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.
Footnotes
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This work was supported by National Institute on Drug Abuse Grants DA-05010 and DA-090454. P.A.Z. is a Hatos scholar and recipient of a predoctoral fellowship from the Howard Hughes Medical Institute.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- HEK
- human embryonic kidney
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- ICI-174864
- N,N-diallyl-Tyr-Aib-Aib-Phe-Leu
- DOR
- δ-opioid receptor
- FITC
- fluorescein isothiocyanate
- BSA
- bovine serum albumin
- PTX
- pertussis toxin
- TIPP
- Tyr-Tic-Phe-Phe
- Received May 18, 2001.
- Accepted January 19, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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