Involvement of Trp-284, Val-296, and Val-297 of the Human δ-Opioid Receptor in Binding of δ-Selective Ligands

Abstract

Given the high homology in amino acid sequence between the ␦-opioid receptor and the two other types ( and ), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human ␦ receptor responsible for the selectivity of ␦-selective ligands, two different ␦/ chimeras were constructed. In the first one, the ␦ sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the ␦ receptor were replaced by the counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different ␦-selective ligands. These results suggested that the region of the third extracellular loop of the ␦ receptor is crucial for the type selectivity. Opioid receptors are cell surface glycoproteins that constitute specific binding sites for a variety of compounds used for treating pain. Extensive pharmacological studies led to the definition of three opioid receptor types, , , and ␦ (1, 2). Indeed, the availability of highly selective ligands permitted a better characterization of these three types of receptors. In the past few years, cDNAs encoding the , , and ␦ of different species have been cloned (reviewed in Ref.3). However, human receptors represent the ultimate therapeutic targets. Thus, the cloning of human , , and ␦ cDNAs (4 -6) provided particularly relevant tools for opioid drug discovery.Analysis of the predicted amino acid sequences of the three human opioid receptors has shown that these receptors have characteristics common to the guanine nucleotide-binding regulatory protein-coupled receptors with an extracellular N-terminal domain, a cytoplasmic C-terminal domain, and seven putative transmembrane domains (7,8). Moreover, given their high degree of identity (approximately 60%) with the highest similarity in the transmembrane domains and intracellular loops, distinct residues in these receptors may confer their selectivity to some ligands. Binding sites of selective ligands most probably reside, at least in part, in the divergent regions that are the extracellular loops and the N-terminal domain. The C-terminal domain is also a divergent region, but its involvement in binding of ligands is unlikely.All of the opioid analgesics currently used clinically interact with the receptor and are known to induce severe side effects and addiction (9, 10). These major disadvantages were less pronounced with the use of a number of agonists selective for the receptor which also display analgesic properties (11-13), suggesting that some effective analgesics with minimal side effects, selectively interacting with one of the three types of opioid receptors, may be developed. Particularly, ␦ receptors that bind enkephalin-like peptides with high affinity have been proposed to mediate analgesia (14 -16) and to induce weak opiate physical dependence (17, 18), making them an interesting t...

Novel “Restoration of Function” Mutagenesis Strategy to Identify Amino Acids of the δ-Opioid Receptor Involved in Ligand Binding

“…This protein binds nonselective opioid ligands but is devoid of affinity for ␦-selective ligands. Our results are in agreement with results from previous studies using /␦ or ␦/ chimeric receptors that have shown that ␦-selective ligands interact mainly with the region containing the sixth transmembrane domain and the third extracellular loop of the ␦-opioid receptor (35)(36)(37)(38)(39)(40)50). In this study, we have delimited this region to 10 amino acids that are located between arginine residue at position 291 and leucine residue at position 300.…”

“…A mutant of hDOR with an alanine residue at position 291 instead of an arginine binds ␦-selective ligand DPDPE and SNC-80 with wild-type affinity suggesting that arginine 291 (␦ residue) is not critical for the binding of ␦-selective ligand (35). However, the adjacent residue at position 292 is also an arginine which may compensate for the substitution at position 291.…”

“…Construction of the ␦/ 291-300 Chimera and Random Mutagenesis-Evidence from different groups has suggested that the third extracellular loop of ␦-opioid receptors is involved in the binding of selective ligands (35)(36)(37)(38)(39)(40)50). A chimeric ␦-opioid receptor in which 10 amino acids of the third extracellular loop (amino acids 291 to 300) were replaced by the corresponding amino acids from the receptor was constructed.…”

“…The construction of chimeric receptors is a powerful approach to investigate the structural basis for the subtype specificity of G protein-coupled receptor (29 -34). Previous studies from our group (35) and others (36 -40) have demonstrated, using chimeras, the importance of the third extracellular loop of the ␦-opioid receptor for ␦ ligand selectivity.…”

The Seventh Transmembrane Domains of the δ and κ Opioid Receptors Have Different Accessibility Patterns and Interhelical Interactions

“…Thus, the TM7 may start at different positions in different GPCRs. The finding that Val7.31(296), Val7.32(297), and Leu7.35(300) are crucial in selectivity of the δOR ligand binding (57,58) is consistent with these residues being part of the binding pocket. (4,59).…”