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G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to -opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human -receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of -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 -receptors, and DsRed-fluorescent protein to GRK2 and GRK3. All fusion proteins retained their biologic activities. Permanent cell lines (HEK 293, were transfected to express either green fluorescent -receptors or to coexpress the tagged receptor and a specific GRKDsRed 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 -receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human -receptor also causes translocation of GRK2-and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent -receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of -receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-␤␥ subunits for -receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of -receptors, we conclude that GRK3 more strongly induces -receptor internalization than GRK2.

Activation of G protein-coupled receptors (GPCRs) may bring about their disappearance from the cell membrane, and it is commonly accepted that G protein-coupled receptor kinases (GRKs) play a key function in this mechanism. Opioid receptors belong to the family of GPCRs and are substrates of GRKs. We examined the fate of ␦-and -opioid receptors and GRK2 and 3 in living cells during the process of receptor sequestration, using laser scanning microscopy. For visualization purposes, receptors and kinases were tagged at their respective C terminus with a fluorophore. The opioid receptors were fused to enhanced green fluorescence protein (EGFP), and the GRKs were linked to red fluorescence protein (DsRed). The cDNAs of these constructs served for transfection of human embryonic kidney 293 cells and neuroblastoma-glioma hybrid cells (NG 108-15), respectively. We report that activation of ␦-opioid-EGFP receptors triggers a rapid translocation of cytosolic GRKDsRed toward the cell membrane, which in turn releases vesicles carrying both green fluorescent ␦-receptors and red fluorescent GRKs. Phosducin, a G␤␥ scavenger, completely prevents translocation of GRKs and the formation of vesicles. In analogous experiments with -opioid

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BackgroundThe Y-box binding protein 1 (YB-1) is considered to be one of the key regulators of transcription and translation. However, so far only limited knowledge exists regarding its cellular distribution in the adult brain.ResultsAnalysis of YB-1 immunolabelling as well as double-labelling with the neuronal marker NeuN in rat brain tissue revealed a predominant neuronal expression in the dentate gyrus, the cornu ammonis pyramidal cell layer, layer III of the piriform cortex as well as throughout all layers of the parahippocampal cortex. In the hilus of the hippocampus single neurons expressed YB-1. The neuronal expression pattern was comparable in the hippocampus and parahippocampal cortex of adult macaques and humans. Double-labelling of YB-1 with the endothelial cell marker Glut-1, the multidrug transporter P-glycoprotein, and the astrocytic marker GFAP did not indicate a co-localization. Following status epilepticus in rats, no induction of YB-1 occurred in brain capillary endothelial cells and neurons.ConclusionIn conclusion, our study demonstrates that YB-1 is predominantly expressed in neurons in the adult brain of rats, macaques and humans. Lack of a co-localization with Glut-1 and P-glycoprotein argues against a direct role of YB-1 in the regulation of blood-brain barrier P-glycoprotein.

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