Chronic Treatment with Morphine Disrupts Acute Kinase-Dependent Desensitization of GPCRs.

Based on studies using mutations of the µ-opioid receptor (MOR), phosphorylation of multiple sites on the C-terminus has been recognized as a critical step underlying acute desensitization and the development of cellular tolerance. The aim of this study is to explore which kinases mediate desensitization of MOR in brain slices from drug-naïve and morphine-treated animals. Whole-cell recordings from locus coeruleus neurons were made, and the agonist-induced increase in potassium conductance was measured. In slices from naïve animals, pharmacological inhibition of G-protein receptor kinase (GRK2/3) with compound 101 blocked acute desensitization. Following chronic treatment with morphine, compound 101 was less effective at blocking acute desensitization. Compound 101 blocked receptor internalization in tissue from both naïve and morphine-treated animals, suggesting that GRK2/3 remained active. Kinase inhibitors aimed at blocking protein kinase C and c-Jun N-terminal kinase had no effect on desensitization in tissue taken from naïve animals. However, in slices taken from morphine-treated animals, the combination of these blockers along with compound 101 was required to block acute desensitization. Acute desensitization of the potassium conductance induced by the somatostatin receptor was also blocked by compound 101 in slices from naïve but not morphine-treated animals. As was observed with MOR, it was necessary to use the combination of kinase inhibitors to block desensitization of the somatostatin receptor in slices from morphine-treated animals. The results show that chronic treatment with morphine results in a surprising and heterologous adaptation in kinase-dependent desensitization. SIGNIFICANCE STATEMENT: The results show that chronic treatment with morphine induced heterologous adaptations in kinase regulation of G protein coupled receptor (GPCR) desensitization. Although the canonical mechanism for acute desensitization through phosphorylation by G protein-coupled receptor kinase is supported in tissue taken from naïve animals, following chronic treatment with morphine, the acute kinase-dependent desensitization of GPCRs is disrupted such that additional kinases, including protein kinase C and c-Jun N-terminal kinase, contribute to desensitization.

Separation of Acute Desensitization and Long-Term Tolerance of µ-Opioid Receptors Is Determined by the Degree of C-Terminal Phosphorylation.

Phosphorylation of sites on the C terminus of the µ-opioid receptor (MOR) results in the induction of acute desensitization that is thought to be a precursor for the development of long-term tolerance. Alanine mutations of all 11 phosphorylation sites on the C terminus of MORs almost completely abolished desensitization and one measure of tolerance in locus coeruleus neurons when these phosphorylation-deficient MORs were virally expressed in MOR knockout rats. In the present work, we identified specific residues that underlie acute desensitization, receptor internalization, and tolerance and examined four MOR variants with different alanine or glutamate mutations in the C terminus. Alanine mutations in the sequence between amino acids 375 and 379 (STANT-3A) and the sequence between amino acids 363 and 394 having four additional alanine substitutions (STANT + 7A) reduced desensitization and two measures of long-term tolerance. After chronic morphine treatment, alanine mutations in the sequence between 354 and 357 (TSST-4A) blocked one measure of long-term tolerance (increased acute desensitization and slowed recovery from desensitization) but did not change a second (decreased sensitivity to morphine). With the expression of receptors having glutamate substitutions in the TSST sequence (TSST-4E), an increase in acute desensitization was present after chronic morphine treatment, but the sensitivity to morphine was not changed. The results show that all 11 phosphorylation sites contribute, in varying degrees, to acute desensitization and long-term tolerance. That acute desensitization and tolerance are not necessarily linked illustrates the complexity of events that are triggered by chronic treatment with morphine. SIGNIFICANCE STATEMENT: In this work, we showed that the degree of phosphorylation on the C terminus of the µ-opioid receptor alters acute desensitization and internalization, and in measures of long-term tolerance to morphine. The primary conclusion is that the degree of phosphorylation on the 11 possible sites of the C terminus has different roles for expression of the multiple adaptive mechanisms that follow acute and long-term agonist activation. Although the idea that acute desensitization and tolerance are intimately linked is generally supported, these results indicate that disruption of one phosphorylation cassette of the C terminus TSST (354-357) distinguishes the two processes.

Regulators of G-Protein Signaling (RGS) Proteins Promote Receptor Coupling to G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channels.

Regulators of G-protein signaling (RGS) proteins negatively modulate presynaptic µ-opioid receptor inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). Paradoxically, we find that G-protein-coupled receptor (GPCR) activation of G-protein-gated inwardly rectifying K+ channels (GIRKs) in the vlPAG is reduced in an agonist- and receptor-dependent manner in transgenic knock-in mice of either sex expressing mutant RGS-insensitive Gαo proteins. µ-Opioid receptor agonist activation of GIRK currents was reduced for DAMGO and fentanyl but not for [Met5]-enkephalin acetate salt hydrate (ME) in the RGS-insensitive heterozygous (Het) mice compared with wild-type mice. The GABAB agonist baclofen-induced GIRK currents were also reduced in the Het mice. We confirmed the role of Gαo proteins in µ-opioid receptor and GABAB receptor signaling pathways in wild-type mice using myristoylated peptide inhibitors of Gαo1 and Gαi1-3 The results using these inhibitors indicate that receptor activation of GIRK channels is dependent on the preference of the agonist-stimulated receptor for Gαo versus that for Gαi. DAMGO and fentanyl-mediated GIRK currents were reduced in the presence of the Gαo1 inhibitor, but not the Gαi1-3 inhibitors. In contrast, the Gαo1 peptide inhibitor did not affect ME activation of GIRK currents, which is consistent with results in the Het mice, but the Gαi1-3 inhibitors significantly reduced ME-mediated GIRK currents. Finally, the reduction in GIRK activation in the Het mice plays a role in opioid- and baclofen-mediated spinal antinociception, but not supraspinal antinociception. Thus, our studies indicate that RGS proteins have multiple mechanisms of modulating GPCR signaling that produce negative and positive regulation of signaling depending on the effector.SIGNIFICANCE STATEMENT Regulators of G-protein signaling (RGS) proteins positively modulate GPCR coupling to GIRKs, and this coupling is critical for opioid- and baclofen-mediated spinal antinociception, whereas µ-opioid receptor-mediated supraspinal antinociception depends on presynaptic inhibition that is negatively regulated by RGS proteins. The identification of these opposite roles for RGS proteins has implications for signaling via other GPCRs.