed via receptor activation. For receptors with known agonists, functional receptor expression is easily monitored using this method. However, for receptors without verified agonist ligands, a lack of signal in receptor activation assays may be due to the ligand of interest not activating the receptor or due to a lack of appropriate receptor expression on the CHO-K1 cells. To address this limitation, Section 3.3 evaluates a variety of transfection conditions, and recommends controls for evaluating transfection technique. Methods to verify gene expression, such as end-point PCR or qPCR may be helpful to determine if the receptor protein is being expressed. However, in practice we have had trouble with PCR-based assays to confirm expression in recombinant systems, as even minute quantities of plasmid contamination after mRNA extraction can yield false positive results. If aiming to verify gene expression by PCR, care must be taken to ensure full digestion of plasmid by DNase treatment prior to reverse transcription reactions. In this case, it is important to incorporate “No-reverse transcriptase” controls to determine if PCR signal may be arising from plasmid contamination. Alternatively, Western blot can be a useful approach to verify protein expression in transiently transfected cell lines. However, antibodies may not be available for all receptors of interest (especially understudied receptors or those from non-model organisms), limiting the generalizability of the Western blot approach. In these cases, fusion of an epitope tag (e.g., FLAG-tag or HA-tag) to the receptor protein may facilitate Western blot analysis in evaluating receptor protein expression.

Another potential limitation is promiscuity of the Gα protein co-transfected in these studies. Although promiscuous Gα proteins have been shown to associate with a large variety of GPCRs (Conklin et al., 1993; Heydorn et al., 2004; Kostenis et al., 2005a; Kostenis et al., 2005b; Offermanns et al., 1995; Wedegaertner et al., 1993) there is no guarantee that a chosen Gα subunit will couple a given GPCR of interest to the PLC pathway. In some cases, it may be beneficial to explore several promiscuous Gα proteins in combination to maximize chances of success. Alternatively, it has been demonstrated that other methods of monitoring receptor function, such as receptor internalization, mass redistribution, and β-arrestin recruitment, can be an effective tool to identifying ligand-receptor interactions, as not all receptors signal through canonical pathways (Foster et al., 2019). Similarly, it is also possible that the GPCR of interest may require co-receptors absent in CHO-K1 cells to signal (Aiyar et al., 1996; Chang et al., 1993; Flühmann et al., 1995). In these cases, it may be beneficial to explore transfection in alternative cell types, or alternative methods of monitoring ligand-receptor interactions (Foster et al., 2019).

5. Summary

This chapter describes our general protocol to develop a new cell-based activity assay for a given GPCR of interest. Steps to isolate the gene of interest from tissue, clone the ORF into an appropriate expression vector, optimize transfection conditions, and generate dose-response experiments are outlined. These methods should allow for evaluation of putative ligand-receptor interactions, and aid laboratories hoping to develop novel GPCR modulators.