A Constitutively Internalizing and Recycling Mutant of the μ-Opioid Receptor

Abstract: Internalization and recycling of G protein-coupled receptors (GPCRs), such as the μ-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in μ-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (³⁵⁴ThrSerSerThrIleGluGlnGlnAsn³⁶²) unique to the C-terminus of the μ-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these μ-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wildtype and mutant receptors. Both the wild-type μ-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress μ-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.     

Regulation of Multiple Effectors by the Cloned δ-Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

Abstract: The δ-opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ-opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin. The inhibitory response of [ d -Pen², d -Pen⁵]enkephalin was dependent on the expression of the δ-opioid receptor and exhibited an EC₅₀ of 1 n M . The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ-opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ-opioid receptor with type II adenylyl cyclase and an activated mutant of α_s converted the δ-opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk⁻ fibroblasts, the cloned δ-opioid receptor was able to stimulate the formation of inositol phosphates (EC₅₀ = 8 n M ). This response was sensitive to pertussis toxin. The opioid-mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ-opioid receptor to couple to G proteins other than G_i was also examined. Cotransfection studies revealed that the δ-opioid receptor can utilize G_z to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.     

Role of Src in ligand-specific regulation of δ-opioid receptor desensitization and internalization

The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of δ-opioid receptor (δOR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the δOR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates β-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted β-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen β-arrestin function by dual regulations: promoting β-arrestin recruitment and increasing β-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize δOR, also behaved as TIPP in failure to utilize Src to regulate δOR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate δOR signaling and reveal the molecular events by which Src modulates δOR responsiveness.     

Coupling of the Cloned μ-Opioid Receptor with the ω-Conotoxin-Sensitive Ca2+ Current in NG108-15 Cells

Abstract: Voltage-dependent Ca²⁺ currents were measured in NG108-15 neuroblastoma × glioma hybrid cells transformed to express the rat μ-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba²⁺ as charge carrier. A μ-opioid receptor-selective agonist, [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin caused significant inhibition of voltage-dependent Ca²⁺ currents in μ-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a δ-opioid receptor-selective agonist, [ d -penicillamine², d -penicillamine⁵]enkephalin, induced inhibition of voltage-dependent Ca²⁺ currents in both control and μ-receptor-transformed cells, which is mediated by the δ-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca²⁺ currents induced by [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin and [ d -penicillamine², d -penicillamine⁵]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω-conotoxin GVIA. These results indicate that the μ-opioid receptor expressed from cDNA functionally couples with ω-conotoxin-sensitive N-type Ca²⁺ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.     

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.     

Overexpression of δ-Opioid Receptors in Recombinant Baculovirus-Infected Trichoplusia ni"High 5" Insect Cells

Abstract: "High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ-opioid receptor. The maximal binding capacity for the narcotic antagonist [³H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [³H][ d -penicillamine², d -penicillamine⁵]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [³H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein G_o, which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by G_o. Quantification of Gα_o and Gα_i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gα_o/mg of membrane protein, and a 20-fold higher concentration for Gα_i. The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors.     

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.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

Differential Coupling of μ-, δ-, and κ-Opioid Receptors to Gα16-Mediated Stimulation of Phospholipase C

Abstract: The μ-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G₁₆ protein. Given the promiscuous nature of G₁₆ and the high degree of resemblance of signaling properties of the three opioid receptors, both δ- and κ-opioid receptors are likely to activate G₁₆. Interactions of δ- and κ-opioid receptors with G₁₆ were examined by coexpressing the opioid receptors and Gα₁₆ in COS-7 cells. The δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ-opioid receptor and Gα₁₆. The δ-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators. 13.   Transfusion 1993 ; 33 : 898 – 901 . [PubMed link] 14.   Butson MJ , Yu PK , Cheung T , et al . Dosimetry of blood irradiation with radiochromic film. Transfus Med 1999 ; 9 : 205 – 8 . [PubMed link] 15.   Nath R , Biggs PJ , Ling CC , et al . AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45. Med Phys     

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.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Regulation of Spontaneous Activity of the δ-Opioid Receptor: Studies of Inverse Agonism in Intact Cells

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [³H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine δ-opioid receptor (clone D2). Basal [³H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d -Ala², d -Leu⁵ enkephalin (DADLE) produced strong inhibition of forskolin-amplified [³H]cyclic AMP production, whereas the δ-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC₅₀ (9.4 ± 0.6 × 10⁻⁸ M ) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated K _i. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.     

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.     

Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation

Morphine and other opiates mediate their effects through activation of the μ-opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, β-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of β-arrestin, β-arrestin1 and β-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting β-arrestin2 specifically. In this study, we examine the different contributions of β-arrestin1- and β-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both β-arrestins. Here we show that morphine only recruits β-arrestin2, whereas the MOR-selective enkephalin [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), recruits either β-arrestin. We show that β-arrestins are required for receptor internalization and that only β-arrestin2 can rescue morphine-induced MOR internalization, whereas either β-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, β-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of β-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and β-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.     

SK-N-BE: A Human Neuroblastoma Cell Line Containing Two Subtypes of δ-Opioid Receptors

Abstract: A human neuroblastoma cell line, SK-N-BE, was shown to express a substantial amount of opioid receptors (200–300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of δ-opioid receptors. Results from sucrose-gradient sedimentation experiments were compared with similar data obtained with the μ-opioid receptor of the rabbit cerebellum and the δ-opioid receptor of the hybrid NG108–15 cell line and have shown that the opioid receptor of the SK-N-BE cell line behaved hydrodynamically as an intermediate between μ-and δ-opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK-N-BE cell membranes could belong to two δ-opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of δ-opioid receptor is negatively coupled to the adenylate cyclase via a G_i protein and that the opiate receptors of the SK-N-BE neuroblastoma cell line undergo a rapid down-regulation when preincubated in the presence of the high-affinity opioid agonist, etorphine.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Phosphorylation of Ser363, Thr370, and Ser375 residues within the carboxyl tail differentially regulates mu-opioid receptor internalization

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.     

Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization

Treatment of HEK293 cells expressing the delta-opioid receptor with agonist [d-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor. We constructed several mutants of the potential phosphorylation sites (Ser/Thr) at the carboxyl tail of the receptor in order to delineate the receptor phosphorylation sites and the agonist-induced desensitization and internalization. The Ser and Thr were substituted to alanine, and the corresponding mutants were transiently and stably expressed in HEK293 cells. We found that only two residues, i.e. Thr(358) and Ser(363), were phosphorylated, with Ser(363) being critical for the DPDPE-induced phosphorylation of the receptor. Furthermore, using alanine and aspartic acid substitutions, we found that the phosphorylation of the receptor is hierarchical, with Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cyclase activity, and receptor internalization are intimately related to phosphorylation of Thr(358) and Ser(363), with Thr(358) being involved in the receptor internalization.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.