Proc. Nat!. Acad. Sci. USA Vol. 89, pp. 12048-12052, December 1992 Neurobiology

The 6opioid receptor: Isolation of a cDNA by expression cloning and pharmacological characterization (G protein-coupled receptor/NG 108-15 cells/transient expression/Tyr-D-Thr-Gly-Phe-Leu-Thr/analgesia)




Ecole Supdrieure de Biotechnologie, 11 rue Humann, 67085 Strasbourg Cedex, France

Communicated by Pierre Chambon, August 31, 1992

A random primed expression cDNA library ABSTRACT was constructed from the RNA of NG 108-15 cells. Pools of plasmid DNA were transfected into COS cells, which were screened for their ability to bind 'H-labeled Tyr-D-Thr-GlyPhe-Leu-Thr, a tritiated agonist for the -opioid receptor. A cDNA was isolated that encodes a 371-amino acid-residue protein presenting all the structural characteristics of receptors that interact with guanine nucleotide-binding proteins. Noticeable features are (i) the high hydrophobicity of the encoded protein, (it) its low sequence similarity to both catecholamine receptors and peptide-binding receptors, although it presents the typical aspartate residue involved in catecholamine binding of the first group and the characteristic short third cytoplasmic loop of the second group. When expressed in COS cells, the receptor exhibits pharmacological properties similar to those of the native receptor: high-affinity binding sites for 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr (Kd = 1.4 nM), stereospecific binding sites for the - enantiomers of levorphanol and naloxone, and the selectivity prorde of a 8 receptor, as determined by competition experiments with a set of EL-, 8-, and K-oploid ligands.

dence that a cDNA encoding an opioid receptor has been cloned. We report here the isolation of a cDNA encoding a 8-opioid receptorA We used a transient expression strategy where a cDNA library derived from NG 108-15 cells was screened using 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET), a tritiated agonist for the 8-opioid receptor. Our pharmacological study of the cloned cDNA expressed in COS cells shows that it possesses all expected properties of a 8 receptor. A comparative analysis of the deduced protein sequence with other members of the GPR family is also presented.

MATERIALS AND METHODS Library Construction and Screening. NG 108-15 cells were provided by B. Foucaud (URA 1836, Facultd de Pharmacie, Strasbourg, France). Cells were harvested at 50% confluency, RNA was prepared by LiCl/urea precipitation (6), and polyadenylylated RNA was purified with an oligo(dT) column (Pharmacia). The cDNA library construction in the mammalian expression vector pCDM8 has been described (7). Plasmid DNA was prepared according to the alkaline lysis method (8) from pools of 3000 bacterial colonies. One-tenth of each plasmid pool DNA was independently transfected into COS-1 cells (American Type Culture Collection CRL/1650) using the DEAE-dextran method. After 72 hr, transfected COS-1 cells were assayed for tritiated DTLET binding (61 Ci/mmol, Commissariat a l'Energie Atomique, Saclay, France; 1 Ci = 37 GBq). Cell monolayers were washed twice with PBS/0.5% bovine serum albumin and incubated 35 min at 370C with PBS/0.5% bovine serum albumin/1 nM [3H]DTLET. Dishes were chilled on ice for 5 min, and the cells were washed four times with ice-cold PBS/0.5% bovine serum albumin. The cells were then solubilized in 1% SDS and added to 7 ml of scintillation liquid for counting. Positive pools were fractionated by diluting an aliquot of the glycerol stock in selective medium, growing the bacteria on nitrocellulose membranes overlaid onto agar plates. Pieces of membrane were then cut to isolate pools of colonies. Each of them was scraped into selective medium and grown to 0.5 OD6wn. unit. An aliquot was stored as a glycerol stock for further fractionation, whereas the rest was used for plasmid DNA preparation and COS cell transfection. Ligand Binding. [D-Ala2,D-Leu5ienkephalin (DADLE), cyclic [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE),

Opioid receptors have long been described as membrane receptors of the nervous system mediating the analgesic effects of opium-derived alkaloids. Endogeneous ligands and their precursors have been characterized, and their role in response to pain and stress has been widely studied (1). Pharmacological studies have shown the existence of three subtypes of receptors, ,u (morphine), 8 (enkephalin), and K (dynorphin), the cellular inhibitory actions of which are linked to G protein activation (2). Opioid receptors are, therefore, believed to be part of the G protein-coupled receptor (GPR) family, a class of membrane-bound receptors that exhibit a seven-transmembrane-spanning domain topology and represent 80% of all known receptors (3). Several attempts to clone cDNAs encoding opioid receptors have been reported. A cDNA encoding an opioid-binding protein (OBCAM) with g selectivity was isolated (4), but the predicted protein lacks transmembrane domains, presumed necessary for signal transduction. More recently, the isolation of another cDNA was reported, which was obtained by expression cloning (5). The deduced protein sequence displays seven putative transmembrane domains and is very similar to the human neuromedin K receptor. However, the affinity of opioid ligands for this receptor expressed in COS cells is two orders of magnitude below the expected value, and no subtype selectivity can be shown. Finally, attempts to clone opioid receptors via approaches relying on sequence similarities in the GPR family, using the PCR technology, were unsuccessful. There is, therefore, no convincing evi-

[D-Ala2,D-Leu51enkephalin; DAGO, [D-Ala2,MePhe4,Gly-ol5lenkephalin; DTLET, Tyr-D-Thr-Gly-PheLeu-Thr; DPDPE, cyclic [D-penicillamine2,D-penicillamine5]en-

Abbreviations: DADLE,

kephalin; G protein, guanine nucleotide-binding regulatory protein; GPR, G protein-coupled receptor; Tm, transmembrane domain; U 50488, trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyllbenzeneacetamide. *To whom reprint requests should be addressed. tDeceased, May 29, 1992. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L06322).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.


Neurobiology: Kieffer et al. [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) and trans-(+)3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U 50488) were obtained from Sigma. (-)Naloxone, (+)-naloxone, levorphanol, dextrorphan, bremazocin, and etonitazen were provided by B. Ilien (20). Binding was assayed with membrane preparations. COS-1 cells were transfected with plasmid K56 or with pCDM8 (mock), as before, and were harvested after 72-hr expression time. NG 108-15 cells were collected at 50%o confluency. The cells (3-10

x 107) were washed two times with PBS, pelleted, and frozen prepared at 40C with a single buffer, 50 mM Tris HCI, pH 7.4/10 mM EDTA. The cell pellet was resuspended in 60 ml of buffer, treated with a Dounce homogenizer, and centrifuged at 1100 x g for 10 min. The pellet was resuspended a second time in buffer (30 ml), homogenized, and centrifuged again. Both supernatants were pooled and centrifuged at 110,000 x g for 15 min. The membrane pellet was resuspended in 5 ml of buffer, aliquoted, and stored at -800C. The protein concentration was determined using the Bradford assay (9). Typical binding assays were done in 1-ml volumes by using membranes diluted in the same Tris/EDTA buffer (15-30 jg of protein per ml), competitors, and [3H]DTLET as the radiolabeled ligand. After a 30-min incubation at 370C, the membrane suspension was rapidly filtered on polyethylenimine-precoated GF/B Whatman filters and washed three times with 3 ml ofcold buffer. For competition studies, [3H]DTLET was used at a 1 nM concentration. The Ki values were derived from the Cheng and Prussof equation: K, = lC50A(1 + L/Kd). at -80'C. The membranes were then

Sequence Analysis. Oligonucleotides were synthesized on Applied Biosystems 394 DNA/RNA synthesizer. The cDNA was sequenced in both directions by using the Sanger dideoxynucleotide chain-termination method (Sequenase kit, United States Biochemical). Data base searching was done on Swiss-Prot (release 21, Feb. 92) with BLASTP. The sequence alignments and the dendrogram were done with the CLUSTAL multiple-alignment program (10). The hydrophobicity analysis was determined by the method of Kyte and Doolittle (11) with a window of 11 residues and the transmembrane-domain prediction according to Eisenberg (12) by using the PC GENE program (IntelliGenetics). PCR on Mouse Genomic DNA and Sequence Analysis. A set of oligonucleotides was synthesized based on the cDNA sequence (position 1857-1877 for the forward primer and position 2154-2172 for the reverse primer). PCR amplification was done on a mouse genomic DNA preparation (13) using standard conditions (94°C 1 min, 500C 1.5 min, 72°C 2 min, 30 cycles). The PCR product was subcloned into EcoRI sites of pBluescript (Stratagene) and sequenced in both an


Proc. Natl. Acad. Sci. USA 89 (1992)


vided twice, leading to a single clone, K56, which conferred strong [3H]DTLET-binding capability to COS cells. The signal was six times above the background value, naloxone sensitive, and ligand-concentration dependent. Pharmacology of the Cloned cDNA Transiently Expressed in COS Cells. We first estimated the affinity of [3H]DTLET for K56-expressing COS cells. Fig. 1A shows that a high specific binding is observed on K56-transfected COS cell membranes, whereas it is negligible for mock-transfected cells. [3H]DTLET binding on NG 108-15 membranes, using the same amount of membrane proteins in the assay, is shown for comparison. Scatchard analysis of [3H]DTLET binding A




c) *-.


log[competitor] V)

.0 0


RESULTS Isolation of cDNA K56 by Expression Cloning. We constructed a random-primed cDNA library from NG 108-15

cells (14) expressing 5 x 104 B-opioid receptor molecules per cell, using the mammalian transient expression vector pCDM8 (15). The library contained 3.5 x 106 primary transformants. A part of it was plated and partitioned into 100 pools of 3000 different recombinant bacterial colonies. COS cells transfected with plasmid DNA from these pools were incubated with [3H]DTLET at a 1 nM concentration, which is equivalent to its Kd value for the native receptor. The bound radioactivity was counted after cell lysis. The background was 700 dpm ± 250, and a single fraction produced a signal that was consistently 10% above background. This fraction was subdivided, and the subfractions were tested in COS cells again. Several subfractions produced a signal that emerged 20% above background and disappeared in the presence of naloxone. One of these subfractions was subdi-








FIG. 1. Pharmacological properties of K56-expressing COS cells. For all figures a representative experiment is presented. (A) Affinity of [3H]DTLET for the expressed receptor. Membranes prepared from K56-(m) and pCDM8-(A) transfected COS cells or from NG108-15 (r) cells were incubated with increased concentration of [3H]DTLET ± (-)-naloxone (10-6 M), the difference being defined as specific binding. Assays were done in triplicates. (Inset) Scatchard analysis: Kd is 1.4 nM for both expressed and native receptors, and B". values are 4.2 and 1.2 pmol/mg, respectively. Three independent experiments yielded a Kd value of 1.45 ± 0.05 nM for [3H]DTLET binding to K56-transfected COS cells. (B and C) Competition of [3H]DTLET binding with unlabeled competitors on K56-transfected COS cells. Assays were done in duplicates, and specific binding without competitors was defined as 1N0.


Neurobiology: Kieffer et al.

shows a single class of binding sites with an apparent Kd of 1.4 nM for both K56-transfected COS cell and NG 108-15 cell membranes. This value is similar to the described value (1.04 nM) (16). Bm. values ranged from 3.9 to 6.4 pmol/mg of proteins, depending on the transfected membrane preparation. Considering that, on average, 10% only of the COS cells are efficiently transfected by the plasmid, the expression level of the cloned receptor could be estimated to 5 x 106 molecules per transfected cell, which is 100 times higher than the value determined for NG 108-15. Opioid receptors are highly stereoselective. Levorphanol and (-)-naloxone are known to bind to opioid receptors with high affinity, whereas their (+) enantiomers, dextrorphan and (+)-naloxone, respectively, do not, irrespective of the receptor subtype under study (5, 17). Competition experiments were done with [3H]DTLET with the two pairs of ligands. Fig. 1B indicates that (+)-naloxone is not a competitor. A slight inhibition is seen with dextrorphan at a 1 gM concentration. This agrees with previous data that report that dextrophan has a threeorders-of-magnitude lower binding potency at opioid-binding sites, as compared with levorphanol. Both (-) enantiomers yielded Ki values that meet their reported affinities for a 8-opioid receptor (29.5 nM for naloxone and 20.9 nM for levorphanol, see Table 1). We further investigated the subtype specificity of the cloned receptor by competition experiments between a set of ,u, 6, and K agonists with [3H]DTLET. Fig. 1C shows that they all compete with an order of potency typical of the 6-opioid receptor (17, 18, 20): bremazocin = DADLE = DPDPE >> etonitazen > DAGO > U 50488 with Ki values of 5.7, 6.2, 10.9, 1800, 5050, and 39,100 nM, respectively. We did the same experiment on NG 108-15 membranes and found the same profile with similar affinities. The Ki values are also comparable with values from the literature (see Table 1). In conclusion, the K56 cDNAencoded receptor expressed in COS cells displays binding characteristics similar to that of the native receptor. Primary Structure. K56 contains a 2.2-kilobase (kb) insert with an open reading frame of 1174 base pairs (bp) starting from the 5' end. The first ATG at position 59 was assigned as the translational initiation codon for the protein. The nucleotides surrounding this ATG are in total agreement with the Kozack nucleotide sequence CCATGG (19). No stop codon was found upstream of this ATG. The predicted protein sequence consists of 371-amino acid residues, with a calcuTable 1. Binding potency of opioid ligands for K56 expressed in COS cells pK; Data from literature (ref.) NG Subtype COS-K56 108-15 (17) (18) (19) preference Ligand K = IL = 6 8.25 ± 0.12 8.75 8.8 9.14 Bremazocin DADLE 8 = IL >>> K 8.22 ± 0.15 8.70 9.1 8.82 6>> ,I >> K 7.73 ± 0.48 8.30 8.9 8.57 DPDPE Levorphanol u >> K = 8 7.68 ± 0.07 ND 7.8 (-)-Naloxone IL > 8 = K 7.53 ± 0.09 ND 7.6 - - >> 8 » 6 > K 5.21 ± 0.17 5.65 6.7 6.46 >> DAGO U 50488 K >>> /A > 8 4.35 ± 0.15 4.24 5.0 5.06 pKj values (- log Ki) are determined by competition of unlabeled opioid ligands with [3HIDTLET on K56-transfected COS cells (mean + SEM of three independent experiments) and NG 108-15 cells, as described (see also Fig. 1 B and C). Values found in the literature [columns (17) and (18)] derive from competition experiments done on guinea pig brain membranes (except for etonitazen tested on rat) and show pKi values for the 6 subtype in this tissue. ND, not determined; -, value not found in cited reference. The ligands, including [3H]DTLET, are agonists, except naloxone, which is an antagonist.

Proc. NatL Acad. Sci. USA 89 (1992)

lated Mr of 40,810. The native receptor is a glycoprotein, and the difference between the predicted size and the 58,000-Da molecular mass of the purified NG 108-15 receptor, as estimated by SDS/PAGE (21) is probably due to the attachment of carbohydrates on the two potential N-glycosylation sites present at the N terminus of the predicted protein sequence. K56 cDNA encodes a protein that belongs to the class of GPRs. The sequence is shown on Fig. 2A aligned (22) with four other representative members of the family: the %2adrenergic receptor, the neuromedin K receptor, and the rhodopsin, as well as the N-formyl peptide receptor, with which it shares the best similarity (30%o identity, 10 gaps) among sequences in the Swiss-Prot Data Bank. The conserved residues that are virtually found in all GPRs are present in K56 protein. The encoded protein also contains the potential sites for posttranslational modifications usually found in GPRs: in addition to putative N-glycosylation sites, three potential phosphorylation sites are present in the third cytoplasmic loop, as well as three others in the C-terminal region. The localization of the seven transmembrane domains, as defined by homology with other receptors, agrees well with the hydrophobicity analysis and meets the Eisenberg prediction (Fig. 2B). The size of the protein is small compared with other GPRs, which range from 324 (mas oncogene) to 744 (thyroid-stimulating hormone receptor) amino acid residues. The sequence contains short intra- and extracellular loops, and the hydrophobic domains are, therefore, predominant. The regions that display the higher hydrophilic character concern short stretches of amino acid residues in the third cytoplasmic loop and in the C-terminal tail, regions which in GPRs presumably interact with the intracellular compartment of the cell and are important for signal transduction. Species Origin of K56 Protein. The NG 108-15 cell line used to prepare the library is a hybrid cell line between mouse N18TG neuroblastoma and rat C6Bu-1 glioma cell lines, and the species origin of K56 DNA remained to be determined. Using mouse and rat genomic DNA as a template, we amplified by PCR a 350-nucleotide fragment corresponding to positions 1857-2172 of the cDNA, a 3' noncoding region presumed not conserved among different species. The PCR on mouse genomic DNA produced a single discrete band of the expected size, which was subcloned and sequenced. We found a 100%6 match between this sequence and the cDNA sequence. We, therefore, conclude that K56 encodes a mouse receptor. Sequence Similarity Analysis Within the GPR Family. The sequence of K56 protein contains an Asp residue (position 128) present in catecholamine GPRs only, but it also displays a short third cytoplasmic loop typical of GPRs that have peptides as ligands. To determine the position of K56 receptor in the GPR family, a dendrogram was established, based on sequence similarities of K56 protein with other members of the family (Fig. 2C). Seventeen mouse and rat sequences (preferably mouse when available except for the N-formyl peptide receptor, which is a human sequence), which displayed the best homology with K56 protein, were selected from the Swiss-Prot Data Base. As expected, the aminergic receptors and the peptidergic receptors appear as two distinct groups, and rhodopsin appears as a single sequence. K56 and the human N-formyl peptide receptor (23) form a group separate from the two main groups. In a second alignment that excluded all extra- and intracellular loops and considered the transmembrane domains only, K56 protein appeared as a group with the N-formyl peptide receptor again but closer to a group including the substance K, substance P, neuromedin K, and neuromedin B receptors (data not shown).


Proc. Natl. Acad. Sci. USA 89 (1992)

Neurobiology: Kieffer et al.






























Mouse rhodopsin Rat neurotensin receptor Mouse thyrotropin-releasing hormone receptor Rat neurokinin A (SK) receptor Rat neuromedin K receptor Rat substance P receptor Rat neuromedin B / bombesin receptor Rat serotonin Ic receptor Rat dopamine D2 receptor Rat alpha 2 adrenergic receptor Rat dopamine D3 receptor Mouse beta 2 adrenergic receptor Rat alpha adrenergic receptor Rat m4 muscarinic acetylcholine receptor Mouse ml muscarinic acetylcholine receptor Human N-formyl peptide receptor K56


-20o -30 -40 -50








C 50 40 30 20




140 210 Residue number





FIG. 2. Sequence analysis of K56. (A) Deduced protein sequence aligned with other GPRs. N FORM P., human N-formyl peptide receptor; NEU K., rat neuromedin K receptor; B2 ADR., mouse P2-adrenergic receptor; RHODOP., mouse rhodopsin. The putative transmembrane domains for these receptors are boxed. *, Highly conserved amino acids among GPR family; #, potential phosphorylation sites; and *, consensus N-linked glycosylation sites. (B) Hydrophobicity analysis (11). Hydrophobic domains of the protein appear above the dotted line. Positions of putative transmembrane domains (12) are indicated by short lines, numbered I-VII, as in A. (C) Homology of K56 with other GPRs (10). This dendrogram represents the relative homology between the sequences. Horizontal distances are inversely proportional to percentage homology.

DISCUSSION Our cloning strategy involved the use of a tritiated ligand (61 Ci/mmol) for the detection of receptor-expressing COS cells. All previously reported ligands used in this approach are radiolabeled with isotopes of high specific activity (>2000 Ci/mmol). Our experiment shows that a ligand with low specific activity can be used successfully in expression cloning, provided that the library is partitioned into pools of limited size. NG 108-15 cells express a single type of opioid receptor that has long been characterized as the subtype (24). Two lines of evidence indicate that we succeeded in isolating a cDNA encoding this receptor: (i) the pharmacological profile exhibited by the K56-expressing COS cells is convincing: the binding assay with [3H]DTLET, a prototypal 8 ligand shows the presence of high-affinity binding sites with Kd similar to the native receptor, and competition experiments demon-

strate that the expressed receptor is stereoselective and binds preferably ligands. (ii) The 8-opioid receptor in NG 108-15 cells has been shown to inhibit adenylate cyclase activity through the activation of a G protein (2) and is, therefore, expected to be a member of the GPR family: the deduced protein sequence of K56 indicates unambiguously that this is the case. Analysis of the K56 protein sequence, with respect to its similarity with other GPRs, brings insight to the understanding of the biochemistry and pharmacology of opioid receptors.

Opioid receptors are sensitive to reducing agents, and the of a disulfide bridge has been postulated as essential for ligand binding (25). For rhodopsin, muscarinic, and 8-adrenergic receptors, the two conserved cysteine residues in each of the two first extracellular loops have been shown critical for stabilizing the functional protein structure and are presumed to do so by forming a disulfide bridge. A occurrence


Proc. Nadl. Acad. Sci. USA 89 (1992)

Neurobiology: Kieffer et al.

S-S bond between Cys-121 and Cys-199 of K56 protein might, therefore, be important for correct protein folding. Structure/function studies of opioid ligands have shown the importance of a protonated. amine group for binding to the receptor with high affinity. The binding site of the receptor might, therefore, possess a critical negatively charged counterpart. Catecholamine receptors display in their sequence a conserved aspartate residue that has been shown necessary for binding the positively charged amine group of their ligands. By analogy, the Asp residue of K56 protein at position 128 might be involved in the interaction of the 6-opioid receptor with its ligands. Compared with other membrane receptors, purified opioid receptors are extremely labile molecules (for review, see refs. 26 and 27). Progress in solubilizing these receptors has long been hampered by their high sensitivity to detergents. Moreover, phospholipids added to detergent extracts have been shown to stabilize the purified receptor conformation in an active state. These experiments suggest the occurrence of critical interactions between the protein and its natural lipidic environment that modulate the protein conformation and, thus, its binding capability. Our finding that the deduced amino acid sequence of K56 is a highly hydrophobic protein, probably almost entirely buried in the membrane, agrees with these observations. The opioid receptors are heterogeneous. The origin of different subtypes is currently a speculative question because no molecular characterization has been achieved. At least three classes of opioid receptors are present in the brain, 1z, 8, and K, which differ in their pharmacology (17) and anatomical distribution (28). However, biochemical characterization of opioid receptors from many groups reports a molecular mass of -60,000 Da for all three subtypes, suggesting that they could be related molecules (27). Moreover, the similarity between the three receptor subtypes is supported by the isolation of (i) antiidiotypic monoclonal antibodies competing with both A& and 8 ligands but not competing with K ligands (29, 30) and (ii) a monoclonal antibody raised against the purified g receptor that interacts with both 1L and K receptors (31). Low-stringency hybridization experiments with K56 cDNA as a probe may allow the isolation ofcDNAs encoding the other opioid-receptor subtypes and shed light on the question of the opioid-receptor heterogeneity. The availability of cDNAs encoding the opioid receptors will then permit detailed studies of the signal-transduction mechanism and reveal the anatomical distribution of the mRNAs of these receptors, providing information on their expression pattern in the nervous system. This information should ultimately allow better understanding of the opioid system in nociception and analgesia and also the design of more specific therapeutic drugs. We are grateful to E. Borrelli, R. Hen, and L. Maroteaux for helpful discussions and support; to F. Plewniak for help with the computer alignments; to F. Ruffenach for the oligonucleotides; and to M. Acker for the cell culture. We thank A. Menez for critical reading of the manuscript. This research was supported by the Ecole Superieure de Biotechnologie, Strasbourg, France (J. F. Lefevre, Director) and by the Laboratoire de Gdndtique Moleculaire des

Eucaryotes Supdrieurs du Centre National de la Recherche Scientifique U/184-Laboratoire de Biologie Mol6culaire et de G6nie Genetique de l'Institut National de la Santc et de la Recherche Medicale, Strasbourg, France (P. Chambon, Director). 1. Akil, H., Watson, S. J., Young, E., Lewis, M. E., Khachaturian, H. & Walker, J. M. (1984) Annu. Rev. Neurosci. 7,

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25. Gioannini, T. L., Liu, Y. F., Park, Y-H., Hiller, J. M. & Simon, E. J. (1989) J. Mol. Recogn. 2, 44-48. 26. Demoliou-Mason, C. D. & Barnard, E. A. (1990) in Receptor Biochemistry:A Practical Approach, ed. Hulme, E. C. (Oxford Univ. Press, New York), pp. 99-122. 27. Loh, H. H. & Smith, A. P. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 123-147. 28. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H. & Watson, S. (1987) J. Neurosci. 7, 2445-2464. 29. Gramsch, C., Sculz, R., Kosin, S. & Herz, A. (1988) J. Biol. Chem. 263, 5853-5859. 30. Coscia, C. A., Szfics, M., Barg, J., Belcheva, M. M., Bem, W. T., Khoobehi, K., Donnigan, T. A., Juszczak, R., McHale, R. J., Hanlley, M. R. & Barnard, E. A. (1991) Mol. Brain Res. 9, 299-306. 31. Bero, L. A., Roy, S. & Lee, N. M. (1988) Mol. Pharmacol. 34,


The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization.

A random primed expression cDNA library was constructed from the RNA of NG 108-15 cells. Pools of plasmid DNA were transfected into COS cells, which w...
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