Novel 'restoration of function' mutagenesis strategy to identify amino acids of the δ-opioid receptor involved in ligand binding

Marie Claude Pepin, Shi Yi Yue, Edward Roberts, Claes Wahlestedt, Philippe Walker

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51 Scopus citations


A novel 'restoration of function' mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind δ-selective ligands to an inactive δ/μ receptor chimera in which 10 amino acids of the third extracellular loop of the δ receptor were replaced by the corresponding amine acids from the μ receptor (δ/μ291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for δ- selective ligands. A library of mutants was generated in which some of the 10 amino acids of the μ sequence of δ/μ291-300 were randomly reverted to the corresponding δ amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for δ-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 δ-opioid receptor were present in all revertants. Single and double point mutations were then introduced in δ/μ291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of δ-selective ligands. An increased affinity for δ-selective ligands was observed when the tryptophan 300 (μ residue) of δ/μ291-300 was reverted to a leucine (δ residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of δ-selective ligands to their docking site.

Original languageEnglish (US)
Pages (from-to)9260-9267
Number of pages8
JournalJournal of Biological Chemistry
Issue number14
StatePublished - Apr 4 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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