Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization

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.

G蛋白偶联受体（G Protein Coupled Receptors, GPCR）可形成二聚体或高阶寡聚体，该过程会显著影响这类受体的生理与药理功能。定量生物发光共振能量转移（Quantitative Bioluminescence Resonance Energy Transfer, qBRET）检测是验证假定GPCR二聚体亚基间直接物理相互作用的金标准。若要正确解读此类实验，需维持能量供体海肾荧光素酶（Renilla luciferase）标记受体的表达量恒定，但在表达系统中难以实现这一点。为分析供体表达量非恒定对qBRET曲线的影响，我们开展了蒙特卡洛（Monte Carlo）模拟。研究结果显示，即便供体与受体标记受体间的相互作用为非特异性，供体表达量的下降也会导致qBRET曲线出现饱和现象，进而会对二聚化状态做出错误判断。在此我们提出一种全新的qBRET数据分析方法：在不同供体受体表达水平下，以BRET比值作为受体标记受体表达量的函数进行绘图。借助该方法，当经典qBRET实验结果模糊不清时，我们能够区分二聚化与非特异性相互作用。我们使用雷帕霉素（rapamycin）诱导异二聚化系统，通过实验验证了模拟结果。我们还利用这一新方法探究了多种GPCR的二聚化情况，数据证实了V2加压素受体（V2 vasopressin receptor）与钙敏感受体（calcium sensing receptors, CaSR）的同源二聚化，同时表明这些受体与其他研究过的GPCR——包括I型和II型血管紧张素受体、β2肾上腺素能受体以及CB1大麻素受体（CB1 cannabinoid receptor）——并不发生异二聚化。