Cabergoline was purchased from Abcam (Cambridge, UK). 5′-N-Ethylcarboxamidoadenosine was obtained from Sigma (Darmstadt, Germany). Isoproterenol (hydrochloride) was from Cayman (Ann Arbor, Michigan, USA). Chlorpromazine (hydrochloride) and MBX2982 was obtained from Medchemexpress (Monmouth Junction, NJ, USA). Neuromedin U-25 was purchased from Phoenix pharmaceuticals (Burlingame, CA, USA). α-MSH was obtained from APExBIO (Houston, TX, USA).
As described previously , the coding region of human arrestin 2 was amplified with 5′ Xbal I and 3′ BamH I sites. TEV NIa protease coding sequence was created by PCR to add a linker at the 5′ end and a hemagglutinin (HA) tag and EcoR I restriction site at the 3′ end. The β-arrestin 2-TEV protease fusion gene was inserted into the CD133-CAR-T2A-puro piggyBac transposon vector (gifted from Dr. Zhu ) to replace the CD133-CAR part.
eGFP was generated by PCR to place a Smal I site at the 5′ end and to place a BamH I site at 3′ end. The eGFP fragment was inserted into the TRE-NLS-3XFlag-CAS9-NLS-P2A-hygro piggyBac transposon vector (gifted from Dr. Zhu, unpublished) to replace the NLS-3XFlag-CAS9-NLS part.
All restriction endonucleases used were obtained from New England BioLabs. All constructs were verified by DNA sequencing.
Generation of stable cell lines
HEK-293T and U87-MG cells were obtained from ATCC (Manassas, Virginia, USA). The cell lines were maintained in DMEM (HyClone, Loga, UT, USA) supplemented with 10% FBS (Gemini, West Sacramento, CA, USA) and 1% penicillin/streptomycin (PS, Gibco, Carlsbad, CA, USA). For generation of stable cell lines, 2 × 105 cells were resuspended in 100 μL of Nucleofector™ solution (Lonza, Visp, Switzerland) and transfected with a combination of 1 μg PB-arrestin2-TEV protease-T2A-Puro, 1 μg PB-TRE-eGFP-P2A-hygro and 1 μg Super piggyBac transposase plasmids (System Biosciences, Palo Alto, CA, USA). Electroporation was performed using the DG-130 (HEK 293T) or DS-126 program (U87-MG) on a 4D-Nucleofector (Lonza, Visp, Switzerland). Immediately after transfection, the cells were transferred into 2 mL prewarmed completed medium in a 6 cm dish. After 3 days, drug selection was performed for two weeks with 2 μg/ml puromycin (Invitrogen, San Diego, CA, USA) and 10 μg/ml hygromycin (Invitrogen, San Diego, CA, USA). HEK 293T cells with stable expression of piggyBac-TANGO (HPT cell lines) and U87-MG cells with stable expression of piggyBac-TANGO(UPT cell lines) were maintained in complete DMEM medium supplemented with 2 μg/ml puromycin and 100 μg/ml hygromycin.
Validation of the piggyBac-TANGO assay
For validation of the piggyBac-TANGO assay, HPT and UPT cell lines were plated overnight in DMEM supplemented with 10% FBS in poly-L-Lysine (Sigma, Darmstadt, Germany) precoated 96-well cell culture plates at a density of 6000 to 8000 cells per well. On the following day (day 2), HPT and UPT cells were transfected with the PRESTO-TANGO expression plasmids (Addgene, Watertown, MA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and incubated in a fresh medium with 10% FBS. On day 3, the corresponding GPCR agonists were added to each well and the cells were incubated for another 24 h. A Nikon microscope (Eclipse Ti-E, Nikon, Tokyo, JP) was used to visualize the fluorescence of HPT and UPT cells at indicated magnification.
Split luciferase biosensor cAMP assay for activation of DRD2
The split luciferase complementation assay based on the Glosensor cAMP biosensor technology (Promega, Madison, WI, USA) was performed as described in the PDSP protocol book available online at http://pdspdb.unc.edu/pdspWeb/content/. To determine the DRD2-mediated cAMP production, HEK 293T cells were transfected with DRD2 and the Glosensor cAMP plasmids. After 24 h, the transfected cells were seeded into 96 well plates and maintained in DMEM supplemented with 1% dialyzed FBS. On the day of the assay, the medium was removed and the cells were loaded with 20 μl of 4 mM luciferin in assay buffer for 60 min at 37 °C. Then, cells were incubated for 15 min prior to isoproterenol exposure with indicated concentrations of the compounds prepared in 1 × HBSS (Gibco, Carlsbad, CA, USA) containing 0.1% BSA. The plate was read to determine chemiluminescence. The results were analysed using the GraphPad Prism 6 software (La Jolla, CA, USA).
Copy number quantification
Single HPT cells were sorted into the wells of a 96-well plate and cultured for 2 weeks. Then, single HPT cells were transfected with the ADORA1-TANGO plasmid (Addgene, Watertown, MA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). On the following day, single HPT cells without GFP background were treated with NECA. A clone with GFP response was identified and expanded for quantification of the plasmid copy number. Four clones were seeded in a 12-well plate in triplicate and were harvested after reaching confluence in an alkaline lysis buffer (0.2 M NaOH/ 1 mM EDTA) at 55 °C overnight. Genomic DNA was extracted by phenol-chloroform followed by ethanol precipitation. RT-qPCR was carried out in an ABI ViiA 7 system (Life, Carlsbad, CA, USA) with a ChamQ SYBR qPCR master mix (low ROX premixed) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The forward and reverse primers for eGFP were 5′-ACGACGGCAACTACAAGACC-3′ and 5′-TTGTACTCCAGCTTGTGCCC-3′, respectively. The forward and reverse oligonucleotide primers for GAPDH were 5′-CATCAATGGAAATCCCATCA-3′ and 5′-TTCTCCATGGTGGTGAAGAC-3′, respectively. qPCR was performed with a preincubation cycle at 95 °C for 10 min followed by 40 cycles of amplification (15 s at 95 °C, 60 s at 60 °C, and 15 s at 95 °C). The copy number of eGFP was determined by the standard curves containing wild type HEK 293T genomic DNA (10 ng/μl final concentration) with a serial dilution of a known amounts of the PB-TRE-eGFP-P2A-hygro plasmid as described previously .
For investigation of stability, 4 cloned cell lines were maintained in DMEM supplemented with 10% FBS, 1% PS and 2 μg/ml puromycin. The cells were cultured for up to 7 weeks and genomic DNA was collected every week to determine the plasmid copy number. The plasmid copy number quantification was performed as described in the preceding paragraph.
DRD2-targeted natural product screening
For preliminary screening, the expression plasmid for DRD2-TANGO was transfected into the HPT cells using Lipofectamine 2000. After 24 h, a DRD2 agonist (cabergoline) and natural products were added to the fresh medium and the cells were incubated for another 24 h. Natural product library was obtained from Shanghai R&D Center for Standardization of Chinese Medicines. The final concentrations of cabergoline and the compounds were 10 μM and 25 μM, respectively. The results were visualized using a Nikon microscope at indicated magnification.