β-Arrestin recruitment

β-arrestin recruitment detailed protocol

Mutants: 

3HA-GHSR1a-C-LargeBiT – Using HiFi DNA assembly we inserted 3HA-GHSR1a (available on cDNA.org: https://www.cdna.org/search.php?mode=search&sort=title&sort_direction=0) into the commercialized pBiT1.2-C vector (Promega: https://www.promega.com/products/protein-interactions/live-cell-protein-interactions/nanobit-ppi-starter-systems/?catNum=N2014) between NheI and EcoRI sites. This vector contain the coding sequence for the LgBiT nanoluc fragment. 

N-smallBiT-β-arrestin-2 – using the same method β-arrestin was inserted 3′ of the coding sequence for the SmlBiT nanoluc fragment in the pBiT2.2-N.

Transfection:

On the first day CHO-K1 are plated into 6 well plates with the goal of obtaining a 80-85% confluency the next day. The plated cells were allowed to adhere and recover overnight in an incubator with moisturized atmosphere, 37°C, and 5% CO2.

Cells were transfected using LipoD293™ (SignaGen: http://signagen.com/In-Vitro-DNA-Transfection-Reagents/SL100668/LipoD293-DNA-In-Vitro-Transfection-Reagent) following the recommended protocol. DNA amounts were as follow:

Assay preparation:

The next day, stimulation media we added 50 µL Optimem with vehicle or a 9X concentration of stimulating peptide in the appropriate wells of a 384 compound plate. The compound plate was then placed in the FLIPR Tetra automated kinetic plate reader (Molecular Devices) along with the tips and the chamber was heated up to 37°C. 

Cells were trypsinized using TrypLE™ express. TrypLE was left on the cells for 50 – 60 seconds at 37^oC, TrypLE was aspirated and the plate was incubated at 37°C for an additional 60 seconds. Cells were then resuspended in 800 µL of warm DMEM-F12 supplemented with 5% FBS and seeded at 20 ml / well of a 384 well plate (white, clear bottom).

The NanoBiT substrate was diluted 1/50 in Opti-MEM™ and 20 ml of substrate was added to each cell-containing well immediately before placing the cell plate in the FLIPR Tetra. At this point each well contains cells mixed with substrate in a 40 ml total volume.

The FLIPR Tetra was programmed to measure luminescence every 2 seconds for 5 minutes; then, inject 5 µL of the stimulating media into each well (either ghrelin 9X or vehicle) and measure the luminescence for every 2 seconds for 15 minutes after injection.

Data processing:

In order to calculate the translocation of b-arrestin, the average luminescence value of the first 110 points of baseline acquisition was subtracted from the entire kinetic for each well. The highest value for each set was denoted 100% and used to normalize the data for each condition.