NOVEL RESTORATION OF FUNCTION MUTAGENESIS STRATEGY TO IDENTIFY AMINO-ACIDS OF THE DELTA-OPIOID RECEPTOR INVOLVED IN LIGAND-BINDING

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 recep tor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids fro m the mu receptor (delta/mu 291-300). This chimera binds a nonselectiv e opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acid s of the mu sequence of delta/mu 291-300 were randomly reverted to the corresponding delta amino acid, Using a ligand binding assay, we scre ened this library to select mutants with high affinity for delta-selec tive ligands, Sequence analysis of these revertants revealed that a le ucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human delta-opioid receptor were p resent in all revertants. Single and double point mutations were then introduced in delta/mu 291-300 to evaluate the contribution of the leu cine 300 and arginine 291 residues for the binding of delta-selective ligands, An increased affinity for delta-selective ligands was observe d when the tryptophan 300 (mu residue) of delta/mu 291-300 was reverte d to a leucine (delta residue). Further site-directed mutagenesis expe riments suggested that the presence of a tryptophan at position 300 ma y block the access of delta-selective ligands to their docking site.