Martikkala Eija, Rozwandowicz-Jansen Anita, Hänninen Pekka, Petäjä-Repo Ulla, Härmä Harri
Laboratory of Biophysics and Medicity, University of Turku, Turku, Finland.
J Biomol Screen. 2011 Mar;16(3):356-62. doi: 10.1177/1087057110397356. Epub 2011 Feb 22.
G-protein-coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)-feasible method was developed to measure changes in cAMP levels in HEK293(i) cells overexpressing either β(2)-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory G(s) or inhibitory G(i) proteins. The reproducibility of the assays was excellent, and Z' values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the G(s)-coupled β(2)AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.
G蛋白偶联受体（GPCRs）是一类重要的药物靶点。需要功能性高通量GPCR检测方法来测试越来越多合成的新型药物化合物及其在信号转导过程中的功能。测量环磷酸腺苷（cAMP）浓度的变化是验证腺苷酸环化酶途径中GPCR激活的一种广泛使用的方法。在此，开发了一种单标记时间分辨荧光且适用于高通量筛选（HTS）的方法，用于测量过表达β(2)-肾上腺素能受体或δ-阿片受体的HEK293(i)细胞中cAMP水平的变化。在猝灭共振能量转移（QRET）技术中，可溶性猝灭剂可降低溶液中未结合的铕(III)标记cAMP的信号，而与抗体结合的部分则发出荧光。通过激动剂介导的与刺激性G(s)或抑制性G(i)蛋白偶联的受体刺激，证明了这种均相竞争检测方法的可行性。检测的重现性极佳，Z'值超过0.7。将该检测方法的动态范围、信噪比和检测限与商业时间分辨荧光共振能量转移（TR-FRET）检测方法进行了比较。在这两种均相检测方法中，当腺苷酸环化酶直接被福斯高林刺激或通过激动剂介导的G(s)偶联β(2)AR激活时，获得了相似的检测参数。使用单标记方法的优势在于，与双标记TR-FRET检测方法相比，QRET系统具有成本效益，因为无需对两个结合伙伴进行标记，从而降低了检测优化的要求。
