Abstract

In this study, we explored the relationship between ligand-induced regulation of surface δ opioid receptors and G protein activation. G protein activation was assessed with [³⁵S]guanosine-5′-O-(3-thio)triphosphate (GTPγS) binding assays conducted at both 37 and 0°C. Ligand-independent (constitutive) activity of the δ-receptor was readily observed when the [³⁵S]GTPγS 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]GTPγS 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]GTPγS 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 δ-opioid receptor trafficking.