Abstract

G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to κ-opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human κ-receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of κ-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 κ-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 κ-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 κ-receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human κ-receptor also causes translocation of GRK2- and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent κ-receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of κ-receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-βγ subunits for κ-receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of κ-receptors, we conclude that GRK3 more strongly induces κ-receptor internalization than GRK2.