Biochemistry
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Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay
Chapters
Summary March 10th, 2020
Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient β-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.
Transcript
The Presto-Tango platform was devised to execute the parallel and simultaneous interrogation of all non-olfactory GPCRs in the human genome, including that orphan targets to profile ligand-induced beta-arrestin 2 recruitment. The Presto-Tango platform is the only open source resource for profiling the entire GPCR genome in a parallel approach, using a G protein independent beta-arrestin recruitment assay. Demonstrating the processor would be Genevieve Laroche a Research Associate in my lab, and Manel Zighal, a PhD candidate in my lab.
Before beginning the procedure add 20 microliters of a 25 microgram per milliliter poly-L-lysine solution to each well of the appropriate experimental number of 384 optical bottom plates and incubate the plates at room temperature for 30 minutes to two hours. At the end of the incubation, flick the plates over the sink to remove the excess poly-L-lysine and add 40 microliters of antibiotic antimycotic solution to each well. Then incubate the plates needed for cell seeding at 37 degrees Celsius and place the rest of the plates at four degrees Celsius for long term storage.
To seed HTLA cells for primary screening, gently rinse confluent 150 millimeters cell cultures with 10 milliliters of PBS before treating the cultures with six milliliters of 0.05%trypsin EDTA per dish. When the cells have detached pool the cell suspensions in a 50 milliliter conical tube containing an equal volume of DMEM. And collect the cells by centrifugation.
Re-suspend the pellet at a 2.2 times 10 to the fifth cells per milliliter of DMEM concentration and flick the plates over the sink to remove the antibiotic antimycotic solution. Tap the plates to dry and seed 45 microliters of cells into each well. Then place the plates at 37 degrees Celsius and 45%carbon dioxide overnight.
To prepare the 384 well DNA source plate for transfection distribute 50 nanograms per milliliter of each plasmid complimentary DNA encoding the GPRC Tango construct of interest in 0.1X Tris-EDTA per well into each well of a 96 well plate. Use a multi-channel pipette to manually transfer 10 microliters of DNA solution from each well of the 96 well plate to individual wells of one 384 well DNA source plate in quadruplicate for each condition as illustrated. Next, transfer 40 microliters of a 0.313 molar calcium chloride solution to each well of the DNA source plate and gently mix the contents of each well.
Add 50 microliters of 2x HEPES buffer to each well of the 384 well DNA source plate with gentle mixing. After one minute transfer 10 microliters of the DNA transfection mixture from the 384 well DNA source plate to each well of seeded HTLA cells and incubate the cells in the cell culture incubator overnight. The next day decant the transfected cell medium and slowly add 40 microliters of starving medium to each well taking care not to touch the cells directly.
Then add 20 microliters of the vehicle buffer for the alternating rows without compound in 20 microliters of the compound of interest at a three times concentration into the alternating rows, before returning the plate to the cell culture incubator. 16 to 24 hours following the stimulation decant the transfected cell medium and add 20 microliters of freshly prepared glow reagent to each well for a five to 20 minute incubation at room temperature protected from light. Then read the plates on a micro-plate luminescence counter with an integration time of one second per well.
For a secondary screening, subculture the HTLA cells in 100 millimeter dishes at a five times 10 to the six cells total cell density in 11 millimeters of complete medium per dish for 24 hours at 37 degrees Celsius. The next day, mixed 10 micrograms of GPCR complimentary DNA with 500 microliters of Tris-EDTA calcium chloride solution with vortexing followed by the addition of 500 microliters of HEPES buffer. After vigorous shaking, incubate the solution for one minute at room temperature and immediately add one milliliter of the solution drop wise onto the cells.
Gently rock to evenly distribute the precipitate and place the plate at 37 degrees Celsius for 24 hours. The next day, check the transfection efficiency under a fluorescent cell imager. Transactions greater than 50%coverage are ideal.
Gently rinse the transfected cells with versene solution before detaching the cells with three milliliters of 0.05%trypsin EDTA. Collect the dissociated cells by centrifugation and re-suspend the pellet at a four times 10 to the fifth cells per milliliter of starving medium concentration. Then seed 45 microliters of cells into each well of a poly-L-lysine coded 384 well plate and place the plate in the cell culture incubator for at least four hours.
To prepare a half log dose-curve response plate add 270 microliters of HBSS supplemented with HEPES and antibiotic antimycotic to all but the last row of a 96 well plate and add 30 microliters of each high and low drug solution to the wells of row H.Transfer the solution from each well of row H into the corresponding well of row G with mixing and continue to serially dilute the drug compounds until the last row of the plate is reached. Using the schematic as a reference, mix 20 microliters of the low column dilutions from rows A through G of the 96 well plate and 20 microliters of the high column dilutions to wells B through H of the 96 well plate to the previously seated 384 well plate. Then incubate the plate at 37 degrees Celsius for a minimum of 16 hours.
16 to 24 hours following the stimulation decant the transfected cell medium and add 20 microliters of glow reagent to each well for a five to 20 minute incubation before reading the plates on a micro plate luminescence counter with an integration time of one second per well. In this representative experiment, out of the 168 GPCRs that were interrogated in the primary screening, only dopamine receptor D3 and opsin 5 were contenders as potential active targets. Dopamine receptor D3 produced a significant log2 fold change of 4.7 whereas opsin 5 produced a slightly lower response of 2.39.
By comparison, dopamine receptor D2, the positive control for the primary screen produced a log2 fold change of 4.58. Those response curves from the secondary screening demonstrated that chromatin granule extract produce similar signal windows in half maximal effective concentration values to quinpirole confirming its validity as an active hit at dopamine receptor D3.A flat dose-curve similar to the negative control was produced for opsin 5 however ruling out this receptor as a possible target for the chromatin granule extract. Presto-Tango requires special care as variabilities in cell distribution, transfection efficiency and serial drug dilutions can result in compounded error, which can affect the accuracy of the data.
Orthogonal methods such as Radioligand-binding approaches, Bioluminescence Resonance Energy Transfer and Cyclic AMP calcium and internalization functional assays can be used to further confirm and characterize ligand receptor interactions.
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