Vol. 298, Issue 3, 1015-1020, September 2001

Agonist-, Antagonist-, and Inverse Agonist-Regulated Trafficking of the -Opioid Receptor Correlates with, but Does Not Require, G Protein Activation

Paulette A. Zaki, Duane E. Keith, Jr., James B. Thomas, F. I. Carroll and Christopher J. Evans

Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, California (P.A.Z., D.E.K., C.J.E.); and Research Triangle Institute, Research Triangle Park, North Carolina (J.B.T., F.I.C.)

In this study, we explored the relationship between ligand-induced regulation of surface  opioid receptors and G protein activation. G protein activation was assessed with [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTPS) binding assays conducted at both 37 and 0°C. Ligand-independent (constitutive) activity of the -receptor was readily observed when the [³⁵S]GTPS binding assay was performed at 37°C. We identified a new class of alkaloid inverse agonists (RTI-5989-1, RTI-5989-23, RTI-5989-25), which are more potent than the previously described peptide inverse agonist ICI-174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu). Treatment with these inverse agonists for 18 h caused up-regulation of surface receptors. Eighteen-hour treatment with etorphine resulted in approximately 90% loss of surface receptor, whereas fentanyl, diprenorphine, and morphine caused between 20 and 50% loss. The abilities of ligands to modulate [³⁵S]GTPS binding at 37°C showed a strong correlation with their abilities to regulate surface receptor number (r² = 0.86). Interestingly, the ability of fentanyl to activate G proteins was markedly temperature sensitive. Fentanyl showed no stimulation of [³⁵S]GTPS binding at 0°C but was as efficacious as etorphine, morphine, and diprenorphine at 37°C. Neither the ligand-induced receptor increases nor decreases were perturbed by pertussis toxin pretreatment, suggesting that functional G proteins are not required for ligand-regulated -opioid receptor trafficking.