Institute of Pharmacy, University of Regensburg, Regensburg, Germany.
AbbVie Deutschland GmbH & Co. KG, Ludwigshafen am Rhein, Germany.
Methods Mol Biol. 2021;2274:69-78. doi: 10.1007/978-1-0716-1258-3_7.
G Protein-coupled receptors (GPCRs) transduce signals elicited by bioactive chemical agents (ligands), such as hormones, neurotransmitters, or cytokines, across the cellular membrane. Upon ligand binding, the receptor undergoes structural rearrangements, which cause the activation of G proteins. This triggers the activation of signaling cascades involving amplification, which takes place after every stage of the cascade. Consequently, signals from early stages can be masked when the activation of the signaling cascade is probed remote (distal) from the receptor. This led to the development of several techniques, which probe the activation of such signaling cascades as proximal to the receptor as possible. However, these methods often require specialized equipment or are limited in throughput. By applying split-luciferase complementation to the interaction between the Gα protein and its effector the phospholipase C-β3 (PLC-β3), we introduce a protocol with a conventional plate reader at high throughput. The method is applicable to live cells and additionally allows imaging of the probe by bioluminescence microscopy.
G 蛋白偶联受体 (GPCR) 跨细胞膜传递生物活性化学物质 (配体) 如激素、神经递质或细胞因子引发的信号。配体结合后，受体发生结构重排，导致 G 蛋白激活。这触发了涉及放大的信号级联的激活，级联的每一个阶段之后都会发生放大。因此，当从受体远程 (远端) 探测信号级联的激活时，早期信号可能会被掩盖。这导致了几种技术的发展，这些技术尽可能接近受体探测此类信号级联的激活。然而，这些方法通常需要专门的设备或在通量方面受到限制。通过将荧光素酶互补应用于 Gα 蛋白与其效应物磷脂酶 C-β3 (PLC-β3) 之间的相互作用，我们引入了一种使用常规平板读数器进行高通量检测的方案。该方法适用于活细胞，并且还允许通过生物发光显微镜对探针进行成像。
