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

Abstract

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 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. 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 evidence 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). Plas...

Dimerization of the δ Opioid Receptor:

“…1A, Ϫ, Ϫ). The size of this protein corresponds to the size previously described for ␦-opioid receptor (19,20). Interestingly, we also observed an additional band of about 120 kDa (Fig.…”

cDNA cloning and regional distribution of a novel member of the opioid receptor family

“…cDNA library derived from rat cerebrum poly(A)' RNA was screened by hybridization with a DNA fragment comprising a part of the mouse S-opioid receptor subtype cDNA [4,5]. A novel class of cDNA clones (ROR-C) was isolated and nucleotide sequence analysis revealed an open reading frame of 1,101 nucleotides encoding a sequence of 367 amino acids.…”

Novel “Restoration of Function” Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding

“…Construction of the Library of Mutants-pcDNA3-hDOR consists of a 1.2-kilobase cDNA EcoRI-XhoI fragment of the ␦-opioid receptor (23) sub-cloned at the EcoRI-XhoI site of pcDNA3 (Invitrogen). Using unique site elimination (USE) mutagenesis (43), pcDNA3-hDOR was mutated to pcDNA3-␦/ 291-300 by replacing 10 amino acids (positions 291-300) of the third extracellular loop of the ␦ receptor with the corresponding amino acids from the receptor.…”

“…The recent cloning of the genes encoding the opioid receptors showed that they are members of the seven transmembrane G protein-coupled receptor family (22)(23)(24)(25)(26)(27)(28). There is about 60% amino acid identity among the sequences of the three subtypes , ␦, and .…”