Novel 'Restoration of Function' Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding
| Autor: | Edward Roberts, Claes Wahlestedt, Philippe Walker, Shi Yi Yue, Marie Claude Pepin |
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| Rok vydání: | 1997 |
| Předmět: |
Models
Molecular Arginine Stereochemistry Recombinant Fusion Proteins Molecular Sequence Data Biology Biochemistry δ-opioid receptor Structure-Activity Relationship Receptors Opioid delta Humans Amino Acid Sequence Molecular Biology Peptide sequence Gene Library chemistry.chemical_classification Analgesics Ligand binding assay Tryptophan Enkephalins Cell Biology Ligand (biochemistry) Amino acid Benzomorphans Genetic Techniques chemistry Mutagenesis Site-Directed Leucine Enkephalin D-Penicillamine (2 5) |
| Zdroj: | Journal of Biological Chemistry. 272:9260-9267 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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