Abstract
The third extracellular loop of the human delta-opioid receptor (hDOR) is known to play an important role in the binding of delta-selective ligands. In particular, mutation of three amino acids (Trp(284), Val(296), and Val(297)) to alanine significantly diminished delta-opioid receptor affinity for delta-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the delta-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the delta-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279-299), hDOR-(283-299), hDOR-(281-297), and hDOR-(283-297). A helical conformation was observed for the longest receptor fragment between Val(283) and Arg(291), whereas a nascent helix occurred in a similar region for hDOR-(281-297). Further removal of N-terminal residues Val(281) and Ile(282) abolished helical conformation completely. Binding of the delta-selective ligand deltorphin II to hDOR-(279-299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the alpha-proton chemical shifts for Trp(284) and Leu(286) in hDOR-(279-299) also accompanied this loss of helical conformation. Large upfield displacement of alpha-proton chemical shifts was observed for Leu(295), Val(296), and Val(297) in hDOR-(279-299) following its interaction with deltorphin II, thus identifying a gain in beta-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281-297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the delta-opioid receptor is presented.
Highlights
The ␦-opioid receptor represents a attractive target for the development of pain therapeutics because of its known ability to mediate analgesia without inducing opiate physical dependence
The results suggest that the interaction of deltorphin II with the human ␦-opioid receptor (hDOR) ECLIII generally destabilizes a helix at the N terminus of the extracellular loop and promotes a -structure at its C terminus
To ensure that the conformation of the hDOR ECLIII peptides were not affected by a change in peptide concentration, CD spectra were obtained for the longest peptide, hDOR(279 –299), in 440 l of hexafluoroisopropyl alcohol (HFIP) and 60 l of H2O using four different peptide concentrations ranging from 2 to 0.1 mM
Summary
The ␦-opioid receptor represents a attractive target for the development of pain therapeutics because of its known ability to mediate analgesia without inducing opiate physical dependence. ␦-selective drugs may possess potential clinical benefits over those currently targeted toward the -opioid receptor (5– 8) These advantages include greater relief of neuropathic pain and reduced respiratory depression and constipation as well as a minimal potential for the development of physical dependence (9). High ␦-receptor selectivity may be attributed to a compact peptide conformation, which is postulated as preferred by the ␦-opioid receptor (17) Evidence gathered from both site-directed mutagenesis and chimeric receptor studies as well as from subsequent molecular modeling studies have pointed to a number of residues located in the transmembrane helices and extracellular loops of human ␦-opioid receptor (hDOR)[1] that are potentially involved in the binding of both ␦-selective agonists and antagonists (18 –21). Four peptides of different length containing the ECLIII and some of the transmembrane amino acids of hDOR were syn-
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