Fisher Gregory W, Adler Sally A, Fuhrman Margaret H, Waggoner Alan S, Bruchez Marcel P, Jarvik Jonathan W
Technology Center for Networks and Pathways, Molecular Biosensor and Imaging Center, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
J Biomol Screen. 2010 Jul;15(6):703-9. doi: 10.1177/1087057110370892. Epub 2010 May 20.
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.
配体依赖性受体内化是众多信号系统的一个特征。在本文中，作者描述了一种新型的受体内化活细胞生物传感器，该传感器利用了荧光激活蛋白（FAP）技术。将在其细胞外N端带有FAP结构域的人β2肾上腺素能受体（β2AR）的重组基因转导到哺乳动物细胞中。将细胞暴露于不能透过细胞膜的荧光团会导致细胞表面发出强烈的荧光信号。通过荧光显微镜或流式细胞术，以均相形式，无需洗涤或分离步骤，即可轻松观察并定量受体从表面到细胞内部的激动剂依赖性转位。此处描述的方法可推广到其他受体和细胞表面蛋白，并且适用于各种基于荧光的高通量筛选平台。
