Coelenterazine h

For studies with heterologously expressed G2A, HEK-293 or COS-1 cells were used. HEK-293 cells were seeded on coverslips and transfected with plasmid containing cDNA for G2A or the empty vector (mock; both obtained from cDNA.org) at a concentration of 1–2 µg DNA/coverslip in DMEM medium containing 10% FCS, 1% penicillin, 1% streptomycin (Invitrogen, Carlsbad, CA, USA) using Turbofect reagent (Thermo Fisher) and following the manufacturer’s instructions. Forty-eight hours later, cells were loaded with FURA-2 (Biotium) for 45 min and measured in calcium imaging experiments as described above.
COS-1 were seeded in white walls-clear bottom 96-well plates and transfected with a plasmid encoding a calcium-sensitive bioluminescent fusion protein between aequorin and GFP^{52 (link)} together with plasmid containing cDNA for G2A or control DNA (empty vector, mock) at a concentration of 50 ng/well by using FuGENE 6 reagent (Promega) following manufacturer’s instructions. Forty-eight hours later, cells were loaded with 5 µM coelenterazine h (Invitrogen) in HBSS containing 1.8 mM CaCl₂ and 10 mM glucose for 2 h at 37 °C. Measurements were performed by using a luminometric plate reader (Flexstation 3). The area under each calcium transient was calculated by using SoftMaxPro software and expressed as area under the curve (AUC)^{53 (link)}.

HEK293 cells were co-transfected with a vector encoding for mouse ASC tagged with Luciferase (C-terminus) or YFP (C-terminus; Martín-Sánchez et al., 2016b (link)). After 24 h, transfected cells were seeded on a poly-L-lysine-coated white 96-well plate the day before the assay. The BRET signal was read 5 min after the addition of coelenterazine-h (5 μM; Invitrogen). Luminescence was detected at 37°C in a Synergy Mx plate reader (Biotek) using two filters for emission at 485 ± 20 and 528 ± 20 nm. The BRET ratio was calculated as the difference between the 528 and 485 nm emission ratio of R-Luc-ASC and YFP-ASC divided by the difference between the 528 and 485 nm emission ratio of the R-Luc-ASC protein alone. Results are expressed in milliBRET (mBRET) units normalized to basal signal as previously described (Martín-Sánchez et al., 2016a (link)).

For studies of heterologously expressed receptors, COS-1 cells were seeded in white walls-clear bottom 96-well plates and transfected with plasmid containing cDNA for a calcium-sensitive bioluminescent fusion protein between aequorin and GFP[14 (link)] and plasmids containing the indicated receptors cDNA or control (empty vector, mock) at a concentration of 50 ng/well by using FuGENE 6 reagent (Promega) following manufacturer’s instructions. Forty eight hours later, cells were loaded with 5 μM coelenterazine h (Invitrogen) in HBSS containing 1.8 mM CaCl₂ and 10 mM glucose for 2h at 37°C. Measurements were performed by using a luminometric plate reader (Flexstation 3). The area under each calcium transient was calculated by using SoftMaxPro software and expressed as area under the curve (AUC).

Molecular biology enzymes were obtained from Fermentas (Vilnius, Lithuania) and Stratagene (La Jolla, CA). The cDNA of the human arginine vasopressin receptor 2 was purchased from S&T cDNA Resource Center (Rolla, MO, USA). Cell culture dishes and plates for BRET measurements were purchased from Greiner Bio-One GmbH (Kremsmunster, Austria). Cell culture media, Lipofectamine 2000 and coelenterazine h were purchased from Invitrogen (Carlsbad, CA). Rapamycin was obtained from Merck (Darmstadt, Germany). HEK293 cells were from American Type Culture Collection (Manassas, VA).

β-arrestin 2 recruitment was determined as previously described [17 (link),36 (link)]. Briefly, BRET experiments were performed in HEK-293T cells 48 h after transfection with the cDNA corresponding to A_2AR–YFP or A₃R–YFP (0.5 μg cDNA each) and 1 μg cDNA corresponding to β-arrestin 2-Rluc. Cells (20 μg protein) were distributed in 96-well microplates (Corning 3600, white plates with white bottom) and were incubated with antagonists (SCH 442416 for A_2AR and/or PSB-10 for A₃R) for 15 min and stimulated with agonists (CGS 21680 for A_2AR and/or IB-MECA for A₃R) for 10 min prior to the addition of 5 μM coelenterazine H (Molecular Probes, Eugene, OR, USA). One minute after coelenterazine H addition, BRET between β-arrestin 2-Rluc and receptor-YFP was determined and quantified. The readings were collected using a Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany) that allows the integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm. To quantify protein-Rluc expression, luminescence readings were also performed 10 min after adding 5 μM coelenterazine H.

HEK-293 T cells were transiently transfected with a constant amount of cDNA for Rluc fusion proteins and increasing amounts of cDNA for YFP fusion proteins. At 48 h after transfection, 20 μg of cell suspension were plated in 96-well black microplates for fluorescence detection or in 96-well white microplates for BRET readings and Rluc quantification. YFP fluorescence at 530 nm was quantified in a Fluoro Star Optima Fluorimeter as described above. BRET signal was collected 1 min after addition of 5 μM coelenterazine H (Molecular Probes, Eugene, OR, USA) using a Mithras LB 940. The integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm was recorded. To quantify protein-RLuc expression, luminescence readings were also performed after 10 minutes of adding 5 μM coelenterazine H. The net BRET is defined as (long-wavelength emission/short-wavelength emission)–Cf, where Cf corresponds to long-wavelength emission/short-wavelength emission for the donor construct expressed alone in the same experiment. BRET is expressed as milli-BRET units (net BRET × 1000). To calculate maximum BRET (BRET_max) from saturation curves, data were fitted to a nonlinear regression equation, assuming a single-phase saturation curve with GraphPad Prism software (San Diego, CA, USA).

Oxytocin, carbetocin and arginine vasopressin were obtained from Bachem (Bubendorf, Switzerland). Coelenterazine h was obtained from Molecular Probes, Invitrogen (Carlsbad, CA, USA) and coelenterazine 400a (CLz400) was from Biotium (Hayward, CA, USA).