title: Visualizing preference of G protein-coupled receptor kinase 3 for the process of kappa-opioid receptor sequestration.
abstract: 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.
authors.affiliation: Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Munich, Germany. schulz@pharmtox.vetmed.uni-muenchen.de
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Schulz
authors.forename: Andrea
authors.initials: A
authors.lastname: Wehmeyer
authors.forename: Karin
authors.initials: K
authors.lastname: Schulz
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004705:Endocytosis; D005136:Eye Proteins / Q000494:pharmacology; D054770:G-Protein-Coupled Receptor Kinase 3; D020555:GTP-Binding Protein Regulators; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010750:Phosphoproteins; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; C065631:phosducin; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; C517850:GRK3 protein, mouse; D051552:beta-Adrenergic Receptor Kinases