Studies on Inhibition of μ and δ Opioid Receptor Binding by Dithiothreitol and N-Ethylmaleimide

Abstract

The sensitivity of and ␦ receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC 50 values ‫؍‬ 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than ␦ receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC 50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His 223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against Nethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the opioid receptor.Three major types of opioid receptor, ␦, , and , have been cloned and characterized extensively (reviewed in Refs. 1 and 2). There is approximately 60% amino acid sequence identity between the opioid receptor types. The ␦, , and opioid receptors have unique ligand specificities, anatomical distributions, and physiological functions (3). Morphine, related opioid drugs, and the endogenous opioid peptides activate signal transduction pathways by binding to opioid receptors (4), which are members of the G protein-coupled receptor family (5). G protein-coupled receptors are seven-transmembrane domain (TM) 1 proteins that mediate signal transduction across the plasma membrane. The ligands approach and engage the receptor from the extracellular side, and receptor activation results in the coupling to heterotrimeric G proteins on the intracellular face of the membrane. Opioid receptor types interact with multiple G proteins (6 -8) to regulate adenylyl cyclase, Ca 2ϩ channels, and K ϩ channels. It has been known from early studies on the characterization of opioid receptors that specific binding is inhibited by sulfhydryl reagents, such as iodoacetamide, N-ethylmaleimide (NEM), and p-hydroxymercuribenzoate (9 -11). Preincubation with opioid ligands protected against receptor inactivation, suggesting that the sensitive sulfhydryl group was located within or near the binding site. Evidence has been obtained that analogs of Leu-enkephalin and morphine, containing activated sulfhydryl groups, form mixed disulfide linkages with opioid receptors (12, 13). The covalently bound agonists caused receptor activation that persisted following extensive washing, yet was naloxone-reversible. The result...

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

“…1B), the secretion signal of the S. cerevisiae c~-mating factor was used to facilitate membrane translocation of the receptor. In both cases, part of the amino-terminus of the receptor was deleted since it has been reported that this had no effect on ligand binding [20][21][22]. DNA fragments comprising the fusion genes flanked by 5' and 3' AOX1 sequences were electro-transformed into the GSll5 (his4) and the protease-deficient SMDl168 (his4, pep4) P. pastoris strains and His + transformants were selected.…”

The N-Terminal End Truncated Mu-Opioid Receptor: from Expression to Circular Dichroism Analysis

“…This was particularly true for the peptidic agonist [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) for which a sevenfold reduction in affinity was observed. In general, deletion of the N-terminal domain of the mu-opioid receptor produced little effect on receptor function when it was expressed in higher eukaryotic cells; however, decreased affinities have also been observed for specific ligands with a more stamped effect for agonists and without effect on signal transduction [15,22,[25][26][27]. In addition, analysis of N-glycosidase Ftreated membranes indicated that N-glycan chains within the amino-terminal domain of MOR did not contribute significantly to ligands affinities [27].…”

Mutational analysis of the structure and function of opioid receptors

“…However, since NEM could alkylate the imidazole group of His in addition to the sulfhydryl group of Cys, another probable site for NEM's effect was identified by the substitution of the His residue within e2 loop of the opioid receptor, His 223 , with Ser. 66 Whether these amino acids are directly involved in the ligand recognition or involved in the maintenance of the structural integrity for highaffinity binding could not be distinguished. The importance of the overall arrangement of the seven-helix bundle in the high affinity opioid ligand binding could be demonstrated further by the introduction of a highaffinity zinc switch in the opioid receptor.…”