研究CB1和CB2大麻素受体β-抑制蛋白招募的实验方案。
Protocol to Study β-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors.
作者信息
Soethoudt Marjolein, van Gils Noortje, van der Stelt Mario, Heitman Laura H
机构信息
Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
Department of Medicinal Chemistry, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands.
出版信息
Methods Mol Biol. 2016;1412:103-11. doi: 10.1007/978-1-4939-3539-0_11.
Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.
大麻素CB1和CB2受体是G蛋白偶联受体（GPCRs），在被（部分）激动剂激活后会募集β-抑制蛋白。β-抑制蛋白的募集是由其C末端尾巴的磷酸化诱导的，并且与GPCR信号传导的终止有关；然而，它也可能激活独立于G蛋白的细胞信号通路。在这里，我们描述了一个详细的方案，通过使用PathHunter®检测法来表征配体诱导或抑制β-抑制蛋白募集到人类CB1和CB2受体的效力和功效。后者是一种细胞检测法，可以在384孔板中进行。PathHunter®检测法利用β-半乳糖苷酶互补，并具有化学发光读数。我们使用该检测法来表征一组人类CB1和CB2受体的参考配体（激动剂和拮抗剂）。
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