Mapping the receptor domains critical for the binding selectivity of δ-opioid receptor ligands

While a good deal has been learned about determinants of high affinity ligand/receptor interactions in G-protein-coupled receptors, less is known about mechanisms of ligand selectivity. The opioid receptors offer an excellent opportunity to study the mechanisms whereby structurally very similar receptors discriminate between different but structurally highly related ligands. In the current study, we use a series of chimeric constructs between the δ-opioid receptor and either the μ- or the κ-opioid receptors to investigate the structural basis of binding selectivity of multiple classes of δ-opioid receptor selective ligands. Our results demonstrate that a region containing the sixth transmembrane domain (TM6) and the third extracellular loop (EL3) in the δ-opioid receptor is absolutely critical for δ-opioid receptor selectivity. The introduction of this region into the κ-opioid receptor is sufficient to impart a δ profile for δ-opioid receptor selective alkaloids such as naltrindole and naltriben. In order to locate the amino acid residues that may be involved in ligand selectivity in TM6 and EL3 of the δ-opioid receptor, several mutations were introduced into that region. These mutations showed differential effects on peptide and alkaloid ligands. In addition, none of the individual mutations alone could account for the changes exhibited by the chimeric receptors. We conclude that the selectivity of most δ-opioid ligands is achieved through their interaction with many different residues in the TM6/EL3 region. Our results also support a view that the extracellular domains of peptide receptors may provide the basis of a sorting mechanism for ligand selectivity.