Opioid Agonists Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and Endocytosis of Mu-opioid Receptors

Abstract

The differential ability of various -opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (I Ca ) as a reporter before and after inactivation of a fraction of receptors by ␤-chlornaltrexamine, 2) produce rapid, homologous desensitization of I Ca inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) > > morphine (0.07) > pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize -opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.Tolerance to the analgesic and other effects of opioid drugs, such as morphine, undermines their use in long term treatment (1). Although analgesic tolerance is likely to be a complex phenomenon, an understanding of how -opioid (MOP) 1 receptors are regulated by opioid agonists will lead to important insights into this phenomenon. MOPs are similar to many other G protein-coupled receptors (GPCR) in that they undergo desensitization within several minutes of stimulation by agonists (2-4), and continued agonist exposure results in removal of receptors from the cell surface (5-7). MOP phosphorylation is increased as a consequence of agonist exposure, leading to a commonly held assumption that receptor phosphorylation is essential for MOP desensitization and internalization (5, 8 -10). The mechanism that may be responsible for uncoupling MOP from G protein activation and then promoting receptor sequestration is as follows: phosphorylation of the agonist occupied MOP by a G protein receptor kinase (GRK), subsequent binding of ␤-arrestins to the phosphorylated receptor, and removal of MOP from the cell surface via a clathrin-dependent process (11-15). The enhanced analgesic potency of morphine and diminished analgesic tolerance in ␤-arrestin2 knockout mice suggest that the capacity of opioid agonists to cause receptor desensitization and endocytosis is one of the key cellular mechanisms underlying tolerance (16). Phosphorylation of MOP by protein kinase C (PKC) or calmodulin-dependent kinase II can also reduce the capacity of MOP to activate G proteins (8,17), also contributing to opioid tole...

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

“…16 The potency and efficacy of the opioid agonists correlate well with previous studies in AtT-20 cells and with studies in native neurons where activation of GIRK channels or inhibition of I Ca was used to determine agonist intrinsic activity. 11,[18][19][20] The data we obtained with the hyperpolarization assay are similar to what we obtained when we measured MOR Figure 5. Treatment of AtT-20 µ-opioid receptor (MOR) cells with nigericin, a K-selective antibiotic ionophore, caused a greater decrease in fluorescent signal than the maximally effective concentration of DAMGO (p < 0.05).…”

“…Many studies have reported the endomorphins as being partial agonists, 18,21 whereas methadone has been reported to be an agonist with a similar efficacy to DAMGO. 11,22,23 The most likely explanation for the discrepancy in our study is that methadone appeared to have a reduced maximal effect because of its propensity to block K channels, including the GIRK channels likely to contribute to the hyperpolarization measured in the present study. 22,24 Conversely, the observation that the maximum effect of the endomorphins was similar to that of well-recognized high-efficacy agonists suggests the presence of some spare receptors in our system, as noted previously with similar cell lines.…”

“…11 The pcDNA3 FLAG-MOR construct was a kind gift from Dr. Lakshmi Devi (Mt. Sinai School of Medicine, New York, NY).…”

“…10 In this study, we sought to develop a minimally invasive assay that reflected a naturalistic coupling of MOR and could potentially be used to readily examine agonist regulation of receptor signaling. In mouse pituitary AtT-20 cells, heterologously expressed MOR inhibit native calcium channels (I Ca ) 11 and activate endogenous G-protein-gated inwardly rectifying potassium channels 12 (GIRKs), and in both cases, this signaling is subject to rapid, agonist-induced regulation. We assessed the suitability of a proprietary fluorescent membrane potential assay to act as a robust reporter of MOR activation and desensitization in AtT-20 cells, with the view to potentially providing an assay for HTS of both these important aspects of MOR function.…”

Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling

“…When compared with other mu agonists, morphine induces less internalization of the mu opioid receptor (Borgland et al, 2003;McPherson et al, 2010;Zheng et al, 2011). This suggests that the prototypical role of b-arrestin 2 in initiating receptor internalization does not explain how the effects of morphine, but not of other mu agonists, are altered in mice lacking b-arrestin 2.…”