RT Journal Article SR Electronic T1 Effect of Phosducin on Opioid Receptor Function JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 599 OP 606 VO 289 IS 1 A1 Rüdiger Schulz A1 Andrea Wehmeyer A1 Karin Schulz A1 John Murphy YR 1999 UL http://jpet.aspetjournals.org/content/289/1/599.abstract AB Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind Gβγ subunits. Because the internalization of opioid receptors as well as the activity of adenylyl cyclase (AC) activity depends on G proteins, we tested Phd on these parameters. NG 108–15 hybrid cells stably expressing the phosphoprotein were challenged with [d-penicillamine2,d-penicillamine5]enkephalin to inhibit cAMP generation, demonstrating an increased efficacy of the opioid on AC. Studying the binding of [35S]guanosine-5′-O-(γ-thio)-triphosphate to membranes from Phd overexpressing cells, we found that [d-penicillamine2,d-penicillamine5]enkephalin failed, in the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108–15 cells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transiently transfected to express μ-opioid receptors fused to green fluorescence protein. In controls and in Phd overexpressing cells confocal microscopy identified fluorescence associated with the membrane. Time-lapse series microscopy of living control cells challenged with etorphine (1 μM) revealed receptor internalization within 30 min. In contrast, Phd overexpressing cells largely failed to respond to the opioid. Thus, in Phd overexpressing cells, opioids exhibit an increased efficacy despite the inhibitory action of the phosphoprotein on opioid-stimulated incorporation of [35S]guanosine-5′-O-(γ-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein Gi-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of Gβγ by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids. The American Society for Pharmacology and Experimental Therapeutics