Schulz Rüdiger, Wehmeyer Andrea, Schulz Karin
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Munich, Germany.
Mol Pharmacol. 2002 Jun;61(6):1444-52. doi: 10.1124/mol.61.6.1444.
G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to kappa-opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human kappa-receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of kappa-opioid receptors, employing laser scanning microscopy as a major technique to examine receptor internalization in living cells. For this reason, we fused green fluorescence protein to kappa-receptors, and DsRed-fluorescent protein to GRK2 and GRK3. All fusion proteins retained their biologic activities. Permanent cell lines (HEK 293, NG 108-15) were transfected to express either green fluorescent kappa-receptors or to coexpress the tagged receptor and a specific GRK-DsRed construct. The localization of fluorescent receptors and GRKs was monitored by confocal microscopy before and after opioid exposure of transfected cells. Activation of the murine kappa-receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human kappa-receptor also causes translocation of GRK2- and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent kappa-receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of kappa-receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-betagamma subunits for kappa-receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of kappa-receptors, we conclude that GRK3 more strongly induces kappa-receptor internalization than GRK2.
G蛋白偶联受体激酶（GRKs）使阿片受体磷酸化，最终导致受体隔离。就κ-阿片受体而言，已知内化以物种特异性方式发生。也就是说，激动剂占据的人κ-受体会被隔离，而鼠类受体则不会。本研究集中于κ-阿片受体的内化，采用激光扫描显微镜作为主要技术来检测活细胞中的受体内化。因此，我们将绿色荧光蛋白与κ-受体融合，并将DsRed荧光蛋白与GRK2和GRK3融合。所有融合蛋白均保留其生物学活性。将永生化细胞系（HEK 293、NG 108-15）转染以表达绿色荧光κ-受体，或共表达标记的受体和特定的GRK-DsRed构建体。在转染细胞暴露于阿片类药物之前和之后，通过共聚焦显微镜监测荧光受体和GRKs的定位。鼠类κ-受体的激活触发标记的GRKs迅速向细胞膜转位，但未观察到受体内化。激动剂占据的人κ-受体也会导致GRK2-和GRK3-DsRed转位，随后形成携带绿色荧光κ-受体的囊泡。此外，绿色荧光囊泡分别始终含有红色荧光GRK2和GRK3。κ-受体内化以及GRKs共内化的现象被磷蛋白抑制，表明G蛋白βγ亚基在κ-受体隔离中起关键作用。比较过表达的GRK2和GRK3对κ-受体隔离的影响，我们得出结论，GRK3比GRK2更强烈地诱导κ-受体内化。
