Analgesic Tolerance to High-Efficacy Agonists But Not to Morphine Is Diminished in Phosphorylation-Deficient S375A μ-Opioid Receptor Knock-In Mice

Abstract

Morphine is one of the most potent analgesic drugs. However, the utility of morphine in the management of chronic pain is limited by its rapid development of tolerance. Morphine exerts all of its pharmacological effects via the -opioid receptor. In many systems, tolerance is associated with phosphorylation and desensitization of G-protein-coupled receptors (GPCRs). In case of the -opioid receptor, phosphorylation occurs in an agonist-selective manner. High-efficacy agonists such as [D-Ala 2 -MePhe 4 -Gly-ol]enkephalin (DAMGO), fentanyl, or etonitazene stimulate the phosphorylation of both C-terminal threonine 370 (T370) and serine 375 (S375). In contrast, morphine promotes the phosphorylation of S375 but fails to stimulate T370 phosphorylation. Here, we have assessed the contribution of S375 phosphorylation to the development of antinociceptive tolerance to high-and low-efficacy agonists in vivo. We show that S375 phosphorylation of the -opioid receptor occurs in intact mouse brain in a dose-dependent manner after administration of morphine, fentanyl, or etonitazene. In knock-in mice expressing the phosphorylation-deficient S375A mutant of the -opioid receptor, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in wild-type mice. However, acute and chronic tolerance to morphine was retained in S375A mutant mice. In contrast, antinociceptive tolerance after repeated subcutaneous application of etonitazene or repeated intracerebroventricular application of DAMGO was diminished. Thus, tolerance to agonists with different efficacies develops through distinct pathways. Whereas tolerance induced by DAMGO or etonitazene requires agonist-driven phosphorylation of S375, the development and maintenance of antinociceptive tolerance to morphine occurs independent of S375 phosphorylation.

Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine

“…For example, serine 375 (S375) of MOR-1 in exon 3-encoded C-terminal sequences shared by all C-terminal variants can be phosphorylated mainly by GRK5 (62), contributing to β-arrestin recruitment (63). A S375A mutant mouse displayed reduced tolerance to fentanyl, but not to morphine (64). Phosphorylation of threonine 394 (T394) in the exon 4-encoded C-terminal sequence was involved in mu agonist-induced receptor internalization and desensitization (65,66).…”

Differentiation of Opioid Drug Effects by Hierarchical Multi-Site Phosphorylation

“…There is already evidence for such a relationship, particularly for opioid tolerance (Grecksch et al, 2011). However, as most of the evidence supporting this concept remains correlative in nature, caution is warranted.…”

“…Chemically diverse opioid drugs, despite similarity in their acute actions, differ remarkably in longerterm regulatory effects, and such differences significantly impact the clinical utility of particular drugs. Drug-induced internalization of opioid receptors has attracted considerable interest because several studies have reported some relationship between the ability of opioids to induce MOR internalization and their tendency to produce tolerance (Grecksch et al, 2006;Martini and Whistler, 2007;Koch and Hollt, 2008;Grecksch et al, 2011;Williams et al, 2013).…”

Regulation ofµ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

“…There is accumulating evidence for biased phosphorylation and subsequent events such as arrestin binding and MAPK (ERK1/2, JNK, p38) activation (reviewed by Bruchas and Chavkin, 2010). There is also evidence supporting the existence of multiple phosphorylated forms of MOR in intact cells, and pronounced differences in the phosphorylation of MORs in response to morphine and peptide agonists such as DAMGO and endomorphin 2 (Yu et al, 1997;McPherson et al, 2010;Grecksch et al, 2011Rivero et al, 2012). However, it remains unknown whether observed differences in the functional regulation of MOR, or other GPCRs, result specifically from agonist-selective stabilization of discrete receptor conformations.…”