Vol. 289, Issue 1, 599-606, April 1999

Effect of Phosducin on Opioid Receptor Function

Rüdiger Schulz, Andrea Wehmeyer, Karin Schulz and John Murphy

Institute of Pharmacology, Toxicology and Pharmacy (R.S., A.W.), Gene Center (K.S.), University of Munich, München, Germany; and Max-Planck-Institute for Biochemistry, Department of Cell Biology (J.M.), Martinsried, Germany

Phosducin (Phd) regulates the function of G proteins by its ability to tightly bind G 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-(-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-(-thio)-triphosphate. We suggest that inhibition of GTPase stabilizes the opioid-induced G protein G_i-GTP complex, which is believed to enhance AC inhibition. Finally, scavenging of G by Phd attenuates internalization of opioid receptors, which may contribute to the efficacy of opioids.