Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

Distinct Differences Between Morphine- and [d-Ala2,N-MePhe4,Gly-ol5]-Enkephalin- μ-Opioid Receptor Complexes Demonstrated by Cyclic AMP-Dependent Protein Kinase Phosphorylation

Abstract: The present study demonstrates a conditional, agonist-dependent phosphorylation of the μ-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC₅₀∼40 n M ) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 µ M morphine and 6 n M PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and β-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent μ-opioid-selective peptide [ d -Ala², N -MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) or other enkephalin analogues such as [ d -Ala²]-Met⁵-enkephalinamide (DALA), [ d -Ala², d -Leu⁵]-enkephalin (DADLE), and Met⁵-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.     

ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis

Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment with the mu receptor agonist DAMGO ([d-Ala(2), Me Phe(4), Glyol(5)]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor.     

Cloning, Characterization, and Distribution of a μ-Opioid Receptor in Rat Brain

Abstract: We report the isolation and characterization of a rat cDNA clone encoding a μ-opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ-and K -opioid receptors. Transient expression of the receptor in COS cells revealed high-affinity binding of μ-selective opioid antagonists and agonists, with a K _D for naloxone ∼1.5 n M , and for [D-Ala², N -Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) and morphine at the high-affinity site of 2–4 n M , confirming a μ-opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ-opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ-opioid receptor.     

Phosphorylation and Agonist-Specific Intracellular Trafficking of an Epitope-Tagged μ-Opioid Receptor Expressed in HEK 293 Cells

Abstract: We expressed the cloned μ-opioid receptor (μR) in high abundance (5.5 × 10⁶ sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-μR) was similar to the untagged μR in ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the μ-specific peptide agonist [ d -Ala²,MePhe⁴,Glyol⁵]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-μR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-μR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at ∼70 kDa with weaker bands at ∼65 kDa. EE-μR was labeled with [γ-³²P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65–70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced ∼1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the μR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.     

Mutagenesis of a Single Amino Acid in the Rat μ-Opioid Receptor Discriminates Ligand Binding

Abstract: To investigate the role of Asp¹¹⁴ in the cloned rat μ-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant μ receptor D114N. The wild-type μ receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The μ-selective agonists [ d -Ala²,MePhe⁴,Gly-ol⁵]enkephalin and morphine and the endogenous peptides Met-enkephalin and β-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type μ receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant μ receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N μ receptors to inhibit adenylyl cyclase. Although the Asp¹¹⁴ to Asn¹¹⁴ mutation reduced full-agonist binding, mutation of His²⁹⁷ to Asn²⁹⁷ in the μ receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the μ receptor than do the prototypical full agonists.     

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.     

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils

Abstract Protein kinase C (PKC) appears to have a central role in the O₂⁻ response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP₂)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling of PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O₂⁻. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.     

Role of Protein Kinase C (PKC) in Agonist-Induced μ-Opioid Receptor Down-Regulation

Abstract : Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express μ- and δ-opioid receptors, we investigated whether μ-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by ³H-phorbol ester and [³H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that μ-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [ d -Ala², N -Me-Phe⁴, Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [³H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.     

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.     

Characteristics of the reverse reaction of NADP+-isocitrate dehydrogenase from castor bean seeds

The reductive carboxylation of α-ketoglutarate by purified NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) from maturing castor bean seeds ( Ricinus communis L. ) has been characterized. The optimum pH for the reaction was 6.5, whereas pH 8.5 was optimum for oxidation of isocitrate (forward reaction). The enzyme utilized NADH as well as NADPH as the reducing agent in the reverse reaction, but only NADP⁺ in the forward reaction. The Km values for NADPH and NADH were 0.044 and 2.8 m M respectively, and for α-ketoglutarate and HCO₃ 4.1 and 3.7 m M. The enzyme was activated by various cations including Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Ni²⁺ and Co²⁺. Km values for Mg²⁺ Mn²⁺, Co²⁺ and Zn²⁺ were 12, 34, 37 and 49μ M respectively.     

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [ND_in(NADPH)] in the oxidation of strictly NAD⁺-linked substrates by pea ( Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 μ M diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of ND_in(NADPH) during glycine oxidation (measured both as O₂ uptake and as CO₂ release) was 40–50 nmol mg⁻¹ protein min⁻¹. No significant activity of ND_in(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD⁺-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 μ M , DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, ND_in(NADPH), and ND_in(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD⁺ and NADP⁺, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.     

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala², N-MePhe⁴,Gly-ol⁵] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.     

Amyloid beta peptide and NMDA induce ROS from NADPH oxidase and AA release from cytosolic phospholipase A2 in cortical neurons

Increase in oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurodegenerative diseases including Alzheimer's disease. There is evidence for involvement of amyloid-β peptide (Aβ) in mediating the oxidative damage to neurons. Despite yet unknown mechanism, Aβ appears to exert action on the ionotropic glutamate receptors, especially the N-methyl-D-aspartic acid (NMDA) receptor subtypes. In this study, we showed that NMDA and oligomeric Aβ_1–42 could induce reactive oxygen species (ROS) production from cortical neurons through activation of NADPH oxidase. ROS derived from NADPH oxidase led to activation of extracellular signal-regulated kinase 1/2, phosphorylation of cytosolic phospholipase A₂α (cPLA₂α), and arachidonic acid (AA) release. In addition, Aβ_1–42-induced AA release was inhibited by d (−)-2-amino-5-phosphonopentanoic acid and memantine, two different NMDA receptor antagonists, suggesting action of Aβ through the NMDA receptor. Besides serving as a precursor for eicosanoids, AA is also regarded as a retrograde messenger and plays a role in modulating synaptic plasticity. Other phospholipase A₂ products such as lysophospholipids can perturb membrane phospholipids. These results suggest an oxidative-degradative mechanism for oligomeric Aβ_1–42 to induce ROS production and stimulate AA release through the NMDA receptors. This novel mechanism may contribute to the oxidative stress hypothesis and synaptic failure that underline the pathogenesis of Alzheimer's disease.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

Biochemical Evidence of Functional Interaction Between μ- and δ-Opioid Receptors in SK-N-BE Neuroblastoma Cell Line

Abstract: Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of μ- and δ-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [ d -Ala²- N -methyl-Phe⁴-Gly-ol⁵]enkephalin (DAMGO), a μ-selective agonist, sensitized the functional response of the δ-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [ d -Pen^2,5]enkephalin (DPDPE), a δ-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for μ receptors, increased the inhibitory effect of [ d -Ala², d -Met⁵]enkephalinamide (DAME), a mixed μ/δ agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between μ and δ receptors in the SK-N-BE cell line.     

C-terminal splice variants of the mu-opioid receptor: existence, distribution and functional characteristics

The distribution of the mRNA of different C-terminal splice variants of the μ-opioid receptor in rat CNS was assessed by RT-PCR. The mRNA species for MOR1, MOR1A and MOR1B were readily detectable and distributed widely throughout the rat CNS, with levels of MOR1 and MOR1A mRNA being overall greater than for MOR1B. We did not find convincing evidence that significant levels of MOR1C, MOR1C1, MOR1C2 and MOR1D are present in rat CNS. To examine possible differences in the agonist-induced regulation of MOR1, MOR1A and MOR1B, we expressed these constructs in HEK293 cells along with G-protein-coupled inwardly rectifying K⁺ channel subunits and measured the rate and extent of desensitisation of ( d -Ala², N -Me-Phe⁴,glycinol⁵)-enkephalin (DAMGO)- and morphine-induced G-protein-coupled inwardly rectifying K⁺ currents. Morphine-induced desensitisation was rapid for all three splice variants ( t _½: 1.2–1.7 min) but DAMGO-induced desensitisation was significantly slower for MOR1B ( t _½ 4.2 min). Inhibition of endocytosis by expression of a dynamin-dominant negative mutant increased the rate of DAMGO-induced desensitisation of MOR1B. These data show that some splice variants of μ-opioid receptor are widely expressed in rat CNS but question the existence of others that have been reported in the literature. In addition, whereas the rate of desensitisation of MOR1 and MOR1A is agonist-independent, that for MOR1B is agonist-dependent.     

Site Mutation in the Rat μ-Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin-Dependent Protein Kinase II in Agonist-Mediated Desensitization

Abstract: The rat μ-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on μ-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the μ-opioid receptor. The increase of desensitization could not be observed with a μ-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser²⁶¹ and Ser²⁶⁶ by CaM kinase II is involved in the desensitization of the μ-opioid receptor.     

Functional Coupling of the δ-, μ-, and κ-Opioid Receptors to Mitogen-Activated Protein Kinase and Arachidonate Release in Chinese Hamster Ovary Cells

Abstract: To examine whether the mitogen-activated protein kinase (MAPK) cascade and phospholipase A₂ (PLA₂) are involved in the signal transduction mechanism of the opioid receptor, the δ-, μ-, and κ-opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the δ-, μ-, and κ-receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42-kDa isoform of MAPK (p42), probably owing to phosphorylation. The opioid receptor-mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12-myristate 13-acetate and pretreatment with GF 109203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the δ-, μ-, and κ-receptors with opioid agonists in the presence of A23187, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA₂ is activated by the opioid receptors when the intracellular Ca²⁺ concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by G_i or G_o types of GTP-binding regulatory proteins.