Figure 1.
(A) Schematic representation of qBRET experiments (based on [12]): In qBRET experiments, constant amount of energy donor labeled receptor is coexpressed with increasing amount of acceptor labeled receptor. BRET ratio is plotted as a function of acceptor/donor expression ratio (left panel). Theoretically specific interactions result in a saturation curve (red and green), while non-specific interaction shows linear relationship (blue). The absolute value of BRET ratio is not indicative of the dimerization state of the receptors, therefore BRET50 value (acceptor/donor ratio at half-maximal BRET ratio) is used to determine the affinity of receptors to form dimers (which is the same for red and green curve, indicating the same likelihood of dimerization despite the different BRETmax values). To correctly interpret qBRET curves, donor labeled receptor expression has to be maintained constant with increasing acceptor expression (right panel). (B) HEK293 cells were transiently transfected with a constant amount V2R-RLuc (donor) coding plasmid and with increasing amounts of either AT1R-Venus, β2AdR-Venus, CB1R-Venus, V2R-Venus or cytoplasmic Venus (acceptor) coding plasmid. Various amounts of empty pcDNA3.1 plasmid was added to maintain constant total transfected plasmid amount. Total luminescence and Venus fluorescence were measured at the beginning of each experiment, and intensity ratio was calculated as fluorescence/total luminescence. Intensity ratio shows not the absolute acceptor/donor expression ratio (see Methods for further details) but is proportional with it. BRET ratio was calculated as Emission530/Emission485, and was plotted as a function of intensity ratio (left panel). Measured total luminescence was plotted as a function of measured fluorescence for the investigated donor - acceptor pairs (right panel). Curves were fitted using non-linear regression equation assuming a single binding site (GraphPad Prism). n = 3.
Table 1.
Calculated BRETmax and BRET50 values for classical qBRET experiments.
Figure 2.
Monte Carlo simulations with various donor amounts.
(A) Graphical representation of our simulations: two small membrane pieces in the case of non-specific (left panel) and specific (right panel) interaction, ‘d’ and ‘a’ represents donor and acceptor molecules, respectively, while ‘−‘ indicates empty hexagons. (B and C) Numbers of donor and acceptor molecules were varied from 100 to 1000 for each. BRET ratio was calculated after 1000 simulation time steps based on the total number of neighboring donor-acceptor pairs. Simulated BRET ratio was plotted as a function of the number of acceptor molecules (B) or acceptor/donor ratio (C). Simulations were performed with association and dissociation probabilities for non-specific (left panels) and specific (right panels) interactions (Table 2). (D) Simulations performed for non-specific and specific interactions, when total amount of donor and acceptor were fixed (insert). Curves were fitted using either linear regression or non-linear regression equation assuming a single binding site (GraphPad Prism). n = 5, mean +/− SEM.
Table 2.
Simulation parameters: Association and dissociation probabilities used for the simulation of non-specific and specific interactions.
Figure 3.
Rapamycin inducible dimerization with various donor amounts.
(A) Schematic representation of the rapamycin inducible dimerization system: PM2-FKBP-RLuc (left molecule) and Venus-FRB-CAAX (right molecule) show non-specific interaction in the absence of rapamycin (left panel), while the presence of rapamycin (right panel) results the dimerization of FKBP and FRB domains, converting the interaction into specific. (B, C and D) HEK293 cells were transiently transfected with increasing amounts of PM2-FKBP-RLuc and Venus-FRB-CAAX, while empty pcDNA3.1 plasmid was added to maintain total transfected plasmid amount constant. Fluorescence-Luminescence plot (B) shows a wide and independent distribution of acceptor and donor expression. Cells were treated with vehicle (left panels) or 100 nM rapamycin (right panels) 30 minutes prior to measurements. Total luminescence and Venus fluorescence were measured at the beginning of each experiment, and intensity ratio was calculated as fluorescence/total luminescence. BRET ratio was calculated as Emission530/Emission485, and was plotted as a function of measured fluorescence (C) or intensity ratio (D). Measured points were sorted into low/medium/high luminescence groups based on the measured total luminescence (B). Curves were fitted using either linear regression or non-linear regression equation assuming a single binding site (GraphPad Prism). n = 3.
Figure 4.
Dimerization of V2 vasopressin receptor and CaSR calcium sensing receptor with various other GPCRs.
HEK293 cells were transiently transfected with increasing amounts of V2R-RLuc (A and B) or CaSR-RLuc (C) and with increasing amounts of either AT1R-Venus, AT2R-Venus, β2AdR-Venus, CaSR-Venus, CB1R-Venus or V2R-Venus. Various amounts of empty pcDNA3.1 plasmid was added to maintain constant total transfected plasmid amount. Total luminescence and Venus fluorescence were measured at the beginning of each experiment. BRET ratio was calculated as Emission530/Emission485, and was plotted as a function of measured fluorescence. (A) Representative Type I plots for V2R-β2AdR interaction (left plot) and V2R-V2R interaction (right plot). Measured points were sorted into low/high luminescence groups based on the total measured luminescence (Figure S2). (B and C) The slope of linear regression was calculated for the low and high luminescence groups of different GPCR pairs, and was plotted as a column diagram. Difference between the slopes of linear regression was determined by ANCOVA. n = 3–8.
Figure 5.
Preferred method to perform qBRET experiments.
In Type I plots BRET ratio is plotted as a function of acceptor expression (fluorescence), while in Type II plots BRET ratio is plotted as a function acceptor/donor expression (fluorescence/luminescence).