Effect of phosducin on opioid receptor function

Phosducin (Phd) regulates the function of G proteins by its ability to tigh tly bind G beta gamma subunits. Because the internalization of opioid recep tors 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-penicillamine(2),D-p enicillamine(5)]enkephalin to inhibit cAMP generation, demonstrating an inc reased efficacy of the opioid on AC. Studying the binding of [S-35]guanosin e-5'-O-(gamma-thio)-triphosphate to membranes from Phd overexpressing cells , we found that [D-penicillamine(2),D-penicillamine(5)]enkephalin failed, i n the presence of Phd (0.1 nM), to elevate incorporation of the nucleotide. Phd also strongly inhibited opioid-stimulated GTPase activity. NG 108-15 c ells were also employed to investigate the effect of Phd on opioid receptor internalization. Control cells and cells overexpressing Phd were transient ly transfected to express mu-opioid receptors fused to green fluorescence p rotein. In controls and in Phd overexpressing cells confocal microscopy ide ntified fluorescence associated with the membrane. Time-lapse series micros copy of living control cells challenged with etorphine (1 mu M) revealed re ceptor 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 th e phosphoprotein on opioid-stimulated incorporation of [S-35]guanosine-5'-O -(gamma-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 beta gamma by Phd attenuates inter nalization of opioid receptors, which may contribute to the efficacy of opi oids.