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

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 [S]guanosine-5 -O-(3thio)triphosphate (GTP S) binding assays conducted at both 37 and 0°C. Ligand-independent (constitutive) activity of the -receptor was readily observed when the [S]GTP S 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 ICI174864 (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 [S]GTP S binding at 37°C showed a strong correlation with their abilities to regulate surface receptor number (r 0.86). Interestingly, the ability of fentanyl to activate G proteins was markedly temperature sensitive. Fentanyl showed no stimulation of [S]GTP S 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. Constitutive activity has become a well described characteristic of many G protein-coupled receptors (GPCRs) and has redefined the concept of how GPCRs function. Ligand-independent activity of GPCRs has been described for a variety of receptors either in their wild-type form or in mutated forms (for reviews, see Milligan et al., 1997; Leurs et al., 1998). With the realization that receptors could be active in the absence of ligand, some ligands have had to be reclassified from being antagonists (ligands that bind to the receptor but do not elicit a response) to being inverse agonists (ligands that elicit a response opposite to that of agonists). One of the first GPCRs to be described as having constitutive activity is the Gi/o-coupled -opioid receptor. Ligand-independent activity of this receptor was first shown in NG108-15 cells, which endogenously express the murine -opioid receptor (Costa and Herz, 1989; Costa et al., 1990). Constitutive activity of the receptor has subsequently been demonstrated in cell lines stably transfected with the -receptor from various species (Chiu et al., 1996; Mullaney et al., 1996; Merkouris et al., 1997; Hosohata et al., 1999; Neilan et al., 1999; Labarre et al., 2000). The regulation of GPCRs after various ligand treatments has been an active area of research with the majority of studies focusing on the effect of agonist treatment on receptor function. This has been particularly important arena in the opioid field due to the desire to understand the basis of tolerance and dependence to opioids that result from repeated administration of the drug (Nestler and Aghajanian, 1997). Although these adaptational processes that occur in animals are obviously complex, in vitro studies on cell lines that express opioid receptors have furthered our understanding of the cellular adaptations that occur after ligand treatment. Generally, opioid receptors have been shown to be phosphorylated and desensitized in response to agonist treatment, although the extent of these processes is dependent on the particular agonist (for review, see Law and Loh, 1999). The extent of opioid receptor internalization is also dependent on the type of agonist. For example, the agonist etorphine is able to cause rapid internalization of and -opioid receptors, whereas morphine does not cause this regulatory event (Keith et al., 1996). Opioid receptors have also been 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; GTP S, guanosine-5 -O-(3-thio)triphosphate; ICI-174864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu; DOR, -opioid receptor; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; PTX, pertussis toxin; TIPP, Tyr-Tic-Phe-Phe. 0022-3565/01/2983-1015–1020$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 3 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3783/923556 JPET 298:1015–1020, 2001 Printed in U.S.A. 1015 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from