Scherrer Grégory, Tryoen-Tóth Petra, Filliol Dominique, Matifas Audrey, Laustriat Delphine, Cao Yu Q, Basbaum Allan I, Dierich Andrée, Vonesh Jean-Luc, Gavériaux-Ruff Claire, Kieffer Brigitte L
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM, Université Louis Pasteur, 1 Rue Laurent Fries, 67404 Illkirch, France.
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9691-6. doi: 10.1073/pnas.0603359103. Epub 2006 Jun 9.
The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the delta-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knock-in mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design.
荧光基因编码蛋白与小鼠工程技术的结合为研究哺乳动物体内动态生物学过程提供了一种引人入胜的方法。目前，绿色荧光蛋白（GFP）小鼠主要用于研究基因表达模式或细胞形态及迁移。在此，我们利用增强型绿色荧光蛋白（EGFP）实现了体内G蛋白偶联受体（GPCR）的功能成像。我们构建了将δ-阿片受体（DOR）替换为活性DOR-EGFP融合蛋白的小鼠。共聚焦成像揭示了敲入小鼠整个神经系统中详细的受体神经解剖结构。原代神经元中的实时成像使药物诱导的受体转运能够动态可视化。在DOR-EGFP动物中，药物治疗引发了与行为反应相关的受体内吞作用。受体内化的小鼠对随后的激动剂给药不敏感，这证明受体隔离限制了体内药物疗效。小鼠体内直接的受体可视化是受体生物学和药物设计的一种独特方法。
