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.
Highlights
G Protein Coupled Receptors (GPCRs) were thought to be monomeric entities for a long time, but results of the last two decades indicate that they can form dimers or higher ordered oligomers [1]
While many functional data support the concept of dimerization, evidence of direct physical interaction is crucial to declare the dimerization of two GPCRs
In our classical quantitative Bioluminescence Resonance Energy Transfer (qBRET) experiments, we found that all of the investigated pairs resulted in saturation qBRET curves with BRET50 values of the same magnitude, except for cytoplasmic Venus (Fig. 1B, left panel)
Summary
G Protein Coupled Receptors (GPCRs) were thought to be monomeric entities for a long time, but results of the last two decades indicate that they can form dimers or higher ordered oligomers [1]. The effects of dimerization on receptor signaling are proposed to have great physiological and pharmacological consequences [2,3,4]. While the dimerization of Class C GPCRs, including GABAB receptors is widely accepted [5], the occurrence and functional consequences of rhodopsin like Class A GPCR dimerization are more controversial. Large amounts of data argue that Class A GPCRs can form dimers, even in native tissues [6,7], and this dimerization has important effects on receptor function [8,9]. While some elegant new methods, such as analysis of receptor mobility [10] and visualization of single fluorescently labeled receptors on cell surface [11] are currently available, the current gold standard to study the quaternary structure of GPCRs is the method of quantitative Bioluminescence Resonance Energy Transfer (qBRET) [12,13]
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