Phosphoproteomic approach for agonist-specific signaling in mouse brains: mTOR pathway is involved in κ opioid aversion.

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.

Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors.

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 ß-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.

K3036·58 in the µ opioid (MOP) receptor is important in conferring selectivity for covalent binding of ß-funaltrexamine (ß-FNA).

ß-funaltrexamine (ß-FNA) is an irreversible µ opioid (MOP) receptor antagonist and a reversible agonist of κ opioid (KOP) receptor. ß-FNA binds covalently to the MOP receptor at Lys233(5.39), which is conserved among opioid receptors. Molecular docking of ß-FNA showed that K303(6.58) in the MOP receptor and E297(6.58) in the KOP receptor played distinct roles in positioning ß-FNA. K303(6.58)E MOP receptor and E297(6.58)K KOP receptor mutants were generated. The mutations did not affect ß-FNA affinity or efficacy. K303(6.58)E mutation in the MOP receptor greatly reduced covalent binding of [(3)H]ß-FNA; however, E297(6.58)K did not enable the KOP receptor to bind irreversibly to ß-FNA. Molecular modeling demonstrated that the ε-amino group of K303(6.58) in the MOP receptor interacted with CO of the acetate group of ß-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 CO of ß-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.