Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, D-93053, Regensburg, Germany.
Department of Chemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Sci Rep. 2018 Nov 21;8(1):17179. doi: 10.1038/s41598-018-35615-w.
The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of Gα with its effector phospholipase C-β3. The resulting probe yielded an excellent Z' value of 0.7 and offers a broad and easy applicability to various Gα-coupling GPCRs (hHR, hMR, hNTSR), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hMR. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging.
在探测 G 蛋白偶联受体 (GPCR) 上 G 蛋白依赖性信号级联的激活时，时间越早，对测量信号的放大效应贡献就越小。这在精确量化激动剂效力时特别有用，并且在确定激动剂相对于β-arrestin 途径的偏向性时至关重要。由于大多数具有中等到高通量的经典测定依赖于第二信使的定量，并且提供更接近的读出的测定通常在通量方面受到限制，因此我们开发了一种具有接近读出和足够高通量的技术，可用于活细胞。分裂萤光素酶互补 (SLC) 被应用于评估 Gα 与其效应物磷脂酶 C-β3 的相互作用。由此产生的探针产生了出色的 Z' 值为 0.7，并为各种表达于 HEK293T 细胞中的 Gα 偶联 GPCR（hHR、hMR、hNTSR）提供了广泛而简单的适用性，允许对激动剂和拮抗剂进行功能表征。此外，所开发的传感器通过发光显微镜实现了活细胞的成像，如 hMR 所示。基于 SLC 的多功能探针广泛适用于 GPCR 配体的筛选和药理学表征以及分子成像。
