title: Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide.
abstract: The sensitivity of mu and delta receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than delta 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 IC50 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 mu 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 His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide 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 mu opioid receptor.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences and New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: M
authors.initials: M
authors.lastname: Shahrestanifar
authors.forename: W W
authors.initials: WW
authors.lastname: Wang
authors.forename: R D
authors.initials: RD
authors.lastname: Howells
mesh_terms: D000595:Amino Acid Sequence; D000700:Analgesics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004229:Dithiothreitol / Q000494:pharmacology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008024:Ligands; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017465:Receptors, Opioid, delta / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012694:Serine; D014162:Transfection
chemical_list: D000700:Analgesics; D017931:DNA Primers; D004745:Enkephalins; D008024:Ligands; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D012694:Serine; D006639:Histidine; D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D005033:Ethylmaleimide; D004229:Dithiothreitol