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

Abstract

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind ␦-selective ligands to an inactive ␦/ receptor chimera in which 10 amino acids of the third extracellular loop of the ␦ receptor were replaced by the corresponding amino acids from the receptor (␦/ 291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for ␦-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the sequence of ␦/ 291-300 were randomly reverted to the corresponding ␦ amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for ␦-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 ␦-opioid receptor were present in all revertants. Single and double point mutations were then introduced in ␦/ 291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of ␦-selective ligands. An increased affinity for ␦-selective ligands was observed when the tryptophan 300 ( residue) of ␦/ 291-300 was reverted to a leucine (␦ residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of ␦-selective ligands to their docking site.The opioid receptors are widely distributed throughout the central nervous system and mediate the diverse effects of endogenous opioid peptides and opiate drugs (1). Pharmacological studies have defined at least three classes of opioid receptors, named ␦, , and , that differ in their affinity for ligands and in their distribution in the nervous system (1-6).The antinociception mediated by supraspinal opioid receptors is thought to act via the -opioid receptor subtype (7-10). The numerous side effects accompanying opioid treatment, including respiratory depression and addiction (11), are generally thought to be mediated by the stimulation of receptors. However, there is growing evidence suggesting that selective stimulation of the ␦ receptor may also mediate antinociception (12-19). The strongest indication of an involvement of ␦-opioid receptors in supraspinal antinociception follows from studies with selective antagonists (14 -16). These studies demonstrated that the antinociception produced by intracerebroventricular injection of morphine and [D-Ala 2 ,MePhe 4 ,Gly-ol 5 ]-enkephalin ( agonists) was antagonized by ␤-funaltrexamine and naloxonazine ( antagonists) but not by ICI-174864 (␦ antagonists). Conversely, the antinociception produced by intracerebroventricular injection of DPDPE 1 (␦ agonist) was antagonized by ICI-174864 but not by ␤-funaltrexamine and naloxonazine. Moreover, studies have shown that an antisense oligodeoxynucleotide to the cloned ␦-opioid receptor given intrathecally lowers ␦ but not or spinal (20) and central (21) analgesia. These studies confirm, at the m...

Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

“…Among them, W284(6.58)K and L300(7.35)W mutations had previously been shown to affect significantly the binding of SNC-80 to the δ-opioid receptor. 27 , 28 …”

Structure of the δ-opioid receptor bound to naltrindole

“…These regions interact with ligand moieties that can target binding to a particular opioid subtype. (c) Residues previously characterized as important for opioid subtype selectivity 18-20 are clustered around the upper part of the binding pocket, delineating an “address” region of the receptor. (d) This region is structurally analogous to the allosteric site in muscarinic receptors (blue) suggesting that the high selectivity of muscarinic ligands occupying this space is also a manifestation of the message-address features structurally encoded within GPCRs.…”

Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides

“…Recent structural studies of antagonist-bound human MOR and DOR [30, 47, 48] show that residue 7.35 (Trp318 and Leu300, respectively) lies at the entrance to the binding site and is likely to be a part of the receptor specificity determinant [30, 47, 49, 50]. The Leu7.35Trp mutation in human DOR resulted in decreased affinity for some of the DOR-specific ligands [51], whereas mutations of the conserved tryptophan in MOR suggested the key role of this residue for MOR specificity [52, 53]. As opioid-like peptides from frog skin are thought to be a natural defense mechanism [40, 41], it would thus be advantageous for frogs that these peptides are inactive, although one would expect them to activate the receptors of potential frog predators.…”

“…The substitution of leucine for tryptophan in DOR at position 7.35 is responsible for the binding selectivity both of the DOR-selective antagonist naltrindole and the conformationally constrained DOR-selective peptide agonist [D-Pen2-D-Pen5]enkephalin (DPDPE) [51]. Additionally, the Trp318Leu (7.35) mutation increased the affinity of both of these ligands at MOR [54].…”