( Alexander C. Zambon 1 Bruc...)Gq-coupled Receptor Assays Using FLEXstation

Alexander C. Zambon¹, Bruce R. Conklin¹, Laurence L. Brunton^2,3, Paul A. Insel^2,3, and Anne T. Ferguson⁴

¹Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94141-9100
Departments of Pharmacology² and Medicine³, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636
⁴Molecular Devices Corp., 1311 Orleans Drive, Sunnyvale, CA 94089

Introduction
G_q-coupled receptors are cell-surface proteins that signal through multiple pathways, resulting in the release of Ca²⁺ from intracellular storage vesicles. They are gaining widespread popularity because they are implicated in critical intra- and intercellular processes. Since abnormal regulation of G_q-coupled receptor signaling pathways can cause various diseases, these receptors are prime targets for drug therapy. For this reason, it is important to study the regulation and specificity of G_q-coupled receptor activity.

Traditionally, G_q-coupled receptor activity is monitored by measuring an increase in intracellular inositol triphosphates (IP₃) and/or calcium ions (Ca²⁺) (Fig. 1). Inositol phosphate measurement using [³H] inositol is labor intensive and requires radioactive waste storage and removal. In comparison, the release of calcium from intracellular stores into the cytoplasm was traditionally monitored in single cells by using fluorescence microscopy image analysis and a calcium-sensitive fluorescent dye. This procedure, while allowing a view of individual cells, is laborious and time-consuming when multiple ligands or drugs are being tested.

This application note describes the use of FLEXstation, a 96-well microplate fluorescence reader with fluid transfer capabilities, to measure the activity of the canine P2Y₁₁ (cP2Y₁₁) G_q-coupled receptor (1). The P2 superfamily of proteins includes the P2X family of ligand-gated ion channels and the P2Y family of G-coupled receptors (2). The P2Y receptors selectively respond to nucleotides and their analogs. To date, six subtypes have been cloned in various organisms, including human (3). All P2Y subtypes couple to phosphoinositide-Ca²⁺ pathway (4). Interestingly, P2Y₁₁ mediates signaling through both phosphoinositide-Ca²⁺ and cAMP pathways (3, 4, 5, Fig. 1). This receptor plays a putative role in differentiation of immunocytes and renal disease (3, 6, 7, 8).

Here, we demonstrate that FLEXstation is an accurate, efficient, and rapid alternative to IP₃ measurement in generating EC₅₀ values for cP2Y₁₁ agonists.

BACKGROUND
Cells. The canine P2Y₁₁ receptor was cloned by rapid amplification of cDNA ends (RACE) from the highly differentiated Madin-Darby canine kidney epithelial cell line MDCK-D1 (1, 9). The cP2Y₁₁ gene was cloned into the retroviral expression vector pLXSN, which was used to transfect PA317 retroviral packing cells. The resulting virus was used to infect the canine thymocyte cell line CF2Th. Infected cells were exposed to 0.5 mg/mL G418 for at least 2 weeks. G418-resistant cells were used in subsequent experiments (1).

Characterization of cP2Y₁₁ by phosphoinositide hydrolysis assay. The receptor was previously characterized, and its agonist selectivity profile is shown below:

Adenosine 5'-(2-O-thio) diphosphate (ADPβS) = 2-methylthioadenosine 5' diphosphate (2MT-ADP)>>ADP>ATP (1). The EC₅₀ value for ADP was approximately 190 M as determined by phosphoinositide hydrolysis assay (1, Fig. 2).

MATERIALS
Characterization of cP2Y₁₁ by Calcium Flux Assay

1. Cells: CF2Th canine thymocyte cells that stably overexpress the canine P2Y₁₁ G_q-coupled receptor (1).

2. Reagents: FLEXstation Calcium Assay Kit (Molecular Devices, cat# R8041; Tel: 1-800-635-5577), ADP (Sigma, cat# A2754; Tel: 1-800-325-3010), probenecid (Sigma, cat# P8761).

3. Tissue culture microplates: 96-well, black-walled, clear-bottomed plates (Costar, cat# 3603; Tel: 1-800-492-1110).

4. Compound plates: 96-well, V-bottom clear polypropylene plates (E&K, cat# 651201; Tel: 408-378-2013).

5. Pipet tips: For FLEXstation: 200 L nonsterile, clear polypropylene tips (Molecular Devices, cat# 9000-0623).

6. Medium and buffers: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum as previously described (9), trypsin-EDTA solution (Gibco/Invitrogen, cat# 25200-072; Tel: 1800-828-6686), Hank's balanced salt solution (HBSS) (10X) (Gibco, cat# 14065056; Tel: 1-800-828-6686), HEPES 1M buffer solution (Gibco, cat#15630-080).

METHODS
Cell Preparation
CF2Th cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. Cells were seeded the night before the experiment at a concentration of 2.55 x 10⁴ cells/well in a volume of 100 L per well of a 96-well microplate. Cells were incubated at 37 C in 5% CO₂ overnight.

Cell Loading Procedure
1X Reagent Buffer was prepared (1X HBSS/20 mM HEPES, pH 7.4) with the reagents described above. Loading buffer was prepared as described on the Molecular Devices web site (www.moleculardevices.com/pages/ reagent_lit.html).

Reagent Preparation
1. Prepare a fresh stock of 250 mM probenecid. Dissolve 710 mg of probenecid in 5 mL of 1M NaOH and then add 5 mL of 1X Reagent Buffer.

2. Add 1:50 dilution of 250 mM probenecid to the Loading Buffer (final concentration, 5 mM) and 1:100 dilution of 250 mM probenecid to 1X Reagent Buffer (final concentration, 2.5 mM). Probenecid inhibits endogenous efflux pumps that would prevent the dye from accumulating inside the cell.

3. Prepare a fresh stock of 5 mM ADP in 1X Reagent Buffer containing 2.5 mM probenecid. Prepare dilutions of ADP with 1X Reagent Buffer containing 2.5 mM probenecid (see Table 1).

Dye Loading of Cells
1. Add 100 L of Loading Buffer containing 5 mM probenecid to 100 L of cells that were plated the night before (final volume, 200 L/well).

2. Incubate cells for 0.5 h at 37 C.

3. In the meantime, prepare compound plates.

Compound Plate Preparation
Fifty microliters of ADP was added by FLEXstation to the 200 L already in the assay plate well. Since this was a 1/5 dilution, 5X stocks of ADP were made, starting with a 5 mM stock, which was subsequently diluted in series at 1:3 in 1X Reagent Buffer (see Table 1). Dead volume for the V-bottomed compound plates is less than 10 L, so keep this is mind when deciding the volume of compound to add. Each row of the compound plate contained 12 replicates of one concentration of ADP. Row A contained 1X Reagent Buffer alone (negative control), and rows B through H contained threefold dilutions of 1 mM ADP beginning with 1 mM ADP in row B as shown in Table 1.

SOFTmax PROSoftware Set Up
The assay parameters were as follows:

1. Wavelengths: The samples were excited at 485 nm, and emission was detected at 525 nm with a 515 nm cutoff filter.

2. Sensitivity: The number of reads/well was set to 6, and PMT was set to high.

3. Timing: Samples were read at 1.52 second intervals for 120 seconds (total of 79 reads/well).

4. Autocalibrate: On

5. Compound transfer: The pipettor height was set to 220 L to transfer a volume of 50 L at a rate of 4 (corresponds to a rate of 64 L/second). Compound transfer was started after 16 seconds of background reading.

RESULTS
The average EC₅₀ value obtained with the FLEXstation Calcium Assay Kit and the FLEXstation was 47 mM (Figs. 3, 4). The values varied by less than twofold for experiments performed on the same day and by less than fourfold for experiments performed on different days. The range of values from all experiments was between 24 and 87 mM. The coefficient of variation of replicate samples within an experiment varied from 5.6 to 13.8% (average 8.6%) for the 1 mM ADP concentration.

Furthermore, the EC₅₀ value obtained with the fluorescence-based whole cell method (47 M) compared favorably to the EC₅₀ value obtained by measuring [³H]IP (~190 M). Thus, the assay is highly reproducible within and between experiments as well as between assay types (IP and calcium accumulation).

CONCLUSIONS
FLEXstation can be used to assess the specificity and sensitivity of G_q-coupled receptors for different ligands and drugs that are meant to target these receptors. Specifically, FLEXstation and SOFTmax PRO software provide easy experimental set-up, gathering of data and data analysis for development of calcium mobilization assays. Therefore, FLEXstation provides a useful tool for life science and drug discovery laboratories.

REFERENCES
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2. Communi, D., R. Janssens, N. Suarez-Huerta, B. Robaye, and J. M. Boeynaems. 2000. Advances in signalling by extracellular nucleotides. The role and transduction mechanisms of P2Y receptors. Cell Signal. 12: 351360.

3. von Kugelgen, I., and A. Wetter. 2000. Molecular pharmacology of P2Yreceptors. Naunyn Schmiedebergs Arch. Pharmacol. 362: 310323.

4. Communi, D., C. Govaerts, M. Parmentier, and J. M. Boeynaems. 1997. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J. Biol. Chem. 272: 3196931973.

5. Communi, D., B. Robaye, and J. M. Boeynaems. 1999. Pharmacological characterization of the human P2Y11 receptor. Br. J. Pharmacol. 128: 11991206.

6. van der Weyden, L., A. D. Conigrave, and M. B. Morris. 2000. Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor. Immunol. Cell. Biol. 78: 369374.

7. van der Weyden, L., D. J. Adams, and B. J. Morris. 2000. Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. Hypertension 36: 10931098.

8. Communi, D., R. Janssens, B. Robaye, N. Zeelis, and J. M. Boeynaems. 2000. FEBS Letters 475: 3942.

9. Post, S. R., L. C. Rump, A. Zambon, R. J. Hughes, M. D. Buda, J. P. Jacobson, C. C. Kao, and P. A. Insel. 1998. ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/ paracrine pathway to define receptor preference of an agonist. J. Biol. Chem. 273: 2309323097.

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