Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Buprenorphine exerts its antinocicepttve activity via μ1-opioid receptors

The mechanism of the antinociceptive effect of buprenorphine was assessed by administering selective μ-, μ₁-, δ- and κ-opioid receptor antagonists in mice. Intraperitoneal administration of buprenorphine, at doses of 0.3 to 3 mg/kg, produced dose-dependent antinociception in the tail-flick test. The antinociceptive activity of buprenorphine did not result from the activation of κ- or δ-opioid receptors, since treatment with either nor-binaltorphimine, a selective κ-opioid receptor antagonist, or naltrindole, a selective δ-opioid receptor antagonist, was completely ineffective in blocking buprenorphine-induced antinociception. However, the antinociceptive effect of buprenorphine was significantly antagonized by β-funaltrexamine, a selective μ-opioid receptor antagonist. Moreover, selective μ₁-opioid receptor antagonists, naloxonazine and naltrexonazine, also significantly antagonized the antinociceptive effect of buprenorphine. Co-administration of κ- and δ-opioid receptor antagonists with the μ-opioid receptor antagonists had no significant effect on the antagonistic profiles of the μ-opioid receptor antagonists on the antinociceptive effect of buprenorphine. These results suggest that buprenorphine acts selectively at μ₁-opioid receptors to induce antinociceptive effects in mice.     

The preference of putative pro-enkephalins for different types of opiate receptors

Certain endogenous opioid peptides, which structurally resemble potential enkephalin precursors (pro-enkephalins), were tested for their interaction with μ-, δ- and κ-type of opiate receptors. These studies employed the technique of the development of tolerance towards selective opioid agonists on the isolated guinea-pig ileum and mouse vas deferens preparations. Additionally, binding studies in rat brain homogenate were performed to determine the affinity of these compounds towards different receptor sites in the CNS. These investigations revealed that dynorphin_1–13 and α-neo-endorphin_1–8 may preferentially interact with the κ-type of opiate receptors, dynorphin_1–9 exhibits both δ- and κ-agonistic activity and met-enkephalin-Arg⁶-Phe⁷ is a ligand with selectivity for δ-receptors.     

Design,synthesis and SAR analysis of novel selective σ1 ligands (Part 2)

In order to investigate the molecular features involved in sigma receptors (σ-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards σ₁- and σ₂-Rs subtypes was evaluated. The most promising compounds were also screened for their affinity at μ-opioid, δ-opioid and κ-opioid receptors. Biological results are herein presented and discussed.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Demonstration of κ3-Opioid Receptors in the SH-SY5Y Human Neuroblastoma Cell Line

Abstract: In addition to the μ- and δ-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of κ₃ receptors, accounting for 40% of total opioid binding, as measured with [³H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in κ₃ binding. Fully 75% of the increased binding corresponds to κ₃ sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the μ antagonist d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP). Naloxone benzoylhydrazone, a κ₃ agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional κ₃ receptors.     

Insulin receptors are bivalent as demonstrated by photoaffinity labeling.

Insulin receptors in human placental membranes were photoaffinity-labeled with a radioactive human insulin-like growth factor I (hIGF-I) photoprobe N epsilon B28-monoazidobenzoyl 125I-hIGF-I either alone or together with a non-radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl insulin. Precipitation of the solubilized receptors with anti-insulin antibody showed that receptors labeled with the radioactive hIGF-I photoprobe were detected in the immunoprecipitate only when photolabeling was carried out in the presence of the non-radioactive insulin photoprobe. Comparable results were obtained in converse experiments using a radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl 125I-insulin, a non-radioactive hIGF-I photoprobe N epsilon B28-monoazidobenzoyl hIGF-I, and an antibody to hIGF-I. The amount of radioactive receptors precipitated by either the anti-insulin antibody or the anti-hIGHF-I antibody was close to the expected amount. These observations demonstrate that the insulin receptor is bivalent being capable of binding two molecules of ligand.     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

Identification of Mammalian Brain and Spinal Cord Opioid Receptor by Photoaffinity Labeling

Abstract

A radioiodinated photoreactive enkephal in derivative, ¹²⁵I(D-Ala² p-N₃-Phe⁴-Met⁵) enkephalin, was used to photoaffinity label the opioid receptor from the membranes of four mammalian brains (without cerebellum) and spinal cords. These included the cat, rabbit, guinea pig and mouse. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 43,000-daltons protein was specifically photolabeled in all the membranes tested, as the specific labeling of this protein was inhibited in the presence of 14.5 uM of (D-Ala² Met⁵) enkephalin. These data suggest that the 43,000-daltons protein is a binding protein of the opioid receptor in the different mammalian neural tissues.     

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.     

Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: A study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain

Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose.After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist. Published in 2004.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by δ1-but not δ2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of δ-opioid receptor agonists [D-Pen^2,5]enkephalin (DPDPE) and [D-Ala²]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective δ₁-opioid receptor antagonist, but not with naltriben (NTB), a selective δ₂- opioid receptor antagonist. There were no significant differences in the anticiceptive effect of [D-Ala²]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala²]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal δ₁-opioid receptor-mediated antinociception, but are normally responsive to activation of δ₂-opiod receptors.     

Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands

Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand–receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (K_i = 2 nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (K_i = 21, 36 and 39 nM).     

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.