title: Novel "restoration of function" mutagenesis strategy to identify amino acids of the delta-opioid receptor involved in ligand binding.
abstract: A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind delta-selective ligands to an inactive delta/mu receptor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids from the mu receptor (delta/mu291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the mu sequence of delta/mu291-300 were randomly reverted to the corresponding delta amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for delta-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human delta-opioid receptor were present in all revertants. Single and double point mutations were then introduced in delta/mu291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of delta-selective ligands. An increased affinity for delta-selective ligands was observed when the tryptophan 300 (mu residue) of delta/mu291-300 was reverted to a leucine (delta residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of delta-selective ligands to their docking site.
authors.affiliation: Astra Research Centre Montreal, Montreal, Québec H4S 1Z9, Canada.
authors.forename: M C
authors.initials: MC
authors.lastname: Pepin
authors.forename: S Y
authors.initials: SY
authors.lastname: Yue
authors.forename: E
authors.initials: E
authors.lastname: Roberts
authors.forename: C
authors.initials: C
authors.lastname: Wahlestedt
authors.forename: P
authors.initials: P
authors.lastname: Walker
mesh_terms: D000595:Amino Acid Sequence; D000700:Analgesics / Q000378:metabolism; D001575:Benzomorphans / Q000378:metabolism; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins; D015723:Gene Library; D005821:Genetic Techniques; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017465:Receptors, Opioid, delta / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D000700:Analgesics; D001575:Benzomorphans; D004745:Enkephalins; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D020881:Enkephalin, D-Penicillamine (2,5)-; C027248:bremazocine