Limits...
Improved methodical approach for quantitative BRET analysis of G Protein Coupled Receptor dimerization.

Szalai B, Hoffmann P, Prokop S, Erdélyi L, Várnai P, Hunyady L - PLoS ONE (2014)

Bottom Line: Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state.With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous.The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary.

ABSTRACT
G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

Show MeSH

Related in: MedlinePlus

Classical qBRET experiments.(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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4201472&req=5

pone-0109503-g001: Classical qBRET experiments.(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.

Mentions: In classical qBRET experiments, cells are transfected with constant amount of plasmid coding donor labeled receptor, and increasing amount of plasmid coding acceptor labeled receptor [12], [13]. To maintain equal transfection efficacy, total transfected plasmid amount must also be held constant, with the addition of a non-coding plasmid. The measured BRET ratio is plotted as a function of acceptor/donor expression ratio. In this plotting, specific interactions result in a saturation curve, while non-specific interactions lead to a linear relationship (Fig. 1A, left panel). The absolute value of BRET ratio is also dependent on the distance between donor and acceptor in the quaternary complex, so it is not thought to be indicative about the oligomerization state. Therefore the so called BRET50 value (acceptor/donor ratio at half-maximal BRET value) is used to determine the presence of dimerization: in experiments conducted with one donor and different acceptor labeled receptors, pairs with low BRET50 value thought to form oligomers, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors. To correctly interpret qBRET curves, it is necessary to hold constant the donor labeled receptor expression, independent of the expression of acceptor labeled receptors (Fig. 1A, right panel).


Improved methodical approach for quantitative BRET analysis of G Protein Coupled Receptor dimerization.

Szalai B, Hoffmann P, Prokop S, Erdélyi L, Várnai P, Hunyady L - PLoS ONE (2014)

Classical qBRET experiments.(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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4201472&req=5

pone-0109503-g001: Classical qBRET experiments.(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.
Mentions: In classical qBRET experiments, cells are transfected with constant amount of plasmid coding donor labeled receptor, and increasing amount of plasmid coding acceptor labeled receptor [12], [13]. To maintain equal transfection efficacy, total transfected plasmid amount must also be held constant, with the addition of a non-coding plasmid. The measured BRET ratio is plotted as a function of acceptor/donor expression ratio. In this plotting, specific interactions result in a saturation curve, while non-specific interactions lead to a linear relationship (Fig. 1A, left panel). The absolute value of BRET ratio is also dependent on the distance between donor and acceptor in the quaternary complex, so it is not thought to be indicative about the oligomerization state. Therefore the so called BRET50 value (acceptor/donor ratio at half-maximal BRET value) is used to determine the presence of dimerization: in experiments conducted with one donor and different acceptor labeled receptors, pairs with low BRET50 value thought to form oligomers, while high BRET50 values indicate weak interaction or the absence of interaction between the investigated receptors. To correctly interpret qBRET curves, it is necessary to hold constant the donor labeled receptor expression, independent of the expression of acceptor labeled receptors (Fig. 1A, right panel).

Bottom Line: Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state.With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous.The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary; MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary.

ABSTRACT
G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

Show MeSH
Related in: MedlinePlus