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Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.

β-funaltrexamine (β-FNA) is an irreversible μ opioid (MOP) receptor antagonist and a reversible agonist of κ opioid (KOP) receptor. β-FNA binds covalently to the MOP receptor at Lys233 5.39 , which is conserved among opioid receptors. Molecular docking of β-FNA showed that K303 6.58 in the MOP receptor and E297 6.58 in the KOP receptor played distinct roles in positioning β-FNA. K303 6.58 E MOP receptor and E297 6.58 K KOP receptor mutants were generated. The mutations did not affect β-FNA affinity or efficacy. K303 6.58 E mutation in the MOP receptor greatly reduced covalent binding of [ 3 H]β-FNA; however, E297 6.58 K did not enable the KOP receptor to bind irreversibly to β-FNA. Molecular modeling demonstrated that the e-amino group of K303 6.58 in the MOP receptor interacted with C O of the acetate group of β-FNA to facilitate covalent bond formation with Lys233 5.39 . Replacement of K303 6.58 with Glu in the MOP receptor resulted in repulsion between the COOH of Glu and the C O of β-FNA and increased the distance between K233 5.39 and the fumarate group, making it impossible for covalent bond formation. These findings will be helpful for design of selective non-peptide MOP receptor antagonists.

It has been shown previously that norbinaltorphimine (norBNI) and 5′-guanidinonaltrindole (5′-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1] , [2] . In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss–Webster mice, zyklophin at doses of 0.1, 0.3 and 1 mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20 mg/kg, i.p.) at 18–20 h before challenge with zyklophin (0.3 mg/kg) did not markedly affect scratching. Additionally, KOPR−/− mice given 0.3 mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR−/− mice was confirmed with ex vivo radioligand binding using [3H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5′-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch.

Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.

Chronic administration of cocaine attenuates delta opioid receptor (DOPR) signaling in the striatum and the desensitization is mediated by the indirect actions of cocaine on dopamine D1 receptors (D1R). In addition, DOPR and D1R co-exist in some rat striatal neurons. In the present study, we examined the underlying mechanism of DOPR desensitization by D1R activation.NG 108-15 cells stably expressing HA-rat D1 receptor (HA-D1R) and Chinese hamster ovary (CHO) cells stably expressing both FLAG-mouse DOPR (FLAG-DOPR) and HA-D1R were used as the cell models. Receptor binding, [(35)S]GTPγS binding, receptor phosphorylation and western blot were conducted to examine DOPR affinity, expression, internalization, downregulation, desensitization, phosphorylation and phosphorylated ERK1/2.Pretreatment with either the DOPR agonist DPDPE or the D1R agonist SKF-82958 for 30min attenuated DPDPE-stimulated [(35)S]GTPγS binding to G proteins, demonstrating homologous and heterologous desensitization of the DOPR, respectively. SKF-82958 pretreatment did not affect the level of DOPR or affinity of DOPR antagonist or agonists, nor did it induce phosphorylation, internalization or down-regulation of the DOPR in the CHO-FLAG-DOPR/HA-D1R cells. Pretreatment of cells with inhibitors of PKA, MEK1 and PI3K, but not PKC, attenuated SKF-82958-induced desensitization of the DOPR. The D1R agonist SKF-82958 enhanced phosphorylation of ERK1/2, and pretreatment with inhibitors of MEK1 and PI3K, but not PKA and PKC, reduced the effect. These results indicate that activation of ERK1/2 and/or PKA, but not PKC, is involved in D1 receptor-induced heterologous desensitization of the DOPR.This study provides possible mechanisms underlying D1R activation-induced DOPR desensitization.