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Abstract：
We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand-receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor-ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor-ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.
摘要：
我们在这里介绍了在其表面上显示G蛋白偶联受体的出芽杆状病毒的实施，用于使用荧光各向异性（FA）研究配体-受体相互作用。黑皮质素4（MC4）受体和荧光配体Cy3B-NDP-α-MSH用作模型系统。反应的实时监测和高测定质量允许应用具有动力学机理模型的全局数据分析，该动力学机理模型考虑了非特异性相互作用的影响和反应过程中荧光配体的消耗。受体浓度，确定了荧光配体结合的亲和力和动力学参数以及不同荧光配体群体的状态各向异性。在低Cy3B-NDP-α-MSH浓度下，一个单位点受体-配体结合模型描述了这些过程，而在较高的配体浓度下观察到该模型的差异，这表明了一种更复杂的相互作用机制，类似于放射性配体实验中发现的那些机制。从我们的动力学实验获得的信息和FA测定的固有灵活性也允许估计几种MC4受体特异性未标记化合物的结合参数。总之,用芽杆状病毒开发的FA测定使实验数据达到了可以解决其他受体系统的受体-配体相互作用的复杂模型的水平，并且将成为筛选药理活性化合物的有价值的工具。
