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）可形成二聚体或更高阶的寡聚体，这一过程会显著影响此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）检测是证实疑似GPCR二聚体亚基间直接物理相互作用的金标准。为正确解读此类实验结果，需维持能量供体——海肾荧光素酶（Renilla luciferase）标记的受体的表达量恒定，但在表达系统中很难实现这一点。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模拟。研究结果显示，即便供体标记受体与标记有能量受体的受体间的相互作用为非特异性相互作用，供体表达量的下降仍可导致qBRET曲线呈现饱和特征，进而会对受体二聚化状态产生错误解读。我们在此提出一种全新的qBRET数据分析方法：在不同供体受体表达水平下，将BRET比值作为标记有能量受体的受体的表达量的函数进行绘图。借助该方法，当经典qBRET实验结果存在歧义时，我们能够有效区分特异性二聚化与非特异性相互作用。该模拟结果通过雷帕霉素（rapamycin）诱导的异源二聚化体系得到了实验验证。我们运用这一新方法探究了多种GPCR的二聚化特性，实验数据证实了V2加压素（V2 vasopressin）受体与钙敏感受体（CaSR calcium sensing receptors）的同源二聚化现象，同时表明此类受体与其他研究中的GPCR（包括I型和II型血管紧张素（angiotensin）受体、β2肾上腺素能（β2 adrenergic）受体以及CB1大麻素（CB1 cannabinoid）受体）并不发生异源二聚化。