Knockin mice expressing fluorescent δ-opioid receptors uncover G protein-coupled receptor dynamics in vivo
Knockin mice expressing fluorescent δ-opioid receptors uncover G protein-coupled receptor dynamics in vivo
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DOI：
10.1073/pnas.0603359103
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发表时间：
2006-06-20
影响因子：
11.1
通讯作者：
Kieffer, Brigitte L.
中科院分区：
文献类型：
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作者：
Scherrer, Gregory;Tryoen-Toth, Petra;Kieffer, Brigitte L.
荧光基因编码蛋白与小鼠工程的结合为研究哺乳动物的动态生物学过程提供了一种迷人的手段。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和迁移。在这里，我们使用增强型绿色荧光蛋白（EGFP），以实现功能成像的G蛋白偶联受体（GPCR）在体内。我们创建了小鼠，其中δ阿片受体（DOR）被活性DOR-EGFP融合体取代。共聚焦成像揭示了整个敲入小鼠神经系统的详细受体神经解剖。原代神经元的实时成像允许药物诱导的受体运输的动态可视化。在DOR-EGFP动物中，药物治疗触发了与行为反应相关的受体内吞作用。具有内化受体的小鼠对随后的激动剂给药不敏感，这提供了受体螯合限制体内药物功效的证据。在小鼠中直接受体可视化是受体生物学和药物设计的独特方法。
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 knockin 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.