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

Phosducin is a member of the large group of proteins that bind to G-protein ␤␥-subunits (G ␤␥ ) and whose biological functions are often unknown. Human A431 cells do not contain detectable amounts of phosducin. We generated A431 cells expressing phosducin at a level of Ϸ1 pmol/mg of cytosolic protein, which is Ϸ10% of the phosducin level in brain. cAMP accumulation in response to ␤ 2 -adrenergic receptor agonists was enhanced at early times in phosducin-expressing cells, but reached a lower plateau than in control cells. Permeabilization of the cells with digitonin did not change this pattern, but allowed the introduction of specific inhibitors: antibodies to phosducin abolished all differences between the two cell lines. Inhibitors of the ␤-adrenergic receptor kinase abolished the differences at early time points. An almost complete loss of ␤ 2 -adrenergic receptor desensitization in the phosducin-expressing cells was also observed when intact cells were desensitized and receptor function was then determined in membrane preparations. Inhibition of protein kinase A accentuated the effects of phosducin, suggesting that also in vivo phosducin is regulated by this kinase. These data indicate that phosducin affects G-protein-mediated signaling in at least two ways: it dampens the overall responsiveness, and it impairs the rapid desensitization mediated by the ␤-adrenergic receptor kinase.

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A proteolipid isolated from a lipid extract of mouse brain demonstrates stereospecific binding properties for levorphanol. It is present only in neuronal tissue and most abundant in the rhombencephalon. One component saturates at a concentration corresponding to maximum pharmacologic effect in vivo. The estimated mass is 60,000 daltons per bound opiate molecule.