Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander University Erlangen-Nürnberg, Schuhstraße 19, 91052, Erlangen, Germany.
Department of Psychiatry and Psychotherapy, Friedrich-Alexander University Erlangen-Nürnberg, Schwabachanlage 6, 91054, Erlangen, Germany.
Sci Rep. 2017 Sep 7;7(1):10894. doi: 10.1038/s41598-017-11436-1.
G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D) and the long (D) isoforms of dopamine D receptors in their ability to undergo agonist-induced internalization.
G 蛋白偶联受体（GPCRs），包括多巴胺受体，是一组重要的药理学靶点。激动剂结合后，GPCR 通常会发生内化，这一过程已知会在长时间暴露于激动剂时减弱功能反应。在这项研究中，通过全内反射荧光（TIRF）显微镜在靠近质膜的离散单个事件水平上以高空间分辨率可视化内化。开发了一种新方法，通过在活 CHO 细胞中进行比较荧光定量来确定内化荧光受体-配体复合物的相对程度。该过程需要用还原剂硼氢化钠处理，该还原剂将膜表面上基于菁染料的荧光配体转化为长寿命的还原形式。由于高度极性的还原剂不能穿过细胞膜，因此位于内化隔室中的荧光受体-配体复合物在 TIRF 照明下仍然具有荧光。我们应用该方法研究了多巴胺 D 受体的短（D）和长（D）异构体在诱导内化的能力方面的差异。
